47
34
J. Chen et al.
1 Introduction
Biotransformation and biocatalysis have gained increasing interest in recent years
due to their mild conditions (physiological pH and ambient temperature), environ-
mentally attractive catalysts, high activities and inherent excellent selectivities
including chemo-, regio- and enantio- selectivities [1, 2]. Biocatalysis is now an
established method in the synthesis of organic compounds and is especially useful
for the production of chiral substances. By virtue of these obvious advantages,
biotransformation is becoming a promising alternative to the traditional acid- or
base-catalysed reactions, so is the case of nitrile hydrolysis. Nitriles, as the sub-
strates, are widespread in the environment and they are produced by plants in various
forms, such as cyanoglycosides, cyanolipids, ricinine, phenylacetonitrile, etc. [3].
Despite the fact that a majority of nitriles are highly toxic, mutagenic and carci-
nogenic in nature [4, 5], they are an important class of compounds for their ability
to afford significant intermediates in the synthesis of acids, amides, amines, amidine,
esters, aldehydes, ketones and so on by chemical and enzymatic hydrolysis.
Chemical hydrolysis of nitriles was extensively applied to synthesize amides and
acids previously; however, these applications may not be suitable for the hydrolysis
of nitriles in the presence of sensitive groups. In sharp contrast, enzymatic hydrolysis
of nitriles could alleviate this problem ascribed to the mild reaction conditions.
Besides, three enzymes, namely nitrile hydratase (EC 4.2.1.84), nitrilase (EC
3.5.5.1) and amidase (EC 3.5.1.4) involved in the transformation of nitriles or
amides exhibit great potential of chemo-, enantio- and regioselective synthesis [6].
Contents
1 Introduction .......................................................................................................................... 34
2 Description of Three Classes of Nitrile-Amide Converting Enzymes ................................. 35
2.1 Nitrile Hydratase ......................................................................................................... 35
2.2 Nitrilase ....................................................................................................................... 37
2.3 Amidase ...................................................................................................................... 39
3 The Isolation and Identification of the Nitrile-Amide Converting
Organisms in China.............................................................................................................. 41
4 Factors Affecting the Activity and Enantioselectivity
of Nitrile-Amide Converting Enzyme.................................................................................. 42
4.1 Inducer ........................................................................................................................ 43
4.2 Metal Ions ................................................................................................................... 43
4.3 Effect of Light on Nitrile Hydratase ........................................................................... 44
4.4 Amidase Inhibitor ....................................................................................................... 44
4.5 Temperature and pH .................................................................................................... 44
4.6 Organic Solvents ......................................................................................................... 45
4.7 Steric and Electronic Factors ...................................................................................... 46
5 Applications of Nitrile-Amide Converting Enzymes ........................................................... 48
5.1 Bioconversion of Various High-Value Amides and Acids .......................................... 48
5.2 Biodegradation and Bioremediation ........................................................................... 69
6 Cloning and Expression of Nitrile-Amide Converting Enzymes ......................................... 70
7 Conclusions and Future Prospects ....................................................................................... 72
References .................................................................................................................................. 73
42
Microbial Transformation of Nitriles to High-Value Acids or Amides
35
As a result, these enzymes have evoked substantial attention and they are becoming
more and more demanding. These enzymes operate either by direct hydrolysis of
nitrile to the corresponding acid (by a nitrilase enzyme) or by sequential action of an
enzyme that hydrates the nitrile to the amide and the latter is transformed to the acid
(by an amidase enzyme) (Scheme 1) [7, 8]. To date, various nitrile-amide converting
organisms isolated from bacteria, fungi and plant have been described [9, 10].
Among them, most of them have been derived from bacterial species by enrichment
strategies with nitriles as the sole nitrogen source [11]. Some reactions mediated by
nitrile-converting enzymes have been applied on a large scale in industry. Productions
of acrylamide [12] and nicotinic acid [13] on an industrial scale have proved the
commercial value of these enzymes. With the fast development of these enzymes, an
upsurge of biotransformation of nitriles has been taking place in China as well.
According to the statistical data, an increasing number of reports have appeared and
several institutes and universities have taken part in this research in recent years. As
a result, a variety of microorganisms harboring nitrile-amide converting activities
have been isolated, identified and characterized in various places, some of which
have already applied with moderate success. Moreover, much work has focused on
the organization and regulation of the genes encoding for nitrile metabolism. For
example, research on the expressions of nitrile-degradation enzymatic system in
recombinant strains has been carried out in China in recent years.
2 Description of Three Classes of Nitrile-Amide
Converting Enzymes
2.1 Nitrile Hydratase
Nitrile hydratase, known as metalloenzyme, is a vital enzyme in the bienzymatic
hydrolysis of nitriles to acids, which transforms nitriles to the corresponding amides.
Asano et al. first reported the occurrence of nitrile hydratase from Rhodocococcus
rhodochrous J-1 (formerly identified as Arthrobacter sp. J-1) to degrade acetonitrile,
which was later applied with excellent success to the production of acrylamide from
Scheme 1
The pathways of nitrile compounds by nitrile-amide converting enzymes
R-CN
R-CONH
2
R-CO
2
H
nitrilase
amidase
nitrile
hydratase
72
36
J. Chen et al.
acrylonitrile on an industrial scale [14]. These findings promoted the intensive inves-
tigation of nitrile hydratase including physiochemical properties, substrate specifici-
ties and the reaction mechanism. According to the presence of metal co-factor, nitrile
hydratase can be classified into two kinds: ferric nitrile hydratase and cobalt nitrile
hydratase. The existence of metal ions in the active site of the enzyme is presumably
effective in enhancing the folding or stabilization of the subunit that is dominantly
consisted of a and b subunits. NHase can also be classified into high and low molecular
weight (H- and L-NHases) on the basis of molecular weight of the enzyme. So far, a
considerable number of microorganisms were successfully screened (Table 1).
Additionally, two new bacterial strains, Pseudomonas marginales MA32 and
Pseudomonas putida MA113, containing nitrile hydratase resistant to cyanide were
isolated from soil samples by an enrichment procedure [40]. In contrast to known
nitrile hydratases, which rapidly lose activity at low to moderate cyanide concentra-
tions, the enzymes tolerated up to 50 mM cyanide. Cyanide-resistant nitrile
hydratase will find great application in the hydration of a-hydroxynitriles for the
production of a-hydroxyamide because cyanide is always present in aqueous solu-
tions of a-hydroxynitriles due to their tendency to decompose to the respective
carbonyl compound and prussic acid.
Table 1
Some previously reported microorganisms with nitrile hydratase activity
Microorganisms
Substrates specificity
Agrobacterium tumefaciens d3 [15]
Arylnitriles, arylalkylnitriles, acrylonitrile
Arthrobacter sp. J-1 [16]
Alipatic nitriles
Bacillus cereus [17]
Acrylonitrile
Bacillus sp. BR 449 [18]
Acrylonitrile
Bacillus smithii SC-J05-1 [19]
Arylnitriles
Brevibacterium imperialis CBS 489-74 [20]
Acrylonitrile
Pseudomonas chlororaphis B23 [21]
Alkylnitrile
Pseudomonas putida [22]
Acetonitrile
Pseudonocardia thermophila JCM 3095 [23]
Acrylonitrile
Rhodococcus rhodochrous J-1 [24, 25]
Alkylnitrile, heterocyclic nitriles, arylnitriles
Rhodococcus rhodochrous R312 [26]
Alkylnitrile, benzonitrile
Rhodococcus rhodochrous LL 100-21 [27, 28] Alkylnitriles, acrylonitrile, arylalkylnitriles,
3-cyanopyridine
Rhodococcus erythropolis BL1 [29]
Alkylnitriles, arylalkylnitriles
Rhodococcus rhodochrous A4 [30, 31]
Alkylnitriles, arylnitriles, cycloalkylnitriles,
arylnitriles, heterocyclic nitriles,
arylalkylnitriles
Rhodococcus sp. AJ270 [32]
Wide spectrum nitrile hydratase
Rhodococcus sp. SHZ-1 [33]
Acrylonitrile
Nocardia sp. 108 [34]
Acrylonitrile
Rhodococcus sp. ZJUT-N595 [35]
Acrylonitrile, glycolonitrile, 2,2-dimethylcy-
clopropanecarbonitrile
Rhodococcus sp. N 774 [36]
Aliphatic nitriles
Candida guilliermondii CCT 7207 [37]
Cycloalkylnitriles, arylnitriles, heterocyclic
nitriles
Candida famata [38]
Alkylnitriles
Cryptococcus flavus UFMG-Y61 [39]
Isobutyronitrile
96
Microbial Transformation of Nitriles to High-Value Acids or Amides
37
To date, many studies focused on the mechanism of NHase mediated catalysis.
Kobayashi et al. put forward a possible mechanism as follows. A water molecule
was activated by the metal-bound hydroxide ion after the combination of the metal
ion and water molecule. Imidate as an intermediate was initially formed via attack-
ing on nitrile carbon by the activated water molecule. The imidate were then tau-
tomerized to form the amide form (Fig. 1) [41].
2.2 Nitrilase
Nitrilase, the first nitrile-converting enzyme, was discovered in barley approxi-
mately 40 years ago and famous for the ability to convert indole-3-acetonitrile to
the auxin indole-3-acetic acid [42]. From then on, several microorganisms harbor-
ing nitrilase activity have been screened, purified and characterized. Bacteria, fungi
as well as plants provide excellent source of nitrilase, with bacterial species as the
main source (Table 2), which are generally derived using enrichments from envi-
ronmental samples. In the case of plants, numerous studies were carried out with
Arabidopsis thaliana, from which four kinds of nitrilase were separated numbered
NIT1, NIT2, NIT3 and NIT4. Although it was found that these enzymes were capable
of transforming indole-3-acetonitrile to indole-3-acetic acid, recent results have
indicated that NIT1, NIT2, NIT3 showed significant preference for 3-phenylpropi-
onitrile, whose product, phenylacetic acid, is found in nasturtium. NIT4, however,
was effective in hydrolyzing b-cyano-l-alanine [45].
Depending on the substrate specificity, nitrilase is differentiated into three sub-
classes: aliphatic nitrilase, aromatic nitrilase that shows preference for aromatic and
heterocyclic nitriles and arylacetonitrilase which is highly specific for arylace-
tonitriles [61].
Fig. 1
Catalytic reaction mechanism of nitrile hydratase
N
S
N
S
Cys
109
Cys
109
M
+x
M
+x
S
O
−
O
−
O
−
O
−
O
−
O
−
Cys
112
Cys
112
Ser
113
Ser
113
Cys
114
Cys
114
OH
RCN
HB
H
2
O
R
C
NH
NH
OH
C
O
−
O
−
C
N
S
N
S
S
OH
C
C
R
C
Rearrangement
+
Amide
O
81
38
J. Chen et al.
Numerous investigations have provided insights into the mechanism of nitrilase
catalyzed reaction. All nitrilases studied contain a cysteine residue in their catalytical
center. The mechanism involves a nucleophilic attack by a thiol group in cysteine
residue on the nitrile C-atom, forming an enzyme-linked tetrahedral thiomidate
intermediate which is then attacked by H
2
O and nitrogen atom is released as NH
3
.
Further addition of H
2
O results in the production of acid and a regenerated enzyme
(Fig. 2) [68].
Table 2
Some previously reported microorganisms with nitrilase activity
Bacteria
Fungi
Plants
Acidovorax facilis 72W [43]
Fusarium solani IMI196840
[44]
Arabidopsis thaliana [45]
Bacillus pallidus Dac521 [46]
Fusarium oxysporum [47]
Barley [42]
Alcaligenes faecalis JM3 [48]
Cryptococcus sp. UFMG-Y28
[49]
Chinese cabbage [50]
Alcaligenes faecalis ATCC8750
[51]
Aspergillus niger [52]
Brassica rapa [53]
Rhodococcus rhodochrous J-1 [54] Penicillium multicolor [55]
Rhodococcus rhodochrous NCIMB
11216 [56]
Exophiala oligosperma R1
[57]
Rhodococcus rhodochrous PA-34
[58]
Rhodococcus rhodochrous K22
[59]
Comamonas testosterone [60]
Pseudomonas fluorescens DSM
7155 [61]
Rhodococcus rubber [62]
Acinetobacter sp. AK 226 [63]
Klebsiella ozaenae [64]
Arthrobacter sp. J1[65]
Streptomyces sp. MTCC 7546 [66]
Bacillus subtilis ZJB-063 [67]
Fig. 2
Mechanism of nitrilase-catalysed reaction
Enz-SH + R-C
N
Enz-S-C-R
NH
H
2
O
H
2
O
Enz-S-C-R
NH
3
Enz-S-C-R
O
Enz-S-C-R
O
−
Enz-SH + R-C-OH
O
Thiomidate
intermediate
NH
2
OH
OH
68
Microbial Transformation of Nitriles to High-Value Acids or Amides
39
2.3 Amidase
Amidase, amide bond-cleaving enzymes, exists ubiquitously in nature in both
prokaryotic and eukaryotic forms. To the best of our knowledge, amidase-mediated
processes have been extensively investigated, especially, the hydrolysis of amides
to the corresponding carboxylic acid and ammonia. Additionally, hydroxamic acids
were formed owing to the acyl transfer activity of amidase in the presence of
hydroxylamine. Two reactions involved are shown in Scheme 2.
Besides, amidase is also capable of catalyzing diverse reactions such as ester
hydrolysis, hydroxamic acid hydrolysis, acid hydrazide hydrolysis, amide transfer on
hydrazine, ester transfer on hydroxylamine, ester transfer on hydrazine and so on [69].
Therefore, amidase turns out to be efficient and promising tools for the synthesis
of various compounds. In addition, with regard to nitrile hydratase in Gram-positive
bacterium, its enantioselectivity was always combined with the amidase’s. Namely,
their cooperation gave rise to the excellently pure products. However, the former
usually displayed almost no stereoselectivity with the amidase being a major con-
tributor. Consequently, significant attention has been paid to the isolation and dis-
covery of amidase-producing organisms including bacteria, yeasts, fungi, plants
and animals (Table 3).
Although amidase catalyzes many reactions, some of them proceed at a
comparative low rate employing esters or carboxylic acids as acyl donors. In
sharp contrast, high amidase activity is achieved in the presence of water
(H
2
O) and hydroxylamine (NH
2
OH) as the cosubstrates when amide is used as
the substrate, indicating that these two compounds functions as efficient acyl
acceptors [69].
Both the amidase-catalyzed hydrolytic reaction and the acyl transfer reaction
share the same reaction mechanism. In view of this, study on the mechanism of
the acyl transfer reaction shed light on that of hydrolytic reaction, in which
case, there is difficulty in investigating the mechanism where water is the
cosubstrate. A possible mechanism suggested the reaction belonged to Ping
Pong Bi Bi type: The carbonyl group of amide undergoes a nucleophilic attack
by the enzyme, leading to the formation of a tetrahedral intermediate, which is
consequently converted to an acyl–enzyme intermediate with the release of
Scheme 2
The pathways of hydrolysis and transfer of amide Amide hydrolysis: RCONH
2
+ H
2
O
→ RCOOH + NH
3
Amide acyl transfer reaction: RCONH
2
+ NH
2
OH → RCONHOH + NH
3
Amide hydrolysis:
RCONH
2
+ H
2
O
RCOOH + NH
3
Amide acyl transfer reaction:
RCONH
2
+ NH
2
OH
RCONHOH + NH
3
86
40
J. Chen et al.
ammonia. The acyl–enzyme complex in turn is subjected to attack by water or
hydroxylamine (Fig. 3) [9, 90].
Importantly, a majority of amidases bear enantioselectivity, which contributed to
the synthesis of chiral carboxylic acid via nitrile hydratase and amidase. However,
these nitrile-hydratase-associated amidases are, surprisingly, mostly S-stereospecific.
R-Enantioselective amidases are gaining more and more interest because of their
potential application in the production of d-amino acids and other optically active
compounds. Hydroxamic acids, products of the acyl transfer reactions, can be
detected easily and fairly specifically by the addition of acidic ferric chloride solu-
tion, which results in the production of a deep magenta color [91]. Therefore, this
Table 3
Some previously reported microorganisms with amidase activity
Microorganisms
Substrate specificity
Rhodococcus erythropolis MP50 [70]
Aromatic amide
Geobacillus pallidus RAPc8 [71]
Aliphatic amide
Sulfolobus tokodaii strain 7 [72]
Aromatic amide
Delftia acidovorans [73]
d-Amino acid amide
Arthrobacter sp. J-1 [74]
Aliphatic amide
Rhodococcus rhodochrous M8 [75]
Aliphatic amide
Xanthobacter flavus NR303 [76]
l-Amino acid amide
Brevibacterium sp Strain R312 [77]
Aryloxypropionamides
Variovorax paradoxus [78]
d-Amino acid amide
Pseudonocardia thermophila [79]
Aliphatic, aromatic and amino acid amide
Pseudomonas sp. MCI3434 [80]
Heterocyclic amide
Klebsiella oxytoca [81]
Aliphatic amide
Brevibacillus borstelensis BCS-1 [82]
Aromatic and aliphatic amide
Ochrobactrum anthropi SV3 [83]
Amino acid amide
Stenotrophomonas maltophilia [84]
Peptide amide
Agrobacterium tumefaciens strain d3 [85]
Aromatic amide
Sulfolobus solfataricus MT4 [86]
Aliphatic and aromatic amide
Klebsiella pneumoniae NCTR1 [87]
Aliphatic amide
Bacillus stearothermophilus BR388 [88]
Wide spectrum amidase
Delftia tsuruhatensis ZJB-05174 [89]
2,2-Dimethylcyclopropanecarboxamide
R-C-NH
2
O
R-C
R-C-XE
NH
H
2
O
H
2
O
H
2
O
R-C-NH
2
XE
O
H
NH
3
R-C-XE
O
R-C-OH + EXH
O
EXH
N
Fig. 3
Mechanism of amidase-catalysed reaction
43
Microbial Transformation of Nitriles to High-Value Acids or Amides
41
acyl transfer reaction can be employed in a colorimetric screening procedure for
active and enantioselective amidases. By employing the amidase-catalyzed acyl
transfer reaction, Delftia tsuruhatensis producing R-enantioselective amidase was
screened in our lab [89, 92].
3 The Isolation and Identification of the Nitrile-Amide
Converting Organisms in China
Biotransformation of nitriles is of great potential in organic synthesis and it pro-
vides green access to various carboxylic acids and amides; thus nitrile-amide con-
verting enzymes are of broad use and commercial interest. Recently, biotransformation
of carboxylic acids and amides via these enzymes has been a hot issue in China.
The scarcity of appropriate nitrile-amide converting biocatalysts and the difficulty
in commercial availability of these enzymes promoted the screening and discovery
of the novel nitrile-amide converting organisms in China. So far, a series of organ-
isms producing nitrile-amide degrading enzymes were isolated, identified and
characterized, some of which were purified. Among the obtained organisms, it was
observed that some harbored nitrile hydratase, some produced nitrilase and others
could form amidase. It was also found that the existences of nitrile hydratases were
always accompanied by amidases, so amides and acids were formed in different
proportions in such kinds of microbes mediated reactions. A prominent example is
Rhodococcus sp. AJ270, which is a powerful and robust nitrile hydratase/amidase-
containing microorganism isolated by Wang et al. Later, its broad applications in
transforming various nitriles were substantially explored [32]. Rhodococcus sp.
AJ270 as well as other nitrile-amide converting organisms was dominantly obtained
by enrichment strategies where nitriles were employed as the sole nitrogen source
ascribed to the highly toxic nature. Owing to the fact that screening a desired organism
for a particular biocatalytic process is always a time-consuming and tedious
job, some direct and sensitive readouts of the nitrile-amide converting enzyme
activity have to be considered and developed. Conventional routes employ high-
performance liquid chromatography, liquid chromatography-mass spectrometry,
capillary electrophoresis, or gas chromatography to determine the enzyme activity,
where determinations have to carry out one by one. Furthermore, those traditional
enrichment strategies usually result in the isolation of a rather restricted group of
microorganisms. A successful instance of application of high throughput screening
method in our research was the isolation of Delftia tsuruhatensis, a R-stereospecific
amidase producing bacterium. R-Enantioselective amidases are of considerable
industrial interest due to potential applications in the production of optically active
compounds [92]. More recently, Zhu et al., driven by the attempt to find a fast,
convenient and sensitive method, reported a more accurate and innovative high
throughput route. In their paper, a novel time-resolved luminescent probe:
o-hydroxybenzonitrile derivatives could be applied to detect the activity of the
nitrilases. By the action of nitrilases, o-hydroxybenzonitrile derivatives could be
47
42
J. Chen et al.
transformed to the corresponding salicylic acid derivatives, which, upon binding
Tb
3+
, served as a photon antenna and sensitized Tb
3+
luminescence. Because of the
time-resolved property of the luminescence, the background from the other proteins
(especially in the fermentation system) in the assay could be reduced and, therefore,
the sensitivity was increased. Moreover, because the detection was performed on a
96- or 384-well plate, the activity of the nitrilases from microorganisms could be
determined quickly [93].
Moreover, some other high throughput methods have been reported as alterna-
tives to conventional screening methods. A critical review on selection and screen-
ing strategy for enzymes of nitrile metabolism based on spectrophotometric and
fluorimetric methods has been published [94]. Recently, convient screening meth-
ods have been developed on the basis of the color variation of indicators which are
added to the mixture in advance. Once the acid formed, the color would have a
dramatic change [95]. Additionally, a new method for nitrilase screening has been
developed to detect nitrilase activity. The ammonia product of nitrilase mediated
conversion of nitriles forms a complex with the cobalt ion results in a color change,
which can readily be quantified using a spectrophotometer at 375 nm. The assay
has the potential to be used for the real-time monitoring of nitrilase-catalyzed reac-
tons [96]. More noticeably, Hu et al. introduced a simple and rapid high-throughput
screening method based on a colorimetric reaction of glycolic acid with b-naphthol
in sulfuric acid solution to isolate glycolonitrile-hydrolyzing microorganisms. Four
strains able to convert glycolonitrile to glycolic acid were isolated from soil sam-
ples using this screening method, among which Rhodococcus sp. ZJUT-N595 dis-
played the highest hydrolytic activity [35].
These soil-derived nitrile-amide hydrolyzing organisms have been currently under
active development and some have even achieved with small to moderate success.
The advantage of applying whole cell biocatalysts lies in that they can be relatively
easily and cheaply prepared and the whole cell catalyzed reactions can be operated
much more easily. Nevertheless, some small aliphatic nitriles, hydroxyl- and amino-
substituted nitriles give lower yields and appear to be alternatively metabolized when
whole cell biocatalysts were applied [32]. Hence, on one hand, careful monitoring of
the reaction is strongly recommended to achieve the maximal desired product. On the
other hand, the use of purified enzymes is of substantial significance and benefit in
case that substrate or product utilization by whole cells exists. Due to this, the purifi-
cation and characterization of nitrile-amide converting are under progress in China.
4 Factors Affecting the Activity and Enantioselectivity
of Nitrile-Amide Converting Enzyme
Numerous factors, such as some culture conditions like carbon source, nitrogen
source, inducer and conversion conditions like temperature, pH, reaction time,
cosolvent and so on, turn out to affect the activity and enantioselectivity, and con-
sequently the biomass production.
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