Biocatalysis for the Pharmaceutical Industry || Applications of Nitrile Hydratases and Nitrilases

8

Applications of Nitrile Hydratasesand Nitrilases

Grace DeSantis1 and Robert DiCosimo2

1Biosite, An Inverness Medical Company, 9975 Summers Ridge Road, San Diego,

CA 92121, USA2DuPont, Experimental Station, PO Box 80328, Wilmington, DE 19880-0328, USA

8.1 Introduction

Existing synthetic methods and commercial processes that employ nitrile hydratases (NHases)

and nitrilases continue to be improved by directed evolution of existing enzymes, or by the

discovery of new enzymes with improved properties, and new applications of these catalysts

have recently been described. Numerous reviews have previously been published that describe

applications of NHase [1–6] and nitrilase [1,4–11], and in this review we present examples of

new applications of these nitrile-utilizing catalysts from journal articles, patent applications,

and issued patents that have been published in the past 2–3 years.

8.2 NHase

8.2.1 New NHases

Many previously-characterized NHases have been shown to have poor temperature stability

above ambient temperature. Geobacillus caldoxylosilyticusM16 [12], a thermophile that can

be grown at 70 �C, produces a thermostable NHase when induced during culture with

valeronitrile, crotononitrile or crotonamide. The microbial NHase is reported to be active at

temperatures up to 75 �C. Hydration of a 1.1wt% solution of acrylonitrile in phosphate buffer

(50mM, pH 7.7) at 20–75 �C using 5UmL�1 Geobacillus caldoxylosilyticus NHase (activity

measured at 10 �C) produced acrylamide in quantitative yield. Similarly, 1.1wt% solutions of

adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, n-hexanenitrile, and

benzonitrile were quantitatively converted to the corresponding amides or diamides at 30 �C.

Biocatalysis for the Pharmaceutical Industry : Discovery, Development, and Manufacturing edited by J. Tao, G.-Q. Lin, and A. L.

© 2009 John Wiley & Sons Asia (Pte) Ltd. ISBN: 978-0-470-82314-9

8.2.2 Applications

8.2.2.1 Acrylamide

A Geobacillus thermoglucosidasius Q-6 NHase was expressed in Rhodococcus rhodochrous

strain M33 [13]. The recovery of NHase activity after 30min at 60 �C, 70 �C, and 80 �C was

88%, 84%, and 32% respectively, and the optimum reaction temperature was about 60 �C.There was no decrease in the microbial NHase activity of this transformant in reactions with

increasing acrylonitrile concentrations up to 6% (w/v). In a batch reaction using unimmobi-

lized cells as catalyst, an initial catalyst loading of 0.11wt% dry cell weight (dcw) Rhodo-

coccus rhodochrous M33/Q6 was used to convert a continuous feed of acrylonitrile (not

exceeding 2% in the reaction mixture) to acrylamide at 20–25 �C, where a final concentrationof 52% (weight/solution) acrylamide was accumulated.

Rhodococcus rhodochrous J-1 has been used for the industrial production of acrylamide

from acrylonitrile, but the J-1 NHase is not very stable above 30 �C. Mutant gene libraries of

Rhodococcus rhodochrous J-1 andRhodococcus rhodochrousM8NHases have been prepared,

where amino acid substitutions were made in both a and b subunits of the enzyme, including

substitutions in the region around the cofactor binding domain of the a-subunit, and in the

b-subunit. Mutant NHases with improved temperature stability, and improved stability in high

concentrations of acrylamide produced by the hydration of 5wt%acrylonitrile, were identified,

and these mutant NHases were expressed in transformed Rhodococcus and Escherichia coli

microbial host strains [14]. A Rhodococcus rhodochrous J-1 NHase with an Eb93G mutation

was one of 16 NHase mutants expressed in Escherichia coli JM109 that showed a significant

improvement in temperature stability at 50–60 �Cwhen comparedwith theNHase of the parent

strain (Table 8.1). Eb93G and Nb167Smutations of theRhodococcus rhodochrousM8NHase

had similarly improved temperature stabilities relative to the NHase of the parent strain.

Additional testing of the mutant NHases for conversion of 5wt% acrylonitrile in 30%

acrylamide also demonstrated improved retention of stability or improvement in reaction

rates, relative to the parent strain NHase under these reaction conditions.

A process for production of high-purity acrylamide from acrylonitrile that has a significant

concentration of acrolein impurity has been demonstrated, where the acrylonitrile contains

�100 ppm acrolein [15]. The process utilizes microbial cell catalysts that convert acryloni-

trile feeds having at least 10 ppm acrolein impurity directly to high-purity acrylamidewith no

detectable levels of acrolein, avoiding the need for purification of the acrylonitrile feed prior

to conversion, or removal of acrolein from the product mixture. High-molecular-weight

polymer with high intrinsic viscosity is produced from acrylamide free from by-products

derived from acrolein. In one example, an acrylonitrile solution containing 50 ppm of

acrolein was continuously fed at 25 �C into a reaction mixture containing Rhodococcus

Table 8.1 Temperature stability of Rhodococcus rhodochrous J1 Eb93Gmutant NHase compared with

J1 NHase, each expressed in Escherichia coli JM109

NHase Recovered activity (%)

Untreated 50 �C, 20min 55 �C, 20min 60 �C, 5min

J1 100 54 9 6

J1 Eb93G 100 80 97 105

154 Biocatalysis for the Pharmaceutical Industry

rhodochrous strain 2368, producing a solution containing about 50wt% acrylamide with no

detectable acrolein.

Acrylonitrile produced industrially via propylene ammoxidation contains trace amounts of

benzene. When using Pseudonocardia thermophila JCM3095 or Rhodococcus rhodochrous

J-1 asmicrobial NHase catalyst for conversion of acrylonitrile to acrylamide, concentrations of

benzene of �4 ppm produced a significant increase in the reaction rate [16]. Maintaining the

concentration of HCN and oxazole at �5 ppm and <10 ppm respectively produced high-

quality acrylamide suitable for polymerization.

Dietzia natronolimnaios,Dietziamaris andDietzia psychralcaliphila produce aNHase and

amidase useful for converting acrylonitrile to the corresponding amide and carboxylic

acid [17]. For example, Dietzia natronolimnaios NCIMB 41 165 is an alkaliphile that grows

at high pH (10) and in culturemedia containing high salt concentrations (40 g L�1). TheNHase

specific activity (induced during growth with urea) was 44 170mmol acrylonitrile/minute/g

dcw at pH 7 and 15 �C.

8.2.2.2 Butyramide

While the production of acrylamide by NHase is a well-established industrial process, only a

first report exists for the production of butyramide from butyronitrile. Using Rhodococcus

rhodochrous PA-34 (at a loading of 1 g dcw), 595 g butyramide was prepared in quantitative

yield from 60% (v/v) butyronitrile in a pH 7.0, 1 L batch reaction, at 10 �C [18].

8.2.2.3 Nicotinamide

Rhodococcus rhodochrous J1 must be induced to produce the NHase used in a commercial

process for conversion of 3-cyanopyridine to nicotinamide, and when used as a microbial

catalyst, a red pigment from themicroorganism contaminates the product mixture. In addition,

J1 has a low heat stability and is inhibited by the 3-cyanopyridine. Several new biocatalysts

have been identified that address some of the disadvantages associated with the use of J1 in this

process [19]. Amycolatopsis NA40 has a pH optimum of pH 6.5, a temperature optimum

between 35 and 40 �C at pH 7.0, and a KM for 3-cyanopyridine of 41.7mM (20 �C, pH 7.0); the

3-cyanopyridineKM for Rhodococcus rhodochrous J1 NHase is significantly higher (200mM).

Rhodococcus GF270 and GF376 had better heat stability than J1, and could accumulate about

8.5 M and 7.5 M nicotinamide respectively within 20 h using 2.3mg dcw/30mL reaction

(pH 7.0) with sequential addition of 500mM 3-cyanopyridine. A disadvantage for Amyco-

latopsis is that the NHase was inactivated during use at elevated temperatures, and it had low

tolerance to 3-cyanopyridine and nicotinamide. Rhodococcus GF270 had a high KM value

(>200mM) with respect to 3-cyanopyridine. More desirable was a microbial catalyst with

significantly improved temperature stability and a relatively low KM for 3-cyanopyridine.

Rhodococcus sp. FZ4 [20] has a KM value of 80.5 mM for 3-cyanopyridine and a temperature

optimum for NHase activity of 60 �C. A comparison of temperature stability and the

influence of substrate concentration on enzyme activity for these enzymes are presented

in Tables 8.2 and 8.3 respectively.

Corynebacterium glutamicum (CGMCC No.1464) cells immobilized in calcium alginate

beads cross-linked with polyethenimine and glutaraldehyde have been employed for the

production of nicotinamide from 3-cyanopyridine [21]. The reaction was run at 10–15 �C,

Applications of Nitrile Hydratases and Nitrilases 155

where an aqueous 3-cyanopyridine solution was added continuously at a gradually decreasing

rate such that the total weight of 3-cyanopyridine added was 10–25wt% of the reaction

mixture; the concentration of unreacted 3-cyanopyridine at the conclusion of the reaction was

80–500 ppm.

A thermally stable NHase from Comamonas testosteroni 5-MGAM-4D (ATCC

55 744) [22] was recombinantly expressed in Escherichia coli, and the resulting transformant

cells immobilized in alginate beads that were subsequently chemically cross-linked with

glutaraldehyde and polyethylenimine. This immobilized cell catalyst (at 0.5%dcwper reaction

volume) was added to an aqueous reaction mixture containing 32wt% 3-cyanopyridine at

25 �C, and a quantitative conversion to nicotinamide was obtained. The versatility of this

catalyst system was further illustrated by a systematic study of substrates, which included

Table 8.2 Comparison of the thermal stability of NHase activities of Rhodococcus sp. FZ4 and

GF270 to Amycolatopsis sp. NA40 and Rhodococcus rhodochrous J1

Incubation

time (min)

Incubation

temp. (�C)Relative activity (%)a

Rhodococcus

sp. FZ4

Rhodococcus

sp. GF270

Amycolatopsis

sp. NA40

Rhodococcus

rhodochrous J1

15 50 100 100 nd 100

15 60 93 95 nd 80

15 70 2 5 nd 0

60 20 100 100 100 nd

60 30 100 100 95 nd

60 40 100 100 80 nd

60 50 100 100 32 nd

60 60 100 89 0 nd

60 70 6 0 0 nd

and¼Not determined.

Table 8.3 Dependence of Rhodococcus sp. FZ4, Rhodococcus sp. GF270, Amycolatopsis sp. NA40

and Rhodococcus rhodochrous J1 NHase activities on 3-cyanopyridine concentration

3-Cyanopyridine

(% w/v)

Rhodococcus

sp. FZ4aRelative activity (%)

Rhodococcus

sp. GF270aAmycolatopsis

sp. NA40bRhodococcus

rhodochrous J1b

0 100 100 100 100

2.5 100 100 74 ndc

5.0 100 100 56 86

7.5 100 100 47 ndc

10.0 100 100 16 63

aIncubation for 60min.bIncubation for 15min.cNot determined.


acrylonitrile, methacrylonitrile, adiponitrile, butyronitrile, 3-hydroxyvaleronitrile, and gly-

colonitrile. Typically, reactions were completed in 1 h and reached 100% conversion even at

nitrile concentrations up to 3 M [23].

Unimmobilized Corynebacterium propinquum (CGMCC No. 0886) cells containing a

cobalt-dependent NHase were employed in either batch or continuous reactions for the

production of nicotinamide from 3-cyanopyridine [24]. In the continuous process, membrane

filtration separated precipitated product (�5wt%) and the microbial cell catalyst from the

reaction mixture, where the catalyst was then recovered and returned to the reactor; using a

continuous addition of aqueous 3-cyanpyridine to maintain substrate concentration at �20%

(w/v), a final conversion of �99% was obtained.

8.2.2.4 a-Hydroxycarboxylic Acids/Amides

The activities of NHases from Rhodococcus sp. Adp12 and Gordonia sp. BR-1 strains have

been partially characterized [25]. In reactions that catalyze the hydration of a-hydroxynitrilessuch as lactonitrile or glycolonitrile, the substrate can dissociate to produce HCN and the

corresponding aldehydes. HCN can inhibit and/or inactivate NHase, and it was determined that

these two enzymes remain active in the presence of cyanide ion at concentrations up to 20mM.

The dependence of theNHase activity of cell-free extracts ofRhodococcus rhodochrous J1 and

Gordonia sp. BR-1 on cyanide ion concentration is illustrated in Figure 8.1, demonstrating the

improved cyanide stability of BR-1 NHase relative to that of J1.

In instances where the nitrile has poor solubility in water, the addition of an organic co-

solvent improved solubility and reaction rate. The recovered activity of Rhodococcus Adp12

NHase after 10min in reaction mixtures containing 10–40% (v/v) organic co-solvent at 20 �Cwas determined (Figure 8.2), demonstrating stability in a single-phase reaction mixture

containing methanol or ethanol, or in a two-phase aqueous–organic mixtures containing ethyl

acetate or n-hexane.

Rhodococcus equi XL-1 has also been demonstrated to have superior stability in solutions

containing up to 20mM HCN when compared with several Rhodococcus erythropolis

0

20

40

60

80

100

120

2520151050

KCN (mM)

rela

tive

activ

ity (

%)

Figure 8.1 Dependence of the NHase activity of cell-free extracts of Rhodococcus rhodochrous J1 (~)

and Gordonia sp. BR-1 (&) on cyanide ion concentration


microbial NHase catalysts [26], as shown in Table 8.4 for reactions run by first adding 0–20mM

KCN to the reaction mixture containing only enzyme and buffer. After the solution was

incubated at 20 �C for 30min, the enzyme reaction was started with the addition of

3-cyanopyridine. 2-Hydroxy-4-methylthiobutyroamide (HMBAm, useful as a feed additive

as amethionine substitute)was produced by suspendingRhodococcus equiXL-1wet cells (1 g)

in 25 g of phosphate buffer (0.05 M, pH 6.5) containing 2-hydroxy-4-methylthiobutyronitrile

(HMTBN; 0.2wt%) and NaCl (0.34wt%). The resulting mixture was incubated at 30 �C for

43 h with stirring, adding aliquots of HMTBN during the reaction. When the initial HMTBN

concentration was 537mM, the concentration of cyanide ion in the reaction solution was

2.33mM. A total HMBAm concentration of 75 g L�1 was produced.

Rhodococcus erythropolis NCIMB 11 540 has been employed as biocatalyst for the

conversion of (R)- or (S)-cyanohydrins to the corresponding (R)- or (S)-a-hydroxycarboxylicacids with an optical purity of up to >99% enatiomeric excess (ee) [27–29]; the chiral

cyanohydrins can separately be produced using hydroxynitrile lyase fromHevea braziliensis or

from Prunus anygdalis [30]. Using the combined NHase–amidase enzyme system of the

Rhodococcus erythropolisNCIMB11 540, the chiral cyanohydrinswere first hydrolyzed to the

0

20

40

60

80

100

120

50403020100

co-colvent (%)

rela

tive

activ

ity (

%)

Figure 8.2 Residual activity ofRhodococcusAdp12NHase after 10min in reactionmixtures containing

10–40% (v/v) methanol (&), ethanol (~), ethyl acetate (.) and n-hexane (~) at 20 �C

Table 8.4 Residual activity of Rhodococcus NHases after incubation in 1–20mM cyanide for 30min

Strain KCN (mM)

0 1 5 10 15 20

Rhodococcus equi XL-1 100 100 100 98 94 89

Rhodococcus erythropolis IFO12 539 100 51 13 9 5 4




Rhodococcus erythropolis ATCC11 048 100 54 30 18 10 7

Rhodococcus erythropolis ATCC33 278 100 61 36 42 14 11


chiral hydroxyamide, which was subsequently converted by the amidase into the correspond-

ing chiral a-hydroxycarboxylic acid. This microbial biocatalyst was used to convert (R)-2-

chloromandelonitrile (>99% ee) to (R)-2-chloromandelic acid with a product ee of >99%

(crude yield of 98%). In addition to the direct use of NHase–amidase of Rhodococcus cells, the

enzymes have both been overexpressed in Escherichia coli in active form; in particular, the

amidase can be expressed at levels significantly greater than found in the native Rhodococcus

cells.

3-Hydroxyvaleric acid can be used as a substitute for e-caprolactone in the preparation of

highly branched copolyesters [31]. Immobilization ofComamonas testosteroni 5-MGAM-4D

ATCC 55 744 cells in alginate beads, followed by chemical cross-linking with glutaraldehyde

and polyethylenimine, produced a catalyst with high specific activity and productivity for the

conversion of 3-hydroxyvaleronitrile acid, 3-hydroxybutryonitrile, and 3-hydroxypropioni-

trile to the corresponding 3-hydroxycarboxylic acids in�99% yields [32,33]. In a series of 85

consecutive batch reactions with biocatalyst recycle, 670 g 3-hydroxyvaleric acid/g dcw was

produced with an initial volumetric productivity of 44 g 3-HVA/(L h) and a final product

concentration in each batch reaction of 118 g L�1.

8.2.2.5 4-Methylthio-a-Hydroxybutryamide

Isolated polynucleotide clusters from Rhodococcus opacus which encode four polypeptides

possessing the activities of aNHase (a andb subunits), an auxiliary protein P15K that activates

the NHase, and a cobalt transporter protein were expressed in Escherichia coli DSM 14 459

cells [34]. Methionine nitrile was added continuously to a suspension of the transformant cells

(5.6% w/v of wet cells) in phosphate buffer (50mM, pH 7.5) at 20 �C, at a rate where the nitrileconcentration did not exceed 15 g L�1 while maintaining the pH constant at 7.5. After 320min,

the nitrilewas completely converted into amide, corresponding to a final product concentration

of 176 gL�1. 4-Methylthio-a-hydroxybutyramide is readily hydrolyzed with calcium hydrox-

ide, where the calcium salt of 4-methylthio-a-hydroxybutyric acid (MHA) can be directly used

as a nutritional supplement in animal feed as an alternative to methionine or MHA.

8.2.2.6 Glycine

High-purity glycine, useful as a food additive and as a raw material for synthesizing pharma-

ceuticals, agricultural chemicals, and detergents, was produced by hydrolysis of glycinonitrile

using a microbial catalyst having nitrilase activity, or a combination of NHase and amidase

activities [35]. By running the reaction in the absence of air or oxygen (limiting oxygen to 5 ppm

or less, preferably less than 0.01 ppm using a continuous nitrogen purge) in the presence of a

slight excess of ammonia, and by using a minimal concentration of added buffer to control pH,

the production of organic impurities that inhibited themicrobial enzymewas reduced, leading to

an improvement in product purity. Microbial nitrile-hydrolyzing catalysts included Acineto-

bacter sp.AK226,RhodococcusmarisBP-479-9,CorynebacteriumnitrilophilusATCC21419,

Alcaligenes faecalis IFO 13 111, Mycobacterium sp. AC777, Rhodopseudomonas spheroides

ATCC 11167 and Candida tropicalis ATCC 20311. Acinetobacter sp. AK226 was preferred,

having stable enzyme activity at temperatures up to 50 �C, and producing as much as 461 g

product/g dcw at a glycine production of 19 g/(g dcwh). The addition of reducing agents such as

sodium sulfite, ascorbic acid, or L-cysteine resulted in a significant reduction in by-product


impurities and discoloration of glycine,where glycineyield andpuritywere as high as 100%and

99.99% respectively after recrystallization.

8.2.2.7 3,3,3-Trifluoro-2-Hydroxy-2-Methylpropionic Acid

(S)-3,3,3-Trifluoro-2-hydroxy-2-methylpropionic acid (S)-2,2-HTFMPS) is an important in-

termediate for the preparation of therapeutic amides [36,37]. The racemic amide (S)-3,3,3-

trifluoro-2-hydroxy-2-methyl-propionamide ((R,S)-2,2-HTFMPA) was first prepared in quan-

titative yield by the hydration of 2-hydroxy-2-methyl-3,3,3-trifluoromethylpropionitrile using

a mutant of Rhodococcus equi TG 328-2 that lacked amidase activity. The preparation of (R)-

2,2-HTFMPS and (S)-2,2-HTFMPA from the racemic amide was subsequently accomplished

using Klebsiella oxytoca PRS1, Klebsiella oxytoca PRS1K17, Klebsiella planticula ID-624,

Klebsiella pneumoniae ID-625, and an Escherichia coli transformant expressing the stereo-

specific amidohydrolase activity-derived Klebsiella oxytoca PRS1K17 [38,39]. The prepara-

tion of (S)-2,2-HTFMPS and (R)-2,2-HTFMPA from the racemic amide was performed using

Pseudomonas sp. DSM 11 010, Rhodococcus opacus ID-622, Arthrobacter ramosus ID-620,

and Bacillus sp. ID-621. For example, a Klebsiella oxytoca PRS1 cell suspension was used to

hydrolyze 1.0 wt% (R,S)-2,2-HTFMPA in 0.05 M phosphate buffer (pH 8.0) at 40 �C; after5.5 h, (R)-2,2-HTFMPAwas completely converted into the corresponding acid in 100% ee and

48% yield (Figure 8.3).

8.2.2.8 (S )-3-(Thiophen-2-Ylthio) Butanoic Acid

After all attempted chemical methods of nitrile hydrolysis failed, a suitable nitrilase catalyst

was identified for the conversion of (S)-3-(thiophen-2-ylthio) butanenitrile to the correspond-

ing acid, a building block for Merck�s MK-00 507 carbonic anhydrase inhibitor, with trade

name Dorzolamine (Figure 8.4). A screen of 53 strains revealed 12 that provided >75%

conversion of the nitrile.Of these, oneyielded the desired acid product at high conversion levels

and one yielded the amide product (strain apparently has NHase but lacks amide hydrolase

activity). These strains are Brevibacterium A4 and Brevibacterium R312 pYG811b (reclassi-

fied as Rhodococcus erythropolis) respectively. The recombinant Rhodococcus erythropolis

strain (formerly Brevibacterium R312 pYG811b) was applied at pH 7.0, 30 �C at a substrate

loading of 5mgmL�1 (although up to 30mgmL�1 was shown to be tolerated) in the presence

of 3% acetonitrile as co-solvent (acetonitrile was confirmed not to be a substrate of this

biocatalyst) to generate (S)-3-(thiophen-2-ylthio) butanoic acid on gram scale. Under these

conditions, 60% yield of product was generated after 5 days� incubation. Contaminating amide

CF3

Klebsiella oxytoca PRS1

50 mM KH2PO4 (pH 8.0),40 oCO

H2NH3 OHC

CF3

O

H2NH3C OH

CF3

O

HOH3 OHC

+

(R,S )-2,2-HTFMPA (S)-2,2-HTFMPA (R)-2,2-HTFMPS,48% yield, 100% ee

Figure 8.3 Preparation of (R)-2,2-HTFMPS and (S)-2,2-HTFMPA from racemic 3,3,3-trifluoro-2-

hydroxy-2-methyl-propionamide using Klebsiella oxytoca PRS1


was reported at 5%. Since the stereogenic center was set earlier in the synthesis, an absolute

requirement for enantioselectivity of the biocatalyst was not necessary in this example [40].

8.2.2.9 Malonic Acid Derivatives

TheNHase and amidase fromRhodococcus rhodochrous IFO 15 564was studied using a series

of a,a-disubstituted malononitriles. This amidase preferentially hydrolyzes the pro (R) amide

of the prochiral di-amide, which is an intermediate resulting from the nonenantiotopic NHase

activity on the dinitrile substrate. This transformation was combined with a Hofmann

rearrangement to generate a key precursor of (S)-methyldopa in 98.2% ee and 95% yield

(Figure 8.5) [41].

8.2.2.10 Cyclopropane Carboxylic Acid Derivatives

By screening 53 Rhodococcus and Pseudomonas strains, an NHase–amidase biocatalyst

system was identified for the production of the 2,2-dimethylcyclopropane carboxylic acid

precursor of the dehydropeptidase inhibitor Cilastatin, which is used to prolong the antibacte-

rial effect of Imipenem. A systematic study of the most selective of these strains, Rhodococcus

erythropolis ATCC25 544, revealed that maximal product formation occurs at pH 8.0 but that

ee decreased above pH 7.0. In addition, significant enantioselectivity decreases were observed

above 20 �C. A survey of organic solvent effects identified methanol (10% v/v) as the

SS

CN

SS

COOH

SS

O H

SS

NCH2CH3

O OSO2NH2

Dorzolamine

nitrilase

Figure 8.4 Nitrilase-catalyzed conversion of (S)-3-(thiophen-2-ylthio)butanenitrile to (S)-3,3,3-

trifluoro-2-hydroxy-2-methyl-propionamide, an intermediate in the synthesis of carbonic anhydrase

inhibitor Dorzolamine�

O

O CNCN

HO

HO COOH

NH2

O

O COOHCNH2

OCH2N2

O

O COOCH3

CNH2

O

O

O COOCH3

NHCOOCH3

R. Rhodochrous

Br2, MeONa/MeOHHofmann Rearrangement

(S)-α-methyldopa

98.2% ee, 95% overall yield

Figure 8.5 Conversion of 2-(1,3-benzodioxol-5-ylmethyl)-2-methyl-propanedinitrile to (R)-a-(aminocarbonyl)-a-methyl-1,3-benzodioxole-5-propanoic acid using the non-enantiotopicNHase activity

and enantioselective amidase activity of Rhodococcus rhodochrous IFO 15564


co-solvent providing the greatest enantioselectivity. Under the optimized conditions reported,

the (S)-2,2-dimethylcyclopropane carboxylic acid was produced with an observed ee of 82%

and with overall conversion yield of 45% (Figure 8.6) [42].

NHase from Rhodococcus. sp. AJ270 was isolated, purified, and applied to the enantiose-

lective transformation of a series of cyclopropane carbonitriles. Amides with moderate ee were

isolated from conversion ofmany of the cyclopropane substrates, to yield the amides: trans-(1R,

2R)-3-phenylcyclopropane carbonitrile (49% conv. 22.7% ee), trans-(1S, 3S)-2,2-dimethyl-3-

phenylcyclopropanecarbonitrile (40% conv. 84.7% ee), trans-(1R, 3R)-2,2-dibromo-3-phenylcy-

clopropanecarbonitrile (11.6% conv. 83.8% ee), cis-(1R, 2S)-3-phenylcyclopropanecarbonitrile

(25.8% conv. 95.4% ee), and cis-(1R, 2S)-2,2-dimethyl-3-phenylcyclopropanecarbonitrile (7.9%

conv. 3.2% ee) [43].

8.2.2.11 Oxirane Carboxylic Acid Derivatives

Rhodococcus sp. AJ270 was applied to the transformation of a number of racemic cis- and

trans-3-aryl-2-methyloxiranecarbonitriles (Figure 8.7). In all cases, the NHase activity

proceeded very rapidly and with poor enantioselectivity. In contrast, the amidase activity

was strongly dependent upon substrate structure. In general, the biocatalyst displays a strong

preference for the unsubstituted phenyl side chain or para-substituted phenyl side chain

compared with ortho- or meta-, and this is manifest both with respect to observed conver-

sion and rate and also observed enantioselectivity. In contrast, the biotransformations of

CN

H3CH3C

HHOOC

H3CH3C

CNH

H3CH3C

pH 7, 20 C, 64 h10% v/v methanol

R. erythropolis

82% ee, 45 % yield

(S)-2,2-dimethylcyclopropane carboxylic acid

+

Figure 8.6 Preparation of (S)-2,2-dimethylcyclopropane carboxylic acid, a precursor of Cilastatin

OCNAr

CH3

OCONH2

Ar

CH3

OCONH2

Ar

CH3

OCOOHAr

CH3

Rhodococcus sp. AJ270phosphate buffer, pH 7.25, 30 C

racemic amideracemic nitriletrans-2-methyl-3-phenyloxiranecarbonitrile

2S,3R-enantioselective amidase

2R, 3S-amide 2S, 3R-acid

+

Summary of selected biotransformations Ar = C6H5 45% yield, > 99.5 % ee Ar = 4-F -C6H5 31% yield, > 99 % ee Ar = 4-Cl-C6H5 49% yield, > 99.5 % ee Ar = 3-Cl-C6H5 32% yield, > 20 % ee Ar = 2-Cl-C6H5 40% yield, < 5 % ee Ar = 4-Me-C6H5 31% yield, > 99.5 % ee Ar = 2-Me-C6H5 44% yield, < 5 % ee Ar = 3,4-OCH2O-C6H5 32% yield, 50 % ee

Figure 8.7 Transformation of trans-3-aryl-2-methyloxiranecarbonitriles using the combined NHase

and amidase activities of Rhodococcus sp. AJ270


cis-2-methyl-3-phenyloxiranecarbonitrile substrates proceeded sluggishly. In addition, the

authors report that, for the 2,3-dimethyl-3-phenyloxiranecarbonitrile series of substrates

(either cis or trans),much lower enantioselectivitieswere observed. The 2R,3S-amide products

thus produced may be further transformed to access a-methylated serine and isoserine

derivatives [44]. In a subsequent study, the authors established that immobilization of this

biocatalyst in alginate capsules permitted efficient reuse of the catalyst and tolerated 5%

acetone or methanol, but not ethyl acetate [45].

8.2.2.12 NHases for Bioremediation

Three cyanide-degrading nitrilases were recently cloned and purified and their kinetic profiles

were evaluated in order to better understand their applicability to cyanide bioremediation.

CynD from Bacillus pumilus C1 and DyngD from Pseudomonas stutzeri exhibit fairly broad

pH profiles with>50% activity retained across pH 5.2 to pH 8.0 while the CHT (NHase) from

Gloeocercospora sorghi exhibited a more alkaline pH activity profile with almost all of its

activity retained at pH 8.5, slightly lower thermal tolerance, and quite different metal tolerance

compared with the two bacterial enzymes [46].

2,6-Dichlorobenzonitrile (dichlobenil) is the active ingredient in herbicides Prefix G and

Casoron G. In soil, dichlobenil is degraded to the persistent metabolite 2,6-dichlorobenzamide

(BAM) by several common soil bacteria. BAM is soluble and readily leached into groundwater

and has a low to moderate toxicity with an LD50 of 1144–2300mgkg�1 in mice. Analysis of a

series of common soil bacteria, including several known to express nitrilases, did not reveal any

which could degrade dichlobenil to its diacid. Variovorax sp. is known to degrade the

nonhalogenated analogue benzamide. Apparently, the steric hindrance created by the ortho-

chlorosubstituents makes this substrate unacceptable to any amidases or nitrilases expressed by

common soil bacteria tested thus far [47].

Similarly, the selective herbicides, bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and

ioxynil (3,5-diiodo-4-hydroxybenzonitrile) are degraded by soil bacteria to their corresponding

amideproducts3,5-dibromo-4-hydroxybenzamide(BrAM)and3,5-diiodo-4-hydroxybenzamide

(IAM) but are not further degraded to the corresponding acids. The identification of amidases or

nitrilases able to effect these transformations, in a soil bacterium, would be of value as a

bioremediation agent [48].

8.3 Nitrilase

8.3.1 New Nitrilases

8.3.1.1 Bradyrhizobium japonicum

Bradyrhizobium japonicum USDA110 is a Gram-negative nitrogen-fixing microbe that

expresses a nitrilase (bll6402) whose function may be to detoxify and utilize hydroxynitriles

produced in the metabolism of cyanogenic glycosides. The nitrilase gene was cloned and

expressed in Escherichia coli [49], and the nitrilase had high activity toward mandelonitrile,

with a Vmax andKm of 44.7 Umg�1 and 0.26mM respectively. Similarly, bll6402 also provided

high conversion of phenyl hydroxyl acetonitrile, but in neither case was any enantioselectivity

observed [50]. Despite the apparent lack of selectivity for a-substituted nitriles, nitrilase

bll6402 catalyzed the enantioselective hydrolysis of aromatic b-hydroxynitriles to give


(S)-enriched b-hydroxycarboxylic acids with recovery of (R)-enriched b-hydroxynitriles(Table 8.5) [51,52]. It also selectively hydrolyzed some a,v-dinitriles to v-cyanocarboxylicacids [53]; for example, nitrilase bll6402 hydrolyzes 1-cyanocyclohexaneacetonitrile to

1-cyanocyclohexaneacetic acid (88% isolated yield), a precursor for the antidepressant

gabapentin (Figure 8.8). This same gabapentin precursor has also been prepared using

microbial nitrilase catalysts such as Acidovorax facilis 72W and Escherichia coli SS1001

(a transformant expressing the Acidovorax facilis 72W nitrilase) [54], where quantitative

conversion of the dinitrile with 100% regioselectivity to the desired product was obtained.

8.3.1.2 Exophiala oligosperma

AnewnitrilasewasdiscoveredfromtheblackfungusExophialaoligosperma (establishedby18S

rRNA) by growth enrichments performedwith glucose and phenylacetonitrile as the sole carbon

and nitrogen sources respectively under acid conditions. The novel nitrilase could convert

phenylacetonitrile to phenylacetamide. Resting cells ofExophiala oligosperma exhibit nitrilase

activity in the range pH 2–9, and have an activity optimum at pH 8–9. In addition, single chloro-

and hyroxy-phenylacetonitrile derivatives were also converted to the corresponding acids at

comparable rates. In contrast to nitrilases from the fungi Aspergillus nidulans and Fusarium

Table 8.5 Enantioselective hydrolysis of b-hydroxynitriles catalyzed by nitrilase bll6402

b-Hydroxynitrile Recovered nitrile (R)-1 Product acid (S)-2 Eb

CNX

HO HCO2H

X

HO H

yield (%)a ee (%) yield (%)a ee (%)

X¼ 4-H 41 53 36 48 5

X¼ 4-F 37 74 38 60 9

X¼ 4-Cl 40 53 32 65 8

X¼ 4-CH3 35 76 40 42 5

X¼ 4-OCH3 57 66 27 90 43

X¼ 2-OCH3 46 75 36 43 5

X¼ 3-OCH3 46 67 35 91 52

X¼ 2-Cl 40 75 32 84 27

X¼ 2,4-Cl2 42 37 39 59 13

aIsolated yield.bEnantiomeric ratio.

CN

CN

CN

CO2H CO2H

NH2

gabapentin

nitrilase bll6402

100 mM KH2PO4 (pH 7.2)30 oC; 88% yield

Figure 8.8 Nitrilase bll6402-catalyzed hydrolysis of 1-cyanocyclohexaneacetonitrile to 1-cyanocyclo-

hexaneacetic acid, a precursor for the antidepressant gabapentin


solanii, this nitrilasewas not effective in hydrolysis of benzonitrile. Since phenylacetamidewas

not observed as an intermediate in the conversion of phenylacetonitrile, and since the amide

substratewas hydrolyzed at a rate threefold less than the nitrile substrate, the authors concluded

that the activity is a true nitrilase rather than nitrile hydrolase–amidase combination [55].

8.3.1.3 Streptomyces sp.

Thermally stable nitrilase from Streptomyces sp. MTCC 7546 was induced by benzonitrile

enrichment. While discovered by induction with aromatic nitrile, the enzyme was shown to

exhibit a strong preference for aliphatic nitriles, with as high as 30-fold greater activity with

aliphatic substrates compared with benzonitrile. The enzyme displays optimal activity at pH 7

and 50 �C [56].

8.3.1.4 Aspergillus niger

Anovel nitrilasewas purified fromAspergillus nigerK10 cultivated on 2-cyanopyridine. It was

found to be homologous to a putative nitrilase from Aspergillus fumigatusAf293. The nitrilase

exhibited maximum activity at 45 �C and pH 8.0 with much less activity observed at slightly

acid pH. Its substrate preference was for 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene,

thio-phen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile.

(�)-2-Phenylpropionitrile was only poorly converted by this enzyme and with minimal

enantioselectivity. The enzyme was shown to be multimeric (>650 kDa) and be stabilized

in the presence of sorbitol and xylitol [57].

8.3.1.5 Pyrococcus abyssi

A nitrilase from the hyperthermophile Pyrococcus abyssi, which exhibits optimal growth at

100 �C, was cloned and overexpressed. Characterization of this nitrilase revealed that it is

operational as a dimer (rather than the more common multimeric structure for nitrilases), with

optimal pHat7.4andoptimal apparent activity at 80 �CwithTm(DSC) at 112.7�C.Thesubstrate

specificity of the nitrilase is narrowand it does not accept aromatic nitriles. The nitrilase converts

the dinitriles fumaronitrile and malononitrile to their corresponding mononitriles [58].

8.3.1.6 Pseudomonas putida

An enantioselective nitrilase from Pseudomonas putida isolated from soil cultured with 2mM

phenylacetonitrile was purified and characterized. This enzyme is comprised of 9–10 identical

subunits each of 43 kDa. It exhibits a pH optimum at 7.0 and a temperature optimum at 40 �C(T1/2¼ 160min) and requires a reducing environment for activity. This nitrilase was shown

to have an unusually high tolerance for acetone as co-solvent, with >50% activity retained

in the presence of 30% acetone. The kinetic profile of this nitrilase reveals: KM¼ 13.4mM,

kcat/KM¼ 0.9 s�1mM�1 for mandelonitrile, KM¼ 3.6mM, kcat/KM¼ 5.2 s�1mM

�1 for

phenylacetonitrile, and KM¼ 5.3mM, kcat/KM¼ 2.5 s�1mM�1 for indole 3-acetonitrile. Pre-

liminary analysis of this enzymewith 5mMmandelonitrile revealed formation of (R)-mandelic

acid with 99.9% ee [59].

A systematic study of the substrate specificity profile of this nitrilase (Table 8.6) illustrated

that arylacetonitriles, including phenylacetonitrile derivatives indole-3-acetonitrile and


Table 8.6 Summary of substrate specificity ofPseudomonas putida nitrilase (data is not comprehensive)

Structural class Structure Substrate Relative activity (%)

Aliphatic nitrile CH3�CN Acetonitrile 2.1

CH3(CH2)2�CH2CN Valeronitrile 10.5

Unsaturated nitriles CH2¼CH�CN Acrylonitrile 4.0

CH2¼CH�(CH2)3�CN 6-Hexene nitrile 8

Dinitrile NC�(CH2)�CN Malononitrile 6.8

NC�(CH2)4�CN Adiponitrile 17.2

Cyclic nitrile

CN

CN

Cyclopropane carbonitrile 4.1

Cyclohexane carbonitrile 12.5

Aromatic nitriles

CN

OH, H

Benzonitrile 12.7

2-Hydroxy benzonitrile 2.3

4-Hydroxy benzonitrile 23.1

Heterocyclic nitriles

NCN 2-Cyanopyridine 136.7

HN

NC( )n n=1,2

Indole 3-carbonitrile

(n¼ 1)

15.7

Indole 3-acetonitrile

(n¼ 2)320

SCN 2-Thiophene acetonitrile 136.7

Aryl acetonitriles CN

OH

Mandelonitrile 100

CN

NH2

Phenyl glycinenitrile 192.5

CN Phenyl acetonitrile 342.3

CN

OH, Cl

4-Hydroxy phenyl acetonitrile 435

2-Chloro phenyl acetonitrile 76.1

Phenyl-substituted

aliphatics

CN3-Phenyl propionitrile 22.8

CN4-Phenyl butyronitrile 7.6


2-thiopheneacetonitrile, are suitable substrates. Interestingly, para substitution of the phenyl

ring dramatically increased activity, such thatp-chloro andp-amino phenylacetonitrile exhibited

higher activity than phenylacetonitrile. However, ortho substituents decreased activity. In

addition, longer chain phenyl nitriles were less preferred by the enzyme: phenylacetonitrile

> 3-phenyl propionitrile> 4-phenyl butyronitrile. Substitutionofphenylacetonitrileat2-position

(for example, �OH, �NH2, �CH3) also resulted in a decrease in activity.

8.3.1.7 Alcaligenes sp.

A newly isolated nitrilase producer, Alcaligenes sp. ECU0401, was isolated from soil using

acetonitrile as the sole nitrogen source. (R)-Mandelic acid, the chiral building block for

production of anti-obesity agents, antitumor agents, penicillins, semisynthetic cephalosporins

and used as a chiral resolving agent, was produced from racemic mandelic acid at pH 7.0 at

20mM substrate loading in 12% isolated yield and 99.9% ee using this biocatalyst. Mande-

lamide was not observed as a side product [60].

8.3.2 Applications

8.3.2.1 Cyanobenzoic Acids

Rhodococcus sp. ATCC 39 484 converts o-phthalonitrile, isophthalonitrile, or terephthaloni-

trile to o-cyanobenzoic acid,m-cyanobenzoic acid or p-cyanobenzoic acid respectively as the

major reaction product, while at the same time producing the corresponding cyanobenzamide

and phthalic acid monoamide as unwanted by-products [61]. By-product formation was found

to be dependent on an NHase–amidase hydrolysis pathway that coexists competitively with a

nitrilase pathway in the microbial catalyst. Chemical mutagenesis was employed to produce a

variant of the parent strain (Rhodococcus sp. SD826) lacking the by-product-forming pathway,

whereby cyanobenzamide and phthalic acid monoamide production were significantly re-

duced. A comparison of reaction products obtained from isophthalonitrile using the parent

strain and the SD826 variant is shown in Table 8.7, demonstrating a reduction in production of

m-cyanobenzamide and isophthalic acidmonoamide of 85% and 82% respectively when using

the SD826 strain; a similar improvement in selectivity to cyanoacid was obtained with

terephthalonitrile. A microbial catalyst with improved nitrilase-specific activity relative to

the SD826 variant was produced by the cloning and expression of the nitrilase gene in an

Escherichia coli transformant.

Table 8.7 Reduction of byproduct formation by elimination ofNHase/amidase pathway inRhodococcus

sp. ATCC 39 484

Strain m-Cyanobenzoic acid m-Cyanobenzamide Isophthalic acid

monoamide

Conc.

(%)

Conversion

(mol%)

Conc.

(%)

Conversion

(mol%)

Conc.

(%)

Conversion

(mol%)

ATCC39 484 5.638 98.22 0.023 0.40 0.087 1.34

SD826 5.721 99.67 0.003 0.06 0.016 0.24


The regioselectivity of a Rhodococcus rhodochrous nitrilase has been demonstrated for the

conversion of 5-fluoro-1,3-dicyanobenzene to 5-fluoro-3-cyano-benzoic acid [62]. The nitri-

lasewas expressed in anEscherichia coli transformant, and a cell-free extract was employed as

catalyst (0.14wt% cell-free extract) in 0.1 M sodium phosphate buffer (pH 7.2) at 25 �Ccontaining 0.18 M 5-fluoro-1,3-dicyanobenzene. After 72 h, the conversion was>98% and the

reactionwas stopped by addition of phosphoric acid (pH2.4) to yield 5-fluoro-3-cyano-benzoic

acid as a crystalline product (97% isolated yield).

8.3.2.2 Glycolic Acid

When used for the treatment of recalcitrant melasma [63], and as amonomer in the preparation

of polyglycolic acid for dissolvable sutures [64], drug-delivery materials [65,66], and gas-

barrier packaging materials [67], a high-purity glycolic acid is required. A chemoenzymatic

process for the production of high-purity glycolic acid has been developed (Figure 8.9), starting

with the reaction of formaldehyde and hydrogen cyanide to produce glycolonitrile in >99%

yield and purity [68]. The resulting aqueous glycolonitrilewas subsequently convertedwithout

further purification to ammonium glycolate using a high-activity biocatalyst based on

Acidovorax facilis 72W nitrilase, where protein engineering and optimized protein expression

in an Escherichia coli transformant host were used to improve microbial nitrilase specific

activity by 33-fold compared with the wild-type culture [69]. A biocatalyst productivity of

>1000 g glycolic acid/g dcw was achieved using a glutaraldehyde/polyethylenimine cross-

linked carrageenan-immobilized Escherichia coli MG1655 transformant expressing the

Acidovorax facilis 72W Phe168Val nitrilase mutant, where 3.2 M ammonium glycolate was

produced in consecutive batch reactions with biocatalyst recycle, or in a continuous stirred-

tank reactor [70–73]. Direct conversion of the unpurified ammonium glycolate product

solution to high-purity (>99% pure) aqueous glycolic acid was accomplished by fixed-bed

ion exchange (IEX). Glycolic acid has also been produced from glycolonitrile using microbial

biocatalysts such as Acinetobacter sp. AK226 [74], Corynebacterium propinquum [75], and

Brevibacterium casei (CGMCC No. 0887) [76].

8.3.2.3 2-Hydroxy-4-Methylthiobutyrate Ammonium Salt

The nitrile-hydrolyzing activity ofArthrobacter spp. NSSC 104was shown to be resistant to the

suppressing effect of a-hydroxy nitriles such as lactonitrile and HMTBN, and accumulated the

corresponding a-hydroxy acid ammonium salt at a high concentration [77]. HMTBN (200mM)

was added to a suspension of Arthrobacter spp. NSSC 104 cells (4% dcw) in phosphate buffer

(0.1 M, pH7.5) and mixed at 30 �C; seven more additions of the same amount of HMTBNwere

addedat1 hintervals, thenafurthereightadditionsmadeat1.5 hintervalsoveratotalreactiontime

of 19 h. At completion of the reaction, the concentration of 2-hydroxy-4-methylthiobutyrate

HCHO + HCN H2CHO

CN H2CHO

CO

O- NH4+ H2C

HOCO

OHNaOH

> 99%> 99%

nitrilase,H2O IEX

Figure 8.9 Chemoenzymatic process for production of high-purity glycolic acid employingAcidovorax

facilis 72W Phe168Val nitrilase


ammonium salt (HMTBS)was 49wt% (96% yield). In a series of consecutive batch reactions at

30 �Cwithrecycleof theArthrobacterspp.NSSC104unimmobilizedcells(3.2%dcw),HMTBN

wascontinuously fed to thecell suspension inwater (noaddedbuffer,pHmaintainedat7.4–7.6by

addition of aqueous ammonia) to produce HMTBS at 36wt% (96–97% yield) in each of 10

consecutive batch reactions. The gene coding for the nitA nitrilase from Arthrobacter sp.

NSSC104 has been cloned and expressed in Escherichia coli [78].

The conversion of HMTBN toHMTBS has been performed using an immobilizedmicrobial

cell catalyst [79]. An Escherichia coliWstrain transformant (BIOCAT 714) that expressed the

Alcaligenes faecalis ATCC 8750 nitrilase was suspended in phosphate buffer (pH 8.0) (12%

dcw final concentration), then glutaraldehyde (6wt% solution, 0.5wt% final concentration)

and polyazetidine (Kymene 617 solution, 12.5 wt%, 2.4wt% final concentration) were

sequentially added to the cell suspension, and the resulting mixture sprayed onto 2.0mm

alumina beads. The resulting catalyst contained 25.5% byweight of dry cells, and the thickness

of the coating was 330mm. The activity of this catalyst was 0.56 kg of HMTBN converted to

HMTBS per hour and per kilogram of catalyst (25 �C, pH 6.6, 0.1 M HMTBN). The im-

mobilizedmicrobial catalyst was charged to a thermostatic column reactor maintained at 35 �Cand fitted with a pump via a recirculation loop, and a 95% conversion of HMTBN to HMTBS

(25wt% of product solution) was achieved. The immobilized catalyst half-life in the presence

of 0.2 M HMTBN was 30 h. An electrodialysis unit and a means of concentrating the final

product of the reaction have also been described [80].

8.3.2.4 Acrylic Acid

A Corynebacterium propinquummicrobial cell catalyst was employed to convert acrylonitrile

to ammonium acrylate, where the final concentration of product was 10–20% and the

concentration of unconverted acrylonitrilewas<30 ppm [81]. The ammonium acrylate solution

was concentrated to 40–60%by falling film evaporation, the resulting solution acidified, and the

acrylic acid extracted with diethyl ether at 0–10 �C to obtain high-purity acrylic acid.

Acinetobacter sp. AK226 microbial cells were immobilized in polyacrylamide gel cross-

linked with N,N0-methylene bisacrylamide, and the resulting nitrilase biocatalyst was em-

ployed for the conversion of acrylonitrile to ammonium acrylate [82]. The initial catalyst

specific activity was 74 g ammonium acrylate/(g dcw h) (30 �C, pH 7). By controlling the feed

rate so as to maintain the concentration of acrylonitrile (stabilized with p-methoxyphenol

(40 ppm) as polymerization inhibitor) in the reaction mixture at <1.5wt% (preferably �1wt

%) over the course of the reaction, a final concentration of 40wt% ammonium acrylate was

produced, with a catalyst productivity of at least 500 g ammonium acrylate/g dcw. The

resulting ammonium acrylate was polymerized with N,N0-methylenebisacrylamide in an

aqueous solution containing glycerin and Rongalit to produce a water-absorbing polymer

with high water-holding capacity (79 g water and 35 g water per gram of polymer under

unpressurized and pressurized conditions respectively) [83].

8.3.2.5 2-Chloromandelic Acid

The nitrilase activity of Arthrobacter sp. F-73 retains substantial activity in aqueous

solutions containing a significant concentration of organic co-solvent [84]. More than

10% of nitrilase activity remains at acetone concentrations up to 60%, whereas no activity


was measured with acetone concentrations of 30% or higher for the nitrilase activity of

Alcaligenes faecalis JM3 (Table 8.8). Improvement in the enantioselectivity for conversion

of 2-chloromandelonitrile to 2-chloromandelic acid was observed when using 20% (v/v) of

acetone, dimethylformamide (DMF), 1,3-propanediol, or tetrahydrofuran (THF); 40mM

substrate; 22 h at 30 �C in 100mM phosphate buffer (pH 8.0)) (Table 8.9) when compared

with the absence of added co-solvent. Three sequential aliquots of 2-chloromandelonitrile

(100mM) were added to a reaction mixture initially containing nitrile (100mM) and

Arthrobacter sp. F-73 (58mg dcw/mL) in Tris–HCl buffer (50mM, pH 9.0) containing

20% (v/v) ethyl acetate at 30 �C; a total of 400mM nitrile was reacted in 9 h to produce

(R)-2-chloromandelic acid in 92% yield and 98.5% ee; recovery and recrystallization of the

product improved the ee to 99.3% (89%yield). Repeating the reaction in the absence of added

organic co-solvent, 2-chloromandelonitrile (200mM) was reacted in 21.5 h to produce (R)-2-

chloromandelic acid in only 78% yield and 85.9% ee.

The substitution of the tyrosine residue corresponding to position 296 in wild-type nitrilases

of Alcaligenes faecalisNit1650, Alcaligenes faecalisNit8750, and Alcaligenes faecalisNit338

with cysteine, alanine, asparagine, glycine, serine, phenylalanine, and threonine produced an

increase in the catalytic activity of the enzyme on substituted mandelonitriles [85]. A

comparison of relative specific activity for conversion of racemic 2-chloromandelonitrile by

various Y296 mutations of Alcaligenes faecalis Nit1650 nitrilase with the Y296 wild-type

Table 8.8 Relative activity (%) of Alcaligenes faecalis JM3 and Arthrobacter sp. F-73 nitrilases in

water–acetone mixtures

Strain Acetone (%)

0 10 20 30 40 50 60

Alcaligenes faecalis JM3 100 85.5 34.8 0 0 0 0

Arthrobacter sp. F-73 100 83.8 77.9 67.9 50.9 38.6 17.1

Table 8.9 Conversion of 2-chloromandelonitrile to 2-chloromandelic acid using

Arthrobacter sp. F-73 nitrilase in water–co-solvent mixtures

Co-solvent (20% v/v) Relative activity (%) Yield (%) ee (%)

None 100 57.9 90.1

Methanol 143 62.8 90.1

Ethanol 87.2 65.1 86.2

2-Propanol 51.3 21.3 75.4

Acetone 36.7 53.6 92.6

Dimethyl sulfoxide 92.4 64.1 89.8

DMF 131 66.6 95.7

1,4-Dioxane 115 65.7 90.9

Ethylene glycol 103 54.1 87.6

1,3-Propanediol 78.0 40.2 93.2

THF 58.2 17.0 94.2


nitrilase as control is depicted in Figure 8.10 (measured at 50 �C); relative specific activity wascalculated by setting the activity of the wild-type (Y296) enzyme to 1.0. Each of the mutants

tested exhibited a higher activity than the wild-type Y296 nitrilase. A comparison of mutant

nitrilase Y296C and wild-type nitrilase Y296 for conversion of various arylacetonitriles is

depicted in Figure 8.11; relative specific activity was calculated by setting the activity against

unsubstitutedmandelonitrile at 100%.A significantly higher activity for each of the substituted

mandelonitriles tested was obtained with the Y296C mutant, while activity against unsub-

stituted mandelonitrile remained unchanged.

0

1

2

3

4

5

6

7

8

9

10

ControlSTNGCFAY296

rela

tive

spec

ific

activ

ity

Figure 8.10 Relative specific activity of Y296 mutations of Alcaligenes faecalis Nit1650 nitrilase for

conversion of racemic 2-chloromandelonitrile: Y296A (A), Y296F (F), Y296C (C), Y296G (G), Y296N

(N), Y296T (T), and Y296S (S)

0

20

40

60

80

100

120

140

4-MeMN4-BMN4-CMN3-CMN2-CMNMN

rela

tive

spec

ific

activ

ity (

%)

Figure 8.11 Relative specific activity of Alcaligenes faecalis Nit1650 nitrilase (&) and Y296C mutant

nitrilase (&) for conversion of unsubstituted and substituted mandelonitrile: mandelonitrile (MN),

2-chloromandelonitrile (2-CMN), 3-chloromandelonitrile (3-CMN), 4-chlormandelonitrile (4-CMN),

4-bromomandelonitrile (4-BMN), and 4-methylmandelonitrile (4-MeMN)


8.3.2.6 Mandelic Acid

While the production of (R)-mandelic acid by nitrilase on an industrial scale has long been

established, no sufficiently selective nitrilase to produce (S)-mandelic acid at the requisite

>95% enantiomeric purity has been reported in the literature. This has spurred creative

workarounds to this challenge (Figure 8.12). Benzaldehyde was converted into enantiomeri-

cally pure (S)-mandelic acid by the sequential additions of HCN catalyzed by cross-linked

enzyme aggregate (CLEA) of the (S)-selective oxynitrilase fromManihot esculenta (MeHnL)

and subsequent hydrolysis of the resultant (S)-mandelonitrile, in the presence of a CLEA of the

nonselective recombinant nitrilase fromPseudomonas fluorescensECB 191 (PfNLase). In this

application, the cyanation reaction was performed in a biphasic aqueous diisopropyl ether

medium at pH 5.5. When MeHnL–CLEA and PFNLase–CLEA were applied in tandem in a

90:10 ratio, (S)-mandelic acidwas provided in nearly complete conversion and in 94%ee.When

the MeHnL and PFNLase were combined during CLEA formation to generate a bi-enzyme

catalyst, and then applied to the reaction, (S)-mandelic acidwas provided in 98%ee.The authors

hypothesize that nitrile intermediate is immediately hydrolyzed in the bi-enzymeCLEA system,

suppressing diffusion into the water phase and possible racemization. However, while starting

material is transformed almost completely and the desired (S)-mandelic acid produced in

high ee, this process is limited by the observed formation of 40% (S)-mandelonitrile as

co-product [86].

The recombinantly expressed nitrilase from Pseudomonas fluorescens EBC 191 (PFNLase)

was applied in a study aimed at understanding the selectivity for amide versus acid formation

from a series of substituted 2-phenylacetonitriles, includinga-methyl,a-chloro,a-hydroxy anda-acetoxy derivatives. Amide formation increased when the a-substituent was electron

deficient and was also affected by chirality of the a- stereogenic center; for example,

2-chloro-2-phenylacetonitrile afforded 89% amide while mandelonitrile afforded 11% amide

from the (R)-enantiomer but 55% amide was formed from the (S)-enantiomer. Relative

amounts of amide and carboxylic acid was also subject to pH and temperature effects [87,88].

8.3.2.7 b-Hydroxy and g-Hydroxy Acids

The nitrilase from cyanobacterium Synechocystis sp. PCC6803 was found to effect the

stereoselective hydrolysis of phenyl-substituted b-hydroxy nitriles to (S)-enriched b-hydroxycarboxylic acids. The enzyme also effected the conversion of g-hydroxynitrile, albeit withlesser enantioselectivity (Table 8.10). Interestingly, this enzyme was also was found to

hydrolyze aliphatic dinitriles, such that for 1,2-dicyanoethane and 1,3-dicyanopropane the

H

O

HCN CN

OH

COOH

OH

CNH2

OH

+

(S)-mandelamide(S)-mandelonitrile (S)-mandelic acid

PfNLase

MeHnL

Figure 8.12 Conversion of benzaldehyde into enantiomerically pure (S)-mandelic acid by the sequen-

tial addition of HCN catalyzed by the (S)-selective oxynitrilase from Manihot esculenta (MeHnL), and

subsequent hydrolysis of the resultant (S)-mandelonitrile by the nitrilase from Pseudomonas fluorescens

ECB 191 (PfNLase)


Table 8.10 Hydrolysis of b- and g-hydroxynitriles by Synechocystis sp. PCC6803

nitrilase

OHCN

OHCOOH

OHCN

( )n ( )n ( )n

nitrilase

Entry R n Yield (%) ee (%)

1 Phenyl 1 51 46

2 4-Fluorophenyl 1 28 61

3 4-Chlorophenyl 1 30 65

4 4-Bromophenyl 1 50 23

5 4-Acetylphenyl 1 56 29

6 4-Methoxyphenyl 1 48 32

7 2-Napthyl 1 93 2

8 tert-Butyl 1 93 0

9 Phenyl 2 52 11

Table 8.11 Conversion of g-hydroxy aliphatic nitriles to corresponding lactone

CNR

OH

COOHR

OHO

O

R

Nitrilase lactonization

Substrate (R ¼)a Enzyme pH Time (h) Temp (�C) % Conv. ee% lactone E

-n-Ethyl (a) NIT 1002 6 21.5 37–38 15 50 3.3

-n-Butyl NIT 1002 6 24 30 40 64 6.9

-n-Pentyl (b) NIT 1002 7 11.5 30 42 70 9.3

-n-Octyl (c) NIT 1002 7 11.25 30 16 58 4.2

-Isobutyl NIT 1002 7 0.5 30 33 48 3.6

-n-Ethyl (a) NIT 1003 7 4.6 30 47 54 1.3

-n-Butyl NIT 1003 6 24 30 26 80 11.8

-n-Pentyl (b) NIT 1003 7 4.6 30 30 88 22.6

-n-Octyl (c) NIT 1003 6 24 30 44 20 1.7

-Isobutyl NIT 1003 7 51 36 28 0 1

-n-Ethyl (a) NIT 1004 6 1.75 30 3 38 2.3

-n-Butyl NIT 1004 6 1.75 30 6 46 2.8

-n-Pentyl (b) NIT 1004 6 1.75 30 8 50 3.1

-n-Octyl (c) NIT 1004 6 4 30 29 26 1.9

-Isobutyl NIT 1004 7 2 35 27.5 70 7.3

Substrate ¼b-Hydroxyheptanitrile NIT 1002 5 13 82 11.4

b-Hydroxyheptanitrile NIT 1003 30 30 50 3.7

b-Hydroxyheptanitrile NIT 1004 18.6 19 40 2.6

a(R)-enantimer lactone of substrates are pheromones of (a) Trogoderma beetle, (b) rice weevil, and

(c) rove beetle.


exclusivelymono-acid productwas observed,whereas for 1,6-dicyano hexane and 1,8-dicyano

octane the di-acid product is formed. For dinitriles of intermediate chain length, 1,4-dicyano-

butane and 1,5-dicyano-pentane, both monoacid and diacid products were isolated [89].

A strategy to access lactones via enzymatic hydrolysis of g- and b-hydroxy aliphatic nitrilesto their corresponding acids with subsequent internal esterification was applied using com-

mercially available enzymes from BioCatalytics Inc. A number of g- and b-hydroxy aliphaticnitrile substrates (Table 8.11) were evaluated, with the greatest selectivity observed with

g-hydroxy nonanitrile, which was converted by nitrilase NIT1003 to the precursor of the rice

weevil pheromone in 30% yield, 88% ee with an enatiomeric ratio of E¼ 23 [90].

8.3.2.8 g-Amino Acids

g-Amino butyric acid (GABA) is themajor neurotransmitter in themammalian central nervous

system. The five- and six-membered carbocyclic g-amino carboxylic acids, 3-aminocyclo-

pentanecarboxylic acid (3-ACPA) and 3-aminocyclohexanecarboxylic acid (3-ACHA), have

been used as stereomeric probes of GABA binding and as GABA uptake inhibitors respec-

tively. Despite their utility, a scarcity of methods for the preparation of 3-ACPA and 3-ACHA

exists, and reported methods have been primarily focused on resolution methodology. Their

preparation via nitrilase-catalyzed biotransformation of their corresponding nitriles was

reported using commercially available nitrilaseNIT-106 andNIT107. Using substrate loadings

of 0.34–0.67mM, at pH 8 and 30 �C, and enzyme loadings of 0.5 to 1.0 g L�1, excellent

enantiomeric purities were achieved for the cis isomers andmoremodest enantiomeric purities

achieved for the trans isomers (Figure 8.13) [91].

8.3.2.9 (S)-3-Cyano-5-Methyl Hexanoic Acid

(S)-3-Cyano-5-methyl hexanoic acid, a key intermediate in the preparation of Pregabalin

(Pfizer Lyrica), a GABA analogue for the treatment of neuropathid pain and seizures, was

prepared from isobutylsuccinonitrile (ISBN) using a regio- and stereo-selective nitrilase from

AtNit1 Arabidopsis thaliana (Figure 8.14). While the nitrilase displayed excellent selectivity,

providing the product in 98% ee at 45% conversion (E> 150), the level of activity rate was

inadequate. The authors report a threefold enhancement in activity without sacrifice in

CN

TsHN

CN

TsHN

NIT-106

NIT-106

COOH

TsHN

COOH

TsHN

CN

NHTs

CN

NHTs

NIT-106

NIT-107COOH

NHTs

COOH

NHTs97 % ee, 45 % yield, 1.75 h

55 % ee, 36 % yield, 30 h

99 % ee, 29 % yield, 9 h

86 % ee, 46 % yield, 256 h

Figure 8.13 Preparation of 3-ACPA and 3-ACHA via nitrilase-catalyzed biotransformation


enantioselectivity by a single mutation C236S which was identified by subjecting the cloned

gene to error-prone polymerase chain reaction-based mutagenesis and screening [92].

8.3.2.10 (R)-4-Cyano-3-Hydroxy-Butyrate

A summary of the industrial-scale process development for the nitrilase-catalyzed [93] route

to ethyl (R)-4-cyano-3-hydroxy-butyrate, an intermediate in the synthesis of Atorvastatin

(Pfizer Lipitor) from epichlorohydrin via 3-hydroxyglutaronitrile (3-HGN) was recently

reported (Figure 8.15) [94]. The reaction conditions were further optimized to operate at 3 M

(330 g L�1) substrate, pH 7.5 and 27 �C. Under these conditions, 100% conversion and

product ee of 99% was obtained in 16 h reaction time with a crude enzyme loading of 6%

(based on total protein, 0.1 Umg�1). It is noted that at pH< 6.0 the reaction stalled at<50%

conversion and at alkaline pH a slowing in reaction rate was observed. Since the starting

material is of low cost and the nitrilase can be effectively expressed in the Pfenex

(Pseudomonas) expression system at low cost, introduction of the critical stereogenic center

CNCN nitrilase

ISBNCOOH

CN

COOH

NH2

CNCN

(S)-3-cyano-5-methyl hexanoic acid

+

Pregabalin (LyricaTM)

(R)-isobutylsuccinonitrile

Figure 8.14 Preparation of (S)-3-cyano-5-methyl hexanoic acid from isobutylsuccinonitrile using a

regio- and stereo-selective nitrilase from AtNit1 Arabidopsis thaliana

Cl OCNNC

OH

N

F

O-

OHOH

HNO

OCO2EtNC

OH

COOHNCOH

1) HCN, base

2) NaCN 40-55 oC

HGN, 3 MepichlorohydrinpH 7.5, NaH2PO4, 27 oC

EtOH/H+

Nitrilase, BD9570

(R)-4-cyano-3-hydroxy-butyric acid99% ee

]2 Ca2+

Atorvastatin (LipitorTM)

Figure 8.15 Nitrilase-catalyzed route to ethyl (R)-4-cyano-3-hydroxybutyrate, an intermediate in the

synthesis of Atorvastatin


into Atorvastatin by nitrilase-catalyzed desymmetrization represents a very attractive

industrial-scale process. The nitrilase used in this study was previously discovered through

a metagenomic effort and further optimized through directed evolution via a GSSM strategy

which identified a single amino acid change which increased enantioselectivity, substrate

tolerance, and volumetric productivity [93].

8.3.2.11 5-Norbornene-2-Carboxylic Acids

5-Norbornen-2-carboxylic acids are useful for the preparation of pharmaceutical intermediates,

pesticides, or perfumes, and in the synthesis of cyclic olefin copolymers. Chemical methods for

the preparation of 5-norbornen-2-carboxylic acids are reported to produce mixtures of isomer

mixtures, requiring complex separation methods for isolation of the individual isomers. A

method to convert endo-5-norbornene-2-carbonitrile or exo-5-norbornene-2-carbonitrile to the

corresponding carboxylic acid without interconversion of exo- and endo-isomers has been

demonstrated using arylacetonitrilases (EC 3.5.5.5) [95]; these enzymes are usually not very

active towards hydrolysis of aliphatic nitriles or benzonitriles, but were demonstrated to have

high activity for conversion of 5-norbornen-2-carbonitriles (Figure 8.16). Of the microbial

nitrilases examined, Alcaligenes faecalis Nit338 exhibited the highest relative activity towards

the endo-nitrile, while also displaying exo-nitrile hydrolyzing activity comparable to the other

nitrilase catalysts evaluated. endo-5-Norbornene-2-carbonitrile (0.53M) was hydrolyzed

(>99% conversion) by microbial Nit338 biocatalyst in sodium phosphate buffer (10mM, pH

7.5) at 40 �C, and the isolated endo-5-norbornene-2-carboxylic acid was >99% pure.

8.3.2.12 Malonic Acid Monoesters

The preparation of malonic acid monoesters has been demonstrated using the microbial

nitrilase activity of Corynebacterium nitrilophilus ATCC 21 419, Gordona terrae MA-1, or

Rhodococcus rhodochrous ATCC 33 025 to hydrolyze methyl cyanoacetate, ethyl cyanoace-

tate, n-propyl cyanoacetate, isopropyl cyanoacetate, n-butyl cyanoacetate, tertbutyl cyanoa-

cetate, 2-ethylhexyl cyanoacetate, allyl cyanoacetate, and benzyl cyanoacetate [96]. By

maintaining the concentration of nitrile in a reaction mixture at �5wt%, significant inactiva-

tion of the nitrilase activity was avoided; for example, a total of 25 g of n-propyl cyanoacetate

was added in sequential 5 g portions to a 100mL suspension of Rhodococcus rhodochrous

ATCC 33 025 cells (OD630¼ 5.6) in 50mM phosphate buffer (pH 7.0) over 30 h at 25 �C to

produce mono-n-propyl malonate in 100% yield (Figure 8.17).

Alcaligenes faecalis Nit338

10 mM NaH2PO4 (pH 7.5),40 oC

endo-5-norbornene-2-carbonitrile

CN

H

CO2H

H

endo-5-norbornene-2-carboxylic acid

Figure 8.16 Conversion of endo-5-norbornene-2-carbonitrile to the corresponding carboxylic acid

using Alcaligenes faecalis Nit338


8.3.2.13 Prochiral Sulfoxide Resolution

A prochiral bis(cyanomethyl) sulfoxide was converted into the corresponding mono-acid with

enantiomeric excesses as high as 99% using a nitrilase–NHase biocatalyst. The whole-cell

biocatalyst Rhodococcus erythropolis NCIMB 11540 and a series of commercially available

nitrilases NIT-101 to NIT-107 were evaluated in this study. As outlined in Figure 8.18, the

prochiral sulfoxide may be transformed into five different products (plus enantiomeric iso-

forms), of which, three are chiral (A, B, and C) and two achiral (D and E). Only products A, B,

andEwere observedwith the biocatalysts employed in this investigation. Both enantiomerically

enriched forms of both A and C could be obtained with one of the catalysts used. The best

selectivities are as follows: (S)-A 99%ee, (R)-A 33%ee, (S)-C 66%ee, and (R)-C 99%ee, using

NIT-104, NIT-103, NIT-108, and NIT-107 respectively. Each of these catalysts produced more

than one product and, thus, they are not chemoselective [97].

8.3.2.14 Polymer Modification

Recently, nitrilases have been applied to polymer modification, specifically to the modification

of polyacrylonitrile (PAN). Nearly 3� 106 tons of PANare produced per annum and used in the

textile industry. However, there is a great need to improve moisture uptake, dyeability with

ionic dyes, and feel of this acrylic fiber. The cyano moieties of PAN have been successfully

modified to carboxylates with the commercial Cyanovacta nitrilase, thus enhancing the

aforementioned properties of PAN [98]. Nitrilase action on the acrylic fabric was improved

NCCH2CO2CH2CH2CH350 mM phosphate (pH 7.0),

25 oC

HO2CCH2CO2CH2CH2CH3

R. rhodochrous ATCC 33025

Figure 8.17 Production of mono-n-propyl malonate from n-propyl cyanoacetate using the microbial

nitrilase activity of Rhodococcus rhodochrous ATCC 33025

SNC + CN

-O ..

A

B

C

SNC + CNH2

-O .. O

H2 SNC + COH

-O .. OO

SNC + COH

-O .. O

D

H2 SNC + CNH2

-O .. OO

SHOC + COH

-O .. OO

chiral achiral

nitrilase ornitrile hydratase

E

Figure 8.18 Conversion of a prochiral bis(cyanomethyl) sulfoxide into the corresponding mono-acid

using a nitrilase–NHase biocatalyst


by addition of 1 M sorbitol and 4%N,N-dimethylacetamide, which serve tomake the fibermore

plastic and, thus, increase surface accessibility. In addition, using PAN polymers as a sole

carbon source, a new nitrilase was discovered from Micrococcus luteus strain BST20, which

was also able to modify PAN fabric [99].

8.4 Conclusions

NHases and nitrilases each continue to find use in reactions where significant improvements in

substrate conversion, product yield and purity, enantioselectivity, and/or regioselectivity are

obtained relative to alternative nonenzymatic reactions. The high substrate conversions and

product yields and purities achieved with these enzymes often drastically reduce or eliminate

the need for downstream purification steps, leading to the commercialization of more cost-

efficient processes with less by-product formation andwaste disposal requirements. The use of

genetic engineering tomodify protein structure, through randommutagenesis or by design, has

now routinely been shown to improve the desired functionality of these enzymes. One

disadvantage of using nitrilases to produce carboxylic acids remains the co-production of

one equivalent of ammonia, and conversion of the initially produced ammonium carboxylate to

the desired carboxylic acid often results in the production of an inorganic ammonium salt as by-

product; finding ways to recycle this by-product stream in the process, or to identify

economically attractive and sustainable uses for by-product streams, remains as a challenge

to adoption of nitrilase-catalyzed production of large-volume commodity chemicals.

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Biocatalysis for the Pharmaceutical Industry || Applications of Nitrile Hydratases and Nitrilases