Hydrolysis of L-Arginine –
Chemical and Enzymatic Catalysis
Pedro Miguel Cabral Campello Duarte Turras
Dissertação para obtenção do Grau de Mestre em
Engenharia Biológica
Júri
Presidente:
Professor Luís Joaquim Pina da Fonseca (IST)
Orientadores: Professor Luís Joaquim Pina da Fonseca (IST)
Professor Maurice Franssen (WUR)
Vogais:
Doutor Pedro Fernandes (IST)
Professor José António Leonardo dos Santos (IST)
Setembro 2008
Acknowledgments
I would like to thank my supervisor, Paul Könst, for teaching, supporting and helping
me through the course of this project.
I would also like to thank Professor Maurice Franssen for his guidance and the
opportunity to accomplish such gratifying work in a great research group.
Finally, I would like to thank everyone at the Valorisation of Plant Production Chains
group at Wageningen University. Not only was their advice and help essential during the
course of the project – a special word to Dr. Elinor Scott and Alniek van Zeeland – but I will
also keep everything I have learned during progress meetings, informal meetings and talks.
i
Resumo
O esgotamento das reservas de combustíveis fosseis e a crescente preocupação
com as emissões de CO2 levaram à procura de uma fonte alternativa para obtenção
químicos funcionalizados em larga escala. O projecto N-ergy tem como principal objectivo a
utilização de resíduos agrícolas como principal matéria-prima na produção simultânea de
etanol e químicos azotados. Um dos passos intermediários no processo previsto é a
conversão de L-arginina em L-ornitina.
A hidrólise de L-arginina em L-ornitina e ureia pode ser catalisada quimicamente ou
enzimaticamente. A catálise quimica da reacção pode ser conseguida na presença de
condições fortemente ácidas ou alcalinas. A biocatálise da reacção pode ser conseguida
através da enzima arginase (EC 3.5.3.1).
A hidrotermólise da L-arginina em condições alkalinas levou à produção de L-ornitina
e de vários produtos secundários. O rendimento máximo de obtenção de L-ornitina foi de
15.2% a 150°C com um pH inicial ajustado a 12.0 com hidróxido de sódio. A adição de um
catalisador básico sólido, zeólito NaY, mostrou um efeito catalítico limitado mesmo a
elevadas concentrações. A adição de sais de diferentes metais não influenciou a reacção.
Arginase de Bacillus subtilis foi imobilizada com sucesso em três suportes activados
com grupos epóxi – Seapabeads EC-HFA, Sepabeads EC-EP e Eupergit C 250 L. Após uma
hora de incubação à temperatura ambiente na presença de cada suporte nenhuma arginase
foi detectada no sobrenadante. A ligação covalente aos três suportes testados não levou a
um aumento da estabilidade térmica da arginase e provocou uma redução na actividade
catalitica de 40% a 60%.
Palavras-chave: hidrólise da L-arginina; zeolito NaY; arginase; immobilização de enzimas.
iii
Abstract
The depletion of fossil feedstocks and growing concern over CO2 emissions has led
to the search for an alternative source for bulk functionalized chemicals. The N-ergy project
has the ultimate aim of utilizing agricultural waste streams as the main raw material for the
simultaneous production of ethanol and bulk nitrogen-functionalized chemicals. One of the
intermediate steps in the projected process is the conversion of L-arginine to L-ornithine.
The hydrolysis of L-arginine to L-ornithine and urea can be chemically or
enzymatically catalysed. The chemical catalysis of the reaction can be achieved in the
presence of strong acidic or alkaline conditions. The biocatalysis of the reaction can be
achieved by the enzyme L-arginase (EC 3.5.3.1).
The hydrothermolysis of L-arginase in alkaline conditions led to the production of Lornithine and numerous secondary products. The maximum L-ornithine yield obtained was of
15.2% at 150°C with initial pH adjusted to 12.0 with sodium hydroxide. The addition of a solid
basic catalyst, NaY zeolite, showed limited catalytic effect even at high concentrations. The
addition of various metal salts did not influence the reaction.
Bacillus subtilis arginase was successfully immobilized in three different epoxyactivated supports – Seapabeads EC-HFA, Sepabeads EC-EP and Eupergit C 250 L. After
one hour of incubation at room temperature in the presence of each support no arginase was
detected in the supernatant. The covalent-binding to the three tested supports did not lead to
a significant increase in arginase’s thermal stability and led to a decrease in catalytic activity
(recovered activity of 40%-60%).
Key words: L-arginine hydrolysis; NaY zeolite; arginase; enzyme immobilization.
v
Table of Contents
1. Introduction
1.1. Motivation and Background
1
1
1.1.1. Biomass as a Source of Bulk Chemicals
1
1.1.2. Petrochemical Approach vs Bio-Refinery Approach
2
1.1.3. The N-ergy Project
3
1.1.4. Arginine Conversion
4
1.1.5. Product Applications
5
1.2. Chemical Catalysis
7
1.2.1. Chemically Catalyzed Hydrolysis of Arginine
7
1.2.2. Heterogeneous Catalysis – the use of Zeolites
9
1.3. Enzymatic Catalysis
10
1.3.1. Enzymatic Hydrolysis of L-Arginine – Arginase
10
1.3.2. Industrial Biocatalysis
14
1.3.3. Biocatalyst Immobilization
15
1.4. Aim of this Study
2. Chemical Hydrolysis of L-Arginine
2.1. Materials and Methods
18
21
21
2.1.1. Reagents
21
2.1.2. Solutions
22
2.1.3. Equipment
22
2.1.4. Analytical Techniques
23
2.1.5. Hydrothermolysis Experiments
24
2.1.6. Metal Salt Catalysis Experiments
25
2.1.7. Zeolite Catalysis Experiment
25
2.2. Results and Discussion
26
2.2.1. Hydrothermolysis Experiments
26
2.2.2. Metal Salt Catalysis Experiments
31
2.2.3. Zeolite Catalysis Experiment
32
2.2.4. Analytical Methods
32
3. Enzymatic Hydrolysis of L-Arginine
3.1. Materials and Methods
35
35
3.1.1. Bacillus Subtilis Arginase
35
3.1.2. Epoxy-activated Supports
35
3.1.3. Reagents
35
3.1.4. Solutions
36
3.1.5. Equipment
36
3.1.6. Analytical Techniques
37
3.1.7. Preparation of Arginase Stock Solution
38
vii
3.1.8. Immobilization of Arginase in Different Epoxy-activated Supports
38
3.1.9. Thermal Stability of Immobilized Arginase
40
3.2. Results and Discussion
40
3.2.1. Characterization of the Arginase Stock Solution
40
3.2.2. Immobilization of Arginase in Epoxy-activated Supports
42
3.2.3. Recovered Activity of Immobilized Arginase
45
3.2.4. Thermal Stability of Immobilized Arginase
46
3.2.4. Analytical Methods
48
4. Conclusions and Future Perspectives
51
5. References
53
Appendix A – Gradient Curves
57
Appendix B – NMR Spectra of L-Arginine
58
Appendix C – NMR Spectra of L-Ornithine
60
Appendix D – NMR Spectra of 3-Aminopiperid-2-one
62
Appendix E – NMR Spectra of Putrescine
64
Appendix F – NMR Spectra of 30 h Reaction Mixture
66
Appendix G – Certificates of Analysis
68
Appendix H – Recovered Support Masses
70
viii
Abbreviations and Symbols
CGP
Cyanophycin Granule Peptide
DEPT
Distortionless Enhancement by Polarization Transfer
DMF
Dimethylformamide
HPLC
High Performance Liquid Chromatography
NMR
Nuclear Magnetic Resonance
TSP
2,2,3,3-d(4)-3-(trimethylsilyl)propionic Acid
Tables Index
Table 1.1. Cost breakdown of functionalized and non-functionalized bulk chemicals based
4
on oil at $40 a barrel (adapted from Sanders et al. ).
2
® 11
Table 1.2. Thermal properties of Nylon-6,6 and Stanyl
.
6
Table 1.3. Techniques for arginase immobilization described in literature. The different
techniques are listed in chronological order, from the oldest work to the most
recent.
13
Table 1.4. Advantages and limitations associated to the use of immobilized enzymes
42
(adapted from Cabral et al. )
15
Table 2.1. Reagents utilized during the course of the chemical hydrolysis of arginine
experiments.
20
Table 2.2. Solutions prepared during the course of the chemical hydrolysis of L-arginine
experiments.
21
Table 2.3. Gradient of eluents applied during HPLC analysis.
23
Table 3.1. Reagents utilized during the course of the enzymatic hydrolysis of L-arginine
experiments.
33
Table 3.2. Solutions prepared during the course of the enzymatic hydrolysis of L-arginine
experiments.
34
Table 3.3. Gradient of eluents applied during HPLC analysis.
35
Table H.1. Initial and recovered masses of the three tested epoxy-activated supports,
including duplicates, during the immobilization and epoxy groups blockage
steps.
67
ix
Figures Index
Figure 1.1. Production of functionalized chemicals from naphta and from biomass
4
(Sanders et al. ).
2
Figure 1.2. Conversion of biomass to N-functionalized chemicals and ethanol – the N-ergy
project.
4
Figure 1.3. Conversion of arginine to 1,4-butanediamine through hydrolysis to ornithine
and decarboxylation.
5
Figure 1.4. Comparison of the petrochemical and bio-based approaches for 1,4-butane
4
diamine bulk production (adapted from Sanders et al. ).
5
Figure 1.5. Synthesis of nylon-4,6 from 1,4-butanediamine and adipic acid.
6
Figure 1.6. Acid catalyzed hydrolysis of arginine.
7
Figure 1.7. Alkali catalyzed hydrolysis of arginine
8
Figure 1.8. Thermohydrolysis of urea: (a), reversible conversion of urea to cyanate and
ammonia; (b), hydrolysis of cynate to ammonia and carbon dioxide.
8
Figure 1.9. Topology diagram of rat liver arginase. Relative location of metal ligands is
28
11
indicated by grey circles (adapted from Kanyo et al.) .
Figure 1.10. Proposed mechanism of rat liver arginase-catalysed arginine hydrolysis by
28
metal-activated solvent . The α-amino and α –carboxylate groups are omitted
for clarity.
12
Figure 1.11. Methods of immobilization of biocatalysts.
16
Figure 1.12. Mechanism of immobilization of proteins on epoxy-activated Supports. The
covalent reaction between soluble enzyme and epoxy support is extremely
slow, but the previous adsorption to the support allows a faster covalent
43
reaction (adapted from Mateo et al. ).
17
Figure 1.13. Functional groups of Sepabeads
45
supports .
®
®
EC-EP and Sepabeads
®
EC-HFA
Figure 2.1. Parr Series 500 Multiple Reactor System with 4871 Process Controller.
18
22
Figure 2.2. Time course of L-arginine consumption under different experimental conditions.
The percentages are based on the total concentration of amino acids (Larginine and L-ornithine) in the reaction mixture at t=0. Error bars calculated
using standard deviation of the two duplicate experiments.
26
Figure 2.3. Time course of L-ornithine formation under different experimental conditions.
The percentages are based on the total concentration of amino acids (Larginine and L-ornithine) in the reaction mixture at t=0. Error bars calculated
using standard deviation of the two duplicate experiments.
27
x
Figure 2.4. Example of a typical HPLC chromatogram obtained after heating a L-arginine
solution for 20h in the described experimental settings. The identified peaks
correspond to: (A) L-aspartic acid (internal standard); (B) hydrolyzed excess
derivatization reagent; (C) L-citrulline; (D) L-arginine; (E) L-ornithine; (F)
ammonia; (G) 3-aminopiperid-2-one; (H) retention time of putrescine
elution(none was detected in the samples). All unidentified peeks correspond
to impurities originating either from the solutions utilized or from the HPLC
system.
28
Figure 2.5. Evolution of the pH during the course of the reaction (125°C, initial pH of 12)
and its comparison with ornithine concentration. Error bars were omitted for
clarity.
29
Figure 2.6. Effect of the presence of different metal salts (equimolar concentrations – 25
mM) on the L-arginine thermohydrolysis reaction. The percentages are based
on the total concentration of amino acids (L-arginine and L-ornithine) in the
reaction mixture at t=0.
30
Figure
2.7. Effect of different NaY zeolite concentrations on the L-arginine
thermohydrolysis reaction. The percentages are based on the total
concentration of amino acids (L-arginine and L-ornithine) in the reaction
mixture at t=0. Error bars calculated using standard deviation of the two
duplicate experiments.
31
Figure 2.8. Detail from a HPLC chromatogram for a 25 mM 3-aminopiperid-2-one solution.
The identified peaks correspond to: (O) L-ornithine; (A) ammonia; (L) 3aminopiperid-2-one. All unidentified peeks correspond to impurities originating
either from the solutions utilized or from the HPLC system.
32
Figure 3.1. Cole-Parmer Roto-Torque model 7637-10 Heavy Duty Rototator.
34
Figure 3.2. SDS-PAGE gel of the original arginase solution. Lane (A) corresponds to a
100X dilution while lane (B) corresponds to a 200X dilution. Lane (M) contains
the molecular markers identified with the corresponding molecular weights in
Daltons.
39
Figure 3.3. Protein concentration in the supernatant during the course of arginase
immobilization in different epoxy-activated supports. The percentages are
based on the initial (t=0) protein concentration. Error bars calculated using
standard deviation of the two duplicate experiments.
40
Figure 3.4. Arginase activity in the supernatant during the course of arginase
immobilization in different epoxy-activated supports. The percentages are
based on the initial (t=0) protein concentration. Error bars calculated using
standard deviation of the two duplicate experiments.
41
Figure 3.5. Protein content in the filtration supernatant after each step of the beads
washing procedure. Error bars calculated using standard deviation of the two
duplicate experiments.
42
Figure 3.6. Recovered activity of arginase immobilized in different epoxy supports.
Percentages are calculated by comparison with the soluble form. Error bars
calculated using standard deviation of the two duplicate experiments.
43
Figure 3.7. Evolution of residual activity of arginase immobilized in different epoxy
supports compared to soluble arginase during the course of 24 hours
incubation at 60°C. The percentages are based on the initial (t=0) enzyme
activity.
45
xi
Figure 3.8. Example of a typical HPLC chromatogram obtained for an activity assay
sample.
46
Figure A.1. Different gradient curves identified by the input number.
13
54
Figure B.1. C-NMR spectrum of L-arginine.
55
Figure B.2. DEPT spectrum of L-arginine.
55
1
Figure B.3. H-NMR spectrum of L-arginine.
Figure C.1.
13
C-NMR spectrum of L-ornithine.
Figure C.2. DEPT spectrum of L-ornithine.
1
Figure C.3. H-NMR spectrum of L-ornithine.
Figure D.1.
13
C-NMR spectrum of 3-aminopiperid-2-one.
Figure D.2. DEPT spectrum of 3-aminopiperid-2-one.
56
57
57
58
59
59
1
60
Figure E.1. C-NMR spectrum of putrescine.
13
61
Figure E.2. DEPT spectrum of putrescine.
61
Figure D.3. H-NMR spectrum of 3-aminopiperid-2-one.
1
Figure E.3. H-NMR spectrum of putrescine.
Figure F.1.
13
C-NMR spectrum of 30h reaction mixture.
Figure F.2. DEPT spectrum of 30h reaction mixture.
1
Figure F.3. H-NMR spectrum of 30h reaction mixture.
xii
62
63
63
64
Introduction
1. Introduction
1.1. Motivation and Background
1.1.1. Biomass as a Source of Bulk Chemicals
Oil and other fossil fuels still are the main sources for energy, transport fuels and
(bulk) carbon-based chemicals, but the need for an alternative is undeniable. The depletion
of fossil feedstocks, the increasing oil and transport fuels prices, together with the growing
concern over climate changes and other consequences of CO2 emissions, has led to the
search for a cheap and environmentally friendly alternative.
Focusing on the production of chemicals, the replacement of fossil feedstocks with
CO2 neutral biomass offers a wide array of advantages: it’s a renewable resource which is
currently being produced in large amounts (170,000 million tones per annum accordingly to
1
1992 estimates by Eggersdorfer et al. ); it’s a relatively cheap resource – protein rich waste
streams are generated by plenty of industries, including bio-fuel production processes; the
use of biomass instead of fossil fuels would considerably reduce greenhouse gases
emissions. Moreover, it should be noted that in the long term, after the complete exhaustion
of fossil resources reservoirs, biomass will be the only available raw material for the bulk
production of organic carbon-based chemicals.
Bio-refinery can be defined as the fractionation of biomass into components that, after
further transformation and separation, can be used as final end products. When producing
chemicals through bio-refinery two options can be considered: the development of “new”
chemicals or the production of chemicals similar to the traditionally obtained from the
petrochemical industry. In the first case the focus is mainly on the use of carbohydrates as
2
raw materials in the production of new polymers, such as thermoplastic resins and multi3
application biodegradable additives . While this option can provide chemicals and materials
with unique structures and properties, the introduction of bio-based alternative products
usually requires the re-engineering and optimization of whole production chains and implies a
considerable financial risk. On the other hand, the bio-based synthesis of existing chemicals
can profit from existing infrastructures. These structures – utilized in the production of bulk
chemicals and materials based on fossil fuels – are already highly optimized and do not
require large capital injections. Thus, in the short to mid-term it is expected that there will be a
strong investment in the development of bio-based bulk chemicals identical to those of
petrochemical origin that can be easily integrated in existing processes.
1
Introduction
1.1.2. Petrochemical Approach vs Bio-Refinery Approach
Despite its increasing prices, oil is still a reliable and inexpensive source of carbonbased chemicals. However, the absence of functionalized substances in oil derivatives like
naphta, which is the main raw material used in the production of many chemicals, makes biorefinery a solid alternative to the petrochemical approach in the production of functionalized
chemicals.
4
Figure 1.1. Production of functionalized chemicals from naphta and from biomass (Sanders et al. ).
As shown in figure 1.1, only non-functionalized chemicals (olefins, parafins, etc.) can
be produced from naphtha without major enthalpy changes that require a significant heat
transfer. The preparation of functionalized chemicals from simple molecules, such as
ethylene, often implies the use of large amounts of energy, additional process steps,
dangerous
working
conditions
(high
temperatures,
high
pressures,
corrosive/toxic
substances) and large amounts of various reagents (ammonia, chlorine). The differences in
production costs of non-functionalized and (O-,N-)functionalized chemicals are illustrated in
table 1.1.
Table 1.1. Cost breakdown of functionalized and non-functionalized bulk chemicals based on
4
oil at $40 a barrel (adapted from Sanders et al. ).
Cost type
Non-functionalized (€/ton)
Functionalized (€/ton)
Raw materials
200
650
Capital
300-500
400-650
Operational
50
50
Recovery
50-100
50-100
Total
725
1300
Comparatively, in the biomass mixture it is possible to find a variety of already
functionalized components, considerably reducing the heat necessary to introduce the
functionality. Additionally, these molecules have chemical compositions and structures similar
to the desired products, decreasing the number of conversion steps required. Hence, a well
2
Introduction
designed process using biomass as a raw material should be able to produce bulk
functionalized chemicals at low costs, without all the ecological complications associated with
the use of fossil resources.
A perfect example is the use of amino acids as raw materials in the production
nitrogen-containing bulk chemicals. This concept is one of the foundations of the N-ergy
project, in which this study is incorporated.
1.1.3. The N-ergy Project
The N-ergy project is a long term research project with the ultimate aim of developing
an economically feasible process for the combined production of ethanol and nitrogen
containing chemicals, utilizing biomass as the main raw material. Both end products can be
used as a resource (ethanol) or as a replacement (N-chemicals) for nitrogen containing
products that are traditionally of petrochemical origin. In addition, the produced ethanol can
be utilized as a transport fuel.
Initially, a fermentation step should convert agricultural waste products to ethanol and
insoluble cyanophycin granule peptide (CGP). This fermentation is performed by recombinant
species capable of simultaneous production of ethanol and CGP. Since cyanobacteria – the
natural producers of CGP – are not suitable for the bulk production of large quantities of
5
CGP , cyanobacteria genes have been heterologously expressed in heterotrophic bacteria
6,7
and plants, with high percentages CGP per cell dry mass being obtained . Therefore, it is
expected that the genes responsible for CGP production can be successfully expressed in
species that are established ethanol producers, such as yeast and filamentous fungi. Within
the N-ergy project, research is being performed in order to obtain recombinant strains of
Saccharomyces cerevisiae and Rhizopus oryzae that are able to accumulate large volumes of
CGP.
The synthesis of CGP by cells is heavily dependent on the presence of certain amino
8
acids, particularly arginine, in the growing medium . This can make the selection of a cheap
and reliable nutrient source a complicated task. However, recent studies have acknowledged
8
protoamylasse as a suitable medium for large-scale cyanophycin production . Protamylasse,
or potato juice concentrate, is an abundant waste stream originating from the industrial
production of starch from potatoes. Its composition includes soluble peptides, amino acids,
with asparagine and aspartate as the main components, organic acids, carbohydrates, salts,
and minerals. The high arginine, aspartate, and asparagine contents of protamylasse are of
particular interest for the production and accumulation of CGP.
Cyanophycin granule peptide is a nonribosomally synthesized biopolymer, which
consists of equimolar amounts of arginine and aspartic acid arranged as a polyaspartate
backbone, with arginine residues linked to the β-carboxyl group of each aspartate by its α9
amino group . In nature this polymer is synthesized by most cyanobacteria, as previously
3
Introduction
stated, and is accumulated as granules in the cells cytoplasm. Although originally thought to
be insoluble in water at neutral pH, later studies detected the formation of a soluble form of
10
CGP with identical chemical composition . Naturally, the insoluble form should allow a much
simpler separation of the polymer from the fermentation broth. The reasoning behind the
different behavior of the two CGP forms is not clear at this point.
After extraction, cyanophycin should be converted to nitrogen-functionalized
chemicals. In a first step, the polymer is completely hydrolyzed to its monomers: aspartic acid
and arginine. After separation, both amino acids should undergo further transformations until
the desired N-functionalized chemicals are obtained. Figure 1.2 illustrates the different steps
in the conversion of biomass to the target products.
O
O
H2N
Ethanol
Acrylamide
O
O
OH
OH
N
H
O
*
BIOMASS
NH2
OH
HO
*
Aspartic Acid
H2N
OH
Aminopropanol
n
NH
O
HO
O
NH2
H2N
O
NH2
Urea
HN
NH2
HN
HN
NH2
Cyanophycin
NH2
HN
H2N
Arginine
1,4-Butanediamine
Figure 1.2. Conversion of biomass to N-functionalized chemicals and ethanol – the N-ergy project.
1.1.4. Arginine Conversion
The focus of this study is on the arginine route. In order to be converted to
1,4–butanediamine (a building block for nylon-4,6), arginine has to undergo two main
transformations: the hydrolysis of arginine to ornithine and urea; and the decarboxylation of
ornithine to 1,4–butanediamine and carbon dioxide (figure 1.3). Both steps can be catalyzed
chemically or enzymatically. The two catalysis methods applied to the first step are explored
and compared during this study.
4
Introduction
HO
NH2
HO
Urea
O
NH2
CO2
O
H2N
H2O
HN
1,4-Butanediamine
H2N
NH2
NH2
HN
Ornithine
Arginine
Figure 1.3. Conversion of arginine to 1,4-butanediamine through hydrolysis to ornithine and
decarboxylation.
Although this work is contained in the N-ergy project, the methods utilized to convert
L-arginine to L-ornithine and 1,4-butanediamine are not limited to the treatment of
cyanophycin and can be applied to any L-arginine source. In fact, many agriculture based
industries, including the production of biofuels, generate waste streams with high protein
content. A good example is the protein rich (50%) soybean meal obtained by grinding the
flakes that remain after extraction of most of the oil by solvent or mechanical process. The
protein can be hydrolyzed to its amino acids which, after separation, can be individually
treated.
1.1.5. Product Applications
Putrescine (commercial name of 1,4-butanediamine) is the product obtained from
arginine with the highest market value. However, secondary products like urea and ammonia
(mainly originating from the spontaneous hydrolysis of urea at high temperatures) can also
provide a significant financial return.
Currently 1,4-butanediamine is produced using chemicals of petrochemical origin:
propylene, ammonia and hydrogen cyanide. This process and its comparison with the
proposed bio-based approach are illustrated in figure 1.4. As can be observed, the suggested
process requires less conversion steps, lower working temperatures and is environmentally
friendly. These advantages were previously discussed in section 1.1.2.
HO
N
+ NH3 + 1.5 O2
+
CH4
+
NH3
HC N
NH2
O
NH2
H2N
1,4-Butanediamine
HN
- Urea
- CO2
NH2
HN
Petrochemical
Products
Arginine
(Biomass)
Figure 1.4. Comparison of the petrochemical and bio-based approaches for 1,4-butanediamine bulk
4
production (adapted from Sanders et al. ).
5
Introduction
Putrescine has the potential to be used as an intermediate in a large array of
industries, including the pharmaceutical industry, the agrochemical industry and the textile
industry, among others. However, at the moment its only relevant application is its use as a
co-monomer, along with adipic acid, in the production of nylon-4,6 (figure 1.5),
®
commercialized by DSM under the trade name of Stanyl .
H2N
NH2
+
COOH
HOOC
1,4-Butanediamine
-H2O
*
H
N
N
H
Adipic acid
CO
CO
n *
Nylon-4,6
Figure 1.5. Synthesis of nylon-4,6 from 1,4-butanediamine and adipic acid.
Stanyl
®
is a high performance, high temperature polyamide characterized for its
strong mechanical properties at high temperatures, excellent resistance to wear, low friction,
easy processing and exceptional design freedom. This properties make this polymer an
excellent alternative to the popular nylon-6,6 in processes where high temperatures are
utilized (table 1.2). Key applications for this material include the substitution of metal
components in the automotive industry and Electric & Electronics11.
Table 1.2. Thermal properties of Nylon-6,6 and Stanyl
Properties
Melting point (°C)
3
Density (kg/m )
Nylon-6,6
Stanyl
265
295
1140
1180
6
>15
® 11
.
®
-1
Crystallinity rate (sec ):
-at 200°C
-at 230°C
0.7
10
Glass transition temperature
65
78
As previously stated, urea and ammonia produced in considerable amounts can
provide a significant source of income. Although some minor applications (like the bulk
chemicals industry) are also relevant, both chemicals are mainly utilized as fertilizers. Urea
and ammonia are an excellent nitrogen source for plant growth that offer several advantages:
are relatively cheap products with inexpensive handling, storage and transportation costs; can
be applied to soil as a solid, solution or even spray to certain crops; involve no fire hazard and
little explosion hazard. The prices of these products are rapidly increasing at the moment,
12
with urea and ammonia prices close to reaching $800/tonne and $600/tonne respectively .
6
Introduction
1.2. Chemical Catalysis
1.2.1. Chemically Catalyzed Hydrolysis of Arginine
The hydrolysis of arginine to ornithine is catalyzed in the presence of strongly acid or
alkaline conditions
13,14
.
The acid hydrolysis is a considerably slow process even at high temperatures –
Murray et al.
13
obtained a conversion of 98% of L-arginine to L-ornithine after 120 hours of
heating at 176°C in the presence of 6 N hydrochloridic acid. The acid catalyzed reaction
(figure 1.6) leads solely to the formation of L-ornithine, no undesirable by-products are
produced.
HO
NH2
HO
O
+
H
NH2
+
O
O
H2O
HN
+
H2N
H2N
NH2
NH2
Urea
HN
Ornithine
Arginine
Figure 1.6. Acid catalyzed hydrolysis of arginine.
Alkaline catalyzed hydrolysis (figure 1.7) can be performed faster and at lower
temperatures, with the disadvantage of the formation of by-products.
For each 5.5
equivalents of L-arginine that is converted to L-ornithine and urea, 1 equivalent is converted
to citrulline and ammonia. The citrulline is further hydrolyzed to ornithine but at a slow rate.
Other by-product comes from the reversible conversion of ornithine to its lactam (3-aminopiperid-2-one) at high temperatures under strong alkali concentration. Despite the existence
of these side-reactions high yields of L-ornithine have been obtained. The heating at 110°C
13
for 24 hours with the pH adjusted to 12 with ammonia
resulted in a yield of 90% L-ornithine
with 8% 3-amino-piperid-2-one and 1% citrulline. It should be noted that these results were
obtained through an experimental procedure that is far from being reproducible in an
industrial setting: the reactions were carried in evacuated, sealed 1 ml tubes and heated with
refluxing toluene (as a way of controlling the heating temperature – 110°C – which is
toluene’s boiling point) .
7
Introduction
Citrulline
HO
NH2
O
HN
HO
-NH3
NH2
NH2
O
O
+
OH-
H2O
HN
-Urea
NH2
HO
HN
NH2
O
O
Arginine
NH2
HN
H2N
3-Amino-piperid-2-one
Ornithine
Figure 1.7. Alkali catalyzed hydrolysis of arginine
Other
possible
by-products
suggested
in
literature
are
15
proline
and
16
diketopiperazine (the product of the reaction between two ornithine molecules).
In both cases, acid and alkaline catalysis, the urea formed is immediately hydrolyzed
14
to ammonia and cyanate due to the high reaction temperature . The cyanate is further
hydrolyzed to ammonia and carbon dioxide (figure 1.8)
O
H2N
O
C
C
N
+
NH4 + O
NH2
+ H3 O
C
N
(a)
+
NH3
+ CO2
(b)
Figure 1.8. Thermohydrolysis of urea: (a), reversible conversion of urea to cyanate and ammonia; (b),
hydrolysis of cynate to ammonia and carbon dioxide.
Other than traditional acid and basic catalysis, other techniques have potential to
produce interesting results and are explored in this study. These include the use of metal
salts and heterogeneous (solid) catalysis, with the second one being specially important if this
reaction is integrated in an industrial process. The background to this last method will be
presented in the next section.
8
Introduction
1.2.2. Heterogeneous Catalysis – the use of Zeolites
Zeolites comprise a group of hydrated aluminosilicate minerals and have a microporous structure. The zeolites are framework silicates consisting of interlocking tetrahedrons
of SiO4 and AlO4. In order to be called a zeolite, a mineral should have a (Si +Al)/O ratio of
1/2. The alumino-silicate structure is negatively charged and attracts the positive cations that
reside within. More than 150 zeolite types have been synthesized and 48 naturally occurring
zeolites are known, natural zeolites are formed when volcanic rocks and ash layers react with
alkaline groundwater.
Since synthetic zeolites were first synthesized in 1949 at Union Carbide
17
Corporation , they have proven to be a versatile material with applications in several
industrial processes. These days, major uses are as detergent builders, as adsorbents, and
as catalysts. Although catalytic application of zeolites represents only 12.5% of the total
tonnage utilization, it is 55% of the total market value for synthetic zeolites. Clearly, catalysis
is the major economic driver in the search for new zeolitic materials.
The main use of zeolites as catalysts has always been in the petroleum industry.
Previously, acid catalysts in the fuel industry included silica-alumina gel, supported
17
phosphoric acid and chlorine treated platinum or alumina
, but since the late 50’s
strong-acid zeolites have been successfully utilized as catalysts in the cracking of petroleum
for gasoline production. In the last 30–40 years, between 30% and 50% of all motor fuels
(gasoline, jet, and diesel) have been produced world wide with Y zeolite catalysts.
Due to their important role in petroleum refinery, heterogeneous acidic catalysts have
attracted much more attention than heterogeneous basic catalysis. The catalytic capabilities
18
of basic catalysts were first reported in the early 70’s. Yashima et al.
reported that side
chain alkylation of toluene was catalyzed by alkali ion-exchanged X and Y type zeolites. In
basic zeolites and other basic heterogeneous catalysts the basic sites are believed to be the
19
surface oxygen atoms . Oxygen atoms existing on any materials may act as basic sites
because the O atoms are able to interact attractively with a proton.
The acid-base properties of these materials can be controlled by selecting the types
of ion-exchanged cations and by the Si/Al ratio of the zeolite framework. Wide variation of
acid-base properties can be achieved by ion-exchange and ion-addition, while relatively small
changes in acid-base properties are yielded by changing the Si/Al ratio. When preparing basic
zeolites, two approaches are possible. One approach is to ion-exchange with alkali metal
ions, and the other is to impregnate the zeolite pores with fine particles that can act as bases
themselves. The former produces relatively weak basic sites, while the latter results in the
strong basic sites. In alkali ion-exchanged zeolites, the type of ions used affects the basic
strength of the resulting zeolites. Effects of the alkali ions on basic strength are in the
+
+
+
+
+
following order: Cs > Rb > K > Na > Li .
In the present study, a Y zeolite ion-exchanged with sodium ions (CBV 100 –
SiO2/Al2O3 ratio of 5:1) is tested as a possible catalyst for the arginine hydrolysis reaction.
9
Introduction
The research on the use of NaY zeolite as a catalyst has been focused on the treatment of
toxic compounds. This includes the catalysis of the conversion of nitriles to primary amides
20
21
and the reduction of nitric oxide . Other studies use this material together with other catalysts
22
22
such as metal ions (platinum , cobalt
23
24
and gold ) and metallocenes .
Concerning the catalysis of the arginine hydrolysis reaction, no work with zeolites can
25
be found in literature. Nonetheless, Ikeda et al.
reported mixed results with another
heterogeneous catalyst. In this study montmorillonite was utilized, this material can adsorb
molecules into its interlamellar layers, in which the regular spacing of clay sheets plays an
important role as a shape-selective catalyst. L-arginine was successfully adsorbed into the
interlamellar layer and the hydrolysis reaction was detected using the pressure-jump
relaxation method with electric conductivity detection. However, it was established that the
release of ornithine from the interlamellar layer was very slow step, making this catalyst not
suitable for industrial applications.
1.3. Enzymatic Catalysis
1.3.1. Enzymatic Hydrolysis of L-Arginine – Arginase
The hydrolysis of L-arginine to L-ornithine and urea is catalyzed by L-arginase (Larginine amidinohydrolase, EC 3.5.3.1). This is accomplished by the cleaving of the
guanidinium group from arginine which yields urea, a small nitrogen rich molecule. Thus,
arginase plays a fundamental role in the nitrogen metabolism. It is widely spread through the
evolutionary spectrum and can be find in significantly distinct organisms such as bacteria,
yeast, plants and animals.
In this study is utilized bacterial arginase from Bacillus subtilis. The information found
in literature concerning this organism’s arginase is scarce. Nevertheless, the structure of
arginase is conserved across bacterial species and even across eukaryotic organisms. All
arginases are multimeric metallo-enzymes comprised of identical or near identical sub-units
26
(although it has been shown for human arginase that the monomer is active ). Each
monomer has a binuclear manganese spin-coupled cluster located in the active site that is
profoundly involved in the reaction mechanism. Both Bacilus caldovelox arginase and rat liver
arginase have been subjected to extensive structural studies
27,28
.
Bewley et al. report a trimeric or hexameric quaternary structure, depending on the
27
pH of the medium, for Bacilus caldovelox arginase . The trimer is comprised of three
identical subunits that associate to form a flat disc-like structure. The monomers interact
through a total of 12 hydrogen bounds between each interface. At pH values above 7.0 a
hexamer is formed when one trimer is rotated 20° with respect to the other about a common
threefold axis.
10
Introduction
The fold of Rat liver arginase is similar to that of B. caldovelox, with 248 structurally
27
equivalent Cα atoms (83% of the sequence ). However, the trimer monomer-monomer
interaction is mediated by an additional “S”-shaped oligomerization motif (14 extra residues)
at the carboxy terminus, the conformation of this segment is stabilized by numerous intermonomer van der Walls interactions, hydrogen bonds and salt links
28
(figure 1.9). The
hexameric form is not found in rat liver arginase.
Figure 1.9. Topology diagram of rat liver arginase. Relative location of metal ligands is indicated by
28
grey circles (adapted from Kanyo et al.) .
The geometry of the active site also appears to be similar in rat liver and bacterial
27
28
arginases . The catalytic mechanism proposed by Kanyo et al.
for rat liver arginase
involves the formation of hydrogen bonds between the guanidinium group of arginine and
carboxylate side chain from the Glu277 residue located in the active site (figure 1.10). Later
studies not only confirmed the existence of these bonds but also indicate that other residues –
His141 and Thr246 – accept hydrogen bonds from the guanidinium group and help stabilize
29
the substrate . This array of hydrogen bonds orients the guanidinium group for nucleophilic
attack by a hydroxide ion that bridges both manganese ions. Several bridging metal ligands
coordinate the two manganese ions and stabilize the solvent bridge between them, including
Asp128 which also hydrogen bonds with the hydroxide ion.
The nucleophilic attack leads to the formation of metastable tetrahedral intermediate.
The metal ions are essential in transition state stabilization by keeping the metal-bridging
29
hydroxide in position, with Christiansson et al.
suggesting that one of the manganese ions
also directly interacts with one of the NH2 groups from the guanidinium. Following a proton
transfer to the leaving amino group mediated by Asp128, the collapse of the tetrahedral
intermediate yields the products L-ornithine and urea. The subsequent addition of a water
molecule to the binuclear manganese cluster facilitates urea departure, which may trigger the
ionization of the metal-bridging water molecule to regenerate the nucleophilic metal-bridging
hydroxide ion.
11
Introduction
Figure 1.10. Proposed mechanism of rat liver arginase-catalysed arginine hydrolysis by metal-activated
28
solvent . The α-amino and α –carboxylate groups are omitted for clarity.
Arginase´s catalytic action is highly specific for the hydrolysis of its natural substrate,
L-arginine. Its enantiomer D-arginase is not a substrate and the use of similarly structured
molecules and derivatives as alternative substrates severely attenuates the catalytic activity
30
of rat liver arginase . Tested molecules include: L-canavanine, L-homoarginine, L-argininic
acid, agmatine and L-argininamide (best alternative substrate tested with a 12-fold increase in
KM and a 14-fold decrease in Vmax).
The best arginase inhibitors are those bearing N-hydroxyguanidinium or boronic acid
ω
“warheads” that bridge the binuclear manganese cluster, including N -hydroxy-L-argininine
and N-hydroxy-L-lysine. The simplest arginase inhibitor is the fluoride ion, an uncompetitive
inhibitor with a Ki of 1.3 mM. The crystal structure of the binding of this inhibitor to the
31
enzyme-substrate complex has been determined
and shows an unusual mode of inhibition:
the metal-binding hydroxide ion is displaced by a fluoride ion and another fluoride ion is
2+
added to the vacant coordination site of one of the manganese ions (Mn
A).
The metal
bound fluoride ions are stabilized by short hydrogen bonds with the guanidinium group of the
substrate, in a typical uncompetitive inhibition substrate binding is required to stabilize the
inhibitor.
The properties of Bacilus subtillis arginase were the subject of only one published
32
study by Nakamura et al. . The enzyme purified during this work has a specific activity of 858
U/mg-protein and a native molecular weight of 115.000 Da. It is also reported an optimum pH
at pH=10, a relatively high KM for L-arginine of 13.5 mM and an increased in thermal stability
2+
through the addition of Mn
ions.
In literature various methods for arginine (usually originating from mammal liver)
immobilization have been studied with mixed results (table 1.3). Several of these studies were
conducted with the intent of using arginase has an arginine detection tool.
12
Introduction
Table 1.3. Techniques for arginase immobilization described in literature. The different techniques are
listed in chronological order, from the oldest work to the most recent.
Immobilization Method
Particular Aspects
Covalent binding to controlled pore glass bead
Arginase and urease were simultaneously
derivative
33
immobilized in the beads and assembled on an
ammonia sensing electrode for arginine detection.
34
Entrapment in a highly porous polymer matrix
The enzyme is immobilized in the matrix trough
radiation induced polymerization of acrylic
monomers.
After immobilization on bead shaped matrix only
30-40% of the initial enzymatic activity was
retained.
Covalent binding on various types of carbodiimide
Optimum pH for the catalytic activity shifted in the
activated carboxyl-functionalized polyacrylamide
acid direction.
beads
35
Optimum temperature for the catalytic activity of
the immobilized arginase much higher than that
for the soluble enzyme.
app
Km
of the immobilized arginase for L-arginine
was an order of magnitude higher than that of the
soluble enzyme.
Entrapment in membrane reactor system
36
Enzyme recovered with a UF membrane after a
batch for re-use in the next batch.
Conversion after 24h decreased from 53% to zero
after 5 runs.
Covalent binding on epoxy-functionalized resin
37
The resin is contained inside a continuous-flow
reactor for arginine detection.
The enzyme reactor is stable for more than 6
months and retains about 80% of its initial activity
after 800 assays.
Entrapment in gelatin gel
38
Arginase immobilized together with urease on the
surface of a pH electrode for arginine detection.
The gelatin matrix has no effect on the system.
Covalent binding on N,N’-disuccinimidyl suberate
activated aminopropyl silica
39
Enzyme immobilized on a chromatography
support to study the binding of nor-NOAH to
arginase.
The arginase column is stable during a long
period of time.
The next sections encompass a brief overview of the use of biocatalysts in industrial
processes and a section on the importance of biocatalysts immobilization, with focus on the
immobilization methods utilized in this study.
13
Introduction
1.3.2. Industrial Biocatalysis
For millennia enzymes have been utilized in the production of several food products.
However, it was only decades ago that biocatalysis started to be regarded as a valuable tool
by the chemical industry. Examples of initial applications include the use of acylases,
hydantoinases, and aminopeptidases in the production of optically pure amino acids, and the
use of nitrile hydratase in the enzymatic production of the bulk chemical acrylamide from
40
acrylonitrile .
Since then the industrial use of biocatalysis has expanded, with enzymes being
utilized as catalysts in the industrial synthesis of bulk chemicals, pharmaceutical and
40
agrochemical intermediates, active pharmaceuticals, and food ingredients . Most these
commercial enzymatic processes share several attributes: high product concentrations and
productivities, no undisirable by-products and the use of enzymes that do not require
41
expensive co-factores . Today, the industrial community sees biocatalysis as a highly
promising area of research, especially for the development of sustainable technologies for the
40
production of chemicals . This sustainable, environmentlaly friendly, production of bulk
chemicals (green chemistry) is the aim of the N-ergy project, and of this study in particular.
However, the number and diversity of the applications of biocatalysis are still modest
when compared with traditional catalysis methods. This happens, in part, because of the
limitations inherent to a process catalysed by enzymes: substrate scope, limited enzyme
40
availability and operational stability . Recent breakthroughs in certain areas should help
overcome these limitations. Advances in genomics, directed evolution and bioinformatics
allow not only the discovery of new enzymes but also the optimization of existing ones. In
addition, the development of a feasible biocatalytic process usually requires a major financial
investment. Depending on the type of biocatalyst to be used, specific reactor and hardware
configurations are needed. Biocatalytic processes are typically highly heterogeneous and
need specific designs of the catalyst–hardware interface to allow efficient immobilization and
41
re-utilization .
The lack of operational stability of certain enzymes when utilized in industrial scale
processes is one of the constraints that has received more attention in recent years. Various
techniques have been developed to improve stability so that enzymes can be used with
40
organic solvents, high temperatures and extreme pHs . Among these techniques are protein
engineering, process modification and, as discussed in the next section, immobilization of the
biocatalysts.
14
Introduction
1.3.3. Biocatalyst Immobilization
Immobilization of biocatalysts consists in its confinement to a defined area
(bioreactor), ensuring the maintenance of the catalytic activity and allowing its repeated or
continuous use. Immobilization methods have been applied to a wide array of biocatalysts,
ranging from pure enzymatic extracts to whole microbial cells or even animal and vegetal
tissues. When applied to enzymatic extracts on an industrial setting, immobilization offers
several advantages and some restrictions, the most relevant of which are listed in table 1.4.
Table 1.4. Advantages and limitations associated to the use of immobilized enzymes (adapted from
42
Cabral et al. )
Advantages
Particular Aspects
Retention of the catalyst inside the reactor
Allows reutilization and continuous processes
Possibility of operating on high dilution rates
without the risk of wash-out
High catalyst concentrations
Allows higher volumetric production rates
Faster bioconvertion, important when secondary
reactions are a issue
Controlled microenvironment of the catalyst
Allows manipulation of enzymatic activity and
specificity
Improves enzyme stability
Protects the enzyme against shear stress
Easy separation between catalyst and product
Minimizes product contamination
Precise control of bioconversion time
Limitations
Particular Aspects
Loss of catalytic activity
May occur during the immobilization process,
during the bioconversion or due to the physical
properties of the immobilization matrix.
Empiric process
Specific optimization is needed for each particular
application
Complex control and modeling
The different types of immobilization of biocatalysts have been the subject of several
42
classification systems. One of them, adapted from Cabral et al. , is presented in figure 1.11.
15
Introduction
Biocatalyst Immobilization
Insoluble
Reticulation
Soluble
Bonding to a Support
With Modification of the
Microenvironment
Gel
Adsorption
Ionic Bond
Without Modification of the
Microenvironment
Microencapsulation
Ultrafiltration
Membranes
Covalent Bond
Microcapsules
Inverted Micelles
Figure 1.11. Methods of immobilization of biocatalysts.
Due to its multimeric structure, arginase is an excellent candidate for immobilization
by covalent bonds. The binding of this enzyme to a support can play an important an
important role in avoiding dissociation of the enzyme by keeping the sub-units together. This
kind of immobilization not only should enhance enzyme stability but also is essential when
utilizing arginase in an industrial setting.
Most protocols for protein immobilization described in literature are difficult to
reproduce on an industrial scale where long support handling may be necessary and some
dangerous substances cannot be utilized, problems that are not considered on a laboratory
scale. Comparatively to other immobilization methods, covalent binding supports, and the
epoxy-activated supports utilized in this study in particular, are almost ideal for performing
easy industrial immobilization of enzymes. Epoxy-activated supports are very stable during
storage and also when suspended in neutral aqueous media. Hence, they can be easily
handled before and during immobilization procedures. In addition, these supports are able to
directly form very stable covalent linkages with different protein groups (amino, thiol, and
43
phenolic ones) under very mild experimental conditions .
The immobilization of enzymes in epoxy-activated supports usually follows a two step
mechanism: first a rapid mild physical adsorption between the protein and the support is
produced, and secondly the covalent reaction between adsorbed protein and epoxy groups
occurs (figure 1.12). In order to adsorb proteins during incubation at high ionic strenghts,
commercial epoxy supports are fairly hydrophobic, in hydrophilic supports (e. g., agarose) this
preliminary physical hydrophobic adsorption is not possible. The remaining epoxy groups may
be easily blocked after the protein immobilization, to stop any kind of undesired covalent
support-protein reaction.
16
Introduction
Figure 1.12. Mechanism of immobilization of proteins on epoxy-activated Supports. The covalent
reaction between soluble enzyme and epoxy support is extremely slow, but the previous adsorption to
43
the support allows a faster covalent reaction (adapted from Mateo et al. ).
Even though other immobilization techniques inside porous supports can increase the
enzyme operational stability by preventing any intermolecular process (proteolysis,
aggregation) and by preserving the enzyme from interactions with external interfaces (air,
oxygen, immiscible organic solvents, etc.), these techniques do not necessarily increase the
43
conformational stability of the enzyme . This kind of stability should be achieved if the
immobilization of each enzyme occurs through several residues. This way, all the residues
involved in immobilization preserve their relative positions and the enzyme is unaffected by
43
conformational changes promoted by heat, organic solvents, or any other distorting agents .
Thus, multipoint covalently immobilized enzymes should become more stable than their
soluble counterparts or than randomly immobilized derivatives. It is important that the reactive
groups that react with the enzyme are bound to the surface of the support by short spacer
arms (two to three carbon atoms), allowing the reaction to occur only with the external
residues of the enzyme but not residues located in internal pockets. The bond to a rigid
support through short spacer arms is vital when dealing with multimeric enzymes. Fernadez44
Lafuente et al.
suggests the use of short spacer arms supports together with cross-linking
agents to achieve the stabilization of the quaternary structure of multimeric enzymes with no
side modifications.
In this study three epoxy supports are tested for arginase immobilization:
Sepabeads
®
EC-EP, Sepabeads
®
EC-HFA and Eupergit C 250 L. All supports are
microporous, epoxy-activated, acrylic polymer matrix spherical beads.
Sepabeads
®
EC-EP is a highly activated support functionalized with short chain
®
epoxy groups while the Sepabeads EC-HFA supports are functionalized with epoxy groups
on a longer, more complex, spacer (figure 1.13). Both are very rigid supports that may be
used in stirred tanks or bed reactors. These supports have low swelling tendency in high
molar solutions and in common solvents. Also, their internal geometry offers large internal
17
Introduction
43
plain surfaces where the enzyme may undergo intense interactions with the support . The
standard grade beads have a diameter of 150-300 µm with an average pore diameter of 3045
40 nm and a specific gravity of 1.13 g/ml . The epoxy group density on the beads is around
43
100 µmol/(g of wet support) .
®
®
45
Figure 1.13. Functional groups of Sepabeads EC-EP and Sepabeads EC-HFA supports .
®
Eupergit C 250 L is activated similarly to Sepabeads EC-EP, having on its surface a
dense monolayer of reactive and stable epoxy groups (200 µmol/(g of dry support) according
to the supplier). The bead diameter is 100-250 nm with an average pore diameter of 100
46
nm . This kind of supports is amongst of the most extensively studied due to their capability
of immobilizing enzymes quickly and easily, both at laboratory and industrial scale.
Examples of the industrial application of epoxy-activated supports are the use of
Sepabeads supports on the production of 6-amino penicillanic acid and on the conversion of
47
cephalosporin C into alpha-keto-adipoyl-7-amino-cephalosporanic acid . The only case of
arginase immobilization on an epoxy-activated support found in literature is the already
37
referred work of Alonso et al. , where arginase was successfully immobilized on a
commercial resin for arginine detection.
1.4. Aim of this Study
The main goal of this project is to establish an industrial viable method of converting
L-arginase to L-ornithine, contributing to the aim of the overall N-ergy project of coverting
biomass to ethanol and bulk N-fuctionalized chemicals. With this objective in mind two
approaches are studied: the chemically catalyzed hydrolysis of L-arginine and the hydrolysis
of L-arginine catalyzed by the enzyme arginase.
Concerning the chemical catalysis, the experimental conditions studied are closer to
13,14,15,24
an industrial setting than previous works found in literature
. The effects of pH and
temperature in the yield of ornithine are studied, as well as the influence of metal ions on the
reaction mixture. NaY zeolite, a heterogeneous catalyst potentially suitable for industrial
application is also tested. It should be noted that all research on the chemically catalysis of
the conversion of arginine is focused on the basic hydrolysis. The acid catalyzed reaction,
regardless of being cleaner, is considerably slower and requires very high temperatures,
being inadequate for a large scale process.
18
Introduction
Regarding the biocatalysis, the hydrolysis of L-arginine to L-ornithine and urea
catalyzed by Bacilus subtillis arginase is studied. In this case, the main objective is to
investigate the effect of immobilization in covalent-binding supports on the performance of the
enzyme. Different commercially available epoxy-activated supports suitable for industrial
48,46
use
are tested for optimum stability/activity. After selecting the best performing enzyme
preparation, the chemical and enzymatic hydrolysis of arginine are compared.
Finally, even though the main goal is the production of ornithine, a secondary
objective of this project is to further contribute to the developing N-ergy project. This includes,
if possible, the collection of data on the conversion of ornithine to 1,4-butanediamine and the
monitoring of the formation of economically important secondary products like ammonia and
urea.
19
Chemical Hydrolysis of L-Arginine
2. Chemical Hydrolysis of L-Arginine
2.1. Materials and Methods
2.1.1. Reagents
Table 2.1. Reagents utilized during the course of the chemical hydrolysis of arginine experiments.
Chemical
Supplier
Purity (min)
1,4-Butanediamine
Sigma-Aldrich
99%
Alfa Aesar
98%
3-Aminopiperid-2-one
Activate Scientifics
95%
Acetic acid
Riedel-de-Haen
99.8%
Acetone
Merck
99.8%
Acetonitrile
Merck
99.9%
Al(NO3)3
Merck
98.5%
Borax
Riedel-de-Haen
99.5%
Boric acid
Merck
99.5%
2,2,3,3-d(4)-3-(trimethylsilyl)propionic
acid sodium salt (TSP)
CuSO4
Sigma-Aldrich
99%
Dabsyl chloride
Sigma-Aldrich
99%
Dimethylformamide (DMF)
Lab-Scan
99.8%
Deuterium oxide
Sigma-Aldrich
99
Ethanol
Merck
99.9%
L-Aspartic acid
Merck
99%
L-Arginine
Sigma-Aldrich
99%
L-Citrulline
Sigma-Aldrich
99%
L-Ornithine
Sigma-Aldrich
99%
MnSO4
Sigma-Aldrich
99%
NaY Zeolite
Zeolyst International
-
Sodium Acetate
Merck
99%
Sulfuric acid-D2
Sigma-Aldrich
99%
ZnCl2
Boom
98%
21
Chemical Hydrolysis of L-Arginine
2.1.2. Solutions
Table 2.2. Solutions prepared during the course of the chemical hydrolysis of L-arginine experiments.
Solution
Composition
Dabsyl chloride solution
24 mg of Dabsyl chloride in 10 ml of acetone
Derivatization Buffer
0.1 M borate buffer, pH=9.0
(addition of a 6.2 g/l solution of boric acid in Milli-Q
water to a 38.15 g/l solution of borax in Milli-Q
water until pH = 9.0 is obtained)
Dilution Buffer
50% Acetonitrile
25% Ethanol
25% Eluent A
Eluent A
96% 20mM sodium acetate in Milli-Q water
4% DMF
pH adjusted to 6.4 with concentrated acetic acid
Eluent B
80% Acetonitrile
20% Milli-Q water
2.1.3. Equipment
High Performance Liquid Chromatography
Reverse-phase HPLC analyses were performed on a Waters™ System consisting of:
Waters™ 600s Controller; Waters™ In-Line Degasser; Waters™ 616 Pump; Waters™
®
717plus Autosampler; Waters™ 484 Tunable Absorbance Detector; Nova-Pak C18 column
®
60 Ǻ 3.9 x 150 mm with a Nova-Pak C18 4 µm Guard-Pak™ pre-column insert.
Nuclear Magnetic Resonance
1
H-NMR and
13
C-NMR analyses were performed on a Bruker AVANCE III 400 MHz
NMR spectrometer.
Multiple Reactor System
All reactions were performed on a Parr
®
Series 500 Multiple Reactor System
equipped with a 4871 Process Controller. This system is fully programmable and allows the
simultaneous heating of up to six 75 ml reactors with internal stirring. The individual reactors
are made of alloy C-276 and tolerate operational temperatures up to 300°C and operational
pressures up to 3000 psi. Each reactor includes: a thermocouple mounted inside the reactor
and protected by stainless steel sheaths; a dip tube with sample valve; an optional glass liner
to protect the metal walls from corrosive substances (using the glass liners reduces the
reactor volume to about 300 ml).
22
Chemical Hydrolysis of L-Arginine
®
Figure 2.1. Parr Series 500 Multiple Reactor System with 4871 Process Controller.
Thermomixer
®
An Eppendorf Thermomixer Comfort was utilized. This thermomixer is equipped with
a rack that allows the simultaneous heating of up to 24 1.5 ml eppendorf Safe-Lock tubes. It is
fully programmable, capable of heating or cooling samples from 1°C to 99°C and of agitating
from 300 rpm to 1500 rpm (also has a no mixing mode).
2.1.4. Analytical Techniques
Dabsyl Chloride Derivatization
Previously to HPLC analysis, a Dabsyl chloride derivatization procedure adapted from
49
Kause et al.
was applied to each sample. This pre-column derivatization method allows the
efficient separation and detection in the visible region of amino acids as well as primary and
secondary amines, including putrescine.
Samples were diluted five times with a 1.25 mM L-aspartic acid (internal standard) in
Milli-Q water solution. The total amino acid concentration of the diluted samples should be
around 5 mM, with a concentration of internal standard of 1 mM.
Aliquots of 20 µl of the diluted samples were further diluted with 180 µl of the
derivatization buffer and, after mixing on a vortex mixer, 100 µl of dabsyl chloride solution was
added to the samples, immediately followed by thorough mixing. Samples were incubated at
70°C for 15 minutes in a thermomixer. The reaction was stopped by placing the samples on
ice for 5 minutes, followed by the addition of 200 µl of dilution buffer for a final volume of 500
µl per sample. Finally, the samples were centrifuged for 5 minutes at 14 000 rpm and 200 µl
of the supernatant were used for HPLC analysis.
HPLC Analysis
After derivatization, the samples were analyzed on the previously described reversephase HPLC Waters™ system. 10 µl per sample were injected and the column was eluted at
50°C with a flow rate of 1 ml/min. The gradient of the eluents utilized is described in table 2.3,
23
Chemical Hydrolysis of L-Arginine
the total time of analysis for each sample was of 80 minutes. UV detection was carried at the
wavelength of 436 nm.
Table 2.3. Gradient of eluents applied during HPLC analysis.
Time
% Eluent A
% Eluent B
Curve
0
92
8
6
2
92
8
5
7
80
20
7
35
65
35
6
45
50
50
6
66
0
100
6
71
0
100
6
75
92
8
6
92
8
6
80
a
a
for the slope of the different curves see Appendix A.
NMR Analysis
The samples for NMR analysis were collected from experimental settings where the
reactions were carried out using D2O as solvent. NMR tubes were filled with 1 ml of each
sample. To each tube was added 15 µl of 10% D2SO4 in D2O and small amounts of TSP.
1
Samples were analyzed for H-NMR,
13
C-NMR and DEPT spectra.
2.1.5. Hydrothermolysis Experiments
These experiments were conducted with the objective of studying the influence of pH
and temperature in the alkaline hydrolysis of L-arginine and identifying the setting that leads
to maximum L-ornithine yield.
A solution of 25 mM L-arginine in Milli-Q water was prepared. Portions of this solution
were adjusted to pH 11 and 12 with a concentrated (0.1 M) sodium hydroxide solution.
Volumes of 60 ml of the three solutions were heated in Parr pressurized stirred reactors at
110°C, 125°C and 150°C for a total of 9 distinct experimental conditions. The reactors were
heated accordingly to a pre-programmed temperature gradient. The clock was started after 30
minutes of heating, corresponding to the time needed for the reactors’ internal temperature to
rise from room temperature to the desired temperature.
3 ml samples were taken from each reactor through a dip tube at 0, 1, 2, 18 and 20
hours. The reaction was stopped by immediately placing the samples on ice. The experiments
were conducted in duplicate.
24
Chemical Hydrolysis of L-Arginine
Before each sample was taken, 4 ml aliquots of the reaction mixture were collected
through the dip tube. This portion is not utilized for analysis and corresponds to the volume of
liquid stored inside the tube which is not at the temperature measured inside the reactor.
The samples were stored in the fridge (5˚C) until dabsyl chloride derivatization and
HPLC analysis.
For NMR analysis 60 ml of 25 mM L-arginine in D2O were heated in the same
reactors at 125°C for 15 and 30 hours. The dip tube was not used, instead the reaction
mixture was collected after the reactor cooled down to room temperature.
2.1.6. Metal Salt Catalysis Experiments
This experiment was conducted with the objective of verifying if the presence of metal
ions has any catalytic effect in the arginine hydrolysis reaction.
Different metal salts were added to solutions of 25 mM L-arginine in Milli-Q in
equimolar concentrations. The following salts were added: Aluminium Nitrate (Al(NO3)3·H2O)
9.38 g/l; Copper(II) Sulfate (CuSO4) 3.99 g/l; Manganese(II) Sulfate (MnSO4·H2O) 4.25 g/l:
Zinc Chloride (ZnCl2) 3.41 g/l. Volumes of 30 ml of the four solutions and a blank solution with
no salts added were heated in Parr pressurized stirred reactors (utilizing a glass liner) at
125°C accordingly to a pre-programmed temperature gradient. The clock was started after 30
minutes of heating, corresponding to the time needed for the reactors’ internal temperature to
rise from room temperature to the desired temperature.
3 ml samples were taken from each reactor through a dip tube at 0, 2 and 4 hours.
The reaction was stopped by immediately placing the samples on ice.
Before each sample was taken, 4 ml aliquots of the reaction mixture were collected
through the dip tube. This portion is not utilized for analysis and corresponds to the volume of
liquid stored inside the tube which is not at the temperature measured inside the reactor.
The samples were stored in the fridge (5˚C) until dabsyl chloride derivatization and
HPLC analysis.
2.1.7. Zeolite Catalysis Experiment
This experiment was conducted with the objective of replicating, or improving, the Lornithine yield values obtained through traditional alkaline catalysis utilizing a solid catalyst
suitable for an industrial process. Different concentrations of NaY zeolite were tested for this
effect.
Different quantities of NaY zeolite were added to solutions of 25 mM L-Arginine in
Milli-Q for final concentrations of 0.5 gz/lsol, 1 gz/lsol, 2 gz/lsol, 5 gz/lsol. Volumes of 30 ml of the
three suspensions and a blank solution with no zeolite added were heated in Parr pressurized
25
Chemical Hydrolysis of L-Arginine
stirred reactors (utilizing a glass liner) at 125°C accordingly to a pre-programmed temperature
gradient. The clock was started after 30 minutes of heating, corresponding to the time needed
for the reactors’ internal temperature to rise from room temperature to the desired
temperature.
3 ml samples were taken from each reactor through a dip tube at 0, 18 and 20 hours.
The reaction was stopped by immediately placing the samples on ice. The experiments were
conducted in duplicate.
Before each sample was taken, 4 ml aliquots of the reaction mixture were collected
through the dip tube. This portion is not utilized for analysis and corresponds to the volume of
liquid stored inside the tube which is not at the temperature measured inside the reactor.
The samples were stored in the fridge (5˚C) until dabsyl chloride derivatization and
HPLC analysis.
2.2. Results and Discussion
2.2.1. Hydrothermolysis Experiments
The alkaline hydrothermolysis of L-arginine was followed during 20 hours at different
temperatures. Previous studies on this reaction under similar pH and temperature conditions
13
reported high L-ornithine yields. Murray et al. , for example, obtained a yield of 90% Lornithine at pH=12 and T=110ºC. Yet, the various works found in literature utilize
experimental setups considerably different from the one that was employed in the present
study.
13
16
15
Murray et al. , Wong et al. , and Vallentyne., et al.
performed similar thermal
degradation experiments in sealed evacuated tubes, with the reaction being stopped at
certain point by cooling the tubes. The experimental setting now employed is closer to an
industrial scale process, the reactions are carried in pressurized reactors and samples are
regularly taken through a dip tube.
Figure 2.2 shows the rate of L-arginine consumption under the diverse experimental
conditions tested.
26
Chemical Hydrolysis of L-Arginine
110 C
pH=11
80
125 C
100
pH=10.6
Relative
Concentration (%)
Relative
Concentration (%)
100
pH=12
60
40
20
pH=10.6
pH=11
80
pH=12
60
40
20
0
0
0
5
10
15
20
25
0
5
10
Time (hours)
20
25
150 C
100
Relative
Concentration (%)
15
Time (hours)
pH=10.6
pH=11
80
pH=12
60
40
20
0
0
5
10
15
20
25
Time (hours)
Figure 2.2. Time course of L-arginine consumption under different experimental conditions. The
percentages are based on the total concentration of amino acids (L-arginine and L-ornithine) in the
reaction mixture at t=0. Error bars calculated using standard deviation of the two duplicate experiments.
The small L-arginine consumption registered at t=0 is due to the 30 minutes of
heating necessary for the reactors to reach the desired work temperature. Accordingly to what
14
is described by Warner et al. , temperature positively influences arginine hydrolysis. The
higher conversion values were detected at 150°C where, after 20 hours of reaction, the high
temperature nullifies the pH effect, with concentrations of remaining arginine reaching 6%.
The influence of pH is also clear, especially at lower temperatures. Although the adjustment
of the initial pH to 11 with sodium hydroxide has almost no effect on the rate of consumption,
the adjustment to pH=12 leads to a decrease of arginine concentration of 25% at 110°C and
125°C after 20h of reaction. This effect was also previously described by Warner et al.
Figure 2.3 shows the rate of L-ornithine formation under the diverse experimental
conditions tested.
27
Chemical Hydrolysis of L-Arginine
110 C
25
125 C
25
pH=10.6
pH=10.6
pH=12
Relative
Concentration (%)
Relative
Concentration (%)
pH=11
20
15
10
5
0
pH=11
20
pH=12
15
10
5
0
0
5
10
15
20
25
0
5
Time (hours)
15
20
25
150 C
25
Relative
Concentration (%)
10
Time (hours)
pH=10.6
pH=11
20
pH=12
15
10
5
0
0
5
10
15
20
25
Time (hours)
Figure 2.3. Time course of L-ornithine formation under different experimental conditions. The
percentages are based on the total concentration of amino acids (L-arginine and L-ornithine) in the
reaction mixture at t=0. Error bars calculated using standard deviation of the two duplicate experiments.
Again, the small L-ornithine formation registered at t=0 are due to the initial 30
minutes of heating before the clock was started. The influence of pH in ornithine formation is
concordant with its influence in arginine degradation: in every temperature higher pH leads to
a higher ornithine yield. However, in this case, the effects of temperature are not as linear.
The maximum yield, 15,2%, was obtained at 125°C pH=12.
Comparing arginine consumption with ornithine yield, it becomes clear that secondary
products are being formed. For example, at 125°C pH=12, after 20 hours a consumption of
85,4% of the arginine leads to the formation of only 15,2% of ornithine. The complex HPLC
chromatograms and NMR spectra (Appendix F) reinforce the idea that multiple products are
present in the reaction mixture. Probable side-products are: citrulline, formed directly from
arginine; 3-aminopiperid-2-one, formed from the lactamization of ornithine; putrescine (1,4butanediamine), formed from the decarboxylation of ornithine. The decrease of ornithine
concentration between 18h and 20h at 150°C suggests that ornithine is being converted to
another product.
28
Chemical Hydrolysis of L-Arginine
Figure 2.4. Example of a typical HPLC chromatogram obtained after heating a L-arginine solution for
20h in the described experimental settings. The identified peaks correspond to: (A) L-aspartic acid
(internal standard); (B) hydrolyzed excess derivatization reagent; (C) L-citrulline; (D) L-arginine; (E) Lornithine; (F) ammonia; (G) 3-aminopiperid-2-one; (H) retention time of putrescine elution(none was
detected in the samples). All unidentified peeks correspond to impurities originating either from the
solutions utilized or from the HPLC system.
After identification of the different peaks on the HPLC chromatogram (figure 2.4), the
presence of citrulline and 3-aminopiperid-2-one in the reaction mixture was established. Still,
citrulline appears only in small amounts and the presence of the lactam reasonable amounts
is not enough to explain the substantial disparity between arginine conversion and ornithine
yield. It should be noted that it was not possible to calculate the exact amount of 3aminopiperid-2-one in solution for reasons explained in section 2.2.4.
Despite not being detected during HPLC analysis, the hypothesis of putrescine
formation can’t be neglected. Putrescine is a relatively volatile product, and there is a
possibility that it escapes as a gas at the moment a sample is being collected. This
supposition is supported by the presence of foul odor felt during sample collection (putrescine
is known for its strong odor).
1
However, the comparison of H-NMR spectra appears to demonstrate that there is no
1
putrescine formed during the experiments. The H-NMR spectum of putrescine (Appendix E)
1
shows a distinct peak at 2.6 ppm that clearly is not present on the H-NMR spectum for the
reaction mixture (Appendix F). Samples for NMR analysis are collected only after the reactors
cool to room temperature, which guarantees that this product is in liquid state.
15
Other possible secondary products referred in literature are proline
and
16
diketopiperazine . The proline detected by Vallentyne et al. is probably citrulline incorrectly
identified, which is an easily understandable mistake considering the less accurate detection
methods available at the time of the study (1968). The formation of diketopiperazine from the
29
Chemical Hydrolysis of L-Arginine
reaction between two molecules of ornithine seems unlikely in diluted solutions, which is the
present case. Thus, new and more precise analytical methods should be employed to follow
the thermohydrolysis of arginine reaction in order to clarify which compounds are being
formed and in what amounts.
Also noticeable in figure 2.3 is the big discrepancy (error bars) between the values of
both measurements (duplicates) for each point at high pH values. The experiments were
repeated more than once, also in duplicate, and the big standard deviation persisted. This can
be explained by the complexity of the reactions occurring in the system. At each moment
multiple reactions are happening in the reaction mixture – lactamization, hydrolysis, formation
and degradation of citrulline – and the reactions are dependent from each other and from the
pH of the medium which, in turn, depends from the concentration of the different products (as
seen in figure 2.5). At high initial pH values this equilibrium is even more fragile so a minor
perturbation, like a valve from the dip tube that is opened too much or too little, can greatly
affect the system. On a lab scale the use of buffers would minimize this problem, but the use
of salts is not desirable in an industrial setting.
Relative
Concentration %
16
12
[ORN]
pH
12
11.5
8
11
4
10.5
0
10
0
5
10
15
20
pH
25
Time (hours)
Figure 2.5. Evolution of the pH during the course of the reaction (125°C, initial pH of 12) and its
comparison with ornithine concentration. Error bars were omitted for clarity.
During analysis no urea was detected. This is easily explained as at temperatures
above 100°C in alkaline conditions urea is almost immediately hydrolyzed to ammonia and
14
carbon dioxide . Considerable amounts of ammonia were detected through HPLC analysis.
Despite being less valuable than urea, ammonia market prices at the moment (August 200)
12
are extremely high, reaching $600/tonne . Bearing in mind that each tonne of arginine as the
potential to produce 195 kg of ammonia, it could be of financial interest to recuperate this
secondary product.
30
Chemical Hydrolysis of L-Arginine
2.2.2. Metal Salt Catalysis Experiments
This experiment was performed based on the idea that the presence of metal ions
could help stabilize the guanidinium group of L-arginine making it a better leaving group. This
would lead to faster arginine conversion and lower working temperatures. The lower
temperatures would also minimize the lactamization of ornithine to 3-aminopiperid-2-one. The
influence of manganese ions was specially anticipated, as this metal has an important role on
28
the catalytic mechanism of arginase .
This premise is based on studies on the successful catalysis of the hydrolysis of
amides using different metal ions including: copper(II)
50,51
52
51
, nickel(II) , cobalt(II)
51
(III) . Curiously, the work developed Meriwether et al.
51
and cobalt
is motivated by the catalytic
mechanism of another metallo-enzyme. In this case, different metals are tested to try to mimic
the catalytic activity of exopeptidases.
Different metal salts were tested at 125°C in equimolar concentrations with arginine:
manganese sulfate, zinc chloride, aluminium nitrate and copper sulfate.
4
MnSO4
Relative
Concentration (%)
3,5
ZnCl2
Al(NO3)3
3
no salts added
2,5
2
1,5
1
0,5
0
0
1
2
3
4
5
Time (hours)
Figure 2.6. Effect of the presence of different metal salts (equimolar concentrations – 25 mM) on the Larginine thermohydrolysis reaction. The percentages are based on the total concentration of amino
acids (L-arginine and L-ornithine) in the reaction mixture at t=0.
Again, the ornithine concentrations registered at t=0 are due to the preliminary
heating (30 minutes) needed for reaching he desired work temperature. As observed in figure
2.6 the presence of metal salts appears to have no significant influence on the arginine
hydrolysis reaction. Copper sulfate interfered in the derivatization reaction (certain amino
acids, including the internal standard aspartic acid weren’t derivatized) and its effect couldn’t
be determined.
31
Chemical Hydrolysis of L-Arginine
2.2.3. Zeolite Catalysis Experiment
This experiment was performed with the objective of replicating or improving the
results previously obtained for ornithine yield, utilizing a catalyst suitable for an industrial
process. In literature, no comparable work is found on the arginine hydrolysis reaction with
zeolites or other similar materials. The reaction was followed for 20 hours (figure 2.7) with
addition of NaY zeolite in different concentrations: 0.5 g/l, 1 g/l, 2 g/l, 5 g/l and no zeolite
added.
1g/l
L-Arginine
100
2g/l
1g/l
L-Ornithine
25
2g/l
0.5 g/l
5 g/l
80
no zeolite added
60
40
20
0
Relative
Concentration (%)
Relative
Concentration (%)
0.5 g/l
5 g/l
20
no zeolite added
15
10
5
0
0
5
10
15
20
Time (hours)
25
0
5
10
15
20
25
Time (hours)
Figure 2.7. Effect of different NaY zeolite concentrations on the L-arginine thermohydrolysis reaction.
The percentages are based on the total concentration of amino acids (L-arginine and L-ornithine) in the
reaction mixture at t=0. Error bars calculated using standard deviation of the two duplicate experiments.
As can be observed in figure 2.7, the effects of the zeolite in the reaction are limited
even at high concentrations of 5 g/l. Nonetheless, there is a mild catalytic effect, the best
results, 11.2%, are obtained with 5 g/l at 18 hours. It should be noted that concentrations this
high (5 g/l) of zeolite are probably unsuitable for application in an industrial process.
The zeolite utilized also showed to be the impractical, even at laboratory scale, due to
its accumulating on the side walls of the glass liners, being extremely difficult to remove.
2.2.4. Analytical Methods
Dabsyl Chloride derivatization and the HPLC analysis method utilized were
appropriate for the experiments performed. With the copper sulfate exception already
mentioned, derivatization and separation of all relevant components of the reaction mixture
was achieved. Nonetheless, ideally, specific methods should be applied for some
components, such as ammonia. It was not possible to determine the exact concentrations of
3-aminopiperid-2-one. During derivatization, the relatively high temperatures (70°C) led to the
32
Chemical Hydrolysis of L-Arginine
13
partial conversion of this lactam to ornithine
(figure 2.8), not allowing a viable standard
calibration curve.
Concerning in the NMR analysis, a similar problem is observed in the 3-aminopiperid2-one spectra (appendix D). The lactam’s spectra show peaks that are also present in the
ornithine NMR spectra (appendix C). In this case the conversion is catalyzed by the addition
13
of sulfuric acid .
Figure 2.8. Detail from a HPLC chromatogram for a 25 mM 3-aminopiperid-2-one solution. The
identified peaks correspond to: (O) L-ornithine; (A) ammonia; (L) 3-aminopiperid-2-one. All unidentified
peeks correspond to impurities originating either from the solutions utilized or from the HPLC system.
33
Enzymatic Hydrolysis of L-Arginine
3. Enzymatic Hydrolysis of L-Arginine
3.1. Materials and Methods
3.1.1. Bacillus Subtilis Arginase
The Bacillus Subtillis arginase utilized during the enzymatic hydrolysis of L-arginine
experiments was supplied with the K-LARGE commercial kit for L-arginine/urea/ammonia
detection from Megazyme International Ireland Ltd. According to the supplier, the solution has
an activity of 8300 U/ml and contains pure Bacillus Subtillis protein with the addition of
manganese chloride (25 mM) for enzyme activation and of lithium sulphate (2.5 M), used for
precipitation of the protein during purification.
3.1.2. Epoxy-activated Supports
Sepabeads
®
EC-EP and Sepabeads
®
EC-HFA were supplied by Resindion Srl
®
(Mitsubishi Chemical Corporation). Eupergit C 250 L was supplied by Sigma-Aldrich. For the
certificates of analysis of the supplied Sepabeads supports please consult Appendix G.
3.1.3. Reagents
Table 3.1. Reagents utilized during the course of the enzymatic hydrolysis of L-arginine experiments.
Chemical
Supplier
Purity (min)
Acetic acid
Riedel-de-Haen
99.8%
Acetonitrile
Merck
99.9%
Borax
Riedel-de-Haen
99.5%
Boric acid
Merck
99.5%
Bradford Dye Reagent
Bio-Rad
-
Merck
99%
Fluorescamine (Fluram )
Sigma-Aldrich
99%
Glycine
Merck
99.7%
L-Arginine
Sigma-Aldrich
99%
L-Ornithine
Sigma-Aldrich
99%
Manganese (II) Chloride
Sigma-Aldrich
98%
Sodium Acetate
Merck
99%
Sodium Azide
Merck
99%
Sodium dihydrogenphosphate
Merck
98%
Triethanolamide
Sigma-Aldrich
98%
di-Sodium hydrogenphosphate
®
35
Enzymatic Hydrolysis of L-Arginine
3.1.4. Solutions
Table 3.2. Solutions prepared during the course of the enzymatic hydrolysis of L-arginine experiments.
Solution
Composition
Activation buffer
50 mM triethanolamide in Milli-Q water
1 Mm manganese (II) chloride added
pH adjusted to 8.0 with glacial acetic acid
Derivatization buffer
0.1 M borate buffer, pH=9.0
(addition of a 6.2 g/l solution of boric acid in Milli-Q
water to a 38.15 g/l solution of borax in Milli-Q
water until pH = 9 .0 is obtained)
Eluent A
50 mM sodium acetate in Milli-Q water
pH adjusted to 4.5 with concentrated acetic acid
Fluorescamine solution
10 mg of fluorescamine in 20 ml of acetonitrile
Substrate solution
250 mM L-Arginine in Milli-Q water
pH adjusted to 9.5 with glacial acetic acid
3.1.5. Equipment
High Performance Liquid Chromatography
Reverse-phase HPLC analyses were performed on a Waters™ System consisting of:
Waters™ 600s Controller; Waters™ In-Line Degasser; Waters™ 616 Pump; Waters™
®
®
717plus Autosampler; Jasco 820-FP Intelligent Spectrofluorometer; Nova-Pak C18 column
®
60 Ǻ 3.9 x 150 mm with a Nova-Pak C18 4 µm Guard-Pak™ pre-column insert.
Rotator
During immobilization experiments a Cole-Parmer Roto-Torque model 7637-10
Heavy Duty Rotator was utilized. This rotator allows the gentle but efficient mixing, at variable
speed, of flasks, bottles, and test tubes with different shapes and sizes. The angle of rotation
is adjustable.
Figure 3.1. Cole-Parmer Roto-Torque model 7637-10 Heavy Duty Rototator.
36
Enzymatic Hydrolysis of L-Arginine
Thermomixer
®
An Eppendorf Thermomixer Comfort was utilized. This thermomixer is equipped with
a rack that allows the simultaneous heating of up to 24 1.5 ml eppendorf Safe-Lock tubes. It is
fully programmable, capable of heating or cooling samples from 1°C to 99°C and of agitating
from 300 rpm to 1500 rpm (also has a no mixing mode).
3.1.6. Analytical Techniques
Fluorescamine Derivatization
Previously to HPLC analysis, a fluorescamine derivatization procedure was applied to
each sample. This pre-column derivatization method allows the efficient separation and
fluorescence detection of amino acids.
To 5 µl aliquots of each sample were added 75 µl of the derivatization buffer and 20
µl of fluorescamine solution, immediately followed by thorough mixing. 100 µl of the
derivatized samples were used for HPLC analysis.
HPLC Analysis
After derivatization, the samples were analyzed on the previously described reversephase HPLC Waters™ system. 10 µl per sample were injected and the column was eluted at
30°C with a flow rate of 1 ml/min. The gradient of the eluents utilized is described in table 3.3,
eluent B consists of pure acetonitrile of liquid chromatography gradient grade. The total time
of analysis for each sample was of 20 minutes.
Table 3.3. Gradient of eluents applied during HPLC analysis.
Time
% Eluent A
% Eluent B
Curve
0
80
20
6
6
80
20
6
6.5
60
40
6
9.5
60
40
6
80
20
6
15
a
a
for the slope of the different curves see Appendix A.
Bradford Assay
5 µl of each sample were pipeted to separate wells of a 96 wells microplate. To each
well was added 250 µl of Quick Start Bradford Dye Reagent (Bio-Rad). Absorbance was
immediately measured at 595 nm and compared with a simultaneously obtained standard
curve from Bovine Serum Albumin. Each assay was performed in triplicate.
37
Enzymatic Hydrolysis of L-Arginine
3.1.7. Preparation of Arginase Stock Solution
An arginase stock solution was prepared diluting 0.8 ml of enzyme solution to a total
volume of 5.5 ml with Milli-Q water. Sodium azide was added (0.05% mass) to inhibit
microbial growth. The solution was filter sterilized (0.2 µm filter) and samples were collected
for Bradford and activity assays.
For determination of the solution’s activity, the L-arginine and L-ornithine
concentrations were monitored during an activity assay. A portion of the enzyme stock
solution was diluted 25 times with Milli-Q Water and 300 µl of the dilution were incubated with
300 µl of Milli-Q water for 5 minutes at 37°C in a thermomixer (agitation of 1200 rpm). After
the incubation time, 400 µl of the substrate solution were added and the clock was started.
The reaction was carried in the thermomixer at 37°C. 10 µl samples were taken at 2, 4, 6 and
8 minutes. The samples were immediately quenched with 150 µl of 1 M acetic acid. 100 µl of
the quenched samples were diluted with 400 µl of Milli-Q water and stored in the fridge (5˚C)
until fluorescamine derivatization and HPLC analysis.
3.1.8. Immobilization of Arginase in Different Epoxy-activated Supports
Immobilization in Sepabeads EC-HFA
For the immobilization in Sepabeads EC-HFA, 0.5 ml of arginase stock solution were
added to 7.5 of filter sterilized (0.2 µm filter) 10 mM sodium phosphate buffer, pH=8.0. After
gentle mixing, the solution was added to a sterile 10 ml test tube containing 2.2 g (wet weight;
1 g dry weight) of support, the tube was immediately placed on the rotator at slow rotation
speed and the clock was started. The immobilization was carried for 24 hours with 60 µl
samples of the supernatant being collected at 0, 1, 2, 4, 6, 23 and 24 hours. 30 µl of each
sample were immediately utilized for an activity assay, while the remaining of the sample was
stored in the fridge (5˚C) for posterior Bradford assay. The immobilization was conducted in
duplicate and in the presence of a blank (also in duplicate) containing the same enzyme
solution/buffer ratio but no support added.
Immobilization in Sepabeads EC-EP
For the immobilization in Sepabeads EC-EP, 0.5 ml of arginase stock solution were
added to 7.5 of filter sterilized (0.2 µm filter) 0.8 M sodium phosphate buffer, pH=8.0. After
gentle mixing, the solution was added to a sterile 10 ml test tube containing 2.4 g (wet weight,
1 g dry weight) of support, the tube was immediately placed on the rotator at slow rotation
speed and the clock was started. The immobilization was carried for 24 hours with 60 µl
samples of the supernatant being collected at 0, 1, 2, 4, 6, 23 and 24 hours. 30 µl of each
sample were immediately utilized for an activity assay, while the remaining of the sample was
stored in the fridge (5˚C) for posterior Bradford assay. The immobilization was conducted in
38
Enzymatic Hydrolysis of L-Arginine
duplicate and in the presence of a blank (also in duplicate) containing the same enzyme
solution/buffer ratio but no support added.
Immobilization in Eupergit
For the immobilization in Eupergit C 250, 0.5 ml of arginase stock solution were
added to 7.5 of filter sterilized (0.2 µm filter) 0.8 M sodium phosphate buffer, pH=8.0. After
gentle mixing, the solution was added to a sterile 10 ml test tube containing 1 g (dry weight) of
support, the tube was immediately placed on the rotator at slow rotation speed and the clock
was started. The immobilization was carried for 24 hours with 70 µl samples of the
supernatant being collected at 0, 1, 2, 4, 6, 23 and 24 hours. The samples were centrifuged (1
minute at 1200 rpm) and 60 µl of the supernatant were collected. 30 µl of each sample were
immediately utilized for an activity assay, while the remaining of the sample was stored in the
fridge (5˚C) for posterior Bradford assay. The immobilization was conducted in duplicate and
in the presence of a blank (also in duplicate) containing the same enzyme solution/buffer ratio
but no support added.
Soluble Arginase Activity Assay
For the activity assay of the samples collected during the immobilizations, 30 µl of the
sample was incubated with 30 µl of Milli-Q water for 5 minutes at 37°C in a thermomixer
(agitation of 1200 rpm). After the incubation time, 40 µl of the substrate solution were added
and the clock was started. The reaction was carried in the thermomixer at 37°C. 10 µl
samples were taken at 2, 4, 6 and 8 minutes. The samples were immediately quenched with
150 µl of 1 M acetic acid. 100 µl of the quenched samples were diluted with 400 µl of Milli-Q
water and stored in the fridge (5˚C) until fluorescamine derivatization and HPLC analysis.
Washing
After the immobilization, all supports containing the immobilized enzyme were
washed and stored following the same protocol. The suspensions were filtered using a
sintered glass filter. The filtrate was rinsed with 8 ml of 50 mM sodium phosphate buffer
(pH=8.0) and filtrated once more. The filtered support was collected on a 10 ml sterile test
tube, to which was added 8 ml of sterile 50 mM sodium phosphate buffer (pH=8.0), and
placed on the rotator at slow rotation speed for 45 minutes. After this washing step, the
filtering/rinsing procedure was repeated and the dry support with the immobilized arginase
was stored in the fridge (5˚C). After each filtration step a 1 ml sample of the supernatant was
collected for posterior analysis.
Blockage of the Remaining Epoxy Groups
After immobilization, immobilized arginase can be submitted to the blockage of the
epoxy groups on the support surface that did not react with the enzyme. With this objective,
portions of the three supports containing the immobilized arginase were submitted to a similar
39
Enzymatic Hydrolysis of L-Arginine
blockage procedure. 0.5 g of each support were placed in a 10 ml sterile test tube to which
were added 4 ml of 3 M glycine in 50 mM sodium phosphate buffer (pH=8, filter sterilized).
The suspensions were place in the rotator at slow rotation speed. After 18h the blocked
supports were washed and stored accordingly to the previously described procedure.
Immobilized Arginase Activity Assay
The activity of the enzyme immobilized in the different supports was assayed through
an activity assay comparable to the one utilized for soluble enzyme. 25 mg of the supports
containing immobilized arginase were incubated with 600 µl of activation buffer for 5 minutes
at 37°C in a thermomixer (agitation of 1200 rpm). After the incubation time, 400 µl of the
substrate solution were added and the clock was started. The reaction was carried in the
thermomixer at 37°C. 10 µl samples were taken at 2, 4, 6 and 8 minutes. The samples were
immediately quenched with 150 µl of 1 M acetic acid. 100 µl of the quenched samples were
diluted with 400 µl of Milli-Q water and stored in the fridge (5˚C) until fluorescamine
derivatization and HPLC analysis.
3.1.9. Thermal Stability of Immobilized Arginase
This experiment was conducted with the objective of studying the effect of
immobilization in the stability of arginase. Arginase immobilized in the three tested supports
and in soluble form was incubated at 60°C for different periods.
Multiple 25 mg portions of each of the three supports containing the immobilized
enzyme (with and without blockage of the remaining epoxy groups) were suspended in 600 µl
of activation buffer water in 1.5 ml test tubes. A set of tubes containing the soluble enzyme
was also prepared by diluting 20 µl of arginase stock solution with 580 µl of activation buffer.
The 7 sets of tubes were placed on a water-bath and the clock was stared. Periodically, a
tube from each set was withdrawn and the remaining activity of the immobilized arginase was
immediately assayed at 60°C as previously described. Tubes were assayed after 1, 2, 4, 6,
16 and 24 hours of incubation at 60°C.
3.2. Results and Discussion
3.2.1. Characterization of the Arginase Stock Solution
The exact protein concentration and activity in the Bacilus subtillis arginase stock
solution where determined by Bradford and activity assays respectively. The Bradford assay
gave a protein concentration of 4.1 mg/ml. This value together with the soluble enzyme
40
Enzymatic Hydrolysis of L-Arginine
activity assay, which reported an activity of 325 U/ml, gives a specific activity of 80 U/mgprotein in the arginase stock solution. One unit is defined as one mol of L-ornithine produced
per minute at pH=9.5 and 37°C.
The value of specific activity obtained is only 9% of the value obtained by Nakamura
32
et al.
for purified Bacilus subtillis arginase (858 U/mg). This low value can not be explained
2+
by the relatively low concentration of manganese ions. The Mn
concentration in the stock
32
solution is close to 7 mM, well above the concentration suggested by Nakamura et al.
for full
enzyme activation (3 mM). A possible explanation can be the use of more aggressive
purification methods that damages the enzyme’s structure. The purification methods
employed were not specified by the supplier.
A SDS-PAGE gel of the original arginase solution was prepared in order to verify its
purity. The run was done in the presence of a reducing agent, guarantying that the enzyme is
dissociated to its sub-units.
Figure 3.2. SDS-PAGE gel of the original arginase solution. Lane (A) corresponds to a 100X dilution
while lane (B) corresponds to a 200X dilution. Lane (M) contains the molecular markers identified with
the corresponding molecular weights in Daltons.
A strong band is observed slightly below 38.000 Da. Due to the presence of reducing
agents, this band should correspond to the monomeric units of arginase and is in accordance
with the range of values reported for the molecular weight of monomers of other bacterial
53,54
arginases, 31.000-34.000 Da
32
Nakamura et al.
.
This value also suggests that the enzyme purified by
was probably in the trimeric form, as a molecular weight of 115.000±5.000
Da was obtained by this author for the purified native arginase.
The only other bands found on the arginase lanes are located around 70.000 Da and
110.000 Da (only observed in the 100X dilution lane). These bands should correspond to the
dimeric and trimeric forms of the enzyme, showing that arginase is not fully dissociated in the
41
Enzymatic Hydrolysis of L-Arginine
presence of the reducing agent. This leads to the conclusion that the arginase solution
supplied is highly pure.
3.2.2. Immobilization of Arginase in Epoxy-activated Supports
The arginase covalent immobilization in Sepabeads EC-HFA, Sepabeads EC-EP and
Eupergit C epoxy-activated supports was followed for 24 hours. Figure 3.3 shows the
evolution of protein concentration in the supernatant during the course of the immobilization
step.
Sepabeads EC-HFA
Sepabeads EC-EP
Protein Concentration (µg/mL)
80%
60%
Sepabeads EC-HFA
40%
Blank
20%
100%
80%
60%
0
5
10
15
20
Sepabeads EC-EP
40%
Blank
20%
0%
0%
25
0
5
10
Time (h)
15
20
Time (h)
Eupergit C
Protein Concentration (µg/mL)
Protein Concentration (µg/mL)
100%
100%
80%
60%
40%
Eupergit C
Blank
20%
0%
0
5
10
15
20
25
Time (h)
Figure 3.3. Protein concentration in the supernatant during the course of arginase immobilization in
different epoxy-activated supports. The percentages are based on the initial (t=0) protein concentration.
Error bars calculated using standard deviation of the two duplicate experiments.
The immobilization course of the enzyme was also followed by measuring the
remaining supernatant activity, as shown on figure 3.4.
42
25
Enzymatic Hydrolysis of L-Arginine
Sepabeads EC-HFA
Sepabeads EC-EP
100%
60%
40%
Sepabeads EC-HFA
Blank
Supernatant Activity (%)
80%
80%
60%
40%
Sepabeads EC-EP
Blank
20%
20%
0%
0%
0
5
10
15
20
25
0
5
10
Time (h)
15
20
Time (h)
Eupergit C
100%
Supernatant Activity (%)
Supernatant Activity (%)
100%
80%
60%
40%
Eupergit C
Blank
20%
0%
0
5
10
15
20
25
Time (h)
Figure 3.4. Arginase activity in the supernatant during the course of arginase immobilization in different
epoxy-activated supports. The percentages are based on the initial (t=0) protein concentration. Error
bars calculated using standard deviation of the two duplicate experiments.
The immobilization of arginase in the three different supports was clearly successful.
After one hour no protein was found in the supernatant of all suspensions (figure 3.3),
indicating that all enzyme is bound to the supports. The analysis of the supernatant activity
(figure 3.4) shows comparable results. After 24 hours of incubation only the suspension with
Sepabeads EC-EP shows slight remaining activity in the supernatant.
Previous studies
48
showed similarly successful results with Sepabeads EC-HFA, the
complete immobilization of different enzymes is observed after 1 to 6 hours. In the case of
Sepabeads EC-EP and Eupergit C, the results obtained for arginase immobilization were
better than the results reported for other enzymes. Using these supports and similar
incubation conditions for covalent immobilization of various enzymes, Mateo et al.
48
observed
an immobilization inferior to 65% of the enzyme after 25 hours of incubation.
After 24 hours, low concentrations of protein are detected on the supernatants of
Sepabeads EC-EP and Eupergit C immobilizations, suggesting that arginase might be only
temporarily bond to this supports. This apparent release of arginase from both short-chained
epoxy group supports is contained in the experimental error margin for this experiment, but
43
25
Enzymatic Hydrolysis of L-Arginine
the immobilization should be followed for a longer period in order to clarify the results. If the
release of arginine from the supports after longer periods of incubation is confirmed, a
possible explanation can be damage inflicted to the structure of the beads by mixing or the
high concentration of sodium phosphate buffer (0.8 M). This seems unlikely, as the mixing
should also affect the Sepabeads EC-HFA beads and previous work with higher
43
concentration of sodium phosphate buffer during incubation did not show similar problems .
The blanks have the exact same composition of the immobilization suspensions
without the addition of support: arginase in 10 mM sodium phosphate buffer for Sepabeads
EC-HFA immobilization; arginase in 0.8 M sodium phosphate buffer for Sepabeads EC-EP
and Eupergit C 250 L immobilization. The evolution of protein concentration and enzyme
activity in the blanks shows a slight decreasing pattern. Again, this apparently abnormal
observation can be due to experimental error. Still, it is possible that 24 hours of incubation at
room temperature in the presence of a buffer can lead to minor degradation of the enzyme,
affecting the Bradford assay and the enzyme’s catalytic activity.
After incubation for 24 hours in the presence of arginase, a washing protocol was
applied to the support beads. During this procedure, the supernatants of the filtration steps
were analyzed in order to detect any protein that might be released from the beads (figure
3.5). In a first observation, the analysis confirms the presence of small amounts of enzyme in
the supernatants of Sepabeads EC-EP and Eupergit C suspensions at the end of incubation
time, similarly to what was observed in figure 3.3. Moreover, during the first rinse step it is
clear that some enzyme is released from the Eupergit C beads, showing that not all arginase
is covalently bound to the support. The protein that is released when the beads are rinsed
with 50 mM sodium phosphate buffer has yet to complete the second step of the binding
mechanism, and is only physically adsorbed to the support’s surface by hydrophobic
interactions. When in contact with a buffer with lower ionic strength (50 mM as opposed to
800 mM), this interactions are weaker and the enzyme is released. The presence of residual,
non-covalently bound enzyme after incubation has previously been reported for Eupergit C
46
supports .
Protein Concentration (µg/mL)
Arginase Immobilization - Rinse/Wash Steps
Sepabeads EC-HFA
60,0
Sepabeads EC-EP
50,0
Eupergit C
40,0
30,0
20,0
10,0
0,0
Supernatant
Rinse 1
Wash
Rinse 2
Figure 3.5. Protein content in the filtration supernatant after each step of the beads washing procedure.
Error bars calculated using standard deviation of the two duplicate experiments.
44
Enzymatic Hydrolysis of L-Arginine
Finally, it should be noted that both Sepabeads supports were easily handled, leading
to minimal support loss during the immobilization of arginase in these supports. However,
Eupergit C 250 L originates viscous suspensions, making the manipulation of the beads more
difficult. This led to a considerable loss of Eupergit C support during the washing steps. The
initial and recovered masses of all supports during the immobilization and blockage steps can
be consulted in Appendix G.
3.2.3. Recovered Activity of Immobilized Arginase
The recovered activity exhibited by the enzyme immobilized in the different supports
was compared with the soluble form. The support beads and soluble enzyme were assayed in
2+
the presence of a 50 mM triethanolamide/acetate buffer (pH=8.0) with 1mM Mn . This buffer
is utilized in order to guarantee that the pH and manganese concentration values are similar
in the different assays, as the soluble enzyme solution is already buffered and contains a
relatively high concentration of manganese ions.
For the calculation of the amount of enzyme immobilized in each milligram of support
the following assumptions were made: 100% of the enzyme in solution during incubation was
immobilized in the Sepabeads EC-HFA support, while only 95% was permanently
immobilized in the Sepabeads EC-EP and Eupregit C supports; 100% of the Sepabeads
supports were recovered after the washing steps, while only 95% of the initial mass of
Eupergit C support was recovered.
Recovered Activity (%)
Recovered Activity of Immobilized Arginase
100
free epoxy groups
epoxy groups
blocked with glycine
80
60
40
20
0
Sepabeads EC-HFA
Sepabeads EC-EP
Eupergit C 250 L
Figure 3.6. Recovered activity of arginase immobilized in different epoxy supports. Percentages are
calculated by comparison with the soluble form. Error bars calculated using standard deviation of the
two duplicate experiments.
As expected, the covalent immobilization greatly influences the catalytic activity of
arginase (figure 3.6). The multi-point reaction between arginase and the support deforms the
enzyme, altering the shape of its active site and, possibly, affecting manganese uptake. The
values of recovered activity obtained for immobilization of arginase in the different epoxy
45
Enzymatic Hydrolysis of L-Arginine
supports – from 43% to 61% – are low but acceptable. The results described in literature for
immobilization of other enzymes on similar supports are extremely irregular. For example, in
the case of ß-galactosidase values of recovered activity range from 15% to 100% depending
48
on the organism of origin and type of epoxy support utilized .
Despite the rather inconsistent results (see error bars), it is clear that the treatment
with glycine decreases the recovered activity of the immobilized arginase. The opposite
results were expected, as glycine was added with the objective of reacting with the epoxy
groups that remained free after incubation time. This would stop the covalent-binding
reaction, preventing excessive enzyme/support interaction that could destabilize the enzymes’
active site. One possible explanation for the abnormal results is that glycine might directly
interact with arginase’s active site, interfering with the catalytic mechanism.
The effect of the type of epoxy-activated support on the activity of immobilized
arginase is not clear. Previous studies show that different supports can have considerably
48
different effect on the immobilized enzyme activity . This may be related to the distinct
hydrophobicity of the surface of the various supports. The stronger or weaker hydrophobic
interactions between the support and the hydrophobic residues can affect the orientation of
the enzyme and of its active site, affecting the catalytic activity. The length of the epoxy
groups’ spacer arms (longer in Sepabeads EC-HFA) can also be a factor on the orientation of
the enzyme.
Finally, it is possible that the immobilization affects the quaternary structure of the
enzyme, resulting in the dissociation of arginase to its sub-units. If this is the case,
immobilized arginase monomers can still show residual activity and previous work
26
indicates
that catalytic activity can be fully restored by the addition of soluble monomers. The
referenced work was performed using human arginase, further experimentation is necessary.
3.2.4. Thermal Stability of Immobilized Arginase
The stability of the immobilized arginase derivatives was analyzed by following the
residual activity of the enzyme during incubation at 60°C. The activity of arginase immobilized
in the three tested supports (with and without blockage with glycine) and of soluble arginase
was assayed after periods of incubation at 60°C up to 24 hours. The results are illustrated in
figure 3.4. To prevent minor pH variations that may have a significant effect in the decrease of
specific activity at high temperatures, the beads were incubated in the presence of a 50 mM
2+
triethanolamide/acetate buffer (pH=8.0) with 1mM Mn . The addition of manganese ions
slows the deactivation of the enzyme (results no shown). In the absence of the metal ion the
deactivation is too fast, not allowing accurate comparison of the influence of immobilization in
the different supports.
46
Enzymatic Hydrolysis of L-Arginine
Figure 3.7. Evolution of residual activity of arginase immobilized in different epoxy supports
compared to soluble arginase during the course of 6 hours incubation at 60°C. The percentages are
based on the initial (t=0) enzyme activity.
Initial interpretation of the results suggests that the blockage of the non-reactive
epoxy groups with glycine has a positive effect in immobilized enzyme stability. The blocked
supports with short-chained epoxy groups (Sepabeads EC-EP and Eupergit 250 C) appear to
retain their activity during the first 6 hours of incubation at 60°C. However, the graphics are
misleading. Even though the activity in these supports remains constant during the monitored
time period, its initial values are low when compared with the non-blocked supports. The low
ornithine concentrations produced not only aggravate the inherent experimental error, but
also difficult HPLC analysis and chromatogram integration. Thus, the residual activity values
47
Enzymatic Hydrolysis of L-Arginine
obtained for the blocked Sepabeads EC-EP and Eupergit 250 C supports are not reliable and
should not be taken in much consideration. Possible solutions for this problem are the use of
more support during activity assays, less dilution of the samples and duplicate experiments.
Concerning the effect of the other tested supports, only the covalent-binding to
Sepabeads EC-EP without glycine blockage shows a mild positive effect in arginase thermal
stability. The immobilization in this supports leads to a 20 % increase in residual activity when
compared with the soluble enzyme after 6 hours of incubation at 60°C.
The immobilization of arginase in Sepabeads EC-HFA and Eupergit C 250 L appears
to have a negative effect in the enzyme’s stability. The deactivation of the immobilized
derivatives for both these supports is faster than the deactivation observed for the free
enzyme, although the blockage of Sepabeads EC-HFA with glycine seems to lead to an
increase in the derivatives stability.
48
The results are not in accordance with the results reported by Mateo et al. . This
author claims that the immobilization of different enzymes in all the three tested supports
consistently increases the enzymes’ thermal stability. Again, the results showed in the present
study are not extremely reliable and future duplicate experiments are essential to draw
objective conclusions.
3.2.4. Analytical Methods
The fluorescamine derivatization and HPLC analysis method utilized were suitable for
the experiments performed. The peeks obtained in the chromatogram show good separation
and are of easy integration (figure 3.5). The concentrations of arginine and ornithine in the
analyzed samples were calculated without difficulties using a linear standard curve.
Figure 3.8. Example of a typical HPLC chromatogram obtained for an activity assay sample.
48
Enzymatic Hydrolysis of L-Arginine
Concerning the Bradford assay, it was verified that high buffer concentrations
interfere with the absorbance reading. This interference was observed during the analysis of
samples from immobilization of arginase in Sepabeads EC-EP and Eupergit C 250 (0.8 M
sodium phosphate buffer). The results were corrected accordingly.
49
Conclusions and Future Perspectives
4. Conclusions and Future Perspectives
Chemical Hydrolysis of L-Arginine
The results obtained show that the chemical catalysis of arginine hydrolysis is not, at
this moment, a suitable method for ornithine production on an industrial scale. The maximum
yield of ornithine obtained was 15.2% at 125°C with pH initially adjusted to pH=12 with
sodium hydroxide. Not only were the yields obtained disappointing when compared to the
13
values mentioned in literature (90% by Murray et al. ), but also the optimization of the
reaction would probably involve the use of buffers or the constant correction of pH with acid or
alkali, techniques undesirable in an industrial process. Nonetheless, on a lab scale study
these changes to the protocol could produce interesting results. The addition of small
quantities of acid or alkali in certain moments of the reaction for pH correction would require
the alteration of the experimental setting, as the reactors utilized do not allow this procedure.
A potential alternative for industry could be manipulation of pH through the continuous
separation of the different components of the reaction mixture.
Unfortunately, the high temperatures employed do not allow the recovery of urea, a
considerably valuable side product. Nonetheless, the production of ammonia can still provide
a substantial source of income in an industrial scale process.
The tested metal salts showed no influence either on arginine consumption rate or
ornithine yield.
The solid catalyst utilized, NaY zeolite, showed limited catalytic effects even at high
concentrations (5 g/l). It also displays the tendency to accumulate in the reactor walls, which
would generate serious complications in industry. As an alternative, other materials with
catalytic properties could be utilized including: zeolites ion-exchanged with metal ions that
+
+
+
generate stronger basic sites (Cs , Rb , K ); alkaline earth oxides; diverse heterogeneous
19
superbasic catalysts .
Enzymatic Hydrolysis of L-Arginine
The results obtained were promising, with Bacillus subtllis arginase being
successfully immobilized in three different supports suitable for industrial application. The
covalent binding to the three tested supports did not show a significant increase in arginase’s
thermal stability and the activity of the immobilized derivatives is considerably low when
compared with the soluble form. The blockage of the un-reactive epoxy groups with glycine
did not show significant increase in stability/activity. Similar results were obtained for all the
tested supports, however, Sepabeads supports are of easier handling and should be
preferred over Eupergit C 250 L.
Further research should focus on identifying the operational conditions that maximize
the production of ornithine and the stability of immobilized arginase. Bacillus subtilis
arginase’s properties have not been the subject of extensive studies. Thus, experimental work
should be realized to determine: optimum pH and temperature; effects of product inhibition;
51
Conclusions and Future Perspectives
2+
optimum Mn
concentrations. The manganese concentration is a particularly important factor
when considering industrial-scale application, as high salt concentrations can interfere with
downstream processing. Naturally, the next research step would be the lab-scale simulation
of a batch or continuous process for ornithine production with recovery and re-utilization of the
immobilized arginase
Finally, it should be noted that any possible industrial application of arginase would
require the large scale production of this enzyme at affordable prices. Presently the enzyme is
only available in diagnostic quantities at high expenses, being usually purified from mammal
liver.
Final Remarks
The research done on the chemical and enzymatic catalysis of the hydrolysis of
L-arginine can not be objectively compared. While the experimental setup utilized to study the
chemically catalyzed reaction is similar to a potential industrial process, the work realized on
the enzymatic conversion was primarily focused on the immobilization of arginase in industrial
suitable supports and not on the optimization of L-ornithine production.
Nonetheless, the biocatalysis approach seems to be the be the most promising. The
arginase catalyzed reaction is very clean when compared to the alkali catalyzed reaction, with
the only secondary product produced being urea. Of course, urea is actually an economically
attractive side product that cannot be obtained from the alkali catalyzed conversion of
L-arginine due to the high temperatures employed. The major drawbacks of arginase
application to a large scale process are the already mentioned dependence on manganese
ions and limited availability.
52
References
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20.
Milic, D. R.; Opsenica, D. M.; Adnadevic, B.; Solaja, B. A., NaY Zeolite: A Useful
Catalyst for Nitrile Hydrolysis. Molecules 200, 5, 118-126.
21.
Schmieg, S. J.; Cho, B. K.; Oh, S. H., Selective catalytic reduction of nitric oxide with
acetaldehyde over NaY zeolite catalyst in lean exhaust feed. Applied Catalysis B:
Environmental 2004, 49, (2), 113-125.
22.
Lu, G., Reducibility and CO hydrogenation over Pt and Pt-Co bimetallic catalysts
encaged in NaY-zeolite. Catalysis Letters 1992, 14, (2), 207.
23.
Salama, T. M., Highly Selective Catalytic Reduction of NO by H2over Au0and Au (I)
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Seong Ihl Woo, Y. S. , Polymerization of ethylene over metallocenes confined inside
the supercage of a NaY zeolite. Macromolecular Rapid Communications 1995, 16,
(7), 489-494.
25.
Ikeda, T.; Yasunaga, T., Kinetic behavoir of L-arginine in the interlamellar layer of
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1253-1257.
26.
Carvajal, N.; Martinez, J.; Fernandez, M., Immobilised monomers of human liver
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Bewley, M. C.; Jeffrey, P. D.; Patchett, M. L.; Kanyo, Z. F.; Baker, E. N., Crystal
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Structure 1999, 7, (4), 435-448.
28.
Kanyo, Z. F.; Scolnick, L. R.; Ash, D. E.; Christianson, D. W., Structure of a unique
binuclear manganese cluster in arginase. Nature 1996, 383, (6600), 554-557.
29.
Christianson, D. W., Arginase: Structure, mechanism, and physiological role in male
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30.
Reczkowski, R. S.; Ash, D. E., Rat liver arginase: Kinetic mechanism, alternate
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31.
Cama, E.; Pethe, S.; Boucher, J.-L.; Han, S.; Emig, F. A.; Ash, D. E.; Viola, R. E.;
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32.
Nakamura, N. F., Masako; Kimura, K., Purification and properties of L-arginase from
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33.
Valle-Vega, P., Arginase-Urease Electrode for Determination of Arginine and Peanut
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34.
Veronese, F. M.; Visco, C.; Benassi, C. A.; Lora, S.; Carenza, M.; Palma, G.,
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1989, 542, 115-120.
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References
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Bommarius, A. S.; Drauz, K., An enzymatic route to L-ornithine from arginine-activation, selectivity and stabilization of L-arginase. Bioorganic & medicinal chemistry
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Alonso, A.; Almendral, M. J.; Baez, M. D.; Porras, M. J.; Alonso, C., Enzyme
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Karacaoğlu, S.; Timur, S.; Telefoncu, A., Arginine selective biosensor based on
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Bagnost, T.; Guillaume, Y. C.; Thomassin, M.; Robert, J. F.; Berthelot, A.; Xicluna, A.;
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Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wubbolts, M.; Witholt, B., Industrial
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Mateo, C.; Abian, O.; Fernandez-Lorente, G.; Pedroche, J.; Fernandez-Lafuente, R.;
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46.
Boller, T.; Meier, C.; Menzler, S., EUPERGIT Oxirane Acrylic Beads: How to Make
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47.
Hilterhaus, L.; Minow, B.; Mu?ler, J.; Berheide, M.; Quitmann, H.; Katzer, M.; Thum,
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References
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Krause, I., Simultaneous determination of amino acids and biogenic amines by
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Meriwether, L., Metal Ion Promoted Hydrolysis of Glycine Amide and of
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Buckingham, D. A., Peptide bond formation and subsequent hydrolysis at a cobalt
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Jenkinson, C. P.; Grody, W. W.; Cederbaum, S. D., Comparative properties of
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Kanda, M.; Ohgishi, K.; Hanawa, T.; Saito, Y., Arginase of Bacillus brevis Nagano:
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56
Appendix A
Appendix A – Gradient Curves
During the elution of a sample through the HPLC system, the rate of change of
solvent composition over time depends on the curve number and the length of the gradient
segment. The gradient curve profile specified in each row of the gradient table (table 2.3)
affects both solvent composition and flow rate. The different curves identified by a specific
number are represented in figure A.1.
Figure A.1. Different gradient curves identified by the input number.
57
Appendix B
Appendix B – NMR Spectra of L-Arginine
-0.0000
30.4308
26.8074
43.4372
57.0779
159.7801
177.0880
ARG-acid (13C NMR in D2O + D2SO4 + TSP)
*** Current Data Parameters ***
NAME
:
abg-acid
EXPNO
:
2
PROCNO
:
1
*** Acquisition Parameters ***
DATE_t
:
10:08:47
DATE_d
:
Aug 07 2008
NS
:
1024
NUCLEUS
:
off
PARMODE :
1D
SW
:
238.8728 ppm
*** Processing Parameters ***
GB
LB
:
:
OFFSET
:
SI
:
0.0000000
1.00 Hz
222.3916 ppm
32768
*** 1D NMR Plot Parameters ***
Height
180
160
140
120
100
(ppm)
Figure B.1.
13
80
60
40
20
:
14.78 cm
Start
:
200.00 ppm
Stop
:
-2.00 ppm
ppm_cm
:
AQ_time
:
NUCLEUS
:
9.14
1.3631490 sec
off
0
C-NMR spectrum of L-arginine.
30.4235
26.8001
43.4299
57.0706
ARG-acid (DEPT135 in D2O + D2SO4 + TSP)
*** Current Data Parameters ***
NAME
:
EXPNO
:
abg-acid
3
PROCNO
:
1
*** Acquisition Parameters ***
DATE_t
:
10:24:32
DATE_d
:
Aug 07 2008
NS
:
256
NUCLEUS
:
off
PARMODE :
1D
SW
:
238.8728 ppm
*** Processing Parameters ***
GB
:
LB
:
OFFSET
:
SI
:
0.0000000
1.00 Hz
222.3916 ppm
32768
*** 1D NMR Plot Parameters ***
Height
180
160
140
120
100
(ppm)
80
60
40
Figure B.2. DEPT spectrum of L-arginine.
58
20
0
:
14.78 cm
Start
:
200.00 ppm
Stop
:
-2.00 ppm
ppm_cm
:
AQ_time
:
NUCLEUS
:
9.14
1.3631490 sec
off
1.7679
1.7510
1.7347
1.7109
1.6877
1.6707
1.6538
1.6444
1.9398
1.9304
1.9141
1.8984
3.2555
3.2386
3.2210
3.8181
3.8030
3.7874
Appendix B
*** Current Data Parameters ***
NAME
:
ABG-ACID
EXPNO
:
1
PROCNO
:
1
*** Acquisition Parameters ***
DATE_t
:
09:07:56
DATE_d
:
Aug 07 2008
NS
:
16
NUCLEUS
:
off
PARMODE :
SW
:
1D
20.5503 ppm
*** Processing Parameters ***
GB
:
LB
:
OFFSET
:
SI
:
0.0000000
0.30 Hz
16.5079 ppm
32768
3.8
3.6
3.4
3.2
3.0
2.8
(ppm)
2.6
2.4
2.2
2.0
Height
:
Start
:
Stop
:
ppm_cm
:
AQ_time
:
NUCLEUS
:
13.78 cm
4.00 ppm
1.50 ppm
0.11
3.9845890 sec
off
2.0677
2.0381
2.0480
1.0000
Integral
*** 1D NMR Plot Parameters ***
1.8
1.6
1
Figure B.3. H-NMR spectrum of L-arginine.
59
Appendix C
Appendix C – NMR Spectra of L-Ornithine
-0.0000
25.5899
30.2413
41.7312
56.9612
176.8911
ORN (13C NMR in D2O + TSP)
*** Current Data Parameters ***
NAME
:
orn
EXPNO
:
2
PROCNO
:
1
*** Acquisition Parameters ***
DATE_t
:
16:57:48
DATE_d
:
Jun 19 2008
NS
:
1024
NUCLEUS
:
off
PARMODE :
SW
1D
:
238.8728 ppm
*** Processing Parameters ***
GB
:
LB
:
OFFSET
:
SI
:
0.0000000
1.00 Hz
222.2822 ppm
32768
*** 1D NMR Plot Parameters ***
Height
180
160
140
120
100
(ppm)
Figure C.1.
13
80
60
40
20
:
14.78 cm
Start
:
200.00 ppm
Stop
:
-4.99 ppm
ppm_cm
:
AQ_time
:
NUCLEUS
:
9.28
1.3631490 sec
off
0
C-NMR spectrum of L-ornithine.
25.5753
30.2340
41.7239
56.9539
ORN (DEPT135 in D2O + TSP)
*** Current Data Parameters ***
NAME
:
EXPNO
:
orn
3
PROCNO
:
1
*** Acquisition Parameters ***
DATE_t
:
17:13:01
DATE_d
:
Jun 19 2008
NS
:
256
NUCLEUS
:
off
PARMODE :
1D
SW
:
238.8728 ppm
*** Processing Parameters ***
GB
:
LB
:
OFFSET
:
SI
:
0.0000000
1.00 Hz
222.2822 ppm
32768
*** 1D NMR Plot Parameters ***
190
180
170
160
150
140
130
120
110
100
(ppm)
90
80
70
60
50
40
30
Figure C.2. DEPT spectrum of L-ornithine.
60
20
10
Height
:
14.78 cm
Start
:
200.00 ppm
Stop
:
ppm_cm
:
AQ_time
:
NUCLEUS
:
0.01 ppm
9.05
1.3631490 sec
off
3.0893
3.0705
3.0523
2.1003
2.0658
2.0508
2.0357
2.0282
2.0025
1.9874
1.9680
1.9523
1.9272
1.9128
1.8940
1.8802
1.8476
1.8288
1.8137
1.7999
0.0000
4.1097
4.0940
4.0784
4.8178
4.7657
4.7187
Appendix C
*** Current Data Parameters ***
NAME
:
OBN-ACID
EXPNO
:
1
PROCNO
:
1
*** Acquisition Parameters ***
DATE_t
:
14:38:06
DATE_d
:
Aug 07 2008
NS
:
16
NUCLEUS
:
off
PARMODE :
SW
:
1D
20.5503 ppm
*** Processing Parameters ***
GB
:
LB
:
OFFSET
:
SI
:
0.0000000
0.30 Hz
16.5223 ppm
32768
8.0
7.0
6.0
5.0
(ppm)
4.0
3.0
2.0
Height
:
13.78 cm
Start
:
10.00 ppm
Stop
:
-0.10 ppm
ppm_cm
:
AQ_time
:
NUCLEUS
:
0.46
3.9845890 sec
off
2.0328
2.0784
9.0
2.0145
1.0000
Integral
*** 1D NMR Plot Parameters ***
1.0
1
Figure C.3. H-NMR spectrum of L-ornithine.
61
Appendix D
Appendix D – NMR Spectra of 3-Aminopiperid-2-one
29.7091
27.5511
25.6264
22.7393
44.2246
41.7021
52.3900
174.3467
171.7950
LAC-acid (13C NMR in D2O + D2SO4 + TSP)
*** Current Data Parameters ***
NAME
:
lac-acid
EXPNO
:
2
PROCNO
:
1
*** Acquisition Parameters ***
DATE_t
:
14:16:38
DATE_d
:
Aug 07 2008
NS
:
1024
NUCLEUS
:
off
PARMODE :
SW
1D
:
238.8728 ppm
*** Processing Parameters ***
GB
:
LB
:
OFFSET
:
SI
:
0.0000000
1.00 Hz
222.4134 ppm
32768
*** 1D NMR Plot Parameters ***
180
160
140
120
Figure D.1.
100
(ppm)
13
80
60
40
20
Height
:
14.78 cm
Start
:
200.00 ppm
Stop
:
ppm_cm
:
AQ_time
:
NUCLEUS
:
-2.00 ppm
9.14
1.3631490 sec
off
0
C-NMR spectrum of 3-aminopiperid-2-one.
55.1167
52.3682
LAC-acid (DEPT135 in D2O + D2SO4 + TSP)
*** Current Data Parameters ***
NAME
:
lac-acid
EXPNO
:
3
PROCNO
:
1
*** Acquisition Parameters ***
DATE_t
:
14:32:05
DATE_d
:
Aug 07 2008
NS
:
256
NUCLEUS
:
PARMODE :
SW
:
off
1D
238.8728 ppm
*** Processing Parameters ***
GB
:
LB
:
OFFSET
:
SI
:
0.0000000
1.00 Hz
222.4134 ppm
32768
*** 1D NMR Plot Parameters ***
180
160
140
120
100
(ppm)
80
60
40
20
Figure D.2. DEPT spectrum of 3-aminopiperid-2-one.
62
0
Height
:
14.78 cm
Start
:
200.00 ppm
Stop
:
ppm_cm
:
AQ_time
:
NUCLEUS
:
-2.00 ppm
9.14
1.3631490 sec
off
Appendix D
0.0000
2.3361
2.3073
2.0847
2.0552
2.0251
1.9605
1.9047
1.8808
1.8582
1.8275
1.2361
3.3509
3.0937
3.0749
3.0567
4.8642
4.8121
4.7620
4.1680
4.1524
4.1367
4.0050
3.9780
LAC-acid (1H NMR in D2O + D2SO4 + TSP)
*** Current Data Parameters ***
NAME
:
LAC-ACID
EXPNO
:
1
PROCNO
:
1
*** Acquisition Parameters ***
DATE_t
:
13:17:00
DATE_d
:
Aug 07 2008
NS
:
NUCLEUS
:
off
PARMODE :
1D
SW
:
16
20.5503 ppm
*** Processing Parameters ***
GB
LB
:
:
OFFSET
:
SI
:
0.0000000
0.30 Hz
16.5524 ppm
32768
9.0
8.0
7.0
6.0
5.0
(ppm)
4.0
3.0
2.0
Height
:
13.28 cm
Start
:
10.00 ppm
Stop
:
ppm_cm
:
AQ_time
:
NUCLEUS
:
-0.10 ppm
0.46
3.9845890 sec
off
0.4046
8.9424
5.5586
3.4375
1.9922
6.7758
3.2915
1.0000
Integral
*** 1D NMR Plot Parameters ***
1.0
1
Figure D.3. H-NMR spectrum of 3-aminopiperid-2-one.
63
Appendix E
Appendix E – NMR Spectra of Putrescine
0.0000
31.7067
43.0070
PUT-acid 08-08-08 (13C NMR in D2O + D2SO4 + TSP)
*** Current Data Parameters ***
NAME
:
pvt-acid
EXPNO
:
2
PROCNO
:
1
*** Acquisition Parameters ***
DATE_t
:
12:55:35
DATE_d
:
Aug 07 2008
NS
:
1024
NUCLEUS
:
off
PARMODE :
1D
SW
:
238.8728 ppm
*** Processing Parameters ***
GB
:
LB
:
OFFSET
:
SI
:
0.0000000
1.00 Hz
221.5384 ppm
32768
*** 1D NMR Plot Parameters ***
180
160
140
120
100
(ppm)
Figure E.1.
13
80
60
40
20
Height
:
14.78 cm
Start
:
200.00 ppm
Stop
:
ppm_cm
:
AQ_time
:
NUCLEUS
:
-2.00 ppm
9.14
1.3631490 sec
off
0
C-NMR spectrum of putrescine.
31.6994
42.9998
PUT-acid 08-08-08 (DEPT135 in D2O + D2SO4 + TSP)
*** Current Data Parameters ***
NAME
:
EXPNO
:
pvt-acid
3
PROCNO
:
1
*** Acquisition Parameters ***
DATE_t
:
13:11:01
DATE_d
:
Aug 07 2008
NS
:
256
NUCLEUS
:
off
PARMODE :
1D
SW
:
238.8728 ppm
*** Processing Parameters ***
GB
:
LB
:
OFFSET
:
SI
:
0.0000000
1.00 Hz
221.5384 ppm
32768
*** 1D NMR Plot Parameters ***
Height
180
160
140
120
100
(ppm)
80
60
40
Figure E.2. DEPT spectrum of putrescine.
64
20
0
:
14.78 cm
Start
:
200.00 ppm
Stop
:
-2.00 ppm
ppm_cm
:
AQ_time
:
NUCLEUS
:
9.14
1.3631490 sec
off
Appendix E
-0.0000
1.4537
1.4456
1.4362
1.4286
1.4205
2.6152
2.5983
2.5826
4.5870
PUT-acid 08-08-08 (1H NMR in D2O + D2SO4 + TSP)
*** Current Data Parameters ***
NAME
:
pvt-acid
EXPNO
:
1
PROCNO
:
1
*** Acquisition Parameters ***
DATE_t
:
11:54:59
DATE_d
:
Aug 07 2008
NS
:
16
NUCLEUS
:
off
PARMODE :
SW
:
1D
20.5503 ppm
*** Processing Parameters ***
GB
:
LB
:
OFFSET
:
SI
:
0.0000000
0.30 Hz
16.2645 ppm
32768
9.0
8.0
7.0
6.0
5.0
(ppm)
4.0
3.0
Height
:
13.28 cm
Start
:
10.00 ppm
Stop
:
-0.10 ppm
ppm_cm
:
AQ_time
:
NUCLEUS
:
0.46
3.9845890 sec
off
1.0088
1.0000
Integral
*** 1D NMR Plot Parameters ***
2.0
1.0
1
Figure E.3. H-NMR spectrum of putrescine.
65
Appendix F
Appendix F – NMR Spectra of 30 h Reaction Mixture
-0.0000
30.2704
30.0590
27.6604
27.4490
26.7637
25.6336
22.7393
46.4701
44.2246
43.3862
41.7968
171.8387
HYDR-ARG30-acid (13C NMR in D2O + D2SO4 +TSP)
*** Current Data Parameters ***
NAME
:
hydr-a~1
EXPNO
:
2
PROCNO
:
1
*** Acquisition Parameters ***
DATE_t
:
16:58:48
DATE_d
:
Aug 07 2008
NS
:
1024
NUCLEUS :
off
PARMODE :
1D
SW
:
238.8728 ppm
*** Processing Parameters ***
GB
:
LB
:
OFFSET
:
SI
:
0.0000000
1.00 Hz
222.3916 ppm
32768
*** 1D NMR Plot Parameters ***
Height
:
14.78 cm
Start
:
200.00 ppm
Stop
:
ppm_cm
:
AQ_time
:
-2.00 ppm
9.14
1.3631490 sec
NUCLEUS :
180
160
140
120
100
80
60
40
20
off
0
(ppm)
Figure F.1.
13
C-NMR spectrum of 30h reaction mixture.
30.0517
27.4344
26.7491
22.7320
46.4628
44.2173
43.3716
41.7823
HYDR-ARG30-acid (DEPT135 in D2O + D2SO4 +TSP)
*** Current Data Parameters ***
NAME
:
EXPNO
:
hydr-a~1
3
PROCNO
:
1
*** Acquisition Parameters ***
DATE_t
:
17:14:14
DATE_d
:
Aug 07 2008
NS
:
256
NUCLEUS
:
PARMODE :
SW
:
off
1D
238.8728 ppm
*** Processing Parameters ***
GB
:
LB
:
OFFSET
:
SI
:
0.0000000
1.00 Hz
222.3916 ppm
32768
*** 1D NMR Plot Parameters ***
180
160
140
120
100
(ppm)
80
60
40
20
Figure F.2. DEPT spectrum of 30h reaction mixture.
66
0
Height
:
14.78 cm
Start
:
200.00 ppm
Stop
:
ppm_cm
:
AQ_time
:
NUCLEUS
:
-2.00 ppm
9.14
1.3631490 sec
off
Appendix F
0.0000
2.3386
2.3123
2.0213
1.9699
1.9555
1.9435
1.9297
1.8852
1.8614
1.8338
1.7510
1.6751
4.7889
4.7475
4.7112
4.2025
3.9046
3.8896
3.8745
3.3490
3.2951
3.2599
3.2436
3.1395
3.0755
3.0567
3.0392
7.2800
HYDR-ARG30-acid (1H NMR in D2O + D2SO4 +TSP)
*** Current Data Parameters ***
NAME
:
HYDR-A~1
EXPNO
:
1
PROCNO
:
1
*** Acquisition Parameters ***
DATE_t
:
15:59:09
DATE_d
:
Aug 07 2008
NS
:
16
NUCLEUS
:
off
PARMODE :
SW
:
1D
20.5503 ppm
*** Processing Parameters ***
GB
:
LB
:
OFFSET
:
SI
:
0.0000000
0.30 Hz
16.5110 ppm
32768
*** 1D NMR Plot Parameters ***
9.0
8.0
7.0
6.0
5.0
(ppm)
4.0
3.0
:
13.28 cm
Start
:
10.00 ppm
Stop
:
-0.10 ppm
ppm_cm
:
AQ_time
:
NUCLEUS
:
0.46
3.9845890 sec
off
37.889
4.1407
3.7722
0.8425
16.636
1.2616
1.0000
0.6784
0.4149
Integral
Height
2.0
1.0
1
Figure F.3. H-NMR spectrum of 30h reaction mixture.
67
Appendix G
Appendix G – Certificates of Analysis
68
Appendix G
69
Appendix H
Appendix H – Recovered Support Masses
Table H.1. Initial and recovered masses of the three tested epoxy-activated supports, including
duplicates, during the immobilization and epoxy groups blockage steps.
Support
Sepabeads EC-HFA
Duplicate I
Duplicate II
Duplicate I Blocked
Duplicate II Blocked
Sepabeads EC-EP
Duplicate I
Duplicate II
Duplicate I Blocked
Duplicate II Blocked
Eupergit C 250
Duplicate I
Duplicate II
Duplicate I Blocked
Duplicate II Blocked
70
Wet Support
Water Content (%)
54
Initial Mass (mg)
Dry Weight
Wet Weight
Recovered Mass
(mg)
1002
1005
230
232
2179
2186
502
505
2152
2123
490
510
979
979
217
213
2331
2332
516
506
2368
2345
488
569
993
990
496
498
-
3938
4024
579
587
58
86