Biodiesel production by enzymatic
transesterification of triglycerides
Francisco António Guilherme Moreira
Thesis to obtain the Master of Science Degree in
Biotechnology
Examination Committee
Chairperson:
Supervisors:
Professor Luís Joaquim Pina da Fonseca
Professor Joaquim Manuel Sampaio Cabral
Doctor Sara Martins Badenes
Member of the Committee:
Professor Frederico Castelo Alves Ferreira
December 2012
ii
Acknowledgments
I would like to thank everyone that supported me throughout this work. However I
would like to give a special thank to some people.
First of all, I want to thank Professor Doutor Joaquim Manuel Sampaio Cabral for
giving me the extraordinary chance of working at Bioprocess Engineering and
Biocatalysis Lab (BEBL), and in particularly under his supervision. It was a pleasure
working at this project, especially after being tailored for what I wanted to work on. All
the availability and guidance given will not be forgotten.
In second place, Doctor Sara Badenes deserves all my gratitude for the dedication
and readiness given to me and my project despite not being in the same workplace.
Also a big thanks for giving me a chance to propose my own ideas without letting me
stray too far from the objective.
For my co-workers Marco Marques, Pedro Fernandes, Nuno Lourenço, Mário
Fonseca and Andreia “Bocelli” a big thanks for all the assist in the lab with more
technical aspects. Your patience was priceless during the whole year.
For keeping the lab a friendly environment where I had a good time working I want
to give a special thanks to Filipe Cota, Andreia Fernandes, Joana Pereira and Sara Rosa.
I would like to acknowledge my good friends, António Soure, João “Pai” Anes,
Bruno Alves and “Rach” Correia, who played a big part in supporting me and whose
friendships will endure for a long time.
For my closest friends, “os verídicos”, huge thanks for being true friends through all
my life and for all the good times still to come.
For the most important person in my academic (and not only) life a big thanks for
Maria João, a “madrinha”, for all the support, caring and love.
Last, but definitely not the least, a huge thanks for my parents, António and Isabel,
and my brother, Tiago. I know I can be a difficult person sometimes but your
unconditional love got me where I am today.
iii
iv
Abstract
The aim of this thesis is to evaluate the use of of cutinase for the production of
biodiesel, especially the mutant T179C, which displays high stability in reserved
micellar organic medium. For this, cutinase mutant T179C is produced by recombinant
Escherichia coli through a 2 l shake flask and a 5 l fermentor approach. The induction
with IPTG is carried out for over-expressing the enzyme for further purification by a
series of downstream processes. The results shows that growth rate (0.407 ± 0.030 h-1)
decreases after induction (0.203 ± 0.020 h-1) as cells spend energy in protein
production. Studies in downstream purification process showed an overall yield of 10%
with a 13.8 purification factor. Moreover, comparing with the 5 l fermentor, this
approach by having a tighter control of parameters, significant improvements were
showed in the outcome of biomass and protein production.
Finally, to infer the possibility of reducing the downstream cost for biodiesel
production, exploratory studies were made using whole-cell biocatalyst for
transesterification of triglycerides, in reversed micellar system, to produce biodiesel.
Several studies to study the effect of surfactant (bis(2-ethylhexyl) sodium
sulfosuccinate), susbtrate (commercial triolein, with 65% purity) and co-substrate
(methanol/buthanol), water content and mass transfer were performed. However, no
conclusive results could be attained as the transesterification reaction did not occur.
Keywords: Cutinase; transesterification; biodiesel; reversed micelles; whole-cell
biocatalyst
v
vi
Resumo
O objetivo desta tese era estudar a utilização da proteína cutinase mutante T179C,
que é estável no sistema de micelas invertidas, para a produção de biodiesel. Esta
protein foi produzida em balão de 2 l e num fermentador de 5 l através da estirpe
recombinante de Escherichia coli. A indução é feita com IPTG que sobre expressa a
produção da proteína cutinase. Os resultados demonstram que o passo de indução
diminui a taxa de crescimento de 0.407 ± 0.030 h-1 para 0.203 ± 0.020 h-1, pois as
células começam a utilizar a energia para produzir cutinase em vez de utilizar para
proliferação. No processo de purificação após produção o rendimento é de 10% com
um factor de purificação de 13.8. Adicionalmente, o estudo em fermentador de 5 l
demonstra uma maior produção de biomassa e de proteína. Isto deve-se ao maior
controlo dos parâmetros da fermentação.
Por fim, a possibilidade de reduzir os custos de produção de biodiesel ao diminuir
os custos de purificação leva ao estudo exploratório da transesterificação com enzima
intracelular. A reação enzimática de transesterificação de triglicéridos é feita num
sistema de micelas invertidas. Foi testado o efeito que o surfactante (dioctil
sulfosuccinate de sódio), o substrato (trioleína a 65% pureza) e co-substrato
(metanol/butanol), o teor de água e possíveis limitações na transferência de massa.
Contudo, os resultados não são conclusivos sendo que a reação não ocorreu utilizando
enzima intracelular.
Palavras-chave: Cutinase; transesterificação; biodiesel; micelas invertidas; enzima
intracelular
vii
viii
List of figures
Figure 1 - World’s production (blue) and consumption (red) of oil from 1990 up to 2009. Green
line represents the tendency of the variation between production and consumption, showing
superior rate of increase consumption than rate of increase production. (EIA 2012) ................. 7
Figure 2 – World’s production (blue) and consumption (red) of biofuel from 2000 up to 2009
(EIA 2012). ..................................................................................................................................... 8
Figure 3 – A) Schematic representation of cutinase tertiary structure. Four α-helices (A, B, C
and F), and five β-sheets represented by black arrows. The active site triad is characterized by
Ser120, Asp175 and His188. B) Three dimensional representation of cutinase fold, highlighting
the active site triad. (Carvalho, et al. 1999b; Egmond and de Vlieg 2000) ................................. 11
Figure 4 – Block diagram representative of the purification steps from production until final
cutinase form ready for utilization.............................................................................................. 20
Figure 5 – General reaction of the transesterification of vegetable oils (Schuchardt, et al. 1998).
..................................................................................................................................................... 22
Figure 6 – Schematic representation of the reversed micelle. Inside the core, facing the polar
heads, is the water droplet, which contains the cutinase for transesterification, and the organic
solvent is in the outside of the system interacting with the hydrophobic chains of the
surfactant (e.g. AOT). .................................................................................................................. 26
Figure 7 – Linear calibration curve of OD measurements (600 nm) vs cell dry weight (mg/mL).
This curve has a slope of 3.672±0.237, and b of 0.238±0.097. Source: Badenes 2008 (Badenes
2010). .......................................................................................................................................... 34
Figure 8 – Structure of 4-nitrophenylbutyrate, also known as p-NPB (SIGMA 2012)................. 35
Figure 9 – Calibration curve of the Pierce Coomassie Bradford method from 5 to 50 µg/mL at
595 nm. It has 0.0063±0.0002 slope and 0.0004±0.0001 intersection. ...................................... 36
Figure 10 – Schematic representation of the hydrolysis reaction of a triglyceride molecule .... 38
Figure 11 – Growth curve of fermentation for the different shake flaks experiments. From
these curves the growth rates were calculated: initial hours of fermentation µ = 0.407 ± 0.030
h-1; after induction with µ = 0.203 ± 0.020 h-1............................................................................. 41
Figure 12 – Representation of the peaks of UV (288 nm) detections (Y axis) versus the volume
buffer through the column for elution (X axis). .......................................................................... 43
Figure 13 – SDS-PAGE analysis of the samples. Gel A wells: 1 and 2 – Molecular weight
markers; 3 – 1st hour of the fermentation; 4- after induction; 5 - 1h after induction; 6 -2h after
induction; 7 - 3h after induction; 8 - 16h after indution; 9 – Osmotic shock with STE; Gel B
wells: 1 – Osmotic shock with water; 2 – Acid precipitation; 3 - Molecular weight markers ; 4 –
Dialysis; 5 – DEAE-Sepharose; 6 – Q-Sepharose; 7 – 20h after induction; 8 – DEAE-Sepharoese
after elution; 9 – Clarified broth ................................................................................................. 44
Figure 14 – Growth curve of fermentation performed in the 5 l Bioflow 3000 reactor. The
induction was performed at 6 h of fermentation. Pre-fermentation was performed as the
explained in section 2.................................................................................................................. 45
Figure 15 – Controlled parameters in the 5 l Bioflow 3000 reactor. From the primary Y axis the
rhombus represents the stirring measures, starting with 200 rpm and in the end with 700 rpm.
For the secondary Y axis there are 3 parameters: the solid triangle represents the pH which
starts at 7.1 oscillating around this value until roughly the 20th hour ending with 5 pH; the circle
stands for temperature and this is the most stable parameter along the fermentation having
ix
no noticeable deviation of the set point, 25 ºC; the solid squares are DOC which starts at 100%,
and reaches optimal condition after 2,5h, then oscillating around this value and decreasing to
0% by the latest hours of fermentation. ..................................................................................... 46
Figure 16 – Chromatogram of samples from the reaction mixture, when using the whole-cell
biocatalyst and the optimal conditions obtained for purified protein. Two overlapped curves
are shown, green line is the chromatogram of the initial time zero sample, and blue line is the
chromatogram of the 24h reaction sample. Blue rectangle – time zone of MG detection; white
rectangle – time zone of AE detection; yellow rectangle – time zone of DG detection; red
rectangle – time zone of TG detection........................................................................................ 50
Figure 17 - Chromatogram of samples from the reaction mixture, when using the whole-cell
biocatalyst permeabilized with CTAB and the optimal conditions obtained for purified protein.
Two overlapped curves are shown, the green line is the chromatogram of the initial time zero
sample, and blue line is the chromatogram of the 24h reaction sample. Blue rectangle – time
zone of MG detection; white rectangle – time zone of AE detection; yellow rectangle – time
zone of DG detection; red rectangle – time zone of TG detection. ............................................ 51
Figure 18 - Chromatogram of samples from the reaction mixture, when using purified protein.
Two overlapped curves are shown, green line is the chromatogram of the initial time zero
curve, and blue line the chromatogram of the 24h reaction sample. Blue rectangle – time zone
of MG detection; white rectangle – time zone of AE detection; yellow rectangle – time zone of
DG detection; red rectangle – time zone of TG detection. ......................................................... 52
Figure 19 - Chromatogram of samples from the reaction mixture, when using using butanol as
a co-substrate instead of methanol. Two overlapped curves are shown, green line is the
chromatogram the initial time zero curve, and blue line the chromatogram of the 24h reaction
sample. Blue rectangle – time zone of MG detection; white rectangle – time zone of AE
detection; yellow rectangle – time zone of DG detection; red rectangle – time zone of TG
detection. .................................................................................................................................... 52
Figure 20 - Chromatogram of samples from the reaction mixture, when using 5% of buffer
instead of 0.73%. A longer period of time was considered for this experiment (48h) .Two
overlapped curves are shown, green line is the chromatogram of the initial time zero curve,
and blue line the chromatogram of the 24h reaction sample. Blue rectangle – time zone of MG
detection; white rectangle – time zone of AE detection; yellow rectangle – time zone of DG
detection; red rectangle – time zone of TG detection. ............................................................... 54
Figure 21 - Chromatogram of samples from the reaction mixture, when using 500 µl of triolein
instead of 399 µl. Two overlapped curves are shown, green line is the chromatogram of the
initial time zero curve, and blue line the chromatogram of the 24h reaction sample. Blue
rectangle – time zone of MG detection; white rectangle – time zone of AE detection; yellow
rectangle – time zone of DG detection; red rectangle – time zone of TG detection. ................. 55
Figure 22 - Chromatogram of samples from the reaction mixture, when using buffer instead of
isoctane. Two overlapped curves are shown, green line is the chromatogram of the initial time
zero curve, and blue line the chromatogram of the 24h reaction sample. Blue rectangle – time
zone of MG detection; white rectangle – time zone of MG detection; yellow rectangle – time
zone of DG detection; red rectangle – time zone of TG detection. ............................................ 55
Figure 23 - Chromatogram of samples from the reaction mixture, when using whole cell
biocatalyst previously sonicated using the optimal conditions obtained for purified protein.
Two overlapped curves are shown, green line is the chromatogram of the initial time zero
curve, and blue line the chromatogram of the 24h reaction sample. Blue rectangle – time zone
of MG detection; white rectangle – time zone of AE detection; yellow rectangle – time zone of
DG detection; red rectangle – time zone of TG detection. ......................................................... 56
x
Figure A1 – Graphic of the conversion to AE representative of chromatogram of figure 16. For
this curve the area under the peaks of the chromatogram were calculated and at 24h the
conversion was 90.572±0.05%.
Figure A2 - Chromatogram of samples from the reaction mixture, without using AOT. Two
overlapped curves are shown, green line is the chromatogram of the initial time zero curve,
and blue line the chromatogram of the 24h reaction sample. Blue rectangle – time zone of MG
detection; white rectangle – time zone of AE detection; yellow rectangle – time zone of DG
detection; red rectangle – time zone of TG detection.
Figure A3 - Chromatogram of samples from the reaction mixture, when using 1% of buffer
instead of 0,73%. Two overlapped curves are shown, green line is the chromatogram of the
initial time zero curve, and blue line the chromatogram of the 24h reaction sample. Blue
rectangle – time zone of MG detection; white rectangle – time zone of AE detection; yellow
rectangle – time zone of DG detection; red rectangle – time zone of TG detection.
Figure A4 - Chromatogram of samples from the reaction mixture, when using 10% of buffer
instead of 0,73%. Two overlapped curves are shown, green line is the chromatogram of the
initial time zero curve, and blue line the chromatogram of the 24h reaction sample. Blue
rectangle – time zone of MG detection; white rectangle – time zone of AE detection; yellow
rectangle – time zone of DG detection; red rectangle – time zone of TG detection.
Figure A5 - Chromatogram of samples from the reaction mixture, when using 20% of buffer
instead of 0,73%. Two overlapped curves are shown, green line is the chromatogram of the
initial time zero curve, and blue line the chromatogram of the 24h reaction sample. Blue
rectangle – time zone of MG detection; white rectangle – time zone of AE detection; yellow
rectangle – time zone of DG detection; red rectangle – time zone of TG detection.
Figure A6 - Graphic of the conversion to AE representative of chromatogram of figure 16. For
this curve the area under the peaks of the chromatogram were calculated and at 24h the
conversion was 54.465±0.07%.
xi
xii
List of tables
Table 1 - Industrial applications of cutinase. Adapted from (Badenes 2010) ............................. 13
Table 2 - Activity calculated, by linearization of the curves and calculated the slope.
Concentration was determined by measuring the OD at 595 nm and applying the calibration
curve represented in figure 8. ..................................................................................................... 43
Table 3 – Different volume percentage of methanol in the reaction media in order to
determine the decrease in activity in whole cell biocatalyst. The incubation period of methanol
was 24h, and the values present are the average of triplicates. ................................................ 53
xiii
xiv
List of abbreviations
AE
Fatty acid alkyl esters
AOT
Bis(2-ethylhexyl) sodium sulfosuccinate
Asp
Aspartic acid
CTAB
cetyltrimethylammonium bromide
Cys
Cysteine
dcw
Dry cell wight (mg/ml)
DEAE
Diethylaminoethanol
DG
Diglycerides
DOC
Dissolved oxygen concentration
EC
Enzyme Commission number
EDTA
Ethylenediaminetetraacetid acid
EIA
Energy Information Administration
Eq.
Equation
FA
Fatty acids
His
Histidine
IPTG
Isopropyl-β-D-thiogalactopyranoside
LB
Luria-Bertani broth
MG
Monoglycerides
NMWCO
Nominal molecular wight cut off (Da)
OD
Optical density
PAGE
Polyacrylamid Gel Elctrophoresis
PDB
Protein Data Bank
PEI
Polyethylenimine
p-NPB
P-Nitrophenylbutyrate
xv
Q
Quaternary
Rpm
Rotations per minute
SDS
Sodium dodecyl sulphate
Ser
Serine
STE
Sucrose, Tris and EDTA
t
Time
TB
Terrific broth
TG/TAG
Triglyceride/Triacylgyceride
TTP
Tween 20, Triton X-100 and Polyethylenimine
UV
Ultra-Violet
Wo
Molar ratio between water and surfactant concentrations
w/w
Weight per weight
wt
Wild type
µ
Specific growth rate (h-1)
xvi
Index
Acknowledgments .........................................................................................................................iii
Abstract ..........................................................................................................................................v
Resumo......................................................................................................................................... vii
List of figures ................................................................................................................................. ix
List of tables ................................................................................................................................ xiii
List of abbreviations ..................................................................................................................... xv
Aim of studies................................................................................................................................ 5
1.
Introduction .......................................................................................................................... 7
1.1.
Overview ....................................................................................................................... 7
1.2.
Cutinase ....................................................................................................................... 10
1.2.1.
Structural characteristics..................................................................................... 10
1.2.2.
Cutinase stability ................................................................................................. 12
1.2.3.
Industrial applications ......................................................................................... 13
1.3.
Production of cutinase ................................................................................................ 14
1.3.1.
Escherichia coli WK-6........................................................................................... 14
1.3.1.1.
1.3.2.
Plasmid pMa/c5-CUF ................................................................................... 14
Production of cutinase in shake flasks ................................................................ 15
1.3.2.1.
Growth media.............................................................................................. 15
1.3.2.2.
Pre-fermentation and fermentation ........................................................... 16
1.3.2.3.
Induction ..................................................................................................... 16
1.3.3.
Production of cutinase in a stirred fermentor .................................................... 17
1.3.3.1.
Control parameters ..................................................................................... 18
1.3.3.1.1. Agitation ................................................................................................... 18
1.3.3.1.2. Oxygen...................................................................................................... 18
1.3.3.1.3. Temperature ............................................................................................ 18
1.3.3.1.4. Anti-foaming ............................................................................................ 19
1.4.
Extraction and purification process ............................................................................ 19
1.4.1.
Recovery .............................................................................................................. 20
1.4.2.
Isolation ............................................................................................................... 20
1.4.3.
Purification .......................................................................................................... 21
1.4.4.
Polishing .............................................................................................................. 21
1.5.
Transesterification process ......................................................................................... 22
1.5.1.
Cutinase microencapsulated in reversed micellar systems ................................ 26
1
1.5.2.
2.
Bioreactors for reversed micellar systems .......................................................... 27
Materials and methods ....................................................................................................... 29
2.1.
Production ................................................................................................................... 29
2.1.1.
Microorganism .................................................................................................... 29
2.1.2.
Culture media ...................................................................................................... 29
2.1.2.1.1. Pre-fermentation medium ....................................................................... 29
2.1.2.1.2. Fermentation medium ............................................................................. 29
2.1.3.
Contaminations ................................................................................................... 30
2.1.4.
Culture conditions ............................................................................................... 30
2.1.5.
Inoculum.............................................................................................................. 30
2.1.6.
Shake flask ........................................................................................................... 30
2.1.7.
Fermentor ........................................................................................................... 31
2.2.
Extraction and purification of cutinase ....................................................................... 32
2.2.1.
Whole-cell biocatalyst ......................................................................................... 32
2.2.1.1.1. Permeabilization TTP................................................................................ 32
2.2.1.1.2. Permeabilization CTAB ............................................................................. 32
2.2.2.
Protein extraction................................................................................................ 32
2.2.2.1.1. Osmotic shock .......................................................................................... 32
2.2.2.1.2. Acid precipitation ..................................................................................... 32
2.2.3.
Dialysis ................................................................................................................. 33
2.2.4.
Ion-exchange chromatography ........................................................................... 33
2.2.5.
Lyophilisation ...................................................................................................... 33
2.3.
Analytical procedures .................................................................................................. 34
2.3.1.
Biomass ............................................................................................................... 34
2.3.2.
Cutinase activity assay......................................................................................... 34
2.3.2.1.1. Methanol induced stress.......................................................................... 35
2.3.3.
SDS-page.............................................................................................................. 36
2.3.4.
Determination of protein concentration ............................................................ 36
2.4.
Enzymatic transesterification ...................................................................................... 37
2.4.1.
Chemicals and enzyme ........................................................................................ 37
2.4.2.
Reversed micelles ................................................................................................ 37
2.4.3.
Transesterification procedure ............................................................................. 37
2.4.3.1.1. Hydrolysis reaction ................................................................................... 38
2.4.4.
2
Analysis ................................................................................................................ 38
3.
Results and Discussion ........................................................................................................ 41
3.1.
Shake flask production and purification ..................................................................... 41
3.1.1.
Growth profile ..................................................................................................... 41
3.1.2.
Quantification of protein and cutinase activity assays ....................................... 42
3.1.3.
Concentration of purified cutinase ..................................................................... 43
3.2.
Stirred fermentor production ..................................................................................... 45
3.2.1.
Growth profile ..................................................................................................... 45
3.2.2.
Parameters control.............................................................................................. 45
3.2.3.
Final product ....................................................................................................... 47
3.3. Whole-Cell Enzymatic transesterification of triglycerides by cutinase ............................... 49
4.
Conclusions and Future Work ............................................................................................. 57
5.
Bibliography ........................................................................................................................ 59
Appendix A .................................................................................................................................. 65
3
4
Aim of studies
This thesis aims to produce cutinase mutante T179C and explore its usage as a
whole-cell biocatalyst for the production of biodiesel.
Firstly, cutinase mutant T179C was produced by recombinant Escherichia coli via
shake flask and purified. This study is centred in the assessment of growth rate and
protein production, as well as the downstream process efficacy. Induction with IPTG
was carried out for protein over-expression. Downstream process for purified cutinase
purification includes centrifugations, osmotic shock, acid precipitation, dialysis,
chromatography and lyophilisation.
Secondly, cutinase mutant T179C was produced as a whole-cell biocatalyst and as
an purified protein in a 5 l fermentor. The objective is to study the scalability of the
production process and the tightness of the control of the parameters such as pH,
temperature, dissolved oxygen concentration and stirring.
Lastly, whole-cell biocatalyst cutinase mutant T179C was used in the
transesterification of triglycerides in a small scale reactor. In industry is given high
importance to low cost production and using whole-cell biocatalyst reduces the
downstream process, which represents a big part of the costs. A commercial triolein
(with 65% purity) is used as a model substrate for the transesterification to simulate
more real complex conditions.
5
6
1. Introduction
1.1. Overview
World’s energy needs are mainly sustained by fossil energy resources, 80% of total
energy supplied, such as petroleum, coal and natural gas (Demirbaş 2008). Fuels are
petroleum-sourced and, due to the increasing demand and depleting supplies, will
soon be exhausted (Manzanera 2008). Statistics from the Energy Information
Administration show that world-wide consumption not only exceeds production, but it
is also increasing at a superior rate than production, as shown in figure 1 (EIA 2012).
90000
Oil - production vs consumption
90000
80000
80000
70000
70000
60000
60000
50000
50000
40000
20000
40000
Consumption
30000
Production
20000
10000
10000
30000
0
0
Figure 1 - World’s production (blue) and consumption (red) of oil from 1990 up to 2009. Green line
represents the tendency of the variation between production and consumption, showing superior rate
of increase consumption than rate of increase production. (EIA 2012)
In addition to the fact of fossil fuel resources are running out, the emissions of
carbon dioxide (CO2) resultant from the combustion of fossil fuels are the major factor
of greenhouse effect and, global warming (Rutz and Janssen 2007). Furthermore, fossil
energies are considered non-renewable, as they cannot be restored as quickly as are
used. However, since the world is highly dependent on energy, one has to consider
alternative and attractive options to fossil fuel.
7
Biodiesel has been considered one of the most prominent alternative energy
resources as it is sustainable, renewable and environmentally friendly (Manzanera
2008). Biodiesel is considered a carbon neutral fuel as it achieves a minimum
greenhouse gas emission effect of less 25% when compared to fossil fuels (Kawano
2006). Furthermore, biodiesel can be used with little or no modification in diesel
engines, which allows small adaptation of the industry to this new fuel source
(Manzanera 2008). Consequently, the usage of biodiesel has grown is the past decade,
as shown in figure 2. Moreover, production can be controlled in order to match
consumption as it is a renewable source and only depends on the extent of how much
is necessary to produce (EIA 2012).
Biofuel - production vs consumption
Thousand of barrels per day
300
250
200
Production
150
Consumption
100
50
0
Years
Figure 2 – World’s production (blue) and consumption (red) of biofuel from 2000 up to 2009 (EIA 2012).
The major drawback of vegetable biodiesel commercialization is the uncompetitive
prices. The production of biodiesel still imposes high costly procedures and the raw
materials, mainly edible oils, are also expensive (Ma and Hanna 1999). Nowadays,
unrefined oils and waste cooking oils are attracting attention to become feedstock,
reducing the costs.
Of the various methods of producing biodiesel, transesterification has gained
interest in the last years. This is the reaction of oil with alcohol yielding alkyl esters
8
(biodiesel) and glycerol, which can be catalyzed by a lipolytic enzyme, namely cutinase.
This enzyme has the ability to degrade short and long chain triacylglycerols (TAGs),
being one of the reasons for the advantage of cutinase, in addition to the ability to
degrade cutin (Lauwereys, et al. 1991). Therefore, this enzyme has a high potential
impact in the biofuels industry specifically biodiesel (Rutz and Janssen 2007).
In 1975, Purdy and Kolattukudy (Purdy and Kolattukudy 1975) defined cutinase as
the enzyme responsible for the hydrolysis of cutin, a insoluble biopolymer
characteristic of the aerial surfaces of higher plants (Kolattukudy, et al. 1981; Matzke
and Riederer 1991). Cutin is a barrier of the plant cuticle and is mostly composed of
esterified fatty acids hydroxylated and epoxy hydroxylated, mainly chains of 16 and 18
atoms of carbon long, cross-linked by ester bonds (Kolattukudy and Purdy 1973;
Matzke and Riederer 1991; Antonio 2003). The role of this barrier is to prevent the
entry of pathogens into plants and therefore, the enzymatic degradation of the cutin
has been proven to be one of the first steps of the infection process (Purdy and
Kolattukudy 1975). This process involves the secretion of purified cutinase by
pathogens, and inhibitors of this enzyme has shown results in the prevention of fungal
entry and hence infection (Purdy and Kolattukudy 1975; Lin and Kolattukudy 1980;
Soliday and Kolattukudy 1983).
Being of such importance to the virulence, cutinase has been greatly studied in
order to improve the understanding of both kinetic and physiological properties
(Crowhurst, et al. 1997; Morid, et al. 2010). The majority of these studies have been
focused on cutinase from the phytopathogenic Fusarium solani pisi, a fungal pathogen
of peas, which has been cloned and expressed in heterologous hosts like Escherichia
coli and Saccharomyces cerevisiae (Carvalho, et al. 1999b).
9
1.2. Cutinase
The reason cutinase has such importance, over other lipophilic enzymes, lies on the
fact that it has activity regardless the presence of an oil-water interface, making
cutinase an important biocatalyst for hydrolysis, esterification and transesterification
reactions (Pio and Macedo 2009).
Cutinase, EC 3.1.1.74, is one of the smallest enzymes that belongs to the class of
serine esterases, and to the superfamily of α/β hydrolases (Ollis, et al. 1992). As it was
mentioned above, the most well studied cutinase is the one from F. solani pisi, also
known as Nectria haematococca, which have properties of both lipase and esterase
(BRENDA 2011). Being able to catalyze the hydrolysis of cutin and a variety of synthetic
esters and triacylglycerols, this enzyme can be potentially used in many industrial
processes, from hydrolyzing milk fat to the oleochemical industry (Carvalho, et al.
1998).
1.2.1. Structural characteristics
Soliday and co-workers first analysed the primary structure of cutinase in 1984
from cloned cDNA (Soliday, et al. 1984). Expressed in E. coli, Martinez and co-workers
were able to solve cutinase structure at 1.6 Å, and afterwards, extended to 1.0 Å by
Longhi and co-workers (Martinez, et al. 1992; Longhi, et al. 1997).
Cutinase is a 197 amino acid long molecule, with a molecular weight of
approximately 22 kDa and an isoelectric point (pI) of 7.6 (Koops, et al. 1999). Being a
one-domain 45x30x30 Å3 size molecule, cutinase consists in a central β-sheet with five
parallel strands, covered by two and three α-helices on either side of the sheet, as
shown in figure 3 (Carvalho, et al. 1998). The folded protein contains an active site
triad, composed by Ser120, His188 and Asp175, which is accessible to the solvent and
is located at one extremity of the protein ellipsoid, surrounded by the loop 80-87 and
by the hydrophobic loop 180-188 (Jelsch, et al. 1998; Egmond and de Vlieg 2000). Up
to 48 different structures regarding cutinase have been published, either wild-type,
mutant forms or inhibitors conjugates (PDB 2011).
10
Figure 3 – A) Schematic representation of cutinase tertiary structure. Four α-helices (A, B, C and F), and five β-sheets represented by
black arrows. The active site triad is characterized by Ser120, Asp175 and His188. B) Three dimensional representation of cutinase fold,
highlighting the active site triad. (Carvalho, et al. 1999b; Egmond and de Vlieg 2000)
Although the backbone structure is fairly rigid, in the regions of substrate binding
and active site triad there is some mobility, being kinetic relevant. However, the
nucleophilic serine appears to be quite rigid, being the other two residues more
flexible (Prompers, et al. 1999).
Lipases exhibit activation in the presence of a lipid-water interface due to its
amphipathic lid. However, cutinase differs from classic lipases and does not exhibit
interfacial activation. The previously mentioned loops, 80-87 and 180-188, bearing
hydrophobic amino acids, may form the interfacial binding site and the catalytic serine
is accessible to solvent and substrate (Martinez, et al. 1992). In another words, the
absence of a hydrophobic lid masking the active-site serine explains why the cutinase
binding to substrate does not require a main-chain rearrangement, as other classic
lipases do, but only the reorientation of few lipophilic side chains (Carvalho, et al.
1998).
11
1.2.2. Cutinase stability
Enzyme stability is affected by temperature, solvents, pH, binding co-factors,
induced stress and the presence of surfactants. The latest is of high importance to
industry given that the detergent area is the largest application of industrial enzymes.
Above all others, the temperature is the most well studied parameter because it can
cause irreversible protein unfolding and consequently enzyme deactivation (Eijsink, et
al. 2005). Thermal and operational cutinase stability have already been assessed in
several studies (Carvalho, et al. 1999a; Gonçalves, et al. 1999; Melo, et al. 2003; de
Barros, et al. 2010).
Despite of setting these parameters to obtain optimal performance, often the
achieved yields are not suitable. Thus, protein engineering is a commonly used
strategy to improve enzymes yields. In the specific case of cutinase, the wild-type is
rapidly inactivated by anionic surfactants (Pocalyko and Tallman 1998). Many single
mutations and post-translation modification were performed on cutinase to
investigate activity improvements (Egmond and de Vlieg 2000). Although some have
shown improvements in activity, the stability of the enzyme was not assessed by this
test. With that objective, Brissos and co-workers screened all 19 possible amino acid
exchanges at each 214 positions, for improved stability in the presence of anionic
surfactant bis(2-ethylhexyl) sodium sulfosuccinate (AOT) (Brissos, et al. 2008a). From
these mutants, three were selected since they showed improvements in stability in the
presence of AOT, namely S54D, L153Q and T179C. The name of the mutants is
assigned according to the amino acid mutated
The S54D mutation, which occurs in the α-helix A, far from the active site,
decreases the distance between helices A and F (Creveld, et al. 1998). The L153Q
mutation changes from a hydrophobic residue to a less hydrophobic amino acid, which
was a weak spot regarding stability (Creveld, et al. 1998). In the T179C mutant, a
cysteine is introduced with the objective of filling space near the active center and next
to another cysteine involved in a disulphide bridge. The stability in presence of AOT is
due to the mutations that prevent the hydrophobic crevices, which are the cause of
the enzyme unfolding induced by this surfactant (Badenes, et al. 2011b).
12
After careful analysis of these three mutants regarding activity, with or without the
presence of AOT, inhibition constants, deactivation, structural stability, and other
parameters, the mutant T179C was chosen In this work for the production of biodiesel
through enzymatic catalysis (Badenes, et al. 2011b).
1.2.3. Industrial applications
As mentioned previously cutinase, as a lipolytic enzyme, has a high value in
industrial applications and it can be used in dairy industry, house hold detergents
industry, oleochemical industry, synthesis of TAGs, polymers and surfactants, and
pharmaceutical industry (Carvalho, et al. 1998). Some applications of cutinase are
summarized in table 1. The biodiesel production is a big area of the oleochemical
industry, with prospects of growing (Korbitz 1999; Manzanera 2008).
Table 1 - Industrial applications of cutinase. Adapted from (Badenes 2010)
Industry
Catalysis reaction
Detergent and laundry
Hydrolysis of
triacylglycerols
Purpose
To decompose fats into
more water-soluble
compounds
Examples
(Okkels 1997;
Flipsen, et al.
1998)
(Barros 2009)
Food
Hydrolysis of milk fat, and
synthesis of esters
To develop flavouring
agents, accelerate
cheese ripening and
lipolysis of butter fat
Chemical:
- Pharmaceutical
- Cosmetics
- Agriculture
Synthesis of esters,
polymers and surfactants,
and preparation of chiral
synthons
To produce ingredients
for personal-care
products,
pharmaceuticals and
agro-chemicals
(Genencor 1988;
Vidinha, et al.
2004)
Oleochemical
Hydrolysis, glycerolysis
and alcoholysis
To produce high value
polyunsaturated fatty
acids and manufacture
of soap
(Mukherjee 1990)
13
Plastic
Hydrolysis of polyesters
To degrade
environmental toxic
pollutants and
biodegradable plastics
(Murphy, et al.
1996; Kim 2002)
1.3. Production of cutinase
1.3.1. Escherichia coli WK-6
In order to obtain cutinase in a large-scale production or even to genetic engineer
the protein, naturally occurring organism cannot achieve high production levels.
Therefore, host microorganisms, such as bacteria or yeast, are used for the production
of heterologous proteins (van Gemeren, et al. 1995).
The most used bacteria model is Escherichia coli, a well studied and characterized
microorganism. Bacteria have the highest growth rates, present high cell density
cultures – enhanced downstream process and low costs, are very stable in terms of
insertion of plasmids of foreign genes and E. coli has many strong signals that induce
protein production. Furthermore, this model can be used in order to direct the protein
to the cytoplasmatic, periplasmic or extracellular space (Brown 2010).
The strain E. coli WK-6 was chosen for host due to the advantages presented
above. This is a used strain with known mutations that proved useful for cutinase
production (Monteiro, et al. 1999).
1.3.1.1.
Plasmid pMa/c5-CUF
In order to make the E. coli WK-6 produce our protein is necessary to introduce a
plasmid containing the gene of the protein of interest into the cells. Plasmids are
circular DNA molecules that lead to an independent existence in the cell, and serve as
cloning vectors capable of using the host replication machinery to produce high
amounts of the desired protein. They vary both in size and copy number, which are
important characteristics for cloning. Reduced size, less than 10 kb, is desirable
14
because larger molecules tend to break down and are more difficult to manipulate.
Normally, the plasmids already code for selective markers in order to be able to isolate
those cells that have been successfully transformed, hence containing the plasmid. As
being well characterized, the sequence of the plasmids is known and therefore, it is
know where restriction enzymes will act. Through these restriction enzymes, the
plasmid is opened and afterwards, through ligases, a segment of DNA coding for the
protein of interest is inserted inside the plasmid (Brown 2010).
The Fusarium solani pisi cDNA library was constructed, and cutinase was expressed
using the pUC19 plasmid in E. coli WK-6 (Soliday, et al. 1984; Lauwereys, et al. 1991).
The gene of interest was cloned behind a signalling peptide for alkaline phosphatise
(phoA), with the objective to direct cutinase to the E. coli periplasm. Throughout a
single oligonucleotide-directed mutagenesis, using a pMa/c type of plasmids, the
fusion of the sequences of phoA and cutinase was obtained. The obtained plasmid,
pMa/c5-CUF, is as cloning vector under transcriptional control of IPTG-inducible Ptac
promoter (Lauwereys, et al. 1991).
1.3.2. Production of cutinase in shake flasks
With the objective of understanding and manipulating the enzyme production, it is
advisable to produce the protein in a simple and unexpensive environment. The
optimal conditions and behaviour of the used microorganism depends on which
protein is producing. Protein production in shake flasks is a commonly used approach
(Gräslund 2008).
1.3.2.1.
Growth media
The majority of bacteria and yeasts can grow in liquid medium, which provides the
essential nutrients to allow efficient growth and proliferation. Growth media can be
divided in defined medium and undefined medium. The first type is when all the
components and their quantities are known, and they provide nitrogen, magnesium,
calcium, carbon source, etc. Whereas, the undefined medium is a complex medium of
15
which the identity and quantity of its components is not known. In the case of LuriaBertani (LB) and Terrific Broth (TB), the components tryptone and yeast extract confer
the uncertainty because they are complex mixtures of unknown chemical compounds.
However, they have proven great efficiency in growing microorganism, higher than
defined media, in growing microorganism. The undefined media does not need any
supplementary additions such as vitamins or growth factors. On the other hand,
undefined mediums cannot be used when precisely controlled conditions are needed
or you want to mark the protein being produced with radioactive amino acids (Brown
2010).
1.3.2.2.
Pre-fermentation and fermentation
After growing the microorganism in culture plates at 37 ºC, in undefined medium
along with agar, the E. coli is inoculated into a pre-fermentation reactor. This step is
performed to guarantee that before the actual fermentation starts we have the cells
are in the mid-to-late log phase of the growth curve, i.e. to make sure that the cells are
in exponential growth with all the production machinery already synthesised. Prefermentation is done in smaller reactors due to only a small amount is going to be used
for the next step (Gräslund 2008; Sousa and Badenes 2008).
Afterwards, for fermentation, the quantity of volume inoculated is calculated
according to the optical density (OD) at the end of the pre-fermentation. Another point
to consider is the lowering of the temperature, for instance from 37 to 25 ºC, hence is
the optimum temperature to achieve higher production while still having good
proliferation. Furthermore, having lower temperatures means slower rates of protein
production allowing produced recombinant proteins time to properly fold (Gräslund
2008; Sousa and Badenes 2008).
1.3.2.3.
Induction
Once the microorganism is in the proper growth stage and in optimum conditions,
the plasmid must be signalled to start producing higher amounts of protein. There are
16
two types of signals that can be sent to the promoter that controls the translation of
the gene coding to the protein of interest: induction and repression. These are the
opposite of each other, while the latest decreases, or even inhibits production, the first
one increases it (Brown 2010). Again, the maintenance of lower temperatures is used
with the same purpose.
As it was mentioned previously, for the plasmid containing cutinase the promoter
Ptac can be induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG). The promoter tac
is naturally induced by lactose but the synthetic inducer IPTG is more functional. This is
due to the ability of penetrating the cell wall freely (Khlebnikov and Keasling 2002).
1.3.3. Production of cutinase in a stirred fermentor
Although shake flaks are a good initial approach, they are not suited for
reproducible and precise controlled environments as it imposes some setbacks. The
shaking bioreactors are mainly used in the first steps of bioprocess development and
afterwards, scientists use more complex stirred tank fermentors (Buchs 2001).
Some of the setbacks related to shake flask cultures are the poor gas transfer, poor
mixing efficiency and non-continuous monitoring. Unless aeration or mixing conditions
are satisfactory, in terms of oxygen limitation or mixing efficiency, studies are carried
out without knowing the true effect of variables under study – medium, aeration,
growth rates, between others (McDaniel and Bailey 1969; Buchs 2001).
Studies concerning the effect of concentration of an added compound to an
occurring reaction were performed. It could be concluded that increasingly added
concentrations affected more the oxygen solubility and mass transfer coefficient,
meaning for example that the mixing was not adequate (Anderlei and Buchs 2001).
Furthermore, Wittmann and co-workers report that intermittent shaking, required for
monitoring growth curves and adding additional compounds, causes oxygen limitations
(Wittmann, et al. 2003).
17
1.3.3.1.
Control parameters
1.3.3.1.1.
Agitation
Accordingly to what was mentioned previously, agitation is a crucial parameter to
be set. Not only uniform mixing is required but also shear stress must be accounted
for. The location and geometry of the impeller and the reactor must be considered as
it influences mixing and flow direction, and hence shear stress. Shear stress is an
important factor as it can disrupt cells, leading to lower protein production. However,
bacterial cells, such as E. coli, have both cell membrane and cell wall, conferring a
higher resistance to shear stress than animal cells, for example (Masters 2000).
The Rushton type impeller induces a strong radial flow and is used for a wide range
of industrial processes. A dual Rushton impeller proved to have a stable flow pattern
with efficient mixing with aeration, showing good oxygen mass transfer (Hudcova, et
al. 1989; Rutherford, et al. 1996).
1.3.3.1.2.
Oxygen
Usually one of the major issues when growing aerobic microorganism is the oxygen
supply (Wittmann, et al. 2003). The aeration of the medium must be optimal,
guaranteeing a good diffusion and oxygen mass transfer coefficients, for proper
culture growth (Buchs 2001). A common approach, besides having a good mixing
system, is the use of airlift bioreactors, where the oxygen input is in the bottom of the
reactor, increasing oxygen transfer and needing less intense agitation (Chisti and
Jauregui-Haza 2002).
1.3.3.1.3.
Temperature
Bioreactors normally operate at temperatures higher than environment
temperatures and have high loss of heat through reactor walls, therefore heating is
necessary. Among the different configurations, water-jacket is the most common and
economic one for small volume reactors. Another advantages are ease of maintenance
18
and no need of sterilization (Fonseca and Teixeira 2007). High volumes will not allow
an efficient heat transfer from the water-jacket to the medium, hence not being
adequate. For these cases cooling coils are a common approach, adding the
disadvantage of thermal stress inside the reactor.
1.3.3.1.4.
Anti-foaming
Cell-bubble attachment could occur leading to cell lysis, due to shear stress while
bubble rupture. Also, cells and culture medium can be lost to the foam, leading to a
decrease in process productivity. Furthermore, foam can reduce the efficiency of gas
exchange as it acts as a barrier between gases and culture surface (Holmes, et al.
2006). In bioreactor, foam can also block exit filter leading to over-pressure inside the
reactor (Routledge, et al. 2011). To prevent this type of events anti-foaming products
are added to the medium, which will not affect the reaction. These anti-foaming
agents include surfactants, as their function is to reduce the surface tension of the
liquid surface (Owen 2000).
1.4. Extraction and purification process
Following the production of the desired protein, the downstream is a crucial part of
the process, even sometimes determines the process viability, either economically or
due to process impossibilities. It is common to use the RIPP (Recovery-IsolationPurification-Polishing) scheme. Accordingly to RIPP firstly it is done the separation of
insolubles, namely the collecting of cells, by means of operations that use broad
characteristics (e.g. size, weight or density), such as filtration, extraction, adsorption or
sedimentation (centrifugation). Afterwards, the product is isolated using other
properties and reducing the overall volume of the sample. For this step operations
such as extraction, ultrafiltration or precipitation are used. The purification step is the
most sensitive one as it uses high specificity from the intended product for removing
impurities, which are hardly removed using chromatography, affinity methods or
fractional precipitation operations. Lastly comes the polishing step where the intent is
19
liquid removal and/or to convert the product to crystalline form by drying,
crystallization of lyophilisation.
lyophilisation Figure 4 represents the overall purification procedure.
Figure 4 – Block diagram representative of the purification steps from production until final cutinase
form ready for utilization.
1.4.1. Recovery
For bioprocesses with intracellular product the first step is centrifugation to
separate
arate cells from culture medium, and then rupture cell wall and cell membrane in
order to recover cutinase. Both physical and non-physical
non physical methods can be considered.
In the first category lie sonication, French press, bead mill and rotor-stator
rotor stator mill. For the
latest category, chemical and physiochemical
physiochemical methods consist in detergents, enzymes,
solvents and osmotic shock. The physical methods are mainly used for cell wall
disruption while chemical and physiochemical methods are used for destabilizing the
cell membrane (Ghosh
Ghosh 2006).
2006
1.4.2. Isolation
One of the most common approaches is to take advantage of the isoelectric point
of the product. Given that cutinase has a relative high isoelectric point (7.6), in order to
remove the majority of proteins,
proteins acid precipitation can be performed.. This process has
20
proven to remove high amounts of protein; unfortunately, the loss of cutinase is also
considerable. Alongside with acid precipitation a dialysis is performed in order to
separate macromolecules from smaller molecules, namely removal of salts, acid or
alkali, to concentrate the product. The principle is again concentration gradient, where
the solute will pass across the membrane, if has lower size than pores, to the more
hypotonic medium, removing undesired compounds from the medium. This is why
multiple outer medium changes, or continuous medium changes, are applied,
increasing the dialysis efficiency (Ghosh 2006).
1.4.3. Purification
In order to separate precisely our product of the various proteins still present in
the medium, a high affinity method has to be used. Ion exchange chromatography is
such technique, which binds to proteins which have affinity and allows others to pass
through (Ghosh 2006). The columns have a Sepharose matrix, where the charged
groups are bound, which determines the efficiency, capacity, chemical stability and
recovery of the columns. Sepharose is an agarose based-matrix that combines
spherical form with high porosity, improving flow properties and capacity (Janson
2011).
1.4.4. Polishing
The goal of the last step is to remove water, improved stability, decrease
temperature sensitivity and enhance shelf life. For this, a common use method is
lyophilisation, which consists in the removal of water by three steps: freezing, primary
drying and secondary drying (Cherian and Corona 2006).
21
1.5. Transesterification process
The usage of vegetable oils directly has proven to be unfeasible, due to high
viscosity, low volatilities and reactivity of unsaturated hydrocarbon chains (Pryde
1983). Therefore, methods have been contemplated to reduce viscosity, such as
dilutions, microemulsifications, pyrolysis, catalytic cracking and transesterification. The
dilution of oils consists in the addition of 4% ethanol or in petroleum diesel. However,
the mixture was not suitable for long-term usage (Demirbaş 2008). Microemulsion of
oils with immiscible liquids, such as methanol and ethanol, causes irregular injector
needle sticking, heavy carbon deposits and incomplete combustion. Pyrolysis or
catalytic cracking, is the conversion of one substance into another, cleavage for
instance, by means of heat and represents a costly technique, which can even yield
more gasoline than diesel fuel (Ma and Hanna 1999). Transesterification, also known
as alcoholysis when the acyl acceptor is an alcohol, is a reversible reaction where
triglycerides are converted to a mixture of alkyl esters (biodiesel) and glycerol, in the
presence of a catalyst. The figure 5 shows the general reaction of the
transesterification of a vegetable oil.
Figure 5 – General reaction of the transesterification of vegetable oils (Schuchardt, et al. 1998).
The reaction is affected by water content, temperature, molar ratio between
glycerides and alcohol, reaction time, TAGs and the catalyst (Carvalho, et al. 1997).
Despite of all the parameters being important, the choice of the catalyst plays a critical
role in the industry. Being a reversible reaction, the catalyst only decreases the time
needed to reach the equilibrium and in order to yield more product, substrate has to
be added to cause an equilibrium shift (Lehninger, et al. 2005). There are many types
of catalyst for this reaction: homogeneous - acids and alkyls, heterogeneous, non-ionic
alkyls and enzymes (Schuchardt, et al. 1998). Although not a catalyst, ultrasounds,
22
microwaves, oscillatory flow reactor, co-solvents (Lam, et al. 2010) and supercritical
conditions (Demirbas 2007) have also been approaches used to improve reaction
yields.
Regarding biodiesel production, alkyl homogeneous catalysis is the most commonly
applied process, using sodium hydroxide (NaOH) or potassium hydroxide (KOH)
(Felizardo, et al. 2006), which shows high efficiency but problems with high free fatty
acid (FFA) concentrations. However, acid homogeneous catalysis, using sulfuric acid
(H2SO4) and hydrochloric acid (HCL), has relatively high yields and is insensitive to FFAs.
Nonetheless, it requires a higher temperature, higher molar ratio of alcohol to oil,
higher reaction times and implies corrosion problems (Komintarachat and Chuepeng
2009). To overcome these problems some approaches using a two-step
transesterification with both acid and alkyl catalyst, have been studied. Despite of
helping with the reaction itself, this two-step approach imposes problems in the
downstream process, where separation and purification becomes more complex and
with more steps (Kulkarni and Dalai 2006).
When referring to heterogeneous catalysis, the reactants and the catalyst are in a
different phase, not only gas, liquid and solid but also as immiscible liquids (Lehninger,
et al. 2005). Solid alkyl catalyst calcium oxide (CaO) is the most used due to low
solubility in methanol, low cost and high alkyl strength (Lam, et al. 2010). Also,
magnesium oxide (MgO) is used due to its high efficiency in transesterification (Di
Serio, et al. 2006). Acid heterogeneous catalysts, such as zirconium oxide (ZrO2) and
titanium oxide (TiO2), have strong potential to replace liquid catalysts, because of their
easier separation and purification downstream processes (Jacobson, et al. 2008).
However, due to slow reaction rate, undesirable reactions and mass transfer
limitations the active usage of solid heterogeneous catalyst is not complete explored
(Badenes 2010; Lam, et al. 2010).
Non-ionic alkyl catalyst, such as 1,5,7-triazabicyclo (4,4,0)dec-5-ene (TBD), has
been studied in order to achieve milder reaction conditions and simpler manipulations
(Schuchardt, et al. 1998). Many set of studies were preformed over this type of catalyst
23
and relatively good yields were obtained; however, they never achieved values as high
as with enzymatic catalyst (Ejikeme 2010).
Of the novel type of approaches, the Oscillatory flow reactor (ORF) is a continuous
flow reactor that creates an oscillatory motion that favors the efficiency of heat and
mass transfer while maintaining a plug flow regime (Harvey, et al. 2003). This ORF
reactor is most used along with heterogeneous catalyst in order to overcome its
drawbacks.
The microwave technology allows a higher yield or cleaner product, less energy
consumption and an environmentally benign compared to conventional heating in
various chemical reactions (Groisman and Gedanken 2008). As a form of
electromagnetic energy, microwave heating process is not dependent on thermal
conductivity of materials, specific heat and density, and therefore is distributed
uniformly and rapid temperature increase is obtained (Lam, et al. 2010). Microwave
technology is used along with both homogeneous (Anan and Danisman 2007) and
heterogeneous catalyst (Perin, et al. 2008), and even has been applied, with promising
results, in the production of biodiesel (Barnard, et al. 2007).
On the other hand, ultrasonic technology has shown to improve mass transfer rate
between immiscible liquid-liquid phases and presents less energy consumption than
the conventional stirring (Ji, et al. 2006). Furthermore, it is a simple technique, with
efficient molar ratio of methanol to triglycerides and shorter reaction times (Singh, et
al. 2007).
Co-solvents, such as tetrahydrofuran (THF) and diethyl ether (DME), are inserted
into the reaction mixture in order to increase the solubility of both triglyceride and
alcohol, and therefore increase the mass transfer rate (Guan, et al. 2009). The problem
of this approach is the addition of steps to the downstream process in order to
separate the co-solvents (Lam, et al. 2010).
Another option is the reaction taken place without catalyst, while the alcohol is in
its supercritical state. In this approach, the water content has a positive effect and
promotes the reaction, in contrary to previously mentioned catalysis. The supercritical
24
state promotes the homogeneity of the medium, accelerating the reaction. In spite of
these advantages, it requires high temperatures (525-675 K) and high pressures of 3560 MPa, becoming impractical in industry scale (Demirbas 2007).
Furthermore, enzymes are highly selective to substrate, enantiomeric selective,
unaffected by the reaction itself, operate at mild temperatures, are easily
manipulated, have simpler downstream process and, can be reutilized or immobilized.
Due to the reasons presented above, enzymes are a suitable alternative to the
processes mentioned previously.
There are two main categories of enzymatic
biocatalysts: purrified and intracellular enzymes. In the first case, the enzyme is
recovered from the microorganism and purified, while in the latest the enzyme is used
while it remains inside or in the walls of the cell (Lehninger, et al. 2005).
Regarding purified lipases, transesterification of sunflower oil with primary alcohols
using lipases from Mucor miehei, nowadays named Rhizomucor miehei, was report by
Mittelbach and co-workers (Mittelbach 1990). Later on, other studies were performed
with lipases from Candida antarctica, Pseudomonas flourescens, Candida rugosa,
Pseudomonas cepacia, Rhizopus oryzae and Thermomyces lanuginosus (Ranganathan,
et al. 2008). The studies performed with the previously mentioned enzyme, cutinase,
belong to this category.
Due to improved immobilization technology, reusability, operational stability and
optimum temperature, conversion rates are higher and reaction time is shorter. Some
of these immobilization methods include adsorption, cross-linkage, entrapment,
encapsulation and covalent bonding (Jegannathan, et al. 2008). Is in this category that,
the previously mentioned enzyme, cutinase belongs.
Nevertheless, purified enzymes have the drawback of needing processes of
extraction and purification of the enzyme. In contrast, intracellular enzymes that
function as a whole-cell catalyst, bypass the extraction and purification improving the
cost effectiveness. However, lower yields are achieved since the enzyme is not fully
available or not so easily reachable. Once more, a common technique is the
immobilization of this whole-cell catalyst in order to become reusable (Gog, et al.
2012). Biomass support particles (BSPs) is one method used for this type of
25
immobilization that has shown good results with 90% of conversion rate (Ban, et al.
2002).
Choosing between purified and intracellular enzymes is to balance the yields and
the costs of the process. If the cost of the catalyst at the highest
highest activity and
stabilization is lowered, the industry of production of biodiesel with enzymatic
catalysis has a bright future (Tan, et al. 2010).
1.5.1. Cutinase microencapsulated in reversed micellar systems
Enzymatic
matic activity in organic solvents can be enhance by a series of strategies that
includes activating substances, surfactant coated lipases or solubilization systems.
The latest is the case of reversed micelles, where an aqueous core faces the polar
heads of the surfactant, while the hydrophobic chains interact with the bulk organic
solvent, figure 6 (Carvalho,
Carvalho, et al. 1999a).
1999a
Figure 6 – Schematic representation of the reversed micelle. Inside the core, facing the polar heads, is
the water droplet, which contains the cutinase for transesterification, and the organic solvent is in the
outside of the system interacting with the hydrophobic chains of the surfactant (e.g. AOT).
26
Lipases are commonly used in reversed micelles due the high stability and activity
in this type of system (Badenes, et al. 2010). Encapsulation in the interior or the
reversed micelles has the particular advantages of forming spontaneously and allowing
a strict control of the water content of the reaction, which is represented by the molar
ratio between water and surfactant concentration, Wo (Carvalho, et al. 1999a; Badenes
2010).
The enzyme stability is affected by pH, buffer molarity, temperature, solvent,
additives and surfactants (Carvalho, et al. 1999a; Gonçalves, et al. 1999; Badenes, et al.
2011b).
Recently, Badenes et al. evaluated the stability of cutinase encapsulated in AOT
reversed micelles (Badenes, et al. 2011b) for the transesterification of oils with alcohol,
and also the effect of the reaction mixture components, separately, on enzyme
stability. As expected, AOT presented a negative effect decreasing 45% over 3h of the
enzymatic activity, and a 90% loss when incubated with methanol over 10 minutes.
1.5.2. Bioreactors for reversed micellar systems
In order to do preliminary studies in the laboratory small reactors are best to test
new conditions and determine optimal parameters. Nonetheless, scalability is not
straightforward, and the small reactors only give the starting point not by-passing
optimization of parameters.
Unlike pharmaceutical industry that relies in batch type tank reactors, the most
commonly used in the oil industry are continuous stirred reactors. These are the most
valuable due to high throughput, more efficiency for not having start-up and shutdown cycles, lower operation costs mainly due to high productivity and reutilization of
the enzyme (Gerpen 2005).
Enzymatic recovery and efficient low cost product separation are crucial
parameters to take in consideration when designing a continuous or fed-batch reactor.
For micellar systems few examples have been encountered in literature and all are
membrane reactors: hydrolysis of olive oil (Prazeres, et al. 1993; Prazeres, et al. 1994)
and lecithin (Morgado, et al. 1996), synthesis of esters (Carvalho, et al. 2000; Carvalho,
27
et al. 2001) and peptides (Serralheiro, et al. 1994; Serralheiro, et al. 1999), and most
recently oil transesterification (Badenes, et al. 2011a).
With an ultrafiltration configuration, the encapsulated enzymes are retained by
the membrane in the interior of the reactor and the products selectively pass through
the membrane (Bódalo, et al. 2001). The preferred membranes are biologically
compatible, resistant to high flows and solvents, good filtration flow and easily washed
(Carvalho, et al. 2001). The retention of reversed micelles by the membrane is affected
by several factors, such as the nature of the surfactant, the size of the micelles, the
membrane properties (NMWCO and chemical compatibility), the operation mode and
the stability of the micelles to the shear forces usually found in membrane devices
(Prazeres, et al. 1994).
For the production of biodiesel, a recent work presented an enzymatic process
using a membrane bioreactor in order to reuse the biocatalyst (Badenes, et al. 2011a).
It is expected that for a whole-cell biocatalysts, which present a higher volume, to be
retained also on the membrane due to the cut off. However, membrane-cell
interactions have not been yet addressed for this particular case.
28
2. Materials and methods
2.1. Production
2.1.1. Microorganism
The over-expression of recombinant cutinase cloned in pMac5-8 was performed
with Escherichia coli WK-6 strain. Protein production and purification were carried out
by adaptation of the procedure in the literature (Lauwereys, et al. 1990).
2.1.2. Culture media
2.1.2.1.1.
Pre-fermentation medium
The composition of the medium for pre-fermentation was 20 g/l LB Broth (from
Sigma) medium (from Becton and Dickson) (pH 7.5). The medium was sterilized by
autoclaving at 121 ºC, during 20 min.
2.1.2.1.2.
Fermentation medium
The culture medium was composed by 250 ml of TB medium by dissolving the
chemical compounds: 12 g/l Bacto™ Triptone and 24 g/l Bacto™ Yeast Extract from
Becton Dickinson, 5 ml/l Glycerol (from Acros), 3.81 g/l KH2PO4 (from Merck) and
12.51 g/l K2HPO4 (from Panreac), necessary to adjust the pH to 7.1. The medium was
sterilized by autoclaving at 121 ºC, during 20 min. 1 M MgSO4.6H2O (from Riedel-de
Haen) solution was prepared and autoclaved separately to avoid the formation of
precipitates, and then added aseptically to the medium after cooling, to obtain a
concentration of 20 mM in the fermentation medium. The TB medium was used for
the induction of cutinase because it allows the cells to growth to high saturation
density.
29
2.1.3. Contaminations
Media were supplemented with previous sterilized ampicillin (from Sarstedt) to a
final concentration of 150 μg/ml. The sterilization of thermolabile ampicillin was
performed by filtration using sterile Milex-GP filter unit (0.22 μm pore diameter, from
Millipore).
2.1.4. Culture conditions
To prevent contaminations during the cutinase production, culture medium
manipulation, inoculation and sampling were performed in a laminar flux chamber
Bioair Instruments (model aura 2000 M.A.C. 4 NF).
2.1.5. Inoculum
Sterile petri dish cultures were inoculated with E. coli WK-6 frozen cells, from the
storage stocks at -80 ºC, and incubated at 37 ºC.
2.1.6. Shake flask
250 ml shake flask with a filling volume of 50 ml of the corresponding prefermentation media, supplemented with sterile ampicillin (150 μg/ml), were
inoculated with cells of inoculum and were left at 37 ºC in an orbital shaker (Agitorb
200) at 250 rpm, for 3 h.
In order to start fermentation with an optical density (OD) of 0.2, a certain volume
of the cells cultured in pre-fermentation medium was transferred to 2 l shake flask
with a filling volume of 250 ml of the fermentation culture medium, supplemented
with ampicillin to a final concentration of 150 μg/ml. The fermentations were
performed in the orbital shaker at 25 ºC and 250 rpm.
At OD (600 nm) approximately 1.0, the sterile inducer IPTG (isopropyl-β-Dthiogalactopyranoside) (from Bioline) was added to a final concentration of 0.1 mM,
with a reinforcement of ampicillin (125 μl of a solution of 100 mg/ml to 250 ml of
30
fermentation media). The sterilization of thermolabile IPTG was perfomed by filtration
using sterile Milex-GP filter unit (0.22 μm pore diameter, from Millipore).
2.1.7. Fermentor
This approach was used solely for the mutant T179C production. 1 l shake flask with
a filling volume of 200 ml pre-fermentation medium, supplemented with sterile
ampicillin (150 μg/ml), was inoculated with cells from inoculum and left in the orbital
shaker at 37ºC and 250 rpm, for 2 h (OD approximately of 2.2). The volume and the
time of pre-fermentation were calculated from studies on fermentation in shake flask.
Batch fermentation culture was performed in a 5 l Bioflow 3000 (New Brunswick
Scientific, equipped with a disk-turbine impeller. The 200 ml of cell cultured in the prefermentation medium were transferred to the culture vessel with a working volume of
3 l of TB medium, supplemented with ampicillin to a final concentration of 150 μg/ml.
The culture medium in the fermentor was maintained at pH value of 7.1 by automatic
control through 2 N NaOH or H2SO4 addition, and at controlled temperature of 25 ºC
by an electrically heated water circulation system and a jacket at the culture vessel.
The dissolved oxygen concentration (DOC), measured as percentage of saturation, was
controlled in cascade mode with the stirring speed, maintaining a constant aeration
rate of 3 l/min (1 vvm). This choice is due to the fact that stirring speed is related to
the power of three to the area per volume of bubbles, making more effective than
adjusting aeration rate. The lower stirring limit was set at 10 % of the motor capacity,
100 rpm, which was the minimum value to guarantee an effective mixture of the
culture medium and pH control. The upper limit was decided to be 700 rpm for safety
reasons.
At OD (600 nm) approximately 1.0, the inducer IPTG was added to a final
concentration of 0.1 mM, with ampicillin reinforcement, and fermentation continued.
Online data acquisition of temperature, pH, agitation and dissolved oxygen
concentration was obtained by BioSTAT 4®.
31
2.2. Extraction and purification of cutinase
2.2.1. Whole-cell biocatalyst
Cell retrieved from the reactor are permeabilized and afterwards either lyophilized or stored at
-20 ºC.
2.2.1.1.1.
Permeabilization TTP
Incubated with 2% (v/v) Tween 20, 2% X-100 Triton, 0,10% Polyethylenimine (PEI)
and 50 mM phosphate buffer pH 7.5, for 20 minutes at 200 rpm and 37 ºC.
2.2.1.1.2.
Permeabilization CTAB
Incubated with 0.3% (w/v) cetyltrimethylammonium bromide (CTAB) and 50 mM
phosphate buffer pH 8.0, for 20 minutes at 200 rpm and 37 ºC.
2.2.2. Protein extraction
2.2.2.1.1.
Osmotic shock
Cutinase sequestered in the periplasm was released by osmotic shock, using first
STE buffer (Tris.HCl pH 8.5 (from Eurobio), Sucrose 20 % (p/v) (from Sigma) and EDTA
12.5 mM (from Sigma)), and afterwards water at 4 ºC, using a ratio (buffer or
water)/(culture medium) of 0.16. The centrifugation steps were done at 9000 rpm, 4
ºC and 30 min, with a SLA-3000 rotor in a Sorvall RC6 centrifuge.
2.2.2.1.2.
Acid precipitation
The pH value of the periplasmic fraction was lowered to 4.7 by adding concentrated
acetic acid (1:2 v/v) (from VWR). After overnight incubation at 4 ºC with vigorous
stirring, the precipitated proteins and cell debris were removed by centrifugation
(9000 rpm, 4 ºC, 40 min), and the supernatant was filtrated under vacuum, using a
vacuum pump (Millipore type XF54 230 50).
32
2.2.3. Dialysis
The dialysis membranes were filled with the enzymatic extract, dipped in 20 mM
Tris.HCl buffer, pH 7.6 (at 4 ºC), and left at 4 ºC with weak stirring for 48 h. The buffer
was changed 3 times.
2.2.4. Ion-exchange chromatography
Further purification was achieved through two anion-exchange chromatographic
columns XK 50, with DEAE-Cellulose® and Q-Sepharose® fast flow media (from
Amersham Biosciences), and using the AKTA purifier 100 and the UNICORN 5.11
software. The resins were pre-equilibrated with 20 mM Tris.HCl buffer (pH 7.6). The
first column is used in a frontal mode of operation and the second in elution mode.
Considering this and since the pI of cutinase is 7.6 and DEAE-cellulose is a weak anionic
exchanger, the enzyme did not bind to this resin and flow through the column,
however contaminant proteins were bound. On the contrary, as Q-Sepharose is a
strong anionic exchanger, the enzyme was bound to the second resin. The elution was
performed with an ionic strength gradient using a 1 M NaCl solution prepared in 20
mM Tris.HCl buffer (pH 7.6). The gradient started at 0 mM and ended at 150 mM NaCl.
In order to remove the large amount of NaCl present in the enzyme solution collected,
a final dialysis against distilled water (4 ºC) for 24 h, with 2 times buffer changes, was
performed.
2.2.5. Lyophilisation
The subsequently lyophilisation was performed in a Chirst Alpha 2-4 lyophilizer from
B. Braun Biotech International, coupled to a Pfeiffer type duo 008B vacuum pump,
during 2 days. At the end, lyophilized cutinase was collected, weighted and stored
dried at -20 ºC. Cutinase purity was confirmed by 15 % SDS-PAGE.
33
2.3. Analytical procedures
2.3.1. Biomass
Culture samples were periodically withdrawn from the shake flask, and the optical
density (OD) was measured at 600 nm, after appropriate dilution, in a double beam
spectrophotometer (Hitachi U-2000). The specific growth rate (μ) was determined,
assuming exponential growth kinetics, by linear regression from the plots of ln(OD) vs
time. A linear calibration curve of dry cell weight versus OD measurements was
performed elsewhere (Badenes 2010), and is shown in figure 7.
Figure 7 – Linear calibration curve of OD measurements (600 nm) vs cell dry weight (mg/mL). This curve
has a slope of 3.672±0.237, and b of 0.238±0.097. Source: Badenes 2008 (Badenes 2010).
2.3.2. Cutinase activity assay
The lipolytic activity of cutinase can be determined through a spectrophometric
method which follows the degradation of p-nitrophenylbutyrate (p-NPB) at 400 nm.
The structure of p-NPB is schematically represented in figure 8.
This ester is
hydrolyzed by cutinase following a Michaelis-Menten kinetics, and one unit of enzyme
activity corresponds to the amount of enzyme required for the production of 1 µmol of
p-NPB per minute (Brissos, et al. 2008b). The activity assay is temperature sensitive,
being necessary to keep the substrate in cold before use, in order to prevent natural
occurring degradation (Tao, et al. 2011). This assay is used in order to determine the
activity of the cutinase obtained throughout the production process, and to calculate
34
the protein quantity for transesterification of different batches needed in order to
have the same activity.
Figure 8 – Structure of 4-nitrophenylbutyrate, also known as p-NPB (SIGMA 2012).
From samples of fermentation, the cutinase activity was measured by analysing the
supernatant and intracellular activities, after cell disruption. Enzyme activity was
determined using p-NPB (p-nitrophenylbutyrate) (from Sigma), delivered from 70 mM
concentrate stock solutions in pure acetronitrile (from Fisher). Samples (15 μl) were
added to reaction mixtures composed by 1470 μl 20 mM Tris.HCl, pH 8.0 and 15 μl of
70 mM p-NPB solution. The reaction rates were determined by monitoring
spectrophotometrically the release of p-nitrophenol at 400 nm (ε = 15400M-1cm-1) and
30 ºC, for one minute. One unit of activity (U) corresponds to 1 μmol of p-nitrophenol
released per minute. Dilutions of the samples were made in order to work in linear
enzyme activity zone with respect to the amount of enzyme.
2.3.2.1.1.
Methanol induced stress
Performing in the same manner as the cutinase activity assay, the methanol was
added to the solution containing the whole-cell biocatalyst 24h prior to the assay at
30ºC with mild agitation. After 24h, the substrate p-NPB was added to measure
spectrophotometrically. Five different methanol concentrations were tested (1, 1.5, 2,
5 and 10% v/v), where the volume of methanol added instead of buffer.
35
2.3.3. SDS-page
Cutinase purity and presence was confirmed by 15 % SDS-PAGE (sodium dodecyl
sulphate-polycrylamidegel) electrophoresis.
2.3.4. Determination of protein concentration
Protein concentration was determined by the microplate procedure Pierce
Coomassie Bradford Protein Assay kit. Preparation of diluted Bovine Serum Albumine
BSA standards, from µg/mL to 0.50 µg/mL, for calibration curve. Addition of 50 µL of
each standard or sample to the blank wells, followed by addition of 200 µL of
Coomassie solution. Plate was mixed on a plate shatter for 30 seconds and afterwards
incubated for 10 minutes at room temperature. The absorbance was read at 595 nm in
a microplate reader (Biorad Spectra MAX 340 pC).
A calibration curve, represented in figure 9, was performed and the linearization
was done within the linear zone, until the 50 µg/mL, and samples measured never
exceeded this value. Specific activities are calculating dividing the activity by the
concentration. Yields determined by ratio between activity of the after and before
each step. Purification factor is calculated by the ratio between specific activity after
Absorbance 595 nm
and before each step.
0,350
0,300
0,250
0,200
0,150
0,100
0,050
0,000
y = 0,0063x + 0,0004
R² = 0,996
0
10
20
30
40
50
60
Concentration (µg/mL)
Figure 9 – Calibration curve of the Pierce Coomassie Bradford method from 5 to 50 µg/mL at 595 nm. It
has 0.0063±0.0002 slope and 0.0004±0.0001 intersection.
36
2.4. Enzymatic transesterification
2.4.1. Chemicals and enzyme
Surfactant bis(2-ethylhexyl) sodium sulfosuccinate, AOT (98 %), triolein (65 %),
methanol (99.9 %), ethanol absolut (99.8 %) and 1-butanol (99 %) were obtained from
Sigma. Isooctane (99.5 %) was purchased from Fluka. All eluents (acetonitrile, 2propanol and n-hexane) were of HPLC grade obtained from LabScan and sodium
phosphate salts (Na2HPO4 and NaH2PO4) were of analytical reagent grade from Merck.
F. solani pisi cutinase was obtained through according to the methods explain in
section 2.2.
2.4.2. Reversed micelles
Cutinase was dissolved in 200 mM phosphate buffer pH 8.0 and was further
microencapsulated in 150 mM AOT in isooctane (molar ratio of water to surfactant,
Wo 2.7 and water concentration is 405 mM), except where indicated otherwise, by the
injection method. This method consisted in adding dropwisely the aqueous solution
with cutinase to the AOT/isooctane solution and strong vortex mixing for 15 s. The
total reaction volume is 3 ml. The conditions buffer molarity, pH, AOT concentration
and Wo were chosen taking into account the desired optimization (Badenes 2010).
Whole-cell biocatalyst reactions were performed using the same default conditions
except when mention some alteration for testing new conditions.
2.4.3. Transesterification procedure
The reactions were performed in a batch stirred reactor with a water jacket
connected to a thermostated bath (Lauda E-100) at 30 ºC. All experiments were
performed using magnetic stirring at 600 rpm. The alcohol initial concentration was
defined at 390 mM and the substrates molar ratio of alcohol to fatty acid chains of 1.6
was used. Reactions were started with the addition of the alcohol (strong vortex
mixing during 20 s) and followed for 24 h. In order to have a baseline for comparison,
37
for all the reactions the amount of cutinase used was the amount required to have 1 U.
This was determined by calculating the activity of cutinase according to section 2.3.2.
2.4.3.1.1.
Hydrolysis reaction
Hydrolysis of triglycerides generates glycerol and fatty acids, as shown in figure 10.
Following the same conditions as the transesterification procedure, with the difference
that isooctane was replaced with the same quantity of buffer. For this assay only
whole-cell biocatalyst was used.
Figure 10 – Schematic representation of the hydrolysis reaction of a triglyceride molecule
2.4.4. Analysis
Samples (50 μL) were taken at specific times and 1 μl of acetic acid (58.5 mM, pH
3.0) was added to stop the reaction along with strong vortex mixting. These samples
were dissolved in 949 μL of n-hexane and were then centrifuged (Sigma 201M) to
remove precipitates. The supernatant was analyzed by high-performance liquid
chromatography (HPLC) using a Chromolith® Performance RP-18 endcapped (100 mm
x 4.6 mm x 2 μm) column. HPLC apparatus (Hitachi LaChrom Elite), equipped with an
autosampler (Hitachi LaCrom Elite L-2200), a HPLC pump (Hitachi LaChrom Elite L2130) and a UV detector (Hitachi LaChrom Elite L-2400) at 205nm, was used. The flow
rate was 1 ml/min and the injection volume was 20 μl. Three mobile phases were
38
employed: phase A consisted of acetonitrile 100 %, phase B consisted of water 100 %
and phase C consisted of n-hexane and 2-propanol (4:5, v/v). The reaction mixture
includes: TG with three combinations of oleic (O) and linoleic (L) acid namely OOO,
OOL and OLL; DG with three combinations of the same fatty acids and couples of sn1,2 and sn-1,3 positions in the glycerol backbone, namely OO-, O-O, LO-, O-L and LL-;
MG of O and L acids in sn-1 and sn-2 positions of the glycerol backbone, namely O--, O-, L-- and -L-; alkyl esters (AE) of the O and L fatty acids and the AOT (Badenes 2010).
In case of hydrolysis, the free oleic and linoleic acids will also appear in the mixture and
are also detected in the HPLC analysis.
39
40
3. Results and Discussion
3.1. Shake flask production and purification
3.1.1. Growth profile
The growth of E. coli was followed for fermentation profile, with samples taken
along the 3h, as shown in figure 11. All four experiments presented same typical
exponential growth reaching an OD of approximately 1.3, after being measured with
proper dilution regarding Lambert-beer law.
Assuming exponential growth kinetics, and according to equation 1, the growth
rates of the four experiments were calculated.
log = log + [1]
The average growth rate calculated was 0.407 ± 0.030 h-1 for the initial hours of
fermentation, which compared to the literature (0.541 h-1) is a lower value than
expected (Badenes 2010). This can be explained by the fact that the cells used were
inocula into petri dish a weak before, which could have affected their ability to
proliferate. Furthermore, the intermittent shaking required for monitoring the OD can
cause setbacks to growth of microorganisms (Wittmann, et al. 2003).
3,5
3
OD (600 nm)
2,5
2
Shake Flask 1
1,5
Shake Flask 2
Shake Flask 3
1
Shake Flask4
0,5
0
0
1
2
3
4
5
Time (hours)
Figure 11 – Growth curve of fermentation for the different shake flaks experiments. From these curves
-1
the growth rates were calculated: initial hours of fermentation µ = 0.407 ± 0.030 h ; after induction with
-1
µ = 0.203 ± 0.020 h .
41
The growth curve after induction, after 2.25h, was also followed, for the first 3
hours and later on the 16th hour. The growth rates were calculated according to
equation 1 and it only was considered the first 3 hours after induction. The average
growth rate after induction was 0.203±0.020 h-1. Once again when compared to the
literature the value is lower than expected (Badenes 2010). However, when analysing
the drop in the growth rate from initial fermentation hours to after induction, one can
see it corresponds to the expected (Badenes 2010). An approximately 50% drop is
observed therefore we can conclude that the cells are using the energy for protein
production instead of using for proliferation.
The calibration curve has a slope of 3.672±0.237, and the intercept of 0.238±0.097.
Applying this relation one can determine that at after induction there was 0.28 mg/mL
of dry cells, and at the 16th hour after induction approximately 3.48 mg/mL of dry cells.
3.1.2. Quantification of protein and cutinase activity assays
A linear regression was performed and the slope corresponds to the activity, where
1 U means 1 µmol of product formed by time unit. The activity values of each sample
are presented in table 2. In order to calculate specific activities, concentrations were
determined by the Pierce Coomassie Bradford method. The quantification of samples
is also shown in table 2.
After each step of purification the cutinase activity drops, as well as the protein
concentration, which means that cutinase is being lost along with other proteins.
However, the specific activity always increases, meaning that the procedures remove
more the other contaminant proteins than cutinase. In spite of this being the desired,
the yields are very low, 40-60%, thus losing too much of the desired protein.
Moreover, only 1.5-3.7 fold increases in purification were observed, which is also very
low. This means that despite of losing more other proteins than cutinase, the loss of
cutinase is still very high. The acid precipitation step is a vigorous step and a high
amount of protein is lost in this step, including cutinase.
42
Table 2 - Activity calculated, by linearization of the curves and calculated the slope. Concentration was
determined by measuring the OD at 595 nm and applying the calibration curve represented in figure 8.
Clarified broth
16th induction hour
Osmotic shock
Acid prepicitation
Dialysis
Overall
Activity
(U)
Concentration
(µg/mL)
1.80
4.80
2.89
1.15
0.460
-
35.2
47. 0
7.68
2.01
0.326
-
Specific
activity
(U/mg)
0.051
0.102
0.376
0.575
1.41
-
Yield (%)
Purification
factor
100
60.3
39.9
39.9
9.60
1.00
3.69
1.53
2.45
13.8
Therefore, the relatively lower yields were probably due to this step. A sudden drop
of concentration after dialysis was due to a rupture of one of the dialysis membranes,
which meant a very high loss of cutinase. After dialysis, only 0.3 µg/mL of protein was
obtained.
3.1.3. Concentration of purified cutinase
During elution of cutinase from column Q-Sepharose, in the chromatographic step,
the absorbance at 288 nm was continuously followed (presented in figure 12). The
protein absorbs at 288, and the peaks in figure represent the elution of the
concentrated cutinase.
Figure 12 – Representation of the peaks of UV (288 nm) detections (Y axis) versus the volume buffer
through the column for elution (X axis).
Several samples from various stages of cutinase production were analysed by a SDSpage electrophoresis and photographs of the gels are presented in figure 13.
43
st
Figure 13 – SDS-PAGE
PAGE analysis of the samples. Gel A wells: 1 and 2 – Molecular weight markers; 3 – 1
hour of the fermentation; 4- after induction;
i
5 - 1h after induction; 6 -2h after induction;
indu
7 - 3h after
induction; 8 - 16h after indution; 9 – Osmotic shock with STE; Gel B wells: 1 – Osmotic shock with water;
2 – Acid precipitation; 3 - Molecular weight markers ; 4 – Dialysis; 5 – DEAE-Sepharose;
pharose; 6 – Q-Sepharose;
7 – 20h after induction; 8 – DEAE-Sepharoese
DEAE
after elution; 9 – Clarified broth
In gel A, the amount of proteins in the samples increases significantly during
fermentation time and after induction, it can be observed a continu
continuous increase on
cutinase 22 kDa band intensity, confirming quantification results. In the last well of gel
A, after osmotic shock with STE, there is a huge decrease in various proteins and the
remaining of the band that corresponds to 22 kDa cutinase, shows
show the initial
purification step. Furthermore, in gel B the first well, still corresponding to osmotic
shock but with water, supports the previous statement. In acid precipitation (second
well) a huge decrease in proteins is observed, as long with a partial loss in the 22 kDa
band intensity. Dialysis sample (well number 4) shows further purification as the bands
become less intense and more separated. Wells number 5 and 6 correspond to the
column DEAE-Sepharose
Sepharose and Q-Sepharose,
Q Sepharose, respectively, and it can be observed
ob
that
during elution, in the latest sample appears the band that corresponds to 22 kDa
cutinase and in the first sample does not. These results are expected since cutinase
only binds to Q-Sepharose
Sepharose column. The 8th well corresponds to a sample taken after
a
elution, to confirm that all protein has been eluted from the column, and none is
unexpectedly bonded. The 7th well shows that as further the induction go more protein
is obtained, up to the 20th hour after induction. The clarified broth sample (9th well),
which corresponds to the supernatant recovered after the initial centrifugation of the
cells, has many undesirable proteins.
44
3.2. Stirred fermentor production
3.2.1. Growth profile
The fermentation scale up is the aim of any producer as it decreases the costs while
obtaining high yields and good quality product. The next step of shake flash production
is the batch type stirred fermentor production, in this case a 3 l work volume reactor.
This study aimed to compare the how straightforward the scalability is, and to achieve
high amount of cutinase mutant T179C for the further studies presented.
In order to compare the yield in biomass, several samples were taken through the
fermentation, which results presented in figure 14. The optimal parameters were
previously established (Badenes 2010).
30
OD (600 nm)
25
20
15
10
5
0
0
5
10
15
20
25
Time (hours)
Figure 14 – Growth curve of fermentation performed in the 5 l Bioflow 3000 reactor. The induction
was performed at 6 h of fermentation. Pre-fermentation was performed as the explained in section 2.
As it is can be observed in figure 14, a much higher OD was obtained in the stirred
fermentor culture then with shake flasks cultures, achieving a 9-fold increase in OD.
3.2.2. Parameters control
The increased production of biomass is expected as parameters such as pH,
temperature and dissolved oxygen concentration are easily controlled, whereas in the
shake flask they are not easy or not even controlled. However, these parameters do
present some variability as the control is made by a detection of shift and adjustment
approach.
30
600
25
RPM
500
20
400
300
15
200
10
100
5
0
0
0
5
10
15
20
pH / Temperature (ºC) / DOC(%)
35
700
RPM
pH
Temperature
DOC
25
Time(h)
Figure 15 – Controlled parameters in the 5 l Bioflow 3000 reactor. From the primary Y axis the
rhombus represents the stirring measures, starting with 200 rpm and in the end with 700 rpm. For the
secondary Y axis there are 3 parameters: the solid triangle represents the pH which starts at 7.1
th
oscillating around this value until roughly the 20 hour ending with 5 pH; the circle stands for
temperature and this is the most stable parameter along the fermentation having no noticeable
deviation of the set point, 25 ºC; the solid squares are DOC which starts at 100%, and reaches optimal
condition after 2,5h, then oscillating around this value and decreasing to 0% by the latest hours of
fermentation.
The temperature has proven to be the most tightly controlled parameter, as it can
be observed in figure 15. However, the other parameters tend to deviate from the
initial conditions. For instance pH that is fairly stable throughout the first hours of
fermentation shows some deviation for the late period. This is due to the huge
increase in the stirring rate, which leads to an increase of foam inside the reactor. As
the base/acid concentrated solutions are added drop-by-drop, the foam prevents
these to reach the liquid and hence destabilizes the pH. The rapid increase in the
stirring rate is related to the decrease of DOC in the reactor. The stirring control was
configured in order to maintain the level of DOC higher than 30%, increasing the
stirring rate as the DOC reduces, but it could never exceed 700 rpm since shear stress
induced to the cells would be too great. Approximately around the 20th hour, the
46
reactor is not in its optimal conditions as the DOC is already lower than 30%,
decreasing rapidly until it reaches 0%. This drop of DOC is due to the increased
biomass production inside the reactor that reaches a saturation level, consuming all
available oxygen.
Since high biomass does not mean high production of the intended protein, the
same activity assays were performed to confirm cutinase production during the
fermentation.
3.2.3. Final product
The end product was 3 l of solution containing the E. coli enriched with cutinase.
Three different paths were chosen afterwards to continue with further studies: 1/3
went through the previously explained extraction and purification steps; 1/3 was dried
and lyophilized; the other 1/3 was dried and preserved at -20 ºC.
47
48
3.3.
Whole-Cell Enzymatic transesterification of triglycerides by
cutinase
With the objective of testing new conditions and to gain insight into the
transesterification of triglycerides with whole-cell biocatalyst, the experiments were
conducted in small-scale reactors (3 ml of working volume). As it has proven higher
stability than wild type, the T179C mutant was used in all assays, except when
indicated otherwise from control purposes.
As the downstream process at any industry comprises a significant part of the cost
of production, the whole-cell biocatalyst would greatly improve the competitiveness of
cutinase biodiesel production (Ranganathan, et al. 2008). Reports have been published
regarding whole-cell biodiesel production with success, using R. Oryzae (Matsumoto,
et al. 2001; Hama, et al. 2004), Pseudomonas sp. (Kaieda, et al. 2001) and Candida sp.
(Shimada, et al. 1999). In some studies permealibilized cells, previously frozen were
used, while in others the enzyme was in the form of a powder. For this reason, in the
previous section, the final product was divided in those three specific ways: the
lyophilized as the powder; the stored at -20 ºC as the freeze cells and the other third as
control biocatalyst.
The first assay was transesterification with whole-cell biocatalyst using the optimal
conditions previously defined (Badenes 2010). As it was an exploratory assay no
changes were made in order to take in account the fact that now cutinase was
intracellular. The results are shown bellow in figure 16.
The figure represents the HPLC chromatogram obtained. The baseline changes in
agreement with the different phases of the eluent gradient. The blue rectangle
represents the usual time zone where the peaks corresponding to the monoglycerides
(MG)are detected, the white rectangle time zone for alkyl esters (AE) detection, the
yellow rectangle the time zone for diglycerides (DG) detection and the red rectangle
the zone for triglycerides (TG) detection. It is expected that in the beginning of the
reaction, time zero, there exists a high concentration of TG, a low concentration of DG
and MG and no alkyl esters present. However, as transesterification promotes the
transformation of TG in alkyl esters, but the intermediates MG and DG, are also
49
present. Itt is expected that the longer the reaction time less concentration of TG,
shown by smaller area under the peaks, and high concentration of AE, and some
evidences of MG and DG.
Figure 16 – Chromatogram of samples from the reaction mixture, when using the whole-cell
whole
biocatalyst
and the optimal conditions obtained for purified protein. Two overlapped curves are shown, green line
is the chromatogram of the initial time zero sample, and blue line is the chromatogram of the 24h
reaction sample.. Blue rectangle – time zone of MG detection; white rectangle – time zone of AE
detection; yellow rectangle – time zone of DG detection; red rectangle – time zone of TG detection.
However, when analysing the figure 16,
1 , which represents only two overlapped time
points of the reaction, time zero and 24h of reaction,
reaction, we conclude that no reaction
occurred as no changes in the peaks were observed. Although there is a minor AE peak
detected, as this peak is present in both the time zero and after 24h reaction it can be
concluded that the substrate
bstrate used already had suffered from some kind of natural
degradation,, most likely light induced degradation.
degradation
Given this result, it was proposed that there might be present an access limitation
of the substrate to the intracellular cutinase in the cells. In fact, there is a study
regarding
biodiesel
production
clearly
stat
stating
that
they
had
performed
permeabilizations treatments to
t the cell membrane (Ban,
Ban, et al. 2002).
2002 Having this in
consideration, after the fermentation step where the end product was divided,
divided two
permeabilizations were performed,
performed one with cetyltrimethylammonium bromide
50
(CTAB) and the other with
wi Tween 20, Triton X-100
100 and Polyethylenimine (TTP).
(TTP)
However, these experiments were also unsuccessful as no AE peak appeared after 24h
reaction, exemplified in figure 17.
1
Figure 17 - Chromatogram of samples from the reaction mixture, when using the whole-cell
whole
biocatalyst
permeabilized with CTAB and the optimal conditions obtained for purified protein. Two overlapped
curves are shown, the green line is the chromatogram of the initial time zero sample, and blue line is the
chromatogram of the 24h reaction sample.
sample Blue rectangle – time zone of MG detection; white
w
rectangle
– time zone of AE detection; yellow rectangle – time zone of DG detection; red rectangle
ctangle – time zone of
TG detection.
Controls were needed in order to attain if the transesterification was not working
due to cutinase folding problems or if it was due to not optimal parameters. The first
control needed was to ensure that the protein produced was in fact able to catalyse
cataly
the transesterification and so, with the cutinase extract and purified from the
fermentation end product a transesterification reaction was performed (figure 18).
In figure 18, in
n contrast with figure 16
1 and 17, the initial chromatogram varies
greatly with the 24h reaction chromatogram, the latest evidencing peaks characteristic
of AE. The conversion in AE is represented in the graphic in figure A1,, where at 24h it
reached a 90.5±0.5% conversion.
conversion With this control we show that purified cutinase has
activity and the whole-cell
cell biocatalyst
catalyst is not having activity due to other effect than
inactive protein. With this new evidence and knowing that methanol is harmful to
cells,, by being a strong denaturating agent, it was proposed that, instead of using such
51
an aggressive substrate, butanol could provide better results (figure
figure 19).
1 Alongside
with this test, the thought that the own cell membrane would
would serve as a microreactor
playing the role of the surfactant AOT was tested (figure A2).
Figure 18 - Chromatogram of samples from the reaction mixture, when using purified protein. Two
overlapped curves are shown, green line is the chromatogram of the initial time zero curve, and blue
b
line the chromatogram of the 24h reaction sample. Blue rectangle – time zone of MG detection; white
w
rectangle – time zone of AE detection; yellow rectangle – time zone of DG detection; red rectangle –
time zone of TG detection.
Figure 19 - Chromatogram of samples from the reaction mixture, when using using butanol as a coco
substrate instead of methanol. Two
T
overlapped curves are shown, green line is the chromatogram the
initial time zero curve, and blue line the chromatogram of the 24h reaction sample.
sample Blue rectangle –
time zone of MG detection; white
hite rectangle – time zone of AE detection; yellow rectangle – time zone of
DG detection; red rectangle – time zone of TG detection.
52
Unfortunately, butanol usage showed no significant different when compared with
methanol. As no reaction occurred in the previously described studies, no assessments
could be made regarding the capability of the membrane to be used instead of the
surfactant.
As a complementary control, the lipolytic activity of whole-cell biocatalyst cutinase
T179C mutant was measured by the p-NPB method when under methanol stress. The
results are shown in table 3.
Table 3 – Different volume percentage of methanol in the reaction media in order to determine the
decrease in activity in whole cell biocatalyst. The incubation period of methanol was 24h, and the values
present are the average of triplicates.
Methanol
0%
1%
1.5%
2%
5%
10%
Activity (U)
0.0124
±0.001
0.0126
±0.002
0.0120
±0.002
0.0117
±0.001
0.0112
±0.003
0.0056
±0.001
Relative
activity (%)
100
102.23
97.38
94.59
90.94
45.02
The standard methanol volume percentage used in the transesterification reaction
in this study is 1.5%. When analysing table 3 it is possible to infer that at 1.5% of
methanol induced stress the whole-cell biocatalyst still maintains 97% of its activity,
not being significantly decreased up until 10%. This means that, at this methanol
concentration, the transesterification reactions with whole-cell biocatalyst do not
suffer inactivation even under 24h methanol stress.
The following step was to confirm if due to the increase contaminants (other
proteins and membrane) in solution, the increase of the water content would be
beneficial. Considering optimal parameters used, the buffer corresponds to 0.73%
(v/v), and then four buffer percentages were tested: 1%, 5%, 10% and 20%. Figure 20
shows the chromatogram of the experiment with 5% water content that occurred for
48h and once again no reaction occurred, while the chromatograms of the reactions
performed with the other percentages are shown in figures A3, A4 and A5.
53
Figure 20 - Chromatogram of samples from the reaction mixture, when using 5% of buffer instead of
0.73%. A longer period of time was considered for this experiment (48h) .Two
.Two overlapped curves are
shown, green line is the chromatogram of the initial time zero curve, and blue line the chromatogram of
the 24h reaction sample. Blue rectangle – time zone of MG detection; white
hite rectangle – time zone of AE
detection; yellow rectangle – time zone of DG detection; red rectangle – time zone of TG detection.
It was also tested if there were mass transfer limitations by increasing substrate
concentrations and decreasing concentration of cells,
cells one
ne of these results is shown in
figure 21. However, in both cases, no reaction occurred. These results directed to
another hypothesis that had been assumed from
f m the beginning, if in fact the triolein,
as a polar molecule, could pass through the cell membrane. To confirm this new
hypothesis, an experiment was thought out using the same instruments but, instead of
methanolysis, it was performed the hydrolysis. The change is only to substitute the
organic media by buffer solution (figure 22).
Analysing the results, it can be seen that there are significant differences in the
chromatograms from time zero and the 24h sample.. Although after 24h there cannot
be detected peaks corresponding to AE, MG or DG, a complete decrease of TG peaks
was accomplished. This iss due to the fact that the analytical procedure is not optimized
to separate fatty acids and so they are eluted in the early minutes of analysis,
analysis blue
rectangle.. With this is possible to confirm that in fact the TG passes through the cell
membrane, being accessible to cutinase.
54
Figure 21 - Chromatogram of samples from the reaction mixture, when using 500 µl of triolein instead of
399 µl. Two overlapped curves are shown, green line is the chromatogram of the initial time zero curve,
and blue line the chromatogram of the 24h reaction sample. Blue rectangle – time zone of MG
detection; white rectangle – time zone of AE detection; yellow rectangle – time zone of DG detection;
red rectangle – time zone of TG detection.
Figure 22 - Chromatogram of samples from the reaction mixture, when using buffer instead of isoctane.
isoctane
Two overlapped
d curves are shown, green line is the chromatogram of the initial time zero curve, and
blue line the chromatogram of the 24h reaction sample. Blue rectangle – time zone of MG detection;
white rectangle – time zone of MG detection; yellow rectangle – time zone of DG detection; red
rectangle – time zone of TG detection.
55
In parallel with the previous control, another was made to confirm that cutinase
was not inactive inside the cell, and that in fact it could perform not only hydrolysis but
also methanolysis. For this experiment,
experiment the cell extract was previously exposed to
sonication in order to rupture the cell membrane. And it was with this complex
mixture, with cell debris, that the reaction was performed (figure 23).
Figure 23 - Chromatogram of samples from the reaction mixture, when using whole cell biocatalyst
previously sonicated using the optimal conditions obtained for purified protein. Two overlapped curves
are shown, green line is the chromatogram of the initial time zero curve, and blue line the
chromatogram of the 24h reaction sample. Blue rectangle – time zone of MG detection; white
w
rectangle
– time zone of AE detection; yellow rectangle – time zone of DG detection; red rectangle – time zone of
TG detection.
Despite of the peaks not representing as high area as the ones obtained
obtai
in the
experiment with purified cutinase (figure
(
18),, it is still visible that transesterification
occurs, confirming the activity of cutinase even when entrapped inside the cell. The
conversion in AE is represented in the graphic in figure A6, where at 24h it reached a
54.5±0.7% conversion.
56
4. Conclusions and Future Work
In conclusion, protein production in shake flask is not optimum for parameters
determination, and culture in stirred tank reactor is advised. These reactors present
improved gas transfer, improved mixing efficiency and continuous monitoring (e.g. pH
and temperature). Growth rates of 0.407 h-1 for initial hours of fermentation and 0.203
h-1 after induction were determined for shake flask, leading to the conclusion that the
cells, after induction, are using the energy to produce protein rather than proliferate.
This hypothesis is supported by the activity assays, which show higher activities after
induction for cutinase production. In addition, the SDS-gel assay shows the increase in
the intensity of the band that corresponds to cutinase. Nonetheless, improvements in
the purification must be done in order to achieve higher yields of protein and higher
purity. Only a 10% yield was obtained in this experiment, with a 14-fold increase in
purification. Other processes of purification can be considered, like two-phase
systems, for faster purification without disregard of purification factor.
The fermentation performed in the stirred fermentor showed significant
improvements in the yield of biomass and hence of cutinase production. Although
much tightly controlled, the parameters such as culture time and foam accumulation
could still be tuned up in order to optimize protein productivity.
The exploratory studies on whole-cell biocatalysis with E. coli entrapping cutinase
proved ineffective for the biodiesel production but enriched with useful information.
Methanol at the concentration used in the whole-cell biocatalyst cutinase T179C
mutant assays does not substantially influence cutinase activity even incubated for
24h, but a step-wise addition for continuous flow-reactors is advised. Several water
contents and mass transfer limitations were address in order to tackle the problem,
but it proved unsuccessful. Using the hydrolysis control over the previous information
it was shown that the oil has the capability to cross the cell membrane and reach
cutinase, but the reason why transesterification does not occur is still elusive.
However, using the sonicated cells, or instead using whole-cell biocatalyst for
hydrolysis, since purification has an overall yield of 10%, this usage might be a good
approach.
57
58
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64
Appendix A
100
90
AE converted (%)
80
70
60
50
40
30
20
10
0
0
50
100
150
200
Time (min)
Figure A1 – Graphic of the conversion to AE representative of chromatogram of figure 16. For this curve
the area under the peaks of the chromatogram were calculated and at 24h the conversion was
90.572±0.05%.
Figure A2 - Chromatogram of samples from the reaction mixture, without using AOT.
AOT Two overlapped
curves are shown, green line is the chromatogram of the
the initial time zero curve, and blue line the
chromatogram of the 24h reaction sample. Blue rectangle – time zone of MG detection; white
w
rectangle
– time zone of AE detection; yellow rectangle – time zone of DG detection; red rectangle – time zone of
TG detection.
65
Figure A3 - Chromatogram of samples from the reaction mixture, when using 1%
% of buffer instead of
0,73%. Two
wo overlapped curves are shown, green line is the chromatogram of the initial time zero curve,
and blue line the chromatogram of the 24h reaction sample. Blue rectangle – time zone of MG
detection; white rectangle – time zone of AE detection; yellow rectangle – time zone of DG detection;
red rectangle – time zone of TG detection.
Figure A4 - Chromatogram of samples from the reaction mixture, when using 10% of buffer instead of
0,73%.. Two overlapped curves are shown, green line is the chromatogram of the initial time zero curve,
and blue line the chromatogram of the 24h reaction sample. Blue rectangle – time zone of MG
detection; white rectangle – time zone of AE detection; yellow rectangle – time zone of DG detection;
red rectangle – time zone of TG detection.
66
Figure A5 - Chromatogram of samples from the reaction mixture, when using 20%
% of buffer instead of
0,73%.. Two overlapped curves are shown, green line is the chromatogram of the initial time zero curve,
and blue line the chromatogram of the 24h reaction sample. Blue rectangle – time zone of MG
detection; white rectangle – time zone of AE detection; yellow rectangle – time zone of DG detection;
red rectangle – time zone of TG detection.
60
AE converted (%)
50
40
30
AE
20
10
0
0
50
100
150
200
Time (min)
Figure A6 - Graphic of the conversion
conversion to AE representative of chromatogram of figure 16. For this curve
the area under the peaks of the chromatogram were calculated and at 24h the conversion was
54.465±0.07%.
67