Biotechnology Advances 20 (2002) 239 – 267
www.elsevier.com/locate/biotechadv
Developments and trends in enzyme catalysis in
nonconventional media
Sajja Hari Krishna*
AK-Technische Chemie und Biotechnologie, Institut für Chemie und Biochemie, Universität Greifswald,
Soldmannstraße 16, D-17487 Greifswald, Germany
Accepted 27 August 2002
Abstract
The conventional notion that enzymes are only active in aqueous media has long been discarded,
thanks to the numerous studies documenting enzyme activities in nonaqueous media, including pure
organic solvents and supercritical fluids. Enzymatic reactions in nonaqueous solvents offer new
possibilities for producing useful chemicals (emulsifiers, surfactants, wax esters, chiral drug
molecules, biopolymers, peptides and proteins, modified fats and oils, structured lipids and flavor
esters). The use of enzymes in both macro- and microaqueous systems has been investigated especially
intensively in the last two decades. Although enzymes exhibit considerable activity in nonaqueous
media, the activity is low compared to that in water. This observation has led to numerous studies to
modify enzymes for specific purposes by various means including protein engineering. This review
covers the historical developments, major technological advances and recent trends of enzyme
catalysis in nonconventional media. A brief description of different classes of enzymes and their use in
industry is provided with representative examples. Recent trends including use of novel solvent
systems, role of water activity, stability issues, medium and biocatalyst engineering aspects have been
discussed with examples. Special attention is given to protein engineering and directed evolution.
D 2002 Elsevier Science Inc. All rights reserved.
Keywords: Nonaqueous media; Enzyme catalysis; Medium engineering; Biocatalyst engineering; Protein
engineering; Directed evolution
* Tel.: +49-3834-864-366; fax: +49-3834-864-373.
E-mail address: [email protected] (S. Hari Krishna).
0734-9750/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved.
PII: S 0 7 3 4 - 9 7 5 0 ( 0 2 ) 0 0 0 1 9 - 8
240
S. Hari Krishna / Biotechnology Advances 20 (2002) 239–267
1. Introduction
According to conventional notion, enzymes are active only in water. Historically,
enzymatic catalysis has been carried out primarily in aqueous systems. Although water is a
poor solvent for preparative organic chemistry, it is the unique specificity of enzymes that
drew the interest of chemists who were seeking highly selective catalytic agents.
Experiments to place enzymes in systems other than aqueous media date back to the end of
the nineteenth century (Hill, 1898; Kastle and Loevenhart, 1900; Bourquelot and Bridel,
1913; Sym, 1936; Dastoli and Price, 1967). Initial studies considered the addition of small
quantities of water-miscible organic solvents like ethanol or acetone to aqueous enzyme
solutions ensuring availability of a high water content to retain the catalytic activity of
enzymes.
Then, biphasic mixtures (aqueous enzyme solution emulsified in a water-immiscible
solvent such as isooctane or heptane) were used, in which substrates from the organic phase
diffuse to the aqueous phase, undergo enzymatic reaction and the products diffuse back. The
size of water droplets may be reduced to facilitate mass transfer resulting in the formation of
microemulsions or reverse micelles, whose stabilization is achieved by adding surfactants
(Martinek et al., 1986; Hari Krishna et al., 2002).
Developments in using enzymes in nearly nonaqueous solvents containing traces of water
have stimulated research in achieving various kinds of enzymatic transformations (Klibanov,
1986, 1989; Schoffers et al., 1996; Bornscheuer and Kazlauskas, 1999; Gandhi et al., 2000;
Giri et al., 2001; Hari Krishna and Karanth, 2002a; Panke and Wubbolts, 2002; Thomas et al.,
2002).
Enzymatic reactions in nonaqueous solvents offer numerous possibilities for the biotechnological production of useful chemicals using reactions that are not feasible in aqueous
media. These reactions include chiral synthesis or resolution (Klibanov, 1990; Collins et al.,
1992; Stinson, 2000; Zaks, 2001); production of high-value pharmaceutical substances (Zaks
and Dodds, 1997; Schulze et al., 1998; McCoy, 1999; Patel, 2001; Rasor and Voss, 2001);
modification of fats and oils (Bornscheuer, 2000a); synthesis of flavor esters and food
additives (Hari Krishna and Karanth, 2001, 2002b; Hari Krishna et al., 1999, 2000a,b,
2001a,b); production of biodegradable polymers (Kobayashi, 1999), peptides, proteins and
sugar-based polymers (Vulfson, 1998).
In nonaqueous solvents, hydrolytic enzymes can carry out synthetic reactions and some
enzymes can also exhibit altered selectivities (Klibanov, 2001), pH memory (Zaks and
Klibanov, 1985, 1988; Klibanov, 1995), increased activity and stability at elevated temperatures (Zaks and Klibanov, 1984; Ahern and Klibanov, 1985), regio-, enantio- and
stereoselectivity (Bornscheuer, 2000a,b), and may be affected by water activity (Halling,
2000).
Currently, there is a considerable interest in the use of enzymes (particularly lipases, esterases
and proteinases) as catalysts in organic synthesis (Schmid and Verger, 1998; Bornscheuer,
2000a,b; Carrea and Riva, 2000; Faber, 2000; Liese et al., 2000; Patel, 2000; Koeller and Wong,
2001). The diversity of potentially useful enzymes at the researcher’s disposal has now become
vast, supplemented by catalytic RNAs (ribozymes) and antibodies (abzymes).
S. Hari Krishna / Biotechnology Advances 20 (2002) 239–267
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Inorganic metal-derived catalysts are capable of carrying out several of the enzymatic
reactions (Noyori, 1994), but the interest in using enzymes as agents for performing various
reactions with high selectivity in both macro- and microaqueous systems has picked up
tremendously during the last two decades. On average, at least one paper dealing with
biocatalysis in organic solvents is published everyday. Since it would be an impossible task to
cover every published account of biocatalysis in one review of reasonable length, this review
focuses on recent significant developments.
2. Enzymes as catalysts—a brief overview
Enzymes are classified into six major groups based on the reaction they catalyze (in the EC
number order): oxido-reductases, transferases, hydrolases, lyases, isomerases and ligases. The
International Union of Biochemistry and Molecular Biology (IUBMB) recognizes almost
4000 enzymes and has categorized them using enzyme nomenclature. The number of existing
enzymes in nature is about 25,000. Latest information on enzymes is available on the Internet
(http://www.expasy.ch/enzyme). Table 1 lists online accessible information resources on
enzymes and proteins and various related aspects.
Enzymes occur widely not only in animals and plants but also in filamentous fungi,
yeast and bacteria. Native or recombinant microorganisms produce a wide spectrum of
useful enzymes with variations in substrate specificity, reaction rate, thermal stability and
optimum pH. Microbial enzymes are relatively easy to obtain by fermentation processes
and with a few purification steps. Several enzymes from a variety of sources are available
from commercial suppliers (Table 2) and few vendors also offer enzyme-screening sets
(Table 3).
Table 1
Unified resource locators (URLs) for online accessible information on proteins and enzymes
Database
URL
ASPDa
BRENDA
ENZYME
Enzyme Structures
ESTHER
KEGG
LIGAND
Lipase Engineering
MDB
MEROPS
Protein Data Bank
PROMISE
SWISS-PROT
UM-BBD
http://www.sgi.sscc.ru/mgs/gnw/aspd/
http://www.brenda.uni-koeln.de
http://www.expasy.ch/enzyme
http://www.biochem.ucl.ac.uk/bsm/enzymes
http://www.ensam.inra.fr/cholinesterase
http://www.genome.ad.jp/kegg
http://www.genome.ad.jp/dbget/ligand.html
http://www.led.uni-stuttgart.de
http://metallo.scripps.edu
http://merops.iapc.bbsrc.ac.uk
http://www.pdb.org
http://bmbsgi11.leeds.ac.uk/promise
http://www.expasy.ch/sprot
http://umbbd.ahc.umn.edu
a
Artificially selected proteins and peptides database.
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Table 2
Major commercial suppliers of enzymes
Supplier
Website URL
Altus Biologics, Cambridge, MA, USA
Amano Pharmaceutical, Nagoya, Japan
Asahi Chemical Industry, Tokyo, Japan
Biocatalysts, Pontypridd, UK
BioCatalytics, Pasadena, CA, USA
Biocon India, Bangalore, India
Biozyme Laboratories, South Wales, UK
Boehringer Mannheim (now merged with Roche)
Calbiochem-Novabiochem, San Diego, CA, USA
http://www.altus.com
http://www.amano-enzyme.co.jp
http://www.asahi-kasei.co.jp
http://www.biocatalysts.com
http://www.biocatalytics.com
http://www.biocon.com
http://www.biozyme.com
http://www.roche.com/diagnostics
http://www.calbiochem.com
http://www.cnbi.com
http://www.diversa.com
http://www.dow.com
http://www.dsm.nl/dfs
http://www.sigma-aldrich.com
http://www.genencor.com
http://www.genzyme.com
http://www.gist-brocades.nl
http://www.aventis.com
http://www.juelich-enzyme.com
http://www.leescientific.com
http://www.mediagalaxy.co.jp/meito
http://www.merck.com
http://www.nagase.co.jp
http://www.neb.com
http://www.novozymes.com
http://www.promega.com
http://www.recordati.it
http://indbio.roche.com
http://www.roehm.de
http://www.sfrl.fr
http://www.serva.de
http://www.sigma-aldrich.com
http://www.thermogen.com
http://www.toyobo.co.jp
http://www.unitica.co.jp
http://search.wako-chem.co.jp
http://www.worthington-biochem.com
Diversa, San Diego, CA, USA
(Innovase LLC, a joint venture of Diversa with Dow Chemical)
DSM Food Specialties, Delft, The Netherlands
Fluka Chemical LLC, Buchs, Switzerland
Genencor International, Rochester, NY, USA
Genzyme Biochemicals, UK
Gist-Brocades, The Netherlands (now DSM group)
Hoechst, Germany (now Aventis, merged with Rhone-Poulenc)
Jülich Enzyme Products, Jülich, Germany
Lee Scientific, St. Louis, MO, USA
Meito Sangyo, Tokyo, Japan
Merck, Germany
Nagase and Co., Japan
New England Biolabs, Beverly, MA, USA
Novozymes, Bagsvaerd, Denmark
Promega, Madison, WI, USA
Recordati, Milan, Italy
Roche Diagnostics, Mannheim, Germany
Röhm, Germany
Seppim, France
Serva, Germany (Invitrogen group)
Sigma-Aldrich-Fluka, St. Louis, MO, USA
ThermoGen, Woodridge, IL, USA
Toyobo, Tokyo, Japan
Unitica, Osaka, Japan
Wako Pure Chemicals Industries, Osaka, Japan
Worthington Biochemical, Lakewood, NJ, USA
Of all the enzymes, hydrolases are the most employed for industrial biotransformations. It
is estimated that approximately 80% of all industrially used enzymes are hydrolases. Oxidoreductases are all cofactor-dependent and the industrial biotransformations have to be
performed coupled with processes for efficient recycling of the expensive cofactors. Transferases and ligases, which play a far larger role in nature than in industry, will gain more
importance in the near future. Lyases and isomerases are already gaining industrial
significance for their unique properties. Special properties and limitations that are specific
S. Hari Krishna / Biotechnology Advances 20 (2002) 239–267
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Table 3
Commercially available enzyme screening kits
Type of enzyme screening kit
Supplier
Alcohol dehydrogenase
Esterases and lipases
Nitrilases
Proteases
Transaminases (aminotransferases)
ThermoGen, BioCatalytics
Altus, Fluka, Roche, ThermoGen
BioCatalytics
Altus
BioCatalytics
for different enzyme classes with one representative of industrial example for each class are
illustrated below.
2.1. Oxido-reductases (EC 1)
These enzymes catalyze oxido-reduction reactions, which means that they act on substrates
through the transfer of electrons. The systematic name is based on donor/acceptor oxido-reductase. The oxidizing substrate is regarded as hydrogen donor and the enzyme is called a
dehydrogenase. If molecular oxygen (O2) is the acceptor, the enzymes may be named as
oxidase.
All oxido-reductases are dependent on cofactors, which either supply or take the reducing or
oxidizing equivalent. The most commonly needed cofactors are NADH/NAD + , NADPH/
NADP + , FADH/FAD + , ATP/ADP and PQQ. Since most of them are expensive, an effective
cofactor regeneration system is required for a cost-effective industrial process. If an isolated
enzyme is being used, it is feasible to employ a second enzyme (in case of NADH, the best
approach is to use formate dehydrogenase that utilizes formate and produces CO2, Scheme 1a)
or by applying a second substrate (Scheme 1b). Application of whole cells instead of isolated
enzymes is also a viable choice. Although a few methods based on electrochemical regeneration
have been detailed (Ruppert et al., 1988), they have not yet developed to commercialization. An
example of industrial processes employing benzoate dioxygenase (EC 1.14.12.10) is depicted
in Scheme 2. Currently, these enzymes are gaining immense attention and their properties are
being engineered mainly by directed evolution (Cirino and Arnold, 2002).
2.2. Transferases (EC 2)
These are enzymes that transfer a chemical group from one compound (donor) to another
(acceptor). The systematic names follow the scheme donor/acceptor group transferase. In
many cases, the donor is a cofactor or coenzyme carrying the often activated-chemical group
to be transferred.
These play a larger role in nature than in industry. Very few transferases are used in
industry due to various reasons: equilibrium reactions often do not attain high yields,
coupling reactions occur and the group-transferring substrates are quite expensive or their
corresponding products are not easily recycled. However, these enzymes would gain
importance in the future if the problems associated with them can be solved. Nonetheless,
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Scheme 1. Different approaches of cofactor regeneration.
high regio- and stereoselectivities in transferase-catalyzed reactions are major causes for their
increasing utility. An industrial process employing D-amino acid transaminase (EC 2.6.1.21)
is presented in Scheme 3.
Scheme 2. Enzymatic transformation catalyzed by an oxido-reductase.
S. Hari Krishna / Biotechnology Advances 20 (2002) 239–267
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Scheme 3. Enzymatic transformation catalyzed by a transferase.
2.3. Hydrolases (EC 3)
These enzymes catalyze the hydrolytic cleavage of C)O, C)N, C)C and some other bonds,
including P)O bonds in phosphates. Their applications are very diverse including hydrolysis
of polysaccharides, nitriles, proteins, lipids, and esterification of fatty acids. Most of these
enzymes are used in processing-type reactions to degrade proteins, carbohydrates and lipids, in
detergent formulations, and in the food industry (Table 4). The term hydrolase is included in
the systematic name, which includes the name of the substrate and the suffix –ase.
Hydrolases are the most commonly used enzymes in industrial processes. Biomass
utilization, with the help of cellulases (a group of hydrolases) to produce fine chemicals
(Hari Krishna et al., 2001c), is also a major thrust area of research in various laboratories. The
literature pertaining to hydrolases is well documented in numerous reviews (Theil, 1995;
Schmid and Verger, 1998; Rao et al., 1998; Gandhi et al., 2000; Koeller and Wong, 2001;
Sharma et al., 2001; Bornscheuer, 2002; Gupta et al., 2002; Hari Krishna and Karanth, 2002a)
and books (Koskinen and Klibanov, 1996; Bornscheuer, 2000a; Bornscheuer and Kazlauskas,
1999; Faber, 2000; Liese et al., 2000; Patel, 2000; Drauz and Waldmann, 2002). A
representative example of industrial processes employing porcine pancreatic lipase (EC
3.1.1.3) is showed in Scheme 4.
2.4. Lyases (EC 4)
These enzymes catalyze the cleavage of C)C, C)O, C)N and a few other bonds in a
different fashion from hydrolysis, often leaving double bonds that may be subjected to further
reactions. Systematic denomination follows the pattern substrate group-lyase. The hyphen is
important to avoid any confusion, e.g., the term hydro-lyase should be used instead of
hydrolyase, which looks quite similar as hydrolase.
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Table 4
Some of the important industrial processes using hydrolasesa
Industry
BASF
Products (application)
Chiral amines and
alcohols (intermediates
for pharmaceuticals
and pesticides, chiral
synthons in asymmetric
synthesis)
Bristol-Myers-Squibb (3R,4S)-Azetidinone
(BMS)
acetate (used in the
synthesis of paclitaxel,
Taxol)
Bristol-Myers-Squibb Hydroxy methyl glutaryl
coenzyme A (HMG-CoA)
reductase inhibitor
(anticholesterol drug)
Chiroscience
Intermediate for the
anti-HIV agent carbovir,
hypocholesteremic
reagents and antifungal
agent brefeldin A
DSM; Tanabe Seiyaku Generation of intermediate
for synthesis of Diltiazem
(antihypertensive drug)
Sepracor
S-Ibuprofen (nonsteroidal
anti-inflammatory
drug, NSAID, candidate)
Uniquema
Isopropyl palmitate and
myristate (used in
production of soaps,
creams, lubricants)
Central del Latte;
Milk lactose hydrolysis to
Sumitomo; others
galactose and glucose
(much sweeter milk,
suitable for lactose
intolerant people)
Toyo Jozo-Asahi
7-Aminocephalosporanic
Chemical; Hoechst acid (intermediate for
semisynthetic penicillins
and cephalosporins)
Various
Glucose from starch
hydrolysis (feed stock
for high fructose
corn syrup)
Yamasa
Ribavirin (antiviral drug)
Enzyme and process
Medium
Remarkb
Burkholderia plantarii
lipase (immobilized);
hydrolysis
MTBE-ethyl
methoxy
acetate
E > 500
(>100 ton)
P. cepacia lipase
(immobilized); hydrolysis
Aqueous
ee>99%
(multi kg)
P. cepacia lipase
(immobilized); acetylation
Toluene
ee = 98%
(multi kg)
P. fluorescens lipase
(soluble); hydrolysis
Aqueous
ee>92%
(multi kg)
Serratia marescens lipase
(immobilized); hydrolysis
Aqueous/toluene
ee = 99%
(multi kg)
Multiphase
C. cylindracea lipase
(hollow fiber membrane);
hydrolysis
C. antarctica lipase (Novo); 2-Propanol
esterification
Milk
b-Galactosidase
(SNAMprogetti and others,
immobilized); hydrolysis
ee = 96%
(multi kg)
99% yield
70 – 80%
Pseudomonas/E. coli
glutaryl amidase
(immobilized); hydrolysis
Aqueous
95% yield
B. licheniformis/A.
niger a-amylase (soluble)
Aqueous
>95%
conversion
Erwinia carotovora
Aqueous
phosphorylase/nucleosidase
(soluble); hydrolysis/group
transfer
–
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247
Table 4 (continued)
Industry
Products (application)
Enzyme and process
Subtilisin carlsberg
(S)-Phenylalanine
(Sigma, soluble);
(intermediate in the
peptide hydrolysis
synthesis of aspartame)
Hoffmann La-Roche Various chiral
Subtilisin carlsberg;
intermediates
hydrolysis
Comamonas acidovorans
Lonza
Intermediate in the
amidase expressed in
synthesis of cilastatin
E. coli (whole cells);
(dehydropeptidase
amide hydrolysis
inhibitor)
Klebsiella terrigena
Lonza
Piperazine-2-carboxylic
amidase (whole cells);
acid (pharmaceutical
amide hydrolysis
intermediate, e.g.,
orally active HIV
protease inhibitor
crixivan from Merck
or precursor for
numerous bioactive
compounds)
a
Data from Liese et al. (2000); Bornscheuer (2000b).
b
E is the E-value (enantioselectivity), ee is enantiomeric excess.
Coca-Cola
Medium
Remarkb
Aqueous two-phase ee = 95%
Aqueous
ee>99%
Aqueous buffer
ee>98%
Aqueous
ee>99%
Lyases have gained significant industrial attention as the predominant and natural bondbreaking (lyase) reactions can be reversed (bond formation, i.e., lyase acting as synthetase)
under nonnatural conditions (i.e., high reactant concentrations), leading to the construction of
new bonds of commercial importance. Often chiral centers are generated during bond
formation. Using protein engineering techniques, the substrate range of these enzymes is
currently being expanded. An industrial example employing phenylalanine ammonia-lyase
(EC 4.1.99.2) is presented in Scheme 5.
2.5. Isomerases (EC 5)
This class represents a small number of enzymes that catalyze geometric or structural
changes within one single molecule and make it possible to employ cheaper substrates to
Scheme 4. Enzymatic transformation catalyzed by a hydrolase.
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Scheme 5. Enzymatic transformation catalyzed by a lyase.
obtain high-value products. Depending on the type of isomerism, these enzymes may be
termed as epimerases, racemases, cis–trans isomerases, tautomerases or mutases. Racemases
are particularly important in kinetic resolutions. The most renowned candidate of this group
is certainly glucose isomerase (EC 5.3.1.5), whose industrial application is depicted in
Scheme 6.
2.6. Ligases (EC 6)
These catalyze a bond formation between two molecules, coupled with hydrolysis of a
pyrophosphate bond in ATP or similar triphosphate. The bonds formed are C)O, C)S and
C)N. The systematic names are written as X:Y ligase. No industrial process is carried out
using ligases at a kilogram scale, but they play a significant role in nature (in ribosomal
peptide synthesis) and also in repairing DNA fragments and in genetic engineering (e.g.,
DNA ligases catalyze C)O bond formation in DNA synthesis).
Extensive information on the potential of different classes of enzymes in organic synthesis
is provided in various books (Koskinen and Klibanov, 1996; Faber, 2000; Liese et al., 2000;
Drauz and Waldmann, 2002).
Scheme 6. Enzymatic transformation catalyzed by an isomerase.
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3. Early developments
3.1. A fascinating history
Enzymes have been in use for thousands of years before their nature was actually
understood. No one really knows when calf stomach was first used as a catalyst for the
first time in the manufacture of cheese. As early as 1783, Spallanzani demonstrated that
gastric juice could digest meat in vitro (Schwan called the active substance responsible for
this as pepsin in 1836 and Berzelius described a ‘catalyst’ as a substance which can breathe
life into slumbering chemical reactions in 1835) (Gutfreund, 1976).
Kirchhoff, in 1814, observed sugar production from starch by malted barley. The active
principle of malt was called diastase and its application was described by Payen and Persoz in
1833. Dubonfout observed invertase activity in 1846. Further to these, Kühne suggested, in
1876, that such nonorganized ferments should be called ‘‘enzymes.’’ The terms ‘‘organized
ferment’’ (e.g., cell-free yeast extract) and ‘‘unorganized ferment’’ (e.g., gastric juice secreted
by cells) are no longer used. Kühne also presented some interesting results from his studies
with trypsin. In 1893, Ostwald defined ‘‘catalyst’’ and classified enzymes as catalysts.
In 1894, Emil Fischer observed that the enzyme called ‘emulsin’ catalyzes the hydrolysis
of b-methyl-D-glucoside, while ‘maltase’ is active towards a-methyl-D-glucoside and formulated his ‘‘lock-and-key’’ theory of enzyme specificity. In 1897, Büchner demonstrated the
conversion of glucose to ethanol using cell-free extract from yeast. Warburg carried out
preparative separation of L-leucine from a racemic mixture by hydrolyzing the propyl ester
with liver extracts in 1906. Rosenberg used D-oxynirerilase from almonds as catalyst for the
synthesis of optically active cyanohydrins in 1908.
In the area of nonaqueous biocatalysis, Hill (1898) was the first to observe that the
biocatalysis of hydrolytic enzymes is reversible. Pottevin (1906) demonstrated that crude
pancreatic lipase could synthesize methyl oleate from methanol and oleic acid in a largely
organic reaction mixture. Bourquelot, Bridel and Verdon described glucoside synthesis in the
presence of high concentration of ethanol or acetone between 1911 and 1913 (Bourquelot and
Bridel, 1913). Sym (1936) improved the enzymatic ester synthesis using pancreatic lipase in
presence of benzene.
Michaelis and Menton (1913) published a theoretical consideration of kinetics of
enzymatic catalysis, which led to the development of the so-called M–M equation. In
1916, Nelson and Griffin demonstrated immobilization of invertase on charcoal with activity
retention. By 1920, about a dozen enzymes were known, but none of them had been isolated.
In 1926, James Sumner crystallized urease from jack bean (Canavalia ensiformis) and
demonstrated that it is a simple protein, which was confirmed by Northrop. By the late 1940s,
many enzymes were available in pure form, but there was still no evidence that proteins
possessed unique amino acid sequences. In 1953, Sanger established the first primary
sequence of a protein (insulin), proving the chemical identity of proteins. Lysozyme was
the first enzyme whose 3-D structure was defined in 1967 with the help of X-ray
crystallography (Phillips, 1967). Further to this, Gutte and Merrifield (1969) synthesized
whole sequence of ribonuclease A in 11,931 steps on a laboratory scale by organic chemical
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methods. Currently, about 4000 enzymes have been catalogued and several hundred of these
can be obtained commercially.
While the first benefit for the industry from the microbiological development had come
very early, enzyme catalysis hardly influenced the industry at that time (Moo-Young et al.,
1985; Davies et al., 1989; Roberts et al., 1995). The commercial use of extracellular microbial
enzymes started in the West around 1890, stimulated by a Japanese entrepreneur named
Takamine, who produced enzymes in the USA based on Japanese technology. The principal
product was ‘Taka-diastase’, which was a mixture of amylolytic and proteolytic enzymes
obtained from Aspergillus oryzae. Röhm (Darmstadt, Germany) applied pancreatic enzymes
in the leather processing for bating of hides in 1908, and in the cleaning of laundry between
1913 and 1915. In France, Boidin and Effront developed bacterial enzymes in 1913 and
found that Bacillus subtilis produced a thermostable a-amylase when grown in still cultures
on a liquid medium prepared by extraction of malt or grain. In 1960, Novo Nordisk
(Bagsvaerd, Denmark) produced protease on a large-scale by cultivating B. licheniformis
in submerged culture.
Invertase was probably the first immobilized enzyme to be used commercially for the
production of Golden Syrup by Tate and Lyle (Decatur, IL, USA) during World War II,
because the preferred reagent, sulfuric acid, was unavailable at that time (Cheetham,
1995). Industrial processes for L-amino acid production based on the batch use of
soluble aminoacylase were already in use in 1954. However, like many batch processes
with soluble enzymes, they had their disadvantages such as high labor costs, complicated product separation, low yields, high enzyme costs and difficulty in recycling the
enzyme.
During the mid-1960s, Tanabe Seiyaku (Tokyo, Japan) tried to overcome these problems
by using immobilized aminoacylases, and eventually produced L-methionine, in 1969, by
aminoacylase immobilized on DEAE-Sephadex in a packed-bed reactor. This became the first
large-scale use of an immobilized enzyme (Trevan, 1980). In 1980, Degussa (Düsseldorf,
Germany) developed a membrane reactor system with native enzymes in homogeneous
solution for the large-scale production of enantiomerically pure L-amino acids (Bommarius et
al., 1992).
Enzymatic isomerization of glucose to fructose represents the largest use of immobilized
enzyme in the manufacture of fine chemicals. High-fructose corn syrup has grown to become
a large-volume biotransformation product. While sucrose is sweet, fructose is about 1.5 times
sweeter and consequently in high demand as a sweetener. However, the food industry took a
long time to become acquainted with the potential of glucose isomerase. The Japanese were
the first to employ soluble glucose isomerase to produce high-quality fructose syrups in 1966.
In 1967, Clinton Corn Processing (Clinton, IA, USA) manufactured enzymatically produced
fructose corn syrup and started, in 1974, the commercial production of fructose syrups using
glucose isomerase immobilized on a cellulose ion-exchange polymers. In 1976, Kato Kagaku
(Kohwa, Nagoya, Japan) was first to manufacture these syrups in a continuous process as
opposed to a batch process and in 1984, to isolate crystalline fructose. The glucose isomerase,
Sweetzyme T (which has a long life because of immobilization), produced by Novo Nordisk
is now largely used in the starch processing industry. Central del Latte (Milan, Italy) was the
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251
first company to commercially hydrolyze milk lactose using SNAMprogetti technology with
immobilized lactase.
Penicillin G, discovered accidentally in 1929 by Fleming in Penicillium notatum,
revolutionized the practice of medicine. Currently, thousands of semisynthetic b-lactam
antibiotics are in production. Most of these are prepared from 6-aminopenicillanic acid (6APA), 7-aminocephalosporanic acid (7-ACA) and 7-amino-des-acetoxycephalosporanic acid
(7-ADCA). Presently, 6-APA is mainly produced by chemical or enzymatic (using penicillin
amidase) deacylation of penicillin G or penicillin V. This process, used since 1973, is the
best-known use of an immobilized enzyme in the pharmaceutical industry. In 1979,
enzymatic production of 7-ACA was realized when Toyo Jozo (now acquired by Asahi
Kasei, Tokyo, Japan), in collaboration with Asahi Chemical Industry (Tokyo, Japan),
developed a chemo-enzymatic two-step process starting from cephalosporin C (Scheme 7).
About 90 tons/annum of 7-ACA are produced using this technology.
Since the 1980s, many industries and research laboratories are applying genetic engineering techniques to improve enzyme production and to alter enzyme properties through
protein engineering and evolutionary design.
3.2. Major technological advances
Five major technological advances are believed to have significantly influenced industry in
adopting enzymatic biotransformations (Lilly, 1994): (i) the development of large-scale
downstream processing techniques for the release of intracellular enzymes from the microorganisms; (ii) improved screening methods for novel biocatalysts (Kieslich et al., 1998;
Demirjan et al., 1999; Miller, 2000; Asano, 2002; Ornstein, 2002); (iii) the development of
immobilized enzymes; (iv) biocatalysis in organic media; and most recently (v) recombinantDNA (r-DNA) technology to produce enzymes at a reasonable cost.
Buckland et al. (1975) examined the use of high proportions of organic solvents to increase
the solubility of reactants in production of cholestenone using isolated cholesterol oxidase.
There seems to be no agreement as to why the biocatalysis in organic media did not take off
earlier (Halling and Kvittingen, 1999; Klibanov, 2000; Kvittingen, 2000). Perhaps the
traditional belief that most enzymes are incompatible with most organic syntheses in
nonaqueous media posed a psychological hurdle. Also, until recently, there was no demand
for enantiopure compounds, and hence, no need to use enzymes. The establishment of
industrial processes (Coleman and Macrae, 1977; Matsuo et al., 1981), and the realization that
most enzymes can function well in organic solvents (Zaks and Klibanov, 1984, 1985, 1986,
1988), have heightened interest in the use of enzymes. Also, the need for enantiomerically
pure drugs is driving the demand for enzymatic processes. This combined with the discovery
of striking new properties of enzymes in organic solvents has led to establishment of organic
phase enzyme processes in industry (Bornscheuer, 2000b; Liese et al., 2000).
Generally, microorganisms isolated from nature produce the desired enzymes at levels that
are too low to allow a cost-effective process. Consequently, a modification of the organism is
desirable for enhancing enzyme production. For this, three approaches are followed for strain
improvement at present. The first one—directed evolution—involves improvement by
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Scheme 7. Chemo-enzymatic two-step process for the production of 7-amino cephalosporanic acid (7-ACA) from
cephalosporin C.
mutation of the gene concerned and selection or screening (Arnold and Moore, 1997;
Bornscheuer et al., 2002). The second method is hybridization, which involves modification
of the cellular genetic information by transferring of DNA from another strain. The third
method is r-DNA technology, whereby genetic information from one strain is manipulated in
vitro and then inserted into the same or another strain.
S. Hari Krishna / Biotechnology Advances 20 (2002) 239–267
253
Random mutagenesis using UV light or chemical mutagens is a well-established general
tool for strain improvement, but the excitement of directed evolution stems from the ability to
target a single gene or region of a gene for achieving the desired effect. Directed evolution
(also called molecular evolution or in vitro evolution) involves using PCR, expressing the
protein and then selecting or screening for strains with improved properties. The DNA
shuffling technique (Stemmer, 1994a,b; Stemmer et al., 1995) places the directed evolution
approach apart from earlier random mutagenesis and screening efforts.
The r-DNA technology has dramatically changed the enzyme production scene. Protein
engineering combined with recombinant expression systems allows one to produce a new
enzyme variant and to rapidly attain concentrations that make industrial production worthwhile. Many microbial enzyme genes have been cloned over the past few years, including
genes for important commercial enzymes. High-level expression has been achieved mainly in
Pichia pastoris, Saccharomyces cerevisiae and Escherichia coli. The secretion is facilitated
by a leader sequence fused to the expressed gene. As P. pastoris does not secrete significant
amounts of proteins into the medium naturally, the a-factor prepro-peptide from S. cerevisiae
is used to induce secretion. Genes will be under the control of strong methanol-inducible AOX
promoter (in case of P. pastoris) or galactose-inducible GAL10 promoter (in case of S.
cerevisiae). In contrast to S. cerevisiae, P. pastoris does not hyperglycosylate the proteins and
can provide up to 100-fold higher expression levels. For the expression of bacterial enzymes,
the genes are placed under the control of the strong temperature-inducible l phage promoter
PL or rhamnose-inducible promoter on the E. coli expression vector. The choice of an
appropriate expression system combined with optimal expression with genetic modifications
as well as suitable fermentation conditions allows the effective production of large amounts of
recombinant enzymes.
4. Recent trends
Enzymes occupy a unique position in synthetic chemistry due to their high selectivities and
rapid catalysis under ambient reaction conditions. Nevertheless, synthetic chemists have been
reluctant to employ enzymes as catalysts, because most organic compounds are waterinsoluble, and the water removal is tedious and expensive. The realization that enzymes can
retain and, in some cases, improve their high specificity in nearly anhydrous media, has
dramatically changed the prospects of employing enzymes in synthetic organic chemistry.
The problems that arise for most biotransformations are low solubility of reactants and
products, and limited stability of biocatalysts.
Carrying out reactions in an aqueous-organic two-phase system would be a solution to
overcome the first problem. This is not always possible due to the limited stability of enzymes
at liquid–liquid interface or in organic solvents. Hence, other approaches are necessary.
These include addition of complexing agents such as dimethylated cyclodextrins or adsorbing
materials like XAD-7 resins (Eli Lilly, Indianapolis, USA), use of membrane-stabilized
interface (Sepracor, Marlborough, MA, USA) and continuous extraction of reaction products
(Forschungszentrum-Jülich, Jülich, Germany).
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The catalyst’s stability can be increased using a variety of methods including the addition of
antioxidants (e.g., dithiothreitol), immobilization, cross-linking, separation from deactivating
reagents, variation of reaction conditions, and by genetic engineering (Burton et al., 2002).
4.1. Solvent systems
Solvent systems used as the reaction media for enzymatic catalysis may be categorized as:
(1) aqueous; (2) water: water-miscible (monophasic aqueous-organic system); (3) water:
water-immiscible (biphasic aqueous-organic system); (4) nonaqueous (monophasic organic
system); (5) anhydrous; (6) supercritical fluids; (7) reversed micelles; (8) solvent-free
systems; (9) gas phase; and (10) ionic liquids. More information can be found in the reviews
and books recommended above. A recent development is the use of ionic liquids for enzyme
catalysis (Madeira-Lau et al., 2000), to improve activity, stability and selectivity (Park and
Kazlauskas, 2002). The most common ionic liquids employed are tetrafluoroborate and
hexafluorophosphate.
4.2. Importance of water activity
Water is critical for enzymes and affects enzyme action in various ways: by influencing
enzyme structure via noncovalent bonding and disruption of hydrogen bonds; by facilitating
reagent diffusion; and by influencing the reaction equilibrium. Too low a water content
generally reduces enzyme activity. A high water content can also reduce reaction rates by
aggregating enzyme particles and causing diffusional limitations. The optimum amount of
water is often within a narrow range. To quantify the amount of water present in the reaction
mixture, the thermodynamic water activity (aw) is the preferred measure.
Optimal water activity is not only important to maintain the catalytic activity of an enzyme,
but also to obtain high reaction rates and yields, and stability of the biocatalyst. Completely
anhydrous solvents do not support enzymatic activity. Some water is always necessary for the
enzyme to retain its native structure responsible for catalysis. The amount of water required to
retain catalytic activity is enzyme dependent. a-Chymotrypsin needs only 50 molecules of
water per enzyme molecule to remain catalytically active (Zaks and Klibanov, 1986).
Enzymes like subtilisin and various lipases are similar in their requirement for trace
quantities of water (Zaks and Klibanov, 1988), while much more water is required for some
enzymes (e.g., polyphenol oxidase requires the presence of about 3.5 ! 107 molecules of
water) (Zaks and Klibanov, 1985). The water present in a biological system can be separated
into two physically distinct categories: the majority (>98%) serving as a true solvent (bulk
water) and a small portion being tightly bound to the enzyme’s surface (bound water). Bound
water is crucial for the enzyme structure and activity.
4.3. Stability aspects
Enzymes inactivate at high temperatures in aqueous media due to both partial unfolding
and covalent alterations in the primary structure (Ahern and Klibanov, 1985). Water is
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255
required for both these mechanisms and hence, the enzyme thermostability in nonaqueous
environments should be high mainly as a consequence of protein rigidity in these systems.
For instance, porcine pancreatic lipase remained stable at 100 !C for >12 h in nonaqueous
solvents (Zaks and Klibanov, 1984). Such stability is seldom possible with other approaches
like chemical cross-linking, immobilization or protein engineering.
Another reason for thermostability, apart from rigidity, is that a number of covalent
processes involved in irreversible or reversible inactivation of proteins such as deamidation,
peptide hydrolysis and cystein decomposition require water, but are extremely slow in
nonaqueous systems. Most of the early work had been restricted to relatively nonpolar
substrates. Stability studies involving polar and water-soluble substrates are also essential to
bring out a clear picture. Presently, enzymatic catalysis is also carried out in gas phase
(Lamare and Legoy, 1993), supercritical fluids (Kamat et al., 1995) and ionic liquids (Park
and Kazlauskas, 2002), for which enhanced thermostability of enzymes is important.
Despite a higher thermostability, the reaction rates of enzyme in nonaqueous systems are
low compared with the reaction rates in aqueous media (Klibanov, 1997). Thermostabilization of enzymes also results sometimes in stabilization towards other denaturing conditions.
Arnold (1990) suggested correlations between enhanced thermostability and stability in
nonaqueous systems. In this respect, proteins from extremophiles do not differ significantly
from their mesophilic counterparts (Haney et al., 1999; Eichler, 2001; Cavicchioli et al.,
2002). However, no general rule or strategy of stabilization has yet been framed, although
several attempts have been made to generalize (Fagain, 1995; Russell and Taylor, 1995;
Shoichet et al., 1995; Hendsch et al., 1996; Jaenicke, 2000; Jaenicke and Böhm, 1998; Kumar
et al., 2000; Lehmann and Wyss, 2001; Lehmann et al., 2002).
During synthetic reactions (e.g., esterification), complications arise because water is a
product. It is observed that, in addition to decreasing the equilibrium yield, accumulation of
water results in decreased enzyme activity. Moreover, accumulated water has also been
shown to adversely affect the long-term stability of the enzyme (Hari Krishna et al., 2001a).
The reduction in water content of the enzymes usually results in an increase of their half-lives.
4.4. Enzyme stabilization strategies
The approaches followed to stabilize enzymes are mainly two: (i) medium engineering and
(ii) biocatalyst engineering.
4.4.1. Medium engineering
Enzymes in nonaqueous systems can be active provided that the essential water layer
around them is not stripped off. Medium engineering in the context of biocatalysis in
nonaqueous media involves the modification of the immediate vicinity of the biocatalyst.
Nonpolar solvents are better than polar ones since the former provide a better microenvironment for the enzyme. If the enzyme’s microenvironment favors high substrate and low
product solubility, the reaction rates would be high.
The solvent effects may not be generalized too far. There are various exceptions of which
lipases are a particular case. For instance, porcine pancreatic lipase is active in anhydrous
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pyridine (Zaks and Klibanov, 1985), suggesting that it can retain the bound water even in a
water-miscible solvent. Since the natural environment for lipases is nonpolar, the ability to
bind essential water tightly might have evolved as a prerequisite condition for catalysis in
hydrophobic environments.
Several aspects have to be considered in choosing an appropriate solvent for a given
reaction. These include compatibility with the selected reaction (substrates and products),
inertness, a low density to minimize mass transfer limitations, and other properties that are
suitable (e.g., surface tension, toxicity, flammability, waste disposal and cost). Halling (1994,
2000) presented a detailed account of predictions that can be made to elucidate the influence
of solvent selection on the equilibrium. Reetz (2002a) reviewed various medium engineering
successes with particular reference to lipase catalysis.
4.4.2. Biocatalyst engineering
Immobilization and protein engineering are long known for improving biocatalyst
efficiency or stability of enzymes in aqueous media. Also, these methods have been used
to improve biocatalyst performance in organic solvents. Laane (1987) used the term
‘‘biocatalyst engineering’’ for these approaches.
Various developments have taken place in this area including the generation of active and
stable homogeneous (soluble) biocatalysts by the covalent or noncovalent modification of the
native enzyme. Covalent techniques are well described in the literature (e.g., attachment of
polyethylene glycol chains to enzymes), while noncovalent modifications are less common,
although they are able to provide highly active and soluble enzyme forms (Okahata and Mori,
1997). These developments have been well documented in various reviews (Khmelnitsky et
al., 1988; DeSantis and Jones, 1999a,b; Govardhan, 1999; Villeneuve et al., 2000).
Enzymes have been employed in nonaqueous systems in various states such as native
enzymes, suspended enzyme powder, solid enzyme adsorbed on support, polyethylene
glycol-modified enzymes soluble in organic solvents (Inada et al., 1986), enzyme entrapped
within a gel or microemulsion or reversed micelle (Bornscheuer et al., 1999a) and
immobilized enzyme. No general guidelines are available yet for choosing the most
appropriate form of the enzyme for a specific purpose.
Enzymes are frequently prepared from aqueous/buffer solution via lyophilization, which
results in undesirable changes in the protein’s secondary structure and about 40% of the active
sites may be denatured. The addition of specific small molecules (excipients) in the freezedrying stage often improves catalytic activity. This formed the basis of molecular imprinting,
which involves the complex formation between a macromolecule and low-molecular-weight
ligand in solution, followed by drying and washing with a selective solvent that removes the
ligand. The protein retains the ligand-induced conformation even after the removal of the
ligand (Russell and Klibanov, 1988).
Activation has been achieved by the addition of crown ethers (Engbersen et al., 1996; van
Unen et al., 2002), lyoprotectants (Dabulis and Klibanov, 1992), transition-state analogues
(Slade and Vulfson, 1998) and substrate or substrate mimics or competitive inhibitors
(Russell and Klibanov, 1988). Nonligand lyoprotectants (sorbitol, sugars and PEG) also
enhanced enzyme activity in organic solvents when present during lyophilization. However,
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257
addition of excipients to suspensions of native enzymes in organic solvents had no
appreciable effect, indicating that the interaction of the excipient with soluble enzyme is
essential to alleviate denaturation of enzymes during lyophilization (Klibanov, 2001).
Addition of surfactants or hydrophobic sol–gel materials before lyophilization enhanced
the lipase activity in organic solvents by up to 100-fold (Reetz, 2002b).
Addition of an inorganic salt has been shown to dramatically enhance the activity of
enzymes in organic solvents (Bedell et al., 1998). Ion pairing of biocatalysts in the presence
of very low concentrations of ionic surfactants resulted in remarkably active ion-paired
enzymes (e.g., subtilisin and a-chymotrypsin with >1000-fold higher activities over native
enzymes were generated). Ion-paired enzymes have also been incorporated into plastic
materials for preparing ‘‘biocatalytic plastics’’ (Wang et al., 1997).
Activated biocatalysts have already found application in the pharmaceutical industry. Saltactivated thermolysin (a bacterial protease) is used to selectively acrylate the 20-hydroxyl
group of taxol in tert-amyl alcohol. In a specific case, taxol was acylated with divinyladipate
to yield taxol 20-vinyladipate, which was used as an acyl donor in Candida antarctica lipasecatalyzed hydrolysis of the terminal vinyl ester to get taxol 20-adipic acid derivative that is
" 1700 times more water-soluble and can be used to design taxol prodrugs with increased
bioavailability (Schmid et al., 2001).
Roche (Mannheim, Germany) and Novo Nordisk market several adsorption-immobilized
enzymes. Altus Biologics (Cambridge, MA, USA) offers various cross-linked enzyme
crystals (CLECs) of different enzymes. Fluka (Buchs, Switzerland) supplies sol – gel
entrapped enzymes.
4.5. Protein engineering
Despite hard competition from chemical modification and related techniques, protein
engineering has become an increasingly important strategy for improving enzymes (Rubingh,
1997; Cedrone et al., 2000; Chen, 2001). The importance of protein engineering in industry
continues to grow with the expanding range of protein applications. Extremophilic proteins
isolated from organisms from extreme environments are emerging as an important source of
new backbones for engineering proteins to attain new properties. The current strategies of
protein engineering include rational design and directed evolution.
4.5.1. Rational design
This was the earliest approach to protein engineering and is still widely employed to
introduce desired characteristics into a target protein. The growing understanding of how to
engineer certain basic enzyme properties (e.g., stability, activity and surface properties) is
beginning to make rational design more efficient. Advances in rational design depend on the
progress made in structure determination, improved modeling protocols and significant new
insights into structure–function relationships (Fersht, 1999). Advances in modeling of free
energy perturbation methods and molecular dynamics calculations also influence this area.
Moult (1996) discussed the state-of-the-art in comparative modeling and ab initio protein
structure predictions.
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Two impressive cases of rational design were reported recently. In the first example,
enantioselectivity of C. antarctica lipase-catalyzed resolution of 1-chloro-2-octanol was
improved (E = 14 to 28) by a single amino acid exchange, Ser-47–Ala, as predicted by
molecular modeling (Rotticci et al., 2001). In the second case (Magnusson et al., 2001), a
transition-state stabilizing threonine near the active site was removed by site-directed
mutagenesis and the lost activity was restored by using chiral substrate (2-hydroxypropanoate) containing the missing functional –OH group, which resulted in improved
enantioselectivity (E = 1.6 to 22). Creating tailored enzymes with opposite enantiopreference
may also be possible with this approach.
Although use of modeling to predict the enantioselectivity is being used by numerous
research groups, much of the available data is empirical and is not easily interpreted at the
molecular level (Kazlauskas, 2000). This is largely due to ambiguity in identifying the
parameters responsible for enzyme–substrate interactions. In addition, properties like activity,
specificity and stability are controlled by multiple sites on protein. Substantial progress is
essential in establishing these features, which would aid application of rational design in a
truly directed manner (Ottosson et al., 2002). A special issue of Biochimica et Biophysica
Acta: Protein Science and Molecular Enzymology has been devoted to protein engineering
spanning the advances achieved with various enzymes (Dalboge and Borchert, 2000),
indicating the importance of this exciting area of research.
4.5.2. Directed evolution
While site-directed mutagenesis and rational protein design are widely practiced, an
alternate method—directed evolution—is gaining increased attention from academic and
industrial laboratories to modify and improve important biocatalysts (Stemmer et al., 1995;
Chirumamilla et al., 2001) and to achieve various other objectives (Patten et al., 1997;
Chartrain et al., 2000). This strategy combines random mutagenesis of target gene with
screening or selection for the desired property and is especially useful for cases like solvent
tolerance or thermostability where available theories are inadequate to make predictions. In
fact, earlier protein engineering studies were targeted to adapt proteins for nonnatural
environments (Arnold, 1988, 1990, 1993a,b; Chen and Arnold, 1991, 1993; Chen et al.,
1991).
The goals currently envisaged are to improve the enzyme’s activity, stability and
selectivity. Much of current research is directed towards increasing the enzyme stability.
Rational design, in this context, is inferior because the molecular basis for increased stability
is ill-defined. Thermostability is also difficult to improve rationally and hence, is a good
target for directed evolution. Random mutagenesis and screening have resulted in more
thermostable subtilisin and lipase (Shinkai et al., 1996). Contrary to these successes, a
rational approach to increase the thermostability of P. camembertii lipase by introducing a
disulfide link was a failure (Yamaguchi et al., 1996).
Numerous choices are available for creating DNA libraries: e.g., error-prone PCR,
combinatorial oligonucleotide mutagenesis, DNA shuffling, exon shuffling, random-priming
recombination, random chimeragenesis on transient templates (RACHITT), staggered extension process (StEP recombination), heteroduplex recombination, incremental truncation for
S. Hari Krishna / Biotechnology Advances 20 (2002) 239–267
259
Table 5
Selected examples for the directed evolution of biocatalysts
Biocatalyst
Evolution target (result)
P. aeruginosa lipase
Increased enantioselectivity (E = 1 to 26)
Staphylococcus hyicus
Altered substrate specificity,
and S. aureus lipases phospholipids versus short-chain
fatty esters (up to 12-fold increase)
P. fluorescens esterase
Increased selectivity (active mutants obtained)
B. subtilis p-nitrobenzyl Increased thermostability (14 !C increase in
esterase
Tm without decrease in original activity at
low temperatures)
p-Nitrobenzyl esterase
Activity in organic solvents (50 – 150-fold
activity in 25 – 30% DMF)
B. sphaericus protease
Increased activity at 10 !C (6-fold increase)
B. lentus subtilisin
Expression level of secreted enzyme (50% increase)
Various subtilisins
Overall improvement of various properties
(active chimaeras with increased activity
and stability)
Subtilisin BPN0
Increased activity at low temperatures
(2-fold increase in rate at 10 !C)
Subtilisin E
Enhanced thermostability
(significant increases reported)
b-Lactamase
Cephalosporinase
Carboxymethyl
cellulase
Galactosidase
Cytochrome P-450
Peroxidase (fungal)
Catalase I
3-Isopropylmalate
dehydrogenase
Lactate dehydrogenase
Aspartate
aminotransferase
Cre recombinase
DNA polymerase
Increased activity toward cefotaxime
(32,000-fold and 2383-fold increase)
Increased activity toward moxalactam
(up to 540-fold increase)
Increased activity
(up to 5-fold increase)
Activity switched to fucosidase
(66-fold increase in specific activity)
Increased activity toward naphthalene
without cofactors (up to 20-fold)
Increased temperature and oxidative
stability (>100-fold in both targets)
Increased peroxidase property
(2% to 58% increase)
Increased thermostability
(>3-fold activity at 70 !C)
Increased activity without cofactor
(70-fold increase)
Increased activity toward
b-branched amino and 2-oxo
acids and valine (>105-fold increase)
Altered specificity to recognize variants of
loxP recombination site (contribution to the
understanding of protein-DNA recognition)
Activity switched to RNA polymerase
(efficient new enzyme activity)
Reference
Reetz et al. (1997)
van Kampen and
Egmond (2000)
Bornscheuer et al. (1999b),
Henke and Bornscheuer (1999)
Giver et al. (1998)
Moore and Arnold (1996)
Wintrode et al. (2000)
Naki et al. (1998)
Ness et al. (1999)
Taguchi et al. (1999)
Zhao and Arnold (1999),
Zhao et al. (1998),
Miyazaki and Arnold (1999)
Stemmer (1994a);
Zaccolo and Gherardi (1999)
Crameri et al. (1998)
Kim et al. (2000)
Zhang et al. (1997)
Joo et al. (1999)
Cherry et al. (1999)
Matsuura et al. (1998)
Akanuma et al. (1998)
Allen and Holbrook (2000)
Yano et al. (1998),
Oue et al. (1999)
Santoro and Schultz (2002)
Xia et al. (2002)
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S. Hari Krishna / Biotechnology Advances 20 (2002) 239–267
the creation of hybrids (ITCHY), recombined extension on truncated templates (RETT),
degenerate oligonucleotide gene shuffling (DOGS) and in vivo recombination.
The mutant genes obtained from directed evolution are transformed and expressed in a
suitable host. Enzyme libraries are selected and/or screened for a range of selection properties
(e.g., substrate range, stability in reactions or in nonaqueous solvents, thermostability). Genes
obtained in the first round may be used as templates for the subsequent evolution cycles. For
sexual evolution, a pool of homologous genes (or genes generated using asexual methods) is
partially digested with DNAse-I and recombined by PCR. Alternatively, homologous
recombination can also be achieved in vivo based on the transformation of S. cerevisiae
with a linearized plasmid and target gene variants yielding a circular plasmid.
Whether mutations should be targeted to specific regions or distributed throughout the
gene and methods of identifying improved variants (screening vs. selection) are the issues of
debate. There is no agreement between different research groups as to a single best approach
and there may never be one, since they all address different needs and situations. Many
reviews on various aspects of directed evolution citing various examples have appeared
(Arnold, 2001; Benhar, 2001; Bornscheuer and Pohl, 2001; Brakmann, 2001; Brakmann and
Johnsson, 2002; Jaeger et al., 2001; Powell et al., 2001; Reetz, 2001). Table 5 illustrates some
examples of important biocatalysts that have been improved using directed evolution. Several
developments have been reported for the high-throughput screening of enzyme libraries
created by directed evolution (Baumann et al., 2001; Reetz, 2002b). Also, several algorithms
and computational models have been proposed to improve the efficiency of directed evolution
(Bolon et al., 2002).
Most protein design efforts have focused on using only a single method. Rational design is
well suited to optimize direct interactions, but is not suitable for identifying distant mutations.
Directed evolution, although optimizing activity, may not be suitable to introduce completely
novel activity. Therefore, it is beneficial to combine the powers of individual techniques.
Some recent work has attempted to combine rational design and directed evolution to
improve biocatalysts (Bornscheuer and Pohl, 2001; Bolon et al., 2002).
Recently, directed evolution has been also applied for metabolic pathway engineering
(Schmidt-Dannert, 2001; Zhao et al., 2002). The ability of directed evolution to simultaneously transfer a large number of genes has been utilized to engineer new products. This
method forms the basis of the emerging area of ‘‘combinatorial biocatalysis,’’ which employs
iterative reactions catalyzed by isolated enzymes or whole cells, in a natural or unnatural
environment, on substrates in solution or on a solid phase and harnesses the natural diversity
of enzymatic reactions to generate libraries of organic compounds. Lipases and proteases
have been employed in nonaqueous media to generate a library of acylated flavonoid
derivatives and dibenzyl-1,2-phenylenedioxy diacetate derivatives (Rich et al., 2002).
5. Concluding remarks
Enzymatic reactions are no longer restricted to aqueous solutions. Chemists can now take
advantage of enzyme specificities under mild conditions to catalyze reactions that were earlier
S. Hari Krishna / Biotechnology Advances 20 (2002) 239–267
261
limited to using chemical catalysis. However, biocatalysis is still not viewed as a first-line
alternative, but only as a last resort when other possible synthetic schemes fail. Nevertheless,
there are numerous examples of industrial biocatalytic processes. Protein engineering is
emerging as a major thrust area in improving enzyme activities and in finding novel
applications. A skillfully selected combination of chemical and biocatalysis is probably the
way forward for many commercial syntheses. The global market for specialty enzymes was
about US$1.5 billion in 1998 and is increasing with a predicted 5–10% growth per annum.
Continuing improvements in biocatalysts and a better understanding of biocatalysis are
expected to greatly influence the production of fine chemicals.
Acknowledgements
The author is grateful to his mentors Prof. Dr. N.G. Karanth (Fermentation Technology and
Bioengineering, Central Food Technological Research Institute, Mysore, India) and Prof. Dr.
Uwe T. Bornscheuer (Technische Chemie und Biotechnologie, Universität Greifswald,
Greifswald, Germany) for their encouragement and suggestions. The Alexander von
Humboldt Foundation (Bonn, Germany) is thanked for the award of Humboldt Research
Fellowship allowing the author to pursue research in Germany.
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