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Microbial Transformation of Nitriles to High-Value Acids or Amides
69
butanoate into monomethyl (R,S)-3-benzoyloxyglutarate and monomethyl (R,S)-3-
benzyloxyglutarate, respectively [168] (Fig. 8). Hydrolysis of the progestin
dienogest (17a-cyanomethyl-17b-hydroxy-estra-4, 9-dien-3-one) was performed
by the nitrile hydratase-containing microorganism R. erythropolis. Along with the
slow hydrolysis of cyano group, aromatization of ring A and hydroxylation
occurred as well. After prolonged fermentation, the 17a-acetamido derivatives of
estradiol and of 9(11)-dehydroestradiol were formed. Three of the metabolites were
also prepared synthetically [169]. Rhodococcus sp. AJ270 also made it possible to
synthesize some carboxylic acids and amides from the nitrile bearing sensitive
groups (Fig. 9) [164].
5.2 Biodegradation and Bioremediation
Due to the intrinsic nature of nitriles as highly toxic, carcinogenic and mutagenic,
it is necessary to control and monitor the discharge of these organonitriles into the
environment. Typical examples of these compounds include acetonitrile, acryloni-
trile and benzonitrile that are widely used in laboratories and industries as solvents
and extractants, or used as an ingredient in pharmaceuticals, plastics, synthetic rub-
bers, drug intermediates (chiral synthons), herbicides and pesticides (e.g., dichlobe-
nil, bromoxynil, ioxynil, buctril), etc. Recent awareness of environmental pollution
caused by chemical-based industries has necessitated the development of enzyme-
based process as alternatives to currently employed chemical processes. Moreover,
bioconversion and biotransformation becomes partial or total replacement of cur-
rently employed toxic chemical process due to the distinct advantages of biotrans-
formation [9]. Hence, their decomposition and detoxification by convenient and
efficient methods is fairly urgent and challenging. More importantly, biodegrada-
tion and bioremediation, a convenient and cost-effective method, has the capability
of eliminating these compounds by degrading them into harmless intermediates or,
to a more desired form, carbon dioxide and water [170].
To the best of our knowledge, the huge potential of nitrile-converting enzymes
has been explored in biodegradation of nitriles by many researchers. A variety of
microorganisms, were demonstrated to be effective on metabolism of some orga-
nonitriles. As previously reported, a considerable number of nitrile-converting
whole-cell biocatalysts have been applied to the removal of acrylonitrile waste
effluents from the manufacture of acrylamide. A mixed culture of bacteria includ-
ing different nitrile hydrolyzing enzymes that degrade effluent from the manufac-
ture of acrylonitrile containing acrylonitrile, fumaronitrile, succinonitrile, etc. are
grown in batch and continuous culture on these components of waste completely
degraded all of the components [171].
Very recently, researchers from Singapore successfully enriched a whole consor-
tium from the activated sludge of a pharmaceutical wastewater treatment plant and
investigated its capability of biodegradation of saturated (acetonitrile), unsaturated
(acrylonitrile), and aromatic (benzonitrile) organonitrile compounds [172].
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J. Chen et al.
Due to the potency and efficiency of biodegradation, similar studies were also
conducted in China, especially in Taiwan, and moreover, some achievements were
obtained [173–175]. Nitrile-converting enzymes have also participated in the
cyano group-containing herbicide decomposition. A bromoxynil-degrading soil
microorganism Agrobacterium radiobacter was used for degradation of the herbi-
cide under nonsterile batch and continuous conditions. The efficacy of degradation
was enhanced by addition of ferrous, cobaltous or cupric ions [176].
6 Cloning and Expression of Nitrile-Amide
Converting Enzymes
Cloning and overexpression stands out as a promising method for the production of
the desirable enzymes in sufficient quantities. So are the nitrile-amide converting
enzymes, and there appeared numerous reports with respect to cloning and expres-
sion of these enzymes which are now accessible in sufficient quantities. As for
nitrile hydratase, both the H- and L-nitrile hydratases genes, composed of two
subunits of different sizes, have been cloned from R. rhodochrous J-1. The amino
acid sequences of each subunit of the H- and L-NHases from R. rhodochrous J-1
showed generally significant similarities to those from Rhodococcus sp. N-774, but
the arrangement of the coding sequences for two subunits is reverse. Each of the
NHase genes was expressed in E. coli cells under the control of lac promoter only
when they were cultured in the medium supplemented with CoCl
2
[177]. The stere-
oselective nitrile hydratase from P. putida 5B has been over-produced in E. coli. A
clearly enhanced enzyme activity six times higher than the native strain and same
stereoselectivity was observed [178].
In China, researchers have carried out similar work and some progress has been
made. To obtain a recombinant Rhodococcus or Nocardia with not only higher
enzymatic activity but also better operational stability and product tolerance for
bioconversion of acrylamide from acrylonitrile, an active and stable expression
system of nitrile hydratase (NHase) was tried to construct as the technical platform
of genetic manipulations. Two NHase genes, NHBA and NHBAX, from Nocardia
YS-2002 were successfully cloned into E. coli and Pichia pastoris system,
however, expression level remained extremely low and the protein was unstable.
To solve this problem, a possible genetic strategy, site-directed mutagenesis of the
a-subunit of the NHase was carried out. After the successful mutagenesis, E. coli
XL1-Blue (pUC18-NHBAM) was screened and the NHase activity was much
higher than that of the prototype [179].
Along with the nitrile hydratase, some amidases have undergone the cloning
and expression. Recently, Cheong et al. have undertaken the research concerning
cloning of a wide-spectrum amidase from Bacillus stearothermophilus BR388 in
E. coli and improving amidase expression using directed evolution. As a desired
result, this mutant, prepared by PCR-based random mutagenesis which resulted
in the substitution of arginine for histidine at position 26, demonstrated a 23-fold
increase in amidase activity compared to the wild-type [88]. The amidase gene
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Microbial Transformation of Nitriles to High-Value Acids or Amides
71
from the hyperthermophilic archaeon Sulfolobus solfataricus has been cloned,
sequenced, and overexpressed in E. coli and the recombinant thermophilic pro-
tein was expressed as a fusion protein with an N-terminus six-histidine-residue
affinity tag [86].
In the case of nitrilase, the cloning of this enzyme first occurred in E. coli and
encoded a bromoxynil-degrading activity from Pneumoniae subsp. ozaenae [180].
In the previous studies, four nitrilases have been cloned from A.thaliana [181].
The corresponding gene of a regioselective aliphatic nitrilase from A. facilis 72W was
cloned and over-expressed in E. coli, yielding a microorganism that efficiently and
regioselectively catalyzes the conversion of aliphatic dinitriles to cyanocarboxylic
acids. However, it was observed that, though a markedly increased quantity of
nitrilase protein was obtained, the majority is present as inclusion bodies and is
inactive. The phenomenon was consistent with the outcome obtained when nitrilase
from C. testosterone was expressed in E. coli, although this was significantly
improved with the co-expression of groESL chaperones [60]. R. rhodochrous J-1,
appeared promising as a versatile nitrile-amide producing bacterium. Hence, it was
investigated extensively, recent studies focusing on the molecular level. Komeda
et al. demonstrated that the 1.4-kb downstream region from a nitrilase gene (nitA)
was found to be required for the isovaleronitrile-dependent induction of nitrilase
synthesis. Sequence analysis of the 1.4-kb region revealed the existence of an open
reading frame (nitR) of 957 codes for a transcriptional regulator in nitA expression
[182]. In China, researchers from Tongji University introduced a series of work
concerning cloning and sequencing of nitrilase from an efficient degrader Nocardia
sp. C-14-1. Southern blotting showed that there was a single nitrilase gene in the
genome of C-14-1. Meanwhile, DNA sequencing and analysis suggested that there
was a fragment of 1,143 bp DNA sequence encoded the nitrilase [183]. Besides, it
was found that the expression of the Vitreoscilla hemoglobin (vgb) gene in vivo
could improve the fermentation density and then contribute the extracellular secre-
tion of the product of bromoxynil-specific nitrilase (bxn) gene. The recombination
plasmid pPIC9K-vgb-bxn was constructed and transformed into P. pastoris GS115.
The results of PCR and SDS-PAGE indicated that the vgb gene and bxn gene had
integrated into the genome of P. pastoris GS115 and expressed in efficient level
[184]. Subsequently, the bxn gene encoding bromoxynil-specific nitrilase was
cloned from genomic DNA of Klebsiella ozaenae by PCR and over-expressed in
E. coli DE3. The recombinant accessibility made it a promising candidate for elimi-
nating bromoxynil to non noxious substances [185]. It could be concluded that the
cloning and over-expression of the encoding genes resulted in a better understand-
ing of enzyme function and the reaction mechanism, which in turn would lead to
improvements in biotechnological applications.
Protein engineering of nitrilase have also been practiced to improve the substrate
and product tolerance and specific activity. A high-activity biocatalyst based on an
A. facilis 72W nitrilase was developed, where protein engineering and optimized
protein expression in an E. coli transformant host were used to improve microbial
nitrilase specific acticity for glycolonitrile by 33-fold compared to the wild-type
strain [186]. Gene site saturation mutagenesis (GSSM) evolution technology was
employed to improve enantioselectivity of nitrilase-catalyzed desymmetrization of
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J. Chen et al.
3-hydroxyglutaryl nitrile to afford (R)-4-cyano-3-hydroxybutyric acid, an interme-
diate to the cholesterol-lowering drug Lipitor [187]. Changing Ala to His in posi-
tion 190 provided a 10% increase in the enantiomeric excess at the commercially
relevant 3 M substrate reaction concentration.
7 Conclusions and Future Prospects
The past few decades have witnessed the fast development of nitrile-amide convert-
ing enzymes, both their reaction mechanisms and applications in manufacture of a
series of pharmaceuticals and chemicals. Formerly, great contribution was made by
hydrolases such as esterases and lipases in the production of enantiopure synthons.
Nowadays, with the discovery of numerous nitrile-amide converting microorgan-
isms and their extremely fast development, these enzymes are becoming more and
more appreciated by organic chemists and are showing competency to compete
with esterases and lipases. Besides their synthetic value, these enzymes also play
an important role in protecting the environment due to the capability of removal
highly toxic nitrile compounds which are rather detrimental to human beings, animals
and plants. In order to exploit fully their biotechnological potential, researches
concerning the following aspects should be carried out in the following ways: (1)
Overcoming some disadvantages of the nitrile-converting biocatalysts, such as nar-
row substrate specificity, low thermostability and pH stability, low tolerance of
substrate and product, undesired and unsatisfied enantioselectivity. (2) Screening
and discovery of new nitrile-amide converting enzymes with promising and attrac-
tive properties. As previously demonstrated, microorganisms producing nitrile-
amide converting enzymes turned out to be dominantly from prokaryotic ones and
eukaryotic organisms constitute only a small part. The latter ones, however, were
always neglected as an excellent source of nitrilase, nitrile hydratase and amidase.
Moreover, these organisms may provide some different properties like excellent
thermostability, regio-, enantio- selectivity, and improved stability in some acidic
and alkaline media, which are favored by some process. (3) Employing genetic
engineering to alter some undesired properties of wild type strain. Bearing these in
mind, researchers would make great progress in techniques related to screening,
cultivation, protein and genetic engineering and hence it is possible to isolate novel
enzymes with extremophilic characteristics.
Despite the advantages of nitrile-amide converting enzymes catalyzed synthesis
of a range of industrially useful acids and amides, a paucity of them have achieved
success in industrial application, with the commercial production of acrylamide and
nicotinamide being the most successful. Manufacture of many other substances has
been proved to be accomplished by these enzymes, especially the processes involve
the regioselective and enantioselective hydrolysis of some prochiral compounds
and racemic nitriles. These compounds, nevertheless, are difficult if not impossible
to convert by means of traditional chemical methods. Chemical hydrolysis of many
nitriles with labile substituents catalyzed by acid or base is also virtually impossible
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