Industrial microbial enzymes: their discovery by screening and use ...

Industrial microbial enzymes: their discovery by screening and
use in large-scale production of useful chemicals in Japan
Jun Ogawa and Sakayu Shimizu*
The application of microbial enzymes to large-scale organic
synthesis is currently attracting much attention, and has been
uniquely developed especially in Japan. The discovery of new
microbial enzymes through extensive and persistent screening
has brought about many new and simple routes for synthetic
processes. The application of these enzymes in so-called
‘hybrid processes’ of enzymatic and chemical reactions,
provide one possible way to solve environmental problems.
Addresses
Division of Applied Life Sciences, Graduate School of Agriculture,
Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku,
Kyoto 606-8502, Japan
*e-mail: sim@kais.kyoto-u.ac.jp
Current Opinion in Biotechnology 2002, 13:367–375
0958-1669/02/$ — see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
DOI 10.1016/S0958-1669(02)00331-2
Abbreviations
CHBE 4-chloro-3-hydroxybutanoate ester
DHase dihydrocoumarin hydrolase
DOPA 3,4-dihydroxyphenylalanine
e.e. enantiomer excess
EPA 5,8,11,14,17-cis-eicosapentaenoic acid
GMP 5′-guanylic acid
IMP 5′-inosinic acid
PUFA polyunsaturated fatty acid
TPL tyrosine phenol-lyase
Introduction
In recent years, the most significant development in the
field of synthetic chemistry has been the application of
biological systems to chemical reactions. Reactions catalyzed
by enzymes or enzyme systems display far greater specificities
than more conventional forms of organic reactions
[1,2]. Accordingly, the industrial use of enzymes has been
developed rapidly [3]; however, in many cases, the substrates
in industrial processes are artificial compounds, and
enzymes known to catalyze suitable reactions for such
processes are still unknown. Therefore, screening for novel
enzymes that are capable of catalyzing new reactions is
constantly needed. One of the most efficient and successful
means of finding new enzymes is to screen large numbers
of microorganisms, because of their characteristic diversity
and versatility [4,5 • ].
Japan locates in the temperate and humid climate zone
and has characteristic changes of the seasons. It has supported
microbial diversity which, for example, sometimes
forces troublesome care in controlling microflora during
traditional fermentations such as rice wine ‘sake’ brewing.
As a result, sophisticated systems for the manipulation of
367
microorganisms were established in this country. Besides
this, the microbial diversity also brought many successes in
developing unique technologies through screening for new
microbial functions. A historical milestone was the discovery
of glutamate fermentation [6]. Following this discovery, a
variety of unique processes using newly screened microorganisms
or their enzymes as biocatalysts have been
invented [7 •]. The hybrid use of enzymes as biocatalysts
together with chemical syntheses is a unique field of
research in Japan. The discovery of novel enzymes with
the required activity and specificity by screening is key to
the establishment of such hybrid processes. Table 1 summarizes
several unique processes recently established in
Japan that use microbial enzymes and which are of great
industrial importance. This article reviews some current
successful industrial applications of microbial enzymes
that were obtained through screening from nature.
Enzymes for amino acid production
Since the development of glutamate fermentation,
research into microbial amino acid production has become
one of the main areas of interest in Japanese microbial
biotechnology [8]. Two major developments have been
made: one is a fermentation process that uses amino-acidoverproducing
mutants and the other is a hybrid process of
enzymatic and chemical reactions. The latter process is
useful for the synthesis of artificial amino acids and recent
results are presented here.
Hydantoinase and carbamoylase for D-amino acid
production
For the industrial production of D-amino acids such as
D-p-hydroxyphenylglycine, a sidechain precursor for semisynthetic
penicillins and cephalosporins, a process consisting
of the chemical synthesis of hydantoin substrates, the
enzymatic stereospecific hydrolysis of hydantoins and the
enzymatic decarbamoylation was developed (Figure 1a)
[9]. This process was originally designed for the synthesis
of L-amino acids, but, in the course of screening, hydrolysis
of various hydantoin compounds was found to occur only
with the D-isomers [10]. This serendipitous outcome
resulted in the commercial production of D-p-hydroxyphenylglycine.
Recent progress was made with the
introduction of stable D-carbamoylase, which was obtained
from Agrobacterium through screening, in place of the
chemical decarbamoylation of N-carbamoyl-D-p-hydroxyphenylglycine.
A triple mutant enzyme created by random
mutagenesis (His57Tyr, Pro203Glu and Val236Ala) yielded
19°C higher heat stability than the wild-type enzyme
[11 •]. This recombinant mutant enzyme was immobilized
and used for the large-scale production of D-p-hydroxyphenylglycine
(~2000 tons/year) with the simultaneous use

368 Protein technologies and commercial enzymes
Table 1
Recent industrial applications of microbial enzymes in Japan.
Item Product Year Organization Process
Amino acids D-p-Hydroxyphenylglycine 1979/1995 Kyoto University and Kaneka Corporation Enzymatic (hydantoinase)
(carbamoylase)
Aspartate 1984 Ajinomoto Co., Inc. Membrane bioreactor
of immobilized D-hydantoinase. This latter step has been
used since 1995 by Kaneka Corporation.
Hydroxylase for hydroxy-L-proline production
Hydroxyproline is a useful chiral substrate for the
chemical synthesis of pharmaceuticals. In particular,
trans-4-hydroxy-L-proline is an important precursor of
antiphlogistics, carbapenem antibiotics, and angiotensinconverting-enzyme
inhibitors. The successful stereoselective
and regioselective hydroxylation of L-proline to
yield hydroxy-L-proline isomers was achieved following the
discovery of two specific proline hydroxylases, 4-hydroxylase
and 3-hydroxylase, in Dactylosporangium sp. RH1 and
Streptomyces sp. TH1, respectively. The enzymes were identified
by extensive screening using a sensitive assay method
capable of separating the eight regioisomers and stereoisomers
of hydroxyproline [12–14]. The enzyme 4-hydroxylase
specifically produces trans-4-hydroxy-L-proline, whereas the
1986 Mitsubishi Chemical Co., Ltd Membrane bioreactor
DOPA 1994 Kyoto University and Ajinomoto Co., Inc. Enzymatic
Hydroxyproline 1997 Kyowa Hakko Kogyo Co., Ltd Enzymatic
Sweetners Paratinose 1984 Shin Mitsui Sugar Co., Ltd Immobilized bioreactor
Aspartame 1987 Tosoh Corporation Enzymatic
Lactosucrose 1990 Hayashibara Co., Ltd Enzymatic
Galacto-oligosaccharide 1990 Nissin Sugar mfg. Co., Ltd Enzymatic
Maltotriose 1990 Nihon Shokuhin Kako Co., Ltd Immobilized bioreactor
Engineered stevia sweetner 1993 Toho Rayon Co., Ltd Enzymatic
Theande-oligosaccharide 1994 Asahi Chemical Industry Co., Ltd Immobilized bioreactor
Trehalose 1995 Hayashibara Co., Ltd Enzymatic
Nigero-oligosaccharide 1998 Nihon Shokuhin Kako Co., Ltd, Kirin Brewery Co., Ltd Enzymatic
and Takeda Food Products, Ltd
Oils Physiologically functional oils 1989 Fuji Oil Co., Ltd Immobilized bioreactor
1990 Kao Corporation Enzymatic
1998 The Nissin Oil Mills, Ltd Enzymatic
Polyunsaturated fatty acids 1998 Kyoto University and Suntory, Ltd Enzymatic
Vitamins Stabilized vitamin C 1990 Hayashibara Co., Ltd Enzymatic
Nicotinamide 1998 Kyoto University and Lonza Group, Ltd Enzymatic
Vitamin C phosphate 1999 Kyowa Hakko Kogyo Co., Ltd Enzymatic
Pantothenate intermediate 1999 Kyoto University and Fuji Chemical Industries, Ltd Enzymatic
Chemicals Acrylamide 1988 Kyoto University and Mitsubishi Rayon Co., Ltd Immobilized bioreactor
Chiral epoxides 1985 Japan Energy Corporation and Canon, Inc. Enzymatic
Pharma Herbesser intermediate 1992 Tanabe Seiyaku Co., Ltd Membrane bioreactor
intermediates
Chiral alcohols 2000 Kyoto University and Kaneka Corporation Enzymatic
Others Casein phosphopeptide 1988 Meiji Seika Kaisha, Ltd Enzymatic
Hypoallergenic rice 1991 Tokyo University and Shiseido Co., Ltd Enzymatic
Hypoallergenic protein 1991 Meiji Milk Product Co., Ltd Enzymatic
3-hydroxylase produces cis-3-hydroxy-L-proline (Figure 1b).
The genes encoding these proline hydroxylases were
cloned in Escherichia coli. The resultant transformants
expressed 1600 times more 4-hydroxylase activity and 1000
times more 3-hydroxylase activity than the original strains.
2-Oxoglutarate, one of the substrates of these hydroxylation
reactions, is supplied from glucose in the reaction medium via
the Embden–Meyerhof pathway and tricarboxylic acid
cycle in E. coli and the product succinate is recycled [15 • ].
Using the E. coli strain for L-proline fermentation
as the host for the hydroxylase, the large-scale transformation
of hydroxy-L-proline from glucose became possible. Kyowa
Hakko Kogyo Co. started production of trans-4-hydroxy-
L-proline using this method in 1997.
Tyrosine phenol-lyase for L-DOPA production
Tyrosine phenol-lyase (TPL) is a pyridoxal 5′-phosphatedependent
multifunctional enzyme that catalyzes the

Figure 1
(a)
(b)
(c)
(d)
O
HN
O
Alkali
racemization
O
HO
HN
NH
N
H
HO
HO
NH
O
OH –
COOH
+
H2O
COOH
H2O
OH HO CH
NHCONH2 HO
D-Hydantoinase
D-Carbamoylase
reversible transformation of tyrosine into phenol, pyruvate
and ammonia. The reverse reaction is available to produce
L-3,4-dihydroxyphenylalanine (L-DOPA), a medicine for
Parkinson’s disease, using pyrocatecol instead of phenol
(Figure 1c). Erwinia herbicola was selected as the most
favorable strain as the source of this enzyme. Optimization
of culture and reaction conditions for L-DOPA production
with E. herbicola cells as the catalysts resulted in the accumulation
of 110 g/l L-DOPA [16]. The enzymatic L-DOPA
production method by E. herbicola TPL was first used in
1993 by Ajinomoto Co., and produces about half of the
worldwide L-DOPA demand of around 250 tons/year.
Recent progress has been made with the construction of a
constitutive mutant of TPL production. A TyrR box, an
OH
HO CH2
O
H H
OH OH
CO2
Succinate
2-Oxoglutarate
O2
Proline-4-hydroxylase
CH 3COCOOH
Base
+
PPi
+ NH 3
N
H
COOH
Tyrosine phenol-lyase
Acid phosphatase/
phosphotransferase
Industrial microbial enzymes Ogawa and Shimizu 369
2-Oxoglutarate
O2
Proline-3-hydroxylase
HO
HO
HO
O
P O
OH
CH2
O
H H
OH OH
Base
CO2
Succinate
COOH
CH CO2
NH2
+ + NH 3
COOH
operator-like region controlled by the binding of TyrR
protein, was found upstream of the TPL gene. The introduction
of a random mutation into TyrR resulted in
overexpression of TPL even under normal culture conditions
in the absence of an essential inducer, L-tyrosine. A
mutation of Val67Ala in the mutant TyrR was found to be
responsible for this TPL overexpression [17 • ].
Enzymes for nucleotide production
As well as amino acid production, microbial nucleotide
production is another key area of interest in Japanese
microbial biotechnology. Nowadays, two processes are
under operation by Kyowa Hakko Kogyo Company for the
production of 5′-guanylic acid (GMP) and 5′-inosinic acid
N
H
OH
COOH
H
+ H2O
NH2 +
Pi
Current Opinion in Biotechnology
Enzymatic production of (a) D-p-hydroxyphenylglycine by D-hydantoinase and D-carbamoylase, (b) hydroxy-L-proline by proline hydroxylase,
(c) L-3,4-dihydroxyphenylalanine by TPL and (d) nucleoside-5′-monophosphate by acid phosphatase/phosphotransferase.

370 Protein technologies and commercial enzymes
Figure 2
(a)
(b)
H2NH2C
(IMP). GMP production involves 5′-xanthylic acid
fermentation by Corynebacterium ammoniagenes coupled
with the energy-requiring amination reaction carried out
Figure 3
Cl
O
H 3CCOS
O
COBE
OH
O
COOR
Current Opinion in Biotechnology
NADPH
NADPH
COOCH3
H3CCOS
+ H3CCOS
COOH
D-β-Acetylthioisobutyrate
(250 g/L, molar yield to D-enantiomer 100%)
COO
Current Opinion in Biotechnology
Lactonase
H2O
Dihydrocoumarin hydrolase
H2O
CH2CH2COOCH3
NADP +
+
NADP
COOR
The stereospecific reduction of 4-chloro-3-oxobutanoate ester
(COBE) to (R)- and (S)-4-chloro-3-hydroxybutanoate ester (CHBE) by
AR1 from Sporobolomyces salmonicolor and S1 from Candida
magnoliae, respectively.
Cl
Cl
H 2NH 2C
(R)-CHBE
H OH
AR1 (S. salmonicolor)
S1 (C. magnoliae)
HO H
OH
H
COOH
OH
D-Pantoic acid
(S)-CHBE
CH3OH
COOR
COO
O
Dihydrocoumarin hydrolase
H2O
Cetraxate
(250 g/L, molar yield 100%)
+
H
OH
O
COOCH 3
CH3OH
CH 2CH 2COOH
Optical resolution of racemic pantolactone by
(a) lactonase from Fusarium oxysporum and
(b) linear ester hydrolysis by dihydrocoumarin
hydrolase. The upper reaction is
stereoselective ester hydrolysis of racemic
methyl β-acetylthioisobutyrate; the lower
reaction is regioselective ester hydrolysis of
cetraxate methylester.
by GMP synthase [18]. IMP production utilizes inosine
phosphorylation, which is carried out by recombinant
E. coli with high activity of guanosine/inosine kinase [19].
Recently, another interesting nucleoside phosphorylation
method, using a novel phosphatase found through
screening, was developed.
Pyrophosphate-nucleoside phosphotransferase for
5′-nucleotide production
Screening for the microbial ability to phosphorylate
inosine at the 5′ position to produce 5′-IMP using
pyrophosphate (PP i ) as the phosphate donor was carried
out by a research group of Ajinomoto Company (Figure
1d). They found that inosine phosphorylation activity
specific to the 5′ position is distributed among the bacteria
belonging to the family Enterobacteriacea. Morganella
morganii was selected as a potential source of the enzyme
[20]. The enzyme was then characterized as an acid
phosphatase/phosphotransferase, which phosphorylates
various nucleosides to the corresponding nucleoside-
5′-monophosphates using a variety of phosphoryl donors
[21]. A mutant enzyme with apparently elevated phosphotransferase
activity and decreased phosphatase activity was
obtained by random mutation. This improvement was
achieved with two mutations, Gly192Asp and Ile171Thr,

Figure 4
Cl
O
O
Cl
O
O
COOH
OH
O
COOEt
O
O
N
Me
i Pr O
O
Cl COOEt
O
Cl
O
O
O
F 3 C
NHCH 3
which resulted in a decreased K m of the enzyme for inosine
[22 • ]. With E. coli overproducing this mutated enzyme,
101 g/l 5′-IMP were synthesized from inosine and PP i in an
88% molar yield.
Enzymes for chiral technology
The need for optically active drugs has increased owing to the
efficacy of these drugs and the market pressure for safe chemical
compounds. Therefore, optically active starting materials
have also been increasingly needed in pharmaceutical and
agrochemical fields. A biological method is often adopted for
the synthesis of optically active compounds, because enzymatic
catalysts express high stereoselectivity [23]. Microbial
enzymes obtained through screening also have important
roles in this organic synthesis field, chiral technology. Here,
such examples of hydrolytic enzymes and cofactor-dependent
enzymes are introduced.
O
CONH
OH
O
CN
CONH
OH O
O
O
O
COOEt Prochiral carbonyl compounds
O
COOEt
O
O
O
N
H
O
O
COOEt
O
Cofactor
regeneration
system
O
Carbonyl
reductase
Industrial microbial enzymes Ogawa and Shimizu 371
O
Cl
Cl
OH
O
iPr
OH
OH
O
OH COOH
Cl
OH
OH
O
N
Me
O
OH
O
OH
N
H
OH
O
iPr
OH
O
NHCH
3
OH
O
H
OH
Lactonase for large-scale optical resolution
We recently reported the discovery of two lactone-ringopening
enzymes, lactonases, with unique properties
[24,25]. The enzyme from the fungus Fusarium oxysporum
catalyzes the reversible hydrolysis of various aldonate lactones
and butyrolactones. The hydrolysis proceeds
stereospecifically by recognizing the configuration at the
2-position carbon; only the lactones carrying a downward
hydroxyl group, when drawn by Haworth projection, at this
position are hydrolyzed. Thus, the enzyme is useful for the
optical resolution of racemic lactones. For example, a
racemic mixture of pantolactone can be well separated into
D-pantoic acid and L-pantolactone (Figure 2a), the D-enantiomer
is useful as the chiral building block for the
commercial production of a vitamin, D-pantothenic acid.
For practical use, the fungal mycelia containing the
enzyme were immobilized into calcium alginate gels.
O
OH
O
Cl COOEt
COOEt
Cl
OH
CONH
OH
OH
F
3
C
OH
Cl
OH
OH
O
COOEt
H
Cl
Cl
COOEt
OH
CONH
OH
COOEt
HO
HO
OH
Chiral alcohol
O
O
NHCH 3
CN
Current Opinion in Biotechnology
Bioreduction system using an E. coli transformant coexpressing carbonyl reductase and a cofactor regeneration system. By selecting the carbonyl
reductase gene corresponding to each substrate, the system is applicable to the production of various useful chiral alcohols.

372 Protein technologies and commercial enzymes
Figure 5
20:3n–6(∆5)
COOH
20:4n–3(∆5) COOH
20:2n–6
20:3n–3
COOH
COOH
18:1n–9 COOH
18:2n–6 COOH
When the gels were incubated in 350 g/L of DL-pantolactone
for 21 h at 30°C with automatic pH control (pH 7.0),
approximately 90% of the D-enantiomer was hydrolyzed
with high optical purity (93–98% enantiomer excess [e.e.]).
After 180 reaction cycles (i.e. 180 days), the gels retained
about 60% of their initial activity [26,27]. This process has
been used since 1999 for the commercial production of
D-pantolactone by Daiichi Fine Chemicals (~3000 tons/year
produced as calcium D-pantothenate).
Another good example showing the diversity and practical
potential of lactonases is dihydrocoumarin hydrolase
(DHase) from Acinetobacter calcoaceticus [25]. The enzyme
was found through screening for aromatic lactonehydrolyzing
enzymes. Although the DHase is highly
specific towards dihydrocoumarin, several other aromatic
lactones, such as 2-coumaranon and homogentisic acid
lactone, and linear esters are also hydrolyzed. The
specificity towards linear esters is very characteristic: the
enzyme is specific toward methyl esters; it recognizes the
configuration at the 2-position; and it hydrolyzes diesters
to monoesters. The recombinant E. coli cells overexpressing
this DHase were shown to be useful for the stereoselective
hydrolysis of methyl β-acetylthioisobutyrate and regioselective
hydrolysis of cetraxate methyl ester, as shown in
Figure 2b [28 • ].
Microbial bioreduction system for large-scale
production of chiral alcohols
In our laboratory we have developed a novel bioreduction
system for the asymmetric reduction of prochiral carbonyl
compounds to the corresponding chiral alcohols [29]. The
catalyst in the system is E. coli transformant cells coexpressing
the genes for an NAD(P)H-dependent carbonyl
reductase and glucose dehydrogenase (GDH), which acts
as a cofactor regenerator. The production of optically
18:0
COOH
∆9
∆12
18:3n–3 COOH
18:2n–9 COOH
18:3n–6 COOH
20:2n–9
20:3n–6
18:4n–3 COOH 20:4n–3
COOH
COOH
COOH
∆5
ALA
EL
∆6
EL
∆5
20:3n–9
20:4n–6
DGLA
ω3 ω3
20:5n–3
EPA
COOH
COOH
COOH
active 4-chloro-3-hydroxybutanoate esters (CHBEs) is a
typical successful example of this bioreduction system.
An aldehyde reductase (AR1) of Sporobolomyces salmonicolor
[30] and a carbonyl reductase (S1) of Candida magnoliae
[31] were found to catalyze NADPH-dependent stereospecific
reduction of 4-chloro-3-oxobutanoate esters
(COBE) to (R)-CHBE and (S)-CHBE, respectively
(Figure 3). These yeast enzymes have been successfully
applied to the E. coli coexpression system for practical
production [32,33 • ]. Some 300–350 g/l (R)- or (S)-CHBE of
high optical purity (92–100% e.e.) was stoichiometrically
obtained on incubation of the transformant cells in a
reaction mixture containing COBE, glucose and a catalytic
amount of NADP + . Very high turnover numbers of
NADPH (13 500 and 35 000 mol CHBE/mol, respectively)
were reported. Based on these results, Kaneka Corporation
started commercial production of (S)-CHBE in 2000.
This bioreduction system is applicable to the production of
many other useful chiral alcohols, by replacing the carbonyl
reductase gene with other appropriate enzyme genes
for carbonyl reduction. A good library of microbial carbonyl
reductases with different substrate specificities and stereospecificities
(i.e. aldehyde reductase isozyme [AR2] from
Sporobolomyces salmonicolor, carbonyl reductases [S4, R, etc]
from C. magnoliae, conjugated polyketone reductases from
Candia parapsilosis and Mucor ambiguus, menadione
reductase from Candida macedoniensis, levodione reductase
from Corynebacterium aquaticum, 1-amino-2-propanol
dehydrogenase from Rhodococcus erythropolis, secondary
alcohol dehydrogenase from Nocardia fusca, etc.) has been
made. Expression of the genes encoding these enzymes
together with the gene for GDH in E. coli cells has been
shown to be practically useful for many chiral alcohol
production processes (Figure 4) [34–42].
MA
AA
Current Opinion in Biotechnology
The biosynthetic pathway for polyunsaturated fatty acids in Mortierella alpina 1S-4 and its mutants. AA, arachidonic acid; ALA, α-linolenic acid;
DGLA, dihomo-γ-linolenic acid; EPA, 5,8,11,14,17-cis-eicosapentaenoic acid; MA, mead acid.

Figure 6
Proposed pathway for conjugated linoleic acid
(CLA) synthesis from linoleic acid by lactic
acid bacteria.
Nitrile-converting enzymes for commodity
chemical production
Microbial enzymes have also been used for the production
of commodity chemicals. The most famous successful
example in this field is the use of nitrile hydratases for
acrylamide production. The success of this process
triggered other application-oriented studies of nitrileconverting
enzymes for regioselective and stereoselective
nitrile conversions and of aldoxime metabolism for nitrile
synthesis and so on. A recent review describes details of
this research field [43 • ].
Fatty-acid-transforming enzyme systems for
polyunsaturated and conjugated fatty acid
production
C 20 polyunsaturated fatty acids (PUFAs), such as 5,8,11-ciseicosatrienoic
acid (mead acid, MA), dihomo-γ-linolenic
acid, arachidonic acid and 5,8,11,14,17-cis-eicosapentaenoic
acid (EPA), exhibit unique biological activities.
Because food sources rich in these PUFAs are limited to a
few seed oils and fish oils, we have screened microorganisms
for alternative sources of PUFAs and isolated an
arachidonic-acid-producing fungus, Mortierella alpina 1S-4
[44]. This fungus produces 30–60 g/l of mycelia (dry
weight) containing about 60% lipids. The lipids mainly
consist of triacylglycerol, which contains arachidonic acid.
The amount of arachidonic acid is 40–70% of lipids;
approximately 13 kg/kl on large-scale fermentation [45].
EPA was produced through the ω3 route from α-linolenic
acid using the same fungus [46]. Further investigation
led to the isolation of desaturase-defective mutants
and opened routes for the production of other
PUFAs (Figure 5) [47 • ,48]. For example, dihomoγ-linolenic
acid and MA can be produced through the
ω6 and ω9 route using ∆5- and ∆12-desaturase-defective
mutants, respectively.
Of recent interest in this field was the selective production
of biologically active conjugated linoleic acid (CLA)
HO
O
C
OH
Industrial microbial enzymes Ogawa and Shimizu 373
Linoleic acid
O
O
HO C
HO C
O
HO C
CLA isomers
O
HO C
isomers using lactic acid bacteria. Through screening for
the ability to isomerize linoleic acid to CLA, a potentially
useful strain, Lactobacillus plantarum, was selected [49 • ,50].
Under optimized conditions, approximately 40 mg/ml
CLA was produced from linoleic acid by the strain via
hydration of linoleic acid and successive dehydrating isomerization
(Figure 6). A hydroxy fatty acid, ricinoleic acid,
was found to be an alternative substrate for CLA production
by the strain.
Environmental aspects of the use of microbial
enzymes
In planning process development for the large-scale
production of useful compounds, it is necessary to
consider both the environmental harmonization of the
process, as well as its economic efficiency. Recent successful
examples (e.g. the Kanena process for D-p-hydroxyphenylglycine
production and the Mitsubishi Rayon process for
acrylamide production) have demonstrated that the introduction
of biochemical reactions into a whole production
process has great potential to improve environmental
harmonization. For example, the industrial enzymatic
process of racemic pantolactone resolution, operated since
1999 by Daiichi Fine Chemicals, has been shown to be
highly satisfactory not only from an economic aspect, but
also from an environmental one (using 49% less water,
producing 30% less CO 2 and with a biological oxygen
demand that is reduced by 62% as compared with the
former chemical resolution method).
Conclusions
Many microorganisms and their enzymes with unique
functions have been discovered by means of extensive
screening and are now commonly used in industrial applications.
Screening for such enzymes, in combination with
current biotechnologies, such as genetic engineering,
protein engineering (including directed and random
mutagenesis), metabolic engineering and so on, will
offer further exciting possibilities for the industrial use of
microbial enzymes.
OH
Current Opinion in Biotechnology

374 Protein technologies and commercial enzymes
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
of special interest
of outstanding interest
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Industrial biocatalysis today and tomorrow. Nature 2001,
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• applications from traditional screening methods. Trends
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This review summarizes the industrial microbial enzymes derived from
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39. Xie SX, Ogawa J, Shimizu S: NAD + -dependent (S)-specific
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A good review providing a general understanding of current research and
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46. Shimizu S, Kawashima H, Akimoto K, Shinmen Y, Yamada H:
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47. Certik M, Sakuradani E, Shimizu S: Desaturase defective fungal
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polyunsaturated fatty acids. Trends Biotechnol 1998, 16:500-505.
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desaturase-defective mutants are summarized.
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49. Ogawa J, Matsumura K, Kishino S, Omura Y Shimizu S: Conjugated
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acid during microaerobic transformation of linoleic acid by
Lactobacillus acidophilus. Appl Environ Microbiol
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A practical scale example of conjugated linoleic acid (CLA) production by
lactic acid bacteria is presented together with the results of pathway
analysis of CLA production form linoleic acid.
50. Kishino S, Ogawa J, Omura Y, Matsumura K, Shimizu S: Conjugated
linoleic acid production from linoleic acid by lactic acid bacteria.
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