"

Journal of Scientific & Industrial Research Vo1.58, December 1 999, pp 925-947

Microbial Metabolism of Nitriles and Its Biotechnological Potential

C Ramakrishna, (Mrs) Heena Dave and M Ravindranathan Research Centre, Indian Petrochemicals Corporation Limited, Vadodara 391 346, India

Biocatalysts display a remarkable capability to function under normal temperature and pressure with high specificity and are poten­tially very economical. Bioconversion of nitri le compounds to a number of economically important compounds is described. A wide variety of microorganisms having the ability to metabolize different nitriles and discovered during the last two decades are described. It is pointed out that the microbial degradation of nitriles proceeds through two distinct enzymatic pathways: nitrilase catalyzes the direct hydrolysis of nitriles to the corresponding carboxylic acids and ammonia, whil e nitrile hydratase catalyzes the hydration of nitriles to

"f- the corresponding amides, followed by their conversion to the corresponding carboxylic acids plus ammonia by amidase. It is men­tioned that the versatile biocatalytic nature and applications of these enzymes are being increasingly recognised for the selective hydrolysis of various types of nitriles for the production of several fine chemicals, pharmaceuticals and optically active nitriles, amides and carboxylic acids, which are not generally feasible by chemical routes. A commercial process involving the multi-kiloton scale synthesis of acrylamide using Rhodococcus rhoc!ochrous J I nitrile hydratase is described, which is the best example of a fully developed industrial application of this biotechnology. Though, recent developments broadened the potential application of these versatile biocatalysts in chemical synthesis and bioremedi ation, further studies are required to fully harness their biotechnological potential.

Introduction

The nitrile compounds, the cyano group (-CN) con­taining organic compounds (organic cyanides) are nu­merous and wide spread in the environment. In nature, nitriles are mainly present in the form of cyanogenic gly­cosides which are produced by plants and animals, such as insects, etc. Plants also produce other type of nitrile compounds like cyanolipids, ricinine, phenylacetonitrile, p-cyanoalanine, etc . l .2 . More importantly, a number of nitrile compounds are manufactured by man for produc­ing a variety of polymers and other chemicals . For ex­ample, acrylonitrile and adiponitrile are produced by the chemical industry on a large scale (world-wide produc­tion : about 45,00,000 and 1 0,00,000 MT per annum, re­spectively3) for the manufacture of polyacrylonitrile and nylon polymers. Some ni tri le compounds, such as bromoxyni l , ioxyni l and dichlorobenil , are herbicides .

Chemical Nature, Uses and Toxicity of Nitrites

Chemical Nature and Uses

The -CN functional group in the nitrile compounds, besides being highly reactive, is able to activate adja­cent C-H bonds, which is the basis of their versatile re­activity. In general, nitriles are important organic com­pounds from economic point of view and exhibit broad IPCL Res Centre Communication No.341

chemical utility including their use as feed stocks, sol­vents, extractants, recrystall izing agents, pharmaceuti­cals, catalysts and pesticides. They are also h ighly sig­nificant intermediates for organic syntheses for prepar­ing amines, ami des, amidines, carboxy acids and esters, aldehydes, ketones including cyclic ketones, imines, het­erocyclic compounds, etc. 1 .3.4. Hydrogenation of nitriles to amines provides some important intermediates for both polyurethanes and polyamides . Acetonitrile is widely used for the preparation of a variety of compounds including pharmaceuticals, perfumes and photographic industry chemicals . It is also used for the separation of butadiene and other olefins from hydrocarbon streams, as a solvent for spinning fibres and for high pressure liquid chromatography (HPLC), etc. a-Aminonitriles are versatile intermediates to obtain amino acids, agrochemi­cals , chelants, radical in itiators and water treatment chemicals . Benzonitrile is used for the production of melamine, in protective coatings and molding resins, as additive in jet fuel, nickel-plating, cotton bleaching baths and for drying acrylic fibre, etc.3. 4 .

Toxicity

Most of the nitriles are highly toxic, mutagenic and carcinogenic in nature. They inhibit cell multipl ication of some algae and sensitive bacteria, such as Pseudomo­nas putida, but inhibitory concentrations vary for dif-

926 J SCI IND RES VOL-58 DECEMBER 1 999

ferent nitriles'. The toxicity and occupational hazard data for the commonly used ni triles are wel l documented45. The general tox icity of nitri les in humans is expressed as gastric distress and vomiting, bronchial irritation, res­piratory distress, convulsions and coma6• The main tox­icity concern for n itriles is their acute lethality, caus­ing a disease, osteolathyrsm, which leads to lameness and skeletal deformities'. The toxic potency of nitri les varies with their chemical stmcture. Their acute toxicity is mainly related to their metabolism in the body that resu lts in the release of the 'cyanide' ion . Though most of the avai lable l iterature ascribes almost all the actions of nitriles to the l iberation of cyanide ions, recent re­ports confirm that both the -CN group and the entire molecule are important for their biological activity. While many of the neural effects are probably due to the -CN moiety, the i lTitating and necrotic effects on the skin, l iver and kidney may be associated with the nature of the alkyl group4 . Recently, the nature and biochemical mechanism of the toxicity and stmcture-activity relation­ships of nitri les have been reviewed by DeVitd'.

Microbial Nitrile Metabolism

The ni trile metabolism is widely distributed in na­ture2•7.K and many microorganisms, plants and some ani­mals can degrade n itriles. A variety of actinomycetes, bacteria, and fungi , that metabolize n itri les as carbon and/or nitrogen source have been described in the litera­ture. Actinomycetes such as Nocardia, Rhodococcus, bacteria such as Acinetobacter, Corynebacterium, Kleb­siella, Pseudomonas and fungi l ike Fusarium and Tri­chodenna are the most prominent genera active in the n itri le metabolism. Three types of biochemical reactions eliminate the -CN group of nitri les: hydrolysis, oxida­tion and reduction2.K •

Hydrolysis

This is the most common reaction for the microbial metabolism of n i triles and it proceeds via the formation of the cOlTesponding carboxylic acid and ammonia. In fact, based on the product two types of hydrolysis reac­tions occur. In the first type, the end products are formed directly without any intermediate, catalyzed by an en­zyme, n itri lase (EC 3 . 5 .5 . 1 nitri le aminohydrolase) as shown in Eq.( J ) .

Nitrilase R-CN ------..

Nitrile R-COOH + NH.l Carboxylic acid . . . ( 1 )

Nitrilases general ly act on aromatic or heterocycl ic nitri les and are shown in plants, fungi and bacleri a L'! 'o

The second type of hydrolysis is by the ac t ion o r a two-enzyme system, consisting of a nitri Ie hydratase ( EC .

4.2. 1 .84) that converts n itrile to amide and an am idase (EC. 3 .5 . 1 .4. ) that converts amide to the correspond ing carboxylic acid and ammonia [Eq.(2») :

N itri le hydratase

R-CN -. R-CON H , Nitrile + Hp Amide

Amida,e

--------. + H ,O

R-COOl-1 + N H , Carboxylic acid . . ( 2 )

Generally, aliphatic nitriles are metabolized through this pathway in several bacteriaL'!. J I l .

A new type of enzyme activity hydrolyzing all types of cyanide compounds, including n itriles, is reported in Acinetobacter sp. RFB J t t . This differs from the hi therto reported enzymes ill that, it is extracellu lar, constitu­tive, located in a l ipid complex and hydrolyzes all the three classes of cyanides such as simple and complex inorganic as well as organic cyanides (nitriles) " .

Oxidation

Many plants and insects oxidize some of the n i triles to cyanohydrins (or a-hydroxynitriles) by an oxygenase enzyme. The cyanohydrins are further decompl)sed spon­taneously or by the action of another enzyme, oxynitri lase or hydroxy n itri le lyase l 2 to an aldehyde and hydrogen cyanide (HCN) .

Oxygenase Oxynitrilase

R-CHl-CN ------� -CHOH-CN ----� N i tri le + O2 a-Hydroxy nitri le

R-CHO + HCN

A ldehyde . . . (3 )

This type of enzyme system, that liberates HCN from n itri les, is almost non-existing in microorganisms. How­ever, a fungus, Trichoderma sp. is reported to degrade diaminomaleonitrile releasing cyanideD .

Reduction

The N2-fixing organisms contain the enzyme ni tro­genase, which hydrogenates N2 and a number of sub-

. '

RAMAKRISHNA et al. : MICROBIAL METABOLISM OF NITRILES 927

strates such as cyanide, nitriles, isonitriles, cyanogens, allenes, azides, etc. using ATP'4. The nitriles are con­verted to hydrocarbons, releasing ammonia. However, it is deactivated in the presence of oxygen [Eq.(4) ] .

R-CN Nitrile

Nitrogenase

---�. R-CH, + N H, + ATP Hydrocarbon

Enzymology of Nitrile Metabolism

. . . (4)

The enzymology of microbial n itrile metabolism is now well understood. The main enzymes that take part in the nitrile metabolism are described below:

NitriLases

Nitrilase, discovered barley about 40 years ago, i s the first nitrile metabol izing enzyme that converted in­dole-3-acetonitrile to indole-3-acetic acid, a plant growth hormone (an auxin) ' ) . Later, a number of microorgan­isms possessing nitrilase activity have been isolated with the capability to metabolize several natural and syn­thetic nitriles l . l s . Based on their substrate specificity, mi­crobial nitrilases are of three types 1 5 :

Aromatic Nitri/ases - These preferentially hydrolyze aromatic or heterocyclic nitriles directly to the corre­sponding acid and ammon ia . M icrobial aromatic n itrilases have been purified and characterized from Pseudomonas Sp. 16. 1 7, Nocardia spp. NCIB 1 1 2 1 5 , 1 1 2 16, and Fusarium soLani ' K , which catabolized ricinine, benzonitrile and p-hydroxy benzonitrile, respectively. In Arthrobacter sp. J- l two types of n itrilases which spe­cifically degrade benzonitrile and p-tolunitrile, have been characterized l 9. In Rhodococcus rhodochrous J 1 cells, high levels (up to 35% of the soluble protein) of aro­matic nitrilase is induced by isovaleronitrile2o.2 1 .

Aliphatic Nitrilases - These nitrilases preferential ly catalyze hydrolysis of aliphatic nitriles directly to acid plus ammonia. This enzyme, first discovered in R. rhodochrous K22, i s a l so s t rong ly i nduced by isovaleronitrile22.23 . Similarly, an aliphatic nitrilase in­duced by £-caprolactam and converting acrylonitrile to acrylic acid, i s described in R. rhodochrous J ] 2\ while another aliphatic n itrilase active on adiponi trile and

cyanovaleric acid forming cyanovaleric acid and adipic ac id , re spec t i ve ly , is iden t i fied in Comamonas testosteroni strain ct2S.

Arylacetonitrilases - These nitrilases preferentially hydrolyze arylacetonitriles. This type of n itrilase, found in A Lca ligenes faecalis JM326. 27 , i s i nduced by isovaleronitrile, does not act upon aromatic or aliphatic nitriles but hydrolyzes only arylacetonitriles such as in­dole-3-acetonitrile, phenylacetonitrile, thiophene-aceto­nitrile, etc . , of which, some are natural precursors for plant hormones, auxins . Yamamoto and coworkers2K character ized an ary l aceton i tr i l ase c apable of enantioselective hydrolysis of mandelonitrile from AL­caligenes faecalis ATCC 8750.

The physicochemical characteristics of ni trilases re­ported i n the literature are l isted in Table I . They are multimeric, composed of one or two types of subunits of different sizes and numbers. Nitrilases do not show the presence of any metal cofactor or prosthetic group unl ike the other nitrile-metabolizing enzymes, nitrile hydratases. All the known nitrilases are reported to be sulphydryl enzymes I 5.2 1 ,29 and the eliminat ion of the -SH groups by chemical modification led to enzyme inacti­vation30. The n itrilases are reported to have a catalyti­cally essential cysteine residue at or near their active site. In fact , the nitrilase from R. rhodochrous K22 is found to have a single cysteine, CYS l70 and when this is replaced by Ala or Ser by site directed mutagenesis, the enzyme lo ses i t s act i v i ty30 . The n i tri l ase from Rhodococcus sp. ATCC 39484 is also found to have two cysteines, one of which is catalytical ly essential" . A possible mechanism for the nitri lase reaction31 ..l2 indi­cates a nucleophilic attack on n itrile carbon atom by a sulphydryl group of enzyme, leading to the formation of a tetrahedral intermediate via enzyme-iminothiol ester format i on . Us i ng ion-spray mass spectroscopy, Stevenson and coworkers32 were able to detect the co­valent enzyme-substrate intermediate in the hydrolysis reaction with the nitrilase purified from Rhodococcus sp. ATCC 39484. Though nitri lase by definition hydro­lyzes nitrile substrates into the carboxylic acids plus am­monia, it must be noted that a small amount of amide is detected when poor substrates are used, possibly due to anomalous breakdown of the reaction intermediate, such as the case with the Rhodococcus sp . ATCC 39484 aro­matic n i tri lase when reacted with a poor substrate, phenylacetonitrile3 l .

928 J SCI IND RES VOL.58 DECEMBER 1 999

Table I - Properties of nitrilases isolated and characterized from different nitrile-metabol izing microorganisms

Source of nitrilase Formation Properties of nitri lasesal Ref. type

Mol. Subunits Opt. Heat Opt. pH Substrate mass and mol. temp. stability pH stabi lity specificity (kDa) mass ( 0c) ( 0c)

(kDa)

Acinetobacter Inducible 5 80 NA b) 50 60 8.0 5 . 8-8 .0 Aliphatic 1 05 sp. AK226 (NA) aromatic &

heterocyclic �. nitriles

inducible 260 6 45 NA 7.5 NA Arylaceto- 27 Alcaligenes (44) nitriles

faecalis 1M3

Arthrobacter induc ible 53 2 40 NA 8.5 NA Aromatic 1 9

sp. J I (a,30) nitriles

(�,23)

Comamonas NA NA Oligomer 25 NA 7.0 NA Adiponi tri le 25 � testosteroni strain ct (38)

Klebsiella Inducible 72 2 35 NA 9.2 NA Bromoxynil 74 ozaenae (38 . 1 )

Nocardia sp.LL I 00-2 1 inducible 530 NA NA NA NA NA Benzonitrile 1 74

sp. NCIB 1 1 2 1 5 inducible 56 1 2 1 0-50 NA 7.0- 7 .0-9.5 Aromatic 1 8 ( NA ) 9.5

Rhodococcus " rhodochrous 11 constitutiv e 78 2 45 45 7 .5 NA A liphatic & 1 34

(4 1 .5 ) arom atic n itri les

rhodochrous K22 constituti ve 650 1 5- 1 6 5 0 40 5.5 NA A l i phati c 2 3 (4 1 ) ni tri les

Fusarium constituti ve 550 M any 4 N A 6- 1 1 N A A liphatic, 1 75 oxysporum

(27) arom atic & . ; heterocyc l i c

n itriles

solani inducible 620 8 1 0-50 N A 7.8- NA A romatic 1 8 (76) 9. 1 n i tri les

a) None of the nitrilases contain any metal cofactor or prosthetic group

b) NA - Not Available.

+

RAMAKRISHNA et at. : MICROBIAL METABOLISM OF NITRILES 929

Recently, a new family of carbon-nitrogen hydrolase enzymes (nitrilase/cyanide hydratase family) has been proposed33 . Extensive aminoacid database searches show that nitrilases are significantly similar to aliphatic ami­dases, cyanide hydratases and �-alanine synthase - all these enzymes appear to have a cysteine residue acting as an active site nucleophile33.

Nitrile Hydratases The reaction sequence for the conversion of nitrile to

amide by a n itrile hydratase and then to carboxylic acid plus ammonia by an amidase, was first proposed by a Japanese research group about 30 years ag034. After­wards, a number of microorganisms containing nitrile hydratase have been isolated and their enzymes have been characterized. These studies show wide ranging physico­chemical properties and substrate specificities of the ni­trile hydratases, which are composed of two types of dissimilar subunits (a and �) varying in number (Table 2). Most importantly, they are metalloenzymes contain­ing iron or cobalt and are classified accordingly.

Ferric Nitrile Hydratases The electron spin resonance (ESR) studies have shown

that the n i tr i l e hydratases from Pseudomonas chloro raphis B 2 3 and Rhodococcus sp. R 3 1 2 (Brevibacterium sp. R3 1 2) are non-heme iron enzymes with low-spin ferric ion35. The ferric nitri le hydratase from Rhodococcus sp. R3 1 2 is the most thoroughly char­acterized enzyme using several spectroscopic techniques including ESR, extended X-ray absorption fine struc­ture (EXAFS) and electron nuclear double resonance (ENDOR) spectroscopies. These studies revealed that the enzyme is a (a �)2 - tetramer that contains two low­spin non-heme ferric (Fe3+) ions which exist in a tet­ragonally distorted octahedral l igand field of three his­tidine imidazoles, two cystein thiolates and the hydrox­ide36-40. The three-dimensional analysis of crystal struc­ture of this enzyme also showed a novel iron centre in a novel fold4 1 . Ferric nitrile hydratases from Rhodococcus spp N-77 4 and R3 1 2 are reported to have identical amino acid sequences to that of Rhodococcus sp. N_77 1 42. The aminoacid sequences of the Rhodococcus sp. N-774 and R. erythropolis JCM 6823 nitrile hydratases are approxi­mately 95% identical, while those of the Rhodococcus sp. N-774 and P. chlororaphis B23 are approximately 60% identical. The visible and ESR spectra associated with the metal centres in the acti ve form of ferric nitrile hydratases from Rhodococcus spp R3 1 2, N-774 and P.

chlororaphis B23 are also virtually identicap5.43.44. Al­though differences in the substrate specificity exist among these enzymes, it is very l ikely that the major features of protein structure, the coordination of the iron etc. are the same. However, one significant difference is that the ferric n itrile hydratases from Rhodococcus spp N-77 I , N-774 and R3 1 2 show the unique characteris­tic of photoreactivity; they lose their activity when stored in the dark as intact cells or crude extracts and this ac­tivity can be recovered by irradiation with UV l ight. Pho­toreactivation of the nitrile hydratase of the strain N-77 1 takes less than 1 Ils and a hypothetical model for this reaction has been proposed on the basis of the re­sults of Mossbauer and ESR spectra42 . Nagamune and his group43 have purified the enzyme from strain N-77 1 in its inactive form and then transformed it to its active form by light irradiation . The absorption and fluores­cence spectra indicates that the chromophore involved in the photoactivation i s an i ron complex and the photoactivation site is found to be located on the a­subunit, which also contains the non-heme iron centre of the enzyme45.47 . Fourier transform infrared (FTIR) spectroscopy has revealed that the inactive n i tri Ie hydratase of strain N-77 1 intrinsically possesses n itric oxide (NO) molecule bound to non-heme iron4x.49 and its photo-dissociation has been suggested to activate the enzyme, while inactivation is due to binding of exog­enous NO with the iron 50.

Some of the nitrile hydratases have been found to be quinoproteins contain ing the pyrroloquinoline quinone (PQQ) as prosthetic group5 1 . This group is suggested to participate in the hydration of the n itrile group by n itrile hydratase, which is a novel function for PQQ9.52 . Earl ier only enzymes like reductases and oxidases are known to have the PQQ as prosthetic group.

Cobalt Nitrile Hydratases Rhodococcus rhodochrous J I , one of the highly stud­

ied and versatile n itrile degrader, has shown the pres­ence of two types of cobalt hydratases: i) Aliphatic ni­trile hydratase. which is a high molecular mass (505 kDa) enzyme, i s heat stable (up to 50°C) and preferen­tially acts on aliphatic nitriles such as acrylonitrile, and i i ) Aromatic nitrile hydratase, which is a low molecular mass ( 1 01 kDa) enzyme that preferentially hydrates aro­matic n itriles such as benzonitrile2X.53. Al iphatic nitrile hydratase is composed of 1 0 a- and 1 0 �-subunits and is selecti vely induced by urea, whi le aromatic n i t ri l e hydratase consists of 2a- and 2�-subunits and is selec-

930 J SCI IND RES VOL.58 DECEMBER 1 999

Table 2 - Properties of nitri le hydratases from different nitrile-metabolizing microorganisms

Source of enzyme For m ation Properties o f N i trile hydratase R e f type r

M etal & M o l . N u . of Opt. H eat Opt . p H S u bstrate PQQ") m ass subun its temp stab il i ty p H stab i l i t y spe�ific i t y

( k D a ) & mo l . ( DC ) ( D C ) mass (k Da )

Agmbacterium tumefaciens inducible Co & Fe 1 0 2 4 N A h ) N A 7 . 5 N A I n d o l e - 3 - 60 IAMB-26 1 (+ ) ( 2 5 ) ac e t o n i tri le

tum�faciens i nducib le Fe 69 4 40 5 0 7 . 0 7 - 1 0 2 - arylpro- 1 2 1

strain d3 ( N A ) (2 7 ) p i o n itrile ( E n antio-select ive)

A rthmbacter i n d u c i b l e N A 420 2 3 5 N A 7 . 0 - N A A l ip hat ic 1 7 6 t sp. J I ( N A ) ( 2 4 ) 7 . 2 n i triles

( 2 7 )

Brevibacterium constitutive Fe 8 5 3 -4 25 20 7 . 8 6.5- 8 .5 A l i p h atic 1 7 7

sp. R 3 1 2') (+ ) ( a , 2 6 ) ni tr i les ( P ,27 . 5 )

sp. ACV2 consti tut ive Fe 80 2 3 5 N A 6.0 NA A l ip hati� 1 5 0

(+) ( 2 6 ) ni triles mutant of R3 1 2 (27.5)

Corynebacterium constitutive Fe 6 1 .4 2 N A N A 8 . 0 - N A A liphat ic 1 7 8 sp. C5 (+ ) 8 . 5 d i n i triles

Pseudomonas Inducible Fe 1 00 4 20 20 7 . 5 6.0 - 7 . 5 A l iphatic 1 7 9

ch/ororaphis B23 ( + ) (a,25 ) n itr i les

( P ,25 ) �

Pseudonocardia Inducible C o N A N A 60 60 N A N A A cryl o - 8 2 thermophila JCM3095 ( N A ) ( 2 9 ) nitrile

( 32 ) RhlldocoCCllS

r/lOdochrotis JI Low mol. mass Inducible Co 1 0 1 1 8 -20 40 N A 8 . 8 N A Preference Enzyme (+) (a,26) Arom atic

(P ,29) n i triles 5 0

High mol. Inducible C o 5 0 5 4-5 3 5 -40 5 0 6 . 5 6.0- 8 . 5 A liphatic mass enzyme A (+) (a,26) n i triles

( P , 2 9 ) sp. Inducible N A 5 2 2 N A N A N A N A A l iph atic 1 8 0

( N A ) ( 26 ) n i tr i les ( 2 3 )

sp. 7 Inducible N A 2 7 8 4 5 2 5 -4 5 7 . 0 5 .0-9.0 A l ip h a t i c & 1 8 1 ( N A ) ( 26 ) arom atic

( 3 2 ) n i tri l es

sp. N -77 1 Con stitutive Fe 60 2 30 0-35 7 . 8 6 . 0-8 .0 A l iphatic 4 2 ( +) (a,27 . S ) n i triles

( P , 2 8 ) Photores-

sp. N-774 p o n s i v e

Const i tu t ive Fe 70 2 < 30 N A 7 . 7 N A A l iph atic 1 8 2 ( + ) (a,2 8 . 5 ) n itri l es

( P , 29) Photores-p o n s i v e

Myrotheciwll verrucaria I n d u c i b l e Zn 1 7 0 6 5 5 N A 7.7 N A C yanam i d e 6 1 (NA ) ( 2 7 . 7 )

a ) PQQ - Pyrroloquinoline quinone prosthetic group (+ , presence)

b) NA - Not Availabl e , c ) same as RhodococclIs sp . R3 1 2

RAMAKRISHNA et at. : MICROBIAL METABOLISM OF NITRILES 93 1

lively induced by cyclohexanecarboxamide2XS1.54 . Ap­proximately 40% of amino; lc id �;eqll �nces hctween th� two R. rhodochrous J I n itri le hydr;ltases ilnd that from Rhodococcus sp. N-774 are ident ical" . Recen t ly, the al i ­phatic nitrile hydratase has been shown to con tai n a low spin, non-corrin cobal t (Co '+) ion with oct ahedral S and N(O) l i gand fi eld'(' . In terest i ng ly, the pre- edge and EXAFS spectra of both cobal t and i ron n i t ri le hydratases have been found to be simi lar. suggest ing the same l igand environment of metal ions in both the enzymes,7

A stereoselecti ve nitri Ie hydratase from Pseudomo­

nas putida NRRL l 8668'x that hydrolyzes (R,S )-2-( 4-chlorophenyl)-3-methylbutyronitrile to the correspond-

t- ing (S)-amide has been purified and characterized for the first time. This stereo-selective enzyme is found to have a- and �-subunits, exists as a� and (a�)l forms and contains a non-corrin low-spin cobalt (C03+) ion in a tetragonally-distorted octahedral ligand field)') . The three dimensional structure of this enzyme appears to be simi­lar to R. rhodochrous J I and Rhodococclls sp. R3 1 2 hydratases59. However, the cobal t enzymes from R. rhodochrous J I and P. putida have threonine in their active site whereas the ferric enzymes from Rhodococcus

-<II sp. N-774 and P. chlororaphis B23 have serine5'J . The difference in the metal cofactor may be attributed to this difference in the active site aminoacid residue54.

A recent reaction model for nitrile hydratase54 shows that the catalysis proceeds without direct coordination of the substrate to the metal ion, where the metal ion activates a water molecule by acting as a Lewis acid and subsequently either the nitrile substrate approaches a metal-bound hydroxide ion that acts as a nucIeophile attacking the n itri le carbon or a metal bound hydroxide ion activates a water molecule, whieh can attack the n i ­trile carbon54. After both these reactions, the nitrile gets converted to an imidate intermediate which finally gives rise to amide.

A novel nitrile hydratase having both cobalt and fer­ric ions, which is involved in the biosynthesis of plant hormone, indole-3-acetic acid is found in Agrobacterium tumefaciens IAMB-26 1 60 . Another new hydratase hav­ing six identical subunits (27 .7 kDa) with zinc as cofac­tor, specifically degrading the herbicide, cyanamide to form urea ha s been reported from the fungus , Myrothecium verrucaria61 • The structural analysis of these enzymes may provide a better understanding of the metal functions in the nitrile metabol izing organisms.

Amidases A number of am idases ac t i ve ly partic ipating in the

hydrolysis or amides, and a l ways found l inked with ni­t ri le hydratases , have bee n puri fied and characterized . They exh ib i t d i f'ferent phy s i coc hemica l c h aracterest ics (Tab le 3 ) w i th a d i verse substrate spec i f ic i ty. Unl ike hydratases, the associat ion or am idases with metals such as cobalt and iro n has been reported only in organ isms l i ke Klebsiella pllellmoniae and RhodococclI.1 Sp.()2!,' . The enantioselect ive amidase characterized from Pseudomo­

nas ch/ororaph is B 2 � has no as soc i ated metalM. Rhodococcus sp.R3 1 2 is reported to have a number of specific amidases: an a-amino amidase which hydrolyzes only L-a-amino amides to the corresponding acids()5, a wide-spectrum amidase hydrolyzing many al iphat ic amides!>" an enantioselec t i ve amidase hydrolyzing ary loxy propionamides6!J , a novel amidase ca l led adipamidase hydrolyzing dinitri les(17 and several en­zymes specific for urea, formamide, L-glutamine, and n icot inamide!>7 . The wide- spectrum amidase from Rhodococcus sp. R3 1 26x is very simi lar to the amidase of Pseudomonas aeruginosa69• Another enantiomer se­lect ive amidase i s descr ibed i n a newly i so lated Rhodococcus s p . , wh i ch is s i m i lar to that i n Rhodococcus sp. R3 1 270. I n R . rhodochrous J I two types of amidases differing in substrate specificity have been reported7 1 . Based on studies with inhibitors the amidases have also been found to be sulphydryl enzymes71. How­ever, no active aminoacid residue has been identified in any of them. Only recently, the real active site residues of R. rhodochrous J I amidase have been identified as Asp ,,! , and Ser I 9" rather than the general ly accepted Cys residue72. Interestingly, besides hydrolyzing amides, different amidases exhibit acyl transfer activity in the presence of hydroxylamine. This reaction has been re­cently described for the amidases of Rhodococcus sp. R3 1 273 , which leads to the formation of a wide range of hydroxamic ac ids.

Genetics of Nitrile Metabolism The first among the n itrilase genes to be cloned was

the Klebsiella ozaenae plasmid born gene bxn, encod­ing a nitrilase that degrades bromoxynil 74 . Later, this gene was used to develop transgenic tobacco or tomato plants resistant to the herbicides 75 . The nitrilase genes (nitA) from Rhodococcus rhodochrous J I , K22 and AL­caligenes faecalis JM3 have been c loned and se­quenced30.76.77 and show significant similarity to bxn gene. Another gene encoding an aliphatic n itrilase, active on

932 ] SCI INO RES VOL.58 DECEMBER 1 999

Table 3 - Properties of amidases purified and characterized from different nitrile-metabolizing microorganisms

Source of enzyme

A r,hrobacter sp. J l

Brevibacterium sp. R 3 1 2h)

sp. R 3 1 2h)

Klebsiella Jineul1loniae NCTR I

Pseudomonas A eruginosa

Formation type

inducible

i nducible

i nducible

Metal

NA

NA

inducible Co & Fe

i nducible NA

Mol. mass (kDa)

320

1 80

1 20

62

200

chLororaphis 823 i nducible no metal 1 05

sp. GDI 2 1 1

Rhodococcl/S

sr·

sr·

i nducib le NA

NA NA

constitutive Fe

43

1 1 8

360

a) NA - Not available ; b) same as Rhodococcus sp. R3 1 2

N o . of subunits & mol. mass (kOa)

8 (39)

4 (43)

2 (4.6)

monomer

8 (38)

2 (54)

NA (26)

2 ( 48 .5 )

NA (44.5)

adiponitri l e and cyanovaleric acid, i s c loned from Comamonas testosteron i ct25.

The genetics of nitrile metabolism to a large extent, has been studied in the industrially important microbial strains having nitrile hydratase and amidase system. M/s Nitto Chemical Industry have patented some of the genes encoding the nitrile metabolizing enzymes from p. chlororaphis B23 and R. rhodochrous J 1 7X.7<J. In all these strains, the amidase genes have been found adja­cent to nitri le f.;,dratase genes in the same operon, ex­cept for aliphatic ni trile hydratase gen': cluster of R.

Properties of amidase

Opt. temp. ( 0C)

55

NA

NA

65

N A

Heat Opt. stabi l i ty p H

(uC)

30-45 7.0-9.0

N A N A

N A N A

30-65 7.0

N A N A

p H stabi l i ty

7.0

N A

N A

5 . 0-8 .5

N A

S ubstrate specificity

A l i phatic amides

Ref.

1 83

A l i phatic 1 84 amides

(wide spectrum)

Aryloxy 66

propionamides (enantio-selective)

A l i phatic amides

A l i phatic amides

63

1 85

50 25-50 7.0-8.6 5 .9-9.9 Al iphatic & 64

NA NA NA

NA NA NA

40 NA 8.5

NA

aromatic ami des

Aromatic amides

1 8 6

NA Aryl propionamide 70

NA Al iphatic ami des 62

rhodochrous J I which does not contain amidase gene. This is the reason why this organism is superior for the production of acrylamide. Overexpression ( 1 .7 times) of R. rhodochrous J I aliphatic nitrile hydratase gene has been achieved using a recombinant Rhodococcl/S host/vector system, in which the presence of amide com­pounds is essential for the assembly of active multi mer nitrile hydrataseRo.

The structural genes coding for the (X- and �-subunits of the stereoselecti ve cobalt n itri l e hydratase from Pseudomonas putida NRRL- 1 8668 have been c loned

.J..

RAMAKRISHNA et at. : MICROBIAL METABOLISM OF NITRILES 933

and sequenced59 . A 6-fold over-production of the active stereoselective enzyme has been obtained by the co-ex­pression of a novel downstream gene encoding a protein (P1 4K) which appears to be a part of an operon that in­cludes the structural genes of (X- and �-subunits of the nitrile hydratase and other potential coding sequencesX I • A nitrile hydratase gene from a moderate thermophile, Pseudonocardia thermophila JCM3095 has been cloned and sequenced for the first timex2. The nitrile hydratase of this organism has (X- and �-subunits, exhibits high optimum temperature (60°C), thermostabi lity and re­quires cobalt for high activity. The nucleotide sequences of this hydratase gene show high homology with the hydratase gene of a mesophilic bacterium, Rhodococcus Sp.70.X2 . Another ni trile hydratase gene cah, has been cloned from the fungus Myrothecium verruca ria, which degrades the herbicide, cyanamide to urea. These fun­gal genes have no sequence similarity with the hydratase genes of Rhodococcus sp . N-774, Pseudomonas chlororaphis B23, R. rhodochrous J I and Rhodococcus Sp.ol and have been used to develop transgenic tobacco plants resistant to high concentration of cyanamidex3.

The advances in the biosynthetic regulation, genetics and better understanding of the structure and reaction mechanism of the n itrile-metabolizing enzymes would not only lead to improved properties such as high activ­ity, high tolerance to substrate and product and thermo­stability, etc . of the biocatalysts used in commercial pro­cesses, but also enable the development of novel nitrile­hydrolyzing enzymes with stereo-, regio- and substrate specif ic i ty for chemica l syntheses or for bioremediation54•

Biotechnological Potential and Applications

The nitrile compounds can be easily prepared by a number of chemical methods and are commonly used as intermediates in the synthesis of several commercially important amides and/or acids X4. For these syntheses, the nitriles are hydrolyzed using traditional chemical methods that have several such drawbacks as : a) reac­tions need be carried out in strongly acidic or basic me­dia, b) h igher reaction temperature, and c) formation of by-products such as toxic HCN or large amount of salt, etc. Compared with the chemical (non-enzymatic) route, the biotechnological route has the following ad­vantages9•X4 : a) less severe (mild) reaction conditions, b) substrate and product specificity, c) formation of prod­ucts with a very h igh purity, d) potential for conducting chemo-, stereo- and regio-selective transformations that

are difficult to achieve through non-enzymatic route, and e) non-polluting.

It has been shown that ni trilases, hydratases and ami­dases hydrolyze a number of structurally diverse nitriles. Several commercially important organic compounds such as p-aminobenzo ic ac id , benzamide , 2 ,6-difluorobenzamide, n icot inamide, i sonicotinamide, pyrazin amide , pyrazino i c ac id , qu i no l i nami de, thiophenamide, etc . have been prepared from the corre­sponding nitri les using the cells of R. rhodochroLis J I containing both nitri lase ' 5.52 and nitrile hydratasex,. The production of acids and amides using R. rhodochrolls J I enzymes has been patented by M/s Nitto Chemical IndustryXo.x7. Camitine has been prepared from the cor­responding nitrile by another patented process using the nitrilase of Corynebacterium sp.xx. Vaughan et af. MY con­verted 3-cyanopyridine into nicotinic acid by n itrilase containing cells of Nocardia rhodochrous induced by benzonitrile, while Eyal and CharlesYO prepared nicoti­namide from cyanopyridine by the whole cell n itri le hydratase. The Novo Industri NS of Denmark has de­veloped two immobilized whole cell biocatalysts, SP36 1 and SP409 containing both nitri le hydratase and ami­dase but lacking nitrilase, for hydrolyzing nitriles. In fact, the immobilized biocatalysts have the advantages that they are very simple to use and can be easily recovered after the reaction for reuse. Using the biocatalyst SP409, Klempier et alYI and deRaadt et al.Y2 achieved mild and selective hydrolysis even in the presence of acid- or base­sensitive groups contained in a broad range of aliphatic, alicyclic, heterocyclic and carbohydrate type nitriles under neutral conditions. Similarly, the biocatalyst is also found to be very effi c i en t and conven ien t for chemoselective and mild hydrolysis of a number of het­erocyclic n itriles to either amides or carboxylic acids. Bulky substituents, insufficient solubil ity and possibly inhibition phenomena are some of the limitations ob­served in this studyY3. Recently, the biotransformation of nitriles into amides and/or acids using R. rhodochrous AJ270 was reportedY4• The range of substrates together with h igh product yields and selec t iv i t ies , makes Rhodococcus as one of the most valuable microbial sys­tems for nitrile hydrolysis. This study has also defined useful limitations for the acceptabi lity of substrates for hydrolysisY4.

The hydrolysis of dinitriles by the nitrile-metaboliz­ing enzymes presents an interesting means of synthe­sizing a wide range of organic compounds not amenable

934 J SCI IND RES VOL.58 DECEMBER 1 999

/ CN R

' CN

d initrile

hydratase

cyanoamide

amidase / COOH R

\ CN

cyano carboxyl ic acid

....

.

....

.

.

.

.

chemical reaction

.

.

.

.........

. ........

..

.

.............

.

..

1 hydratase hydratase

.� / CONH2 R

" CONH2

diamide

..•.

chemical reaction

.....

amidase

....... ...

.......

..•.

.-

-

.

.

�

/ COOH R

' CONHz

amido carboxylic acid

� amidase

/ COOH R

, COOH

dicarboxylic acid

Figure I - Formation of di fferent products from dinitriles through enzymatic and chemical route

for preparation i n h igh y ields by conventional chemical syntheses�·�5. Scient ists from Novo Industri AIS , Den­

mark, found that a d in i tr i le could be converted i nto the corresponding amido-carboxyl ic ac id by applyi ng, in se­

quence, a monoacid generating stra in and an amidase

free preparation from a diac id generati ng strain . About

five types of products coul d be prepared in h igh yie lds by apply ing these enzymes as shown in Figure I , whi le

only two products, a d iam i de or a d iacid are possible with chemical synthesis9.�5. Novo Scientists are able to synthesize precursors for ny lon-6 and nylon-6,6 poly­

mers, adipic acid and caprolactam, from adiponi tri le by combini ng both chemical and enzymatic conversions')5.

The al i phat ic n i tri l ase from R. rhodochroll.\' K22 cata­lyzes the conversion of various a l iphatic n itri les includ­ing dini tri les l ike g lutaroni tri le which i s converted to 4-

cyano butyric acid (Figure 2a). Other d in i tri les such as b u t a d i e n e n i t r i l e , fu maron i t r i l e , ( F i g u re 2 b - c ) malononi tri le, succinonitri le, adiponi tri le, p imelonitrile, etc. are also attacked by K22 n itri l ase, forming the COITe­sponding cyano carboxyl ic acids%. The aromatic n itri lase

from R. rhodochrous J I is capable of conversion of

terephthalon itr i l e and i sophtha lon i tr i l e to 4- and 3-cyanobenzoic acids (Figure 2d-e) without formation of any d i ac i d�7, w h i le the b i ocata ly s t SP36 1 con verts i sophthalonitri le to 3-cyanobenzoic acid , from which a d iacid can also be prepared by esterification of the car­

boxyl ic group fol lowed by re-exposure to the biocata­

lyst for the hydrolys is of the second n itri l e group (Fig­

ure 2e)X4.9x. DuPont company patented an enzymatic process for the b iocatalytic convers ion of azobi sn itri les to cyanoamides or diamides us ing n itri l e hydratases of Pseudomonas, Rhodococcus, or Brevibacleriul11')�.

Another very interesting aspect of n itri l e bioconver­sions is the capab i l i ty of these enzymes to carry out stereoselect ive transformations. First among the earl iest reported stereose lect i ve n itri le conversion is the produc­t ion of optical ly act ive L-a-hydroxy acids from racemic a-hydroxy n itri les by Torulopsis candid({ I IX I . S i m i l arly, Rhodococcus sp. R3 1 2 has been u sed to produce opti­

cal l y act ive L- or D-amino acids from racemic a-amino n i tri les 1 1 1 1

. Macadam and Knowles 1 1 12 showed the con­version of a-ami nopropioni tr i le to L-al an ine us ing im­mobi l ized cel l s of Acinetohacter sp. APN. Hydrolys is

-t

.....

RAMAKRISHNA ef al. : MICROBIAL METABOLISM OF NITRILES 935

Ca) Rhodococcus rhodochrous K22

glutaronitrile

(b)

NC� CN

1 ,3-butadiene nitrile

(e)

� /eN NC"'- -...:/

fumaronitrile

(d)

nitrilase or SP 361

K 22 nitrilase or SP 361

K 22 nitrilase

4-cy3nobutyric acid

NC, /='\ \=.I \COOH

5-cyanopenta 2 ,4-dienoic acid

NC�COOH 3-cyanofumaric acid

6 Rhodococcus rhodochrous J 1

• nitrilase CN

terephthalonitrile 4-cyanobenzoic acid

(e)

CN Rhodococcus COOH COOM. Q rhodochrous J 1 C\N CH2N, and Q � � CN or SP 361 SP 361 COOH

isophthalonitrile 3-cyanobenzoic acid isophthalic acid monoester

Figure 2 - Conversion of aliphatic dinitriles, (al glutaronitrile, (b) I ,3-butadiene nitrile and (cl fumaronitrile by Rhodococcus rhodochrolls K22 nitrilase and the biocatalyst SP3 6 1 and aromatic dinitri lcs, (d) terephthalonitrile, and (el isophthalonitrilc by Rlwdococcus rhodochrolls J I nitrilase and SP 36 1

of (R,S)-( + )-ibuprofen nitri le leading to the formation of the non-steroidal anti-inflammatory drug, (S)-( +)­ibuprofen (Figure 3a) was achieved by the nitrilase of Acinetobacter sp. AK226 with an enantiomer excess (e.e) of above 95% for the (S)-( + )-product acid and the recovered (R)-( + )-nitrile'o:1. (R)-( -)-Mandelic acid, a com­mercially important bui lding block for preparation of some semi-synthetic cephalosporins, is prepared from racemic mandelonitrile (Figure 3b) by an enantioselective nitrilase from Alcaligenes faecalis ATCC 8750104 10' . In a strain of Pseudomonas sp. enantioselective nitrilase catalyzes hydrolysis of racemic O-acetylmandelonitrile (Figure 3c) to (R)-(-)-acetylmandelic acid ,06. Synthesis of optically active aminoacids (L-form, except for ala­n ine) from a-aminonitriles by a n i trilase system of R. rhodochrous PA-34 has been reported2Y• Gradley and

Knowles l()7 showed the enantioselective potential of R. rhodochrous NCIMB 1 1 2 1 6 nitrilase using a range of chiral nitriles including (R,S)-2-methylhexanitrile, which is converted to (S)-( + )-2-methyl hexanoic acid.

With the aid of enzyme system of R. butanica ATCC 2 1 1 97, kinetic resolution of a-arylpropionitriles has been shown successful ly for the first time, resulting in the formation of optically pure (R)-amides and (S)-carboxy­l ic acids (Figure 4a), some of which are important as an t i - i n fl ammatory drugs l OX . Opt i c a l l y pure 2 -

arylhydroxypropionic acids (Figure 4b) which have high herbicidal activity have been prepared from correspond­ing racemic nitriles using Brevibacterium imperiale CBS 49874 cell s with nitrile hydratase and amidase lOY. Dur­ing this study, it was observed that the hydratase con­verted the racemic n itrile substrate by a fast nonspe-

936 J SCI IND RES VOL.58 DECEMBER 1 999

(a) ,. �COOH (S)-(+)-ibuprofen

Acinetobacter sp, AK226 � +

(R,S)-ibuprofen nitrile [2-(4-isobutyl phenyl) propionitrile

S-selective nitrilas�e Me

I � CN �

(R)-(-)-ibuprofen nitrile

(b) OH Alcaligenes faecalis A TCC 8750 OH OH �CN . • M"'CN · +

R-selective nitrilase U � ���H (R,S)-mandelonitrile (S)-mandelonitrile

(c)

(R)-(-)-mandelic acid

/Ac o

Pseudomonas sp. �COOH (R,S)-O-acetyl mandelonitrile

R-selective nitrilase (R)-(-)- acetyl mandelic acid

Figure 3 - Preparation of (a) (R)-ibuprofen, (b) (R)-( -)-mandelic acid, and (c) (R)-( - )-acetyl mandelic acid, using stereo-specific nitrilase enzyme systems

cific hydrolysis while the amidase catalyzed a slow stereoselective conversion into the (R)-acids. The (S)­amides were also recovered with h igh optical purity. The pharmaceutically active, non-steroidal anti-inflammatory substances, 2-arylpropionic acid such as (S)-( + )-2-phe­nyl propionic acid (e.e, 99.4%) was prepared by the combined action of a nitrile hydratase and stereoselective amidase system of Rhodococcus equi TG238 from (R,S)-2-phenylpropionitrile (Figure 4c) and the (R)-( -)-2-phe­nyl propionamide (e.e, 99%) is also isolated "0. Simi­larly, the enant ioselect ive hydrolysis of racemic 2-arylpropionitriles suC;;h as naproxen nitrile, ketoprofen nitrile, etc . was accomplished using the nitrile hydratase and amidase contain ing organisms, Agrobacterium tumefaciens d3 1 1 1 , Rhodococcus sp. C3II, R. erythropolis MPSO1 12. I I J and R. equi A4 "4, as shown in Figure Sa-d. The capab i l i ty of Rhodococcus sp . • C3I I and R. erythropolis MPSO for the regio- and stereo-selecti ve hy­drolysis of various aliphatic and aromatic n itriles and acid amides has been elucidated in detail recently l " . The nitri le hydratase and amidase system of a Pseudomonas

sp. carried out enantioselective hydrolysis of (R,S)-2-i sopropyl-( 4-chlorophenyl)-acetoni trile to the corre­sponding S-acid"6. Pseudomonas sp. BC- 1 8 having ni­trile hydratase and amidase enzyme system accomplished enantioselective hydrolysis of (R,S)-3-phenyllactonitrile to (S)-( -)-3-phenyllactic acid as shown in Figure 6a 1 17 . This compound is a versatile precursor for the synthesis of several pharmacophores such as rennin inhibitors, protease inhibitors and anti-human immunodeficiency virus reagents l l 7 .

In the case of stereoselective hydrolysis reactions of nitrile hydratase and amidase system, it is general ly ob­served that the stereoselectivity resides primarily in the amidase, not in the hydratase"o. 1 I 6. However, some of the recent studies have revealed that the hydratase can also carry out stereoselective transformations. For ex­ample, Blakely and coworkers" x have reported the for­mation of (R)-( + )-2-phenyl butyramidc with an e.e. of 83% from the corresponding n itri le by Rhodococcus sp. AJ270. During this transformation, the (R)-amide inter­mediate could not be hydrolyzed further, as it is not a

t

RAMAKRISHNA et al. : MICROBIAL METABOLISM OF NITRILES

(a)

R R R �CN RJ() hydratase

• �ON�2 . S-specific �OOH

Rvll� � ----II ... R1� (R,S)-aryl substituted

alkane nitrile

(b)

R'

(R)-amide

R = Me, Et R' = iso-bu, CI, OMe

Brevibacterium imperiale

... nonspecific hydratase

(R,S)-2-aryloxy n itrile

(S)-2-aryloxy amide

(e)

Rhodococcus equi TG 328

nonspecific hydratase

(R,S)-2-phenyl propionitrile

(R)-(-)-2-phenyl propionamide

amidase

(S)-acid

R' �:&OyCONH' (R,S)-2-aryloxy amide

I R-selective t amidase

(R)-2-aryloxy carboxylic acid

(R,S)-2-phenyl propionamide � S�I""'e a"",,,, �H3 VeOOH (S)-(+)-2-phenyl propionic acid

Figure 4(a) - General reaction showing the transformation of racemic aryl substituted alkane nitriles to optically pure R­amides and S-acids by Rhodococcus butallica or SP36 1

Figure 4(b) - Preparation of 2-aryloxy carboxylic acids by nonspecific hydratase and R-specific amidase system of Brevibacterium imperiale

Figure 4(c) - Bioconversion of racemic phenyl propionitri le to (R)-( -)-amide and (S)-( +)- acid by the nitrile hydratase and S-selective amidase system of Rhodococcus equi TG328

937

938 J SCI IND RES VOL.58 DECEMBER 1 999

(a) �H3 ofCN Agrobacterium tumefaciens d3

S'specific hydratase

�eONH2 (R.S)-2-phenyl propionitrile (S)-2-phenyl propionamide

� S-specific amidase

�H3 VeOOH (S)-2-phenyl propionic acid

(b)

Rhodococcus equi A4

hydratase + amidase

�eN RAJ . (R ,S)-aryl propionitrile (S)-aryl propionic acid (R)-aryl propionitrile

(c) CH �' Rhodococcus sp.

I '" '<::: CN C311 or MP 50 h h • H,CO

S-specific hydratase

(R,S)-naproxen nitrile [2-(6-methoxy 2-naphthy l propionitrile]

(d) CH, � Rhodococcus I '<::: I '"

. CN erythropolis MP50

h h -------i.�

(R,S)-ketoprofen nitrile [2-(3-benzyl phenyl) propionitrile]

S-specific hydratase

S-naproxen amide � S-specific amidase

�H3 �COOH H3CO� (S)-naproxen (acid)

(S)-ketoprofen amide

+ S-specific amidase

�H, o _ �COOH

V V (S)-ketoprofen (acid)

Figure 5 - Stereospecific biotransformation carried out by nitrile hydratase and amidase system leading to the formation of (a) (S)- 2-pheny[ propionic acid, (b) (S)-aryl propionic acid, (c) naproxen, and (d) ketoprofen

-

RAMAKRISHNA et al. : MICROBIAL METABOLISM OF NITRILES 939

(a)

eN eOOH � Pseudomonas sp. BC-1 8 � V OH . ---------i.� V &H hydratase + amidase

(R,S)-3-phenyl lactonitrile (S)-(-)-3-phenyl lactic acid

(b)

""I X eN Pseudomonas putida

eN '

S-selective hydratase (R,S)-2-(4-chlorophenyl)-3-methyl

butyronitrile (S)-2-(4-<:hlorophenyl)-methyl

butyramide

Figure 6 - Stereo-specitic hydrolysis of (a) phenyllactonitrile, and (b) 2-(4-chlorophenyl)-3-methyl butyronitrile

substrate for the amidase of this bacterium. This obser­vation led to the recognition of the stereoselective na­ture of the nitrile hydratase activity l lx. S imi larly, Fal lon et al. 5x also reported the S-selective nitrile hydratase of p. putida NRRL l 8668, capable of hydrolyzing 2-(4-chlorophenyl)-3-methyl butyronitrile to the correspond­ing (S)-amide (Figure 6b). The S-selective hydratase from A. tumefaciens d3 involved in the hydration of 2-aryl propionitriles has been purified and characterized very recently I 1 9 . In fact, this organism also harbours S­specific amidase I I I . A detai led i nvestigation of the stereoselective hydrolysis of both racemic and prochiral n i tr i les has been made w i th Novo ' s b i ocata lys t S P3 6 1 XOX. 1 211 . 1 2 1 . A seri es of proc h i ral 3 -hydroxyglutaronitrile derivatives have been hydrolyzed u s i ng t h i s ca ta lys t to t he correspond ing ( S )­cyanocarboxylic acids (Figure 7a) with e.e ranging from 22 to 84%1211. With the same biocatalyst, Beard and co­workers l2 1 also carried out stereoselective hydrolysis of several racemic and prochiral nitrile substrates . 2-Alkyl­arylacetonitriles were hydrolyzed to (S)-acids and (R)­amides whereas the c lo se ly rel ated (±) -2 - ( 4-isobutylphenyl)-propionitri le gave the (R)-acid. In some of these conversions the recovered nitrile was also found to be optically active. Regioselective hydrolysis of aro­matic dinitriles also has been demonstrated using SP36 1 with fluoro dinitriles and their methyl or amino substi­tuted compounds as substratesK4 • The R. rhodochrous IFO 1 5564 mediated stereo-selective hydrolysis of ali-

cyclic nitriles and amides and its use for kinetic resolu­t i on and asymmetrizat ion h as been described 1 22 . Rhodococcus sp. R3 l 2 has been reported to have the capabil i ty to transform various prochiral dinitriles to enantiomerically pure (S)-cyanoacids 1 23 . For example, the lactone moiety of mevinic acids, which are pharma­ceutically important as effective hypocholestrolemic agents, can be conveniently synthesized by combination of chemical and enzymatic reactions (Figure 7b) in­volving the hydratase and amidase system of the strain R3 l 2 or the biocatalyst SP36 1 from the corresponding prochiral dinitri le124 .

A new b ioc ata lys t , Ochrobactru111 a n th ropi NCIMB4032 I , which has a broad-spectrum L-specific amidase activity hydrolyzing a large variety of amides ranging from a-H-a-amino-, a-alkyl, a-amino, N-hy­droxy-a-amino acid amides to a-hydroxy-acid ami des has been reported 1 25 . Interestingly, the presence of an enantioselective amidase converting several racemic aro­matic amides such as 2-phenylpropionamide etc . to the corresponding optical ly pure carboxylic acids has been shown in P. chlororaphis B2364. 5-Hydroxy- pyrazine-2-carboxylic acid, a versati le building block for the syn­thesis of new antituberculous agents has been prepared by whole cell biotransformation from 2-cyanopyrazine via pyrazine carboxylic acid (Fig. 7c) using the nitrilase and regioselective dehydrogenase enzymes present in the bacterium Agrobacterium sp. DSM 6336 126.

940 J SCI IND RES VOL.58 DECEMBER 1 999

(a) OR SP 361

�R NC�COOH NC�CN hydratase + amidase

Substituted 3-hydroxy glutaronitrile

(b)

(S)-cyano carboxylic acid

08z

(j S-specific hydratase + amidase

08z

Brevibacterium sp. R312 or

SP 361

o A Chemical Synthes: + 0 I I k " " 0 HOOC CN SP 361 non ,- ,' 0 NC CN

3-(benzyloxy) g lutaronitrile

(e)

(S)-3-cyano acid

specific hydratase + amidase

lactone moiety of mevinic acids

�NyCN __ A

_

9/1

_

0b

_

a

_

c

_

te

_

riu

_

m

--l

s

.�

p .

�N) nitrilase . fNYCOOH regiospecific

. fNYCOOH �N) dehydrogenase HO�N)

2-cyanopyrazine pyrazine-2-carboxylic acid

5-hydroxy pyrazine-2-carboxylic acid

Figure 7 - Enantioselective syntheses of (a) S-cyanoacids, and (b) lactone moiety of mevinic acids using nitri le hydrolyzing enzymes and chemical synthesis, and (c) Synthesis of 5-hydroxy pyrazine-2-carboxylic acid by nonspecific hydrolysis followed by regio-specific hydroxylation of 2-cyanopyrazine

Enzymatic nitri le hydrolysis in low water systems using nitri le hydratase from Rhodococcus sp. DSM 1 1 397 and nitrilase from Pseudomonas sp. DSM 1 1 387 has been studied by Layh and Wil lets 1 27 . This is a very fascinating and emerging area in which information is highly essential . In the patent li terature also several stereoselective whole-cel l biocatalysts for acid produc­tion from nitriles have been described 'O I . 1 2x . i 32, Thus, a great commercial potential is envisaged for the nitrile­metabol izing enzymes as catalysts for converting ni­triles to higher value amides and/or acids on an indus­trial scale. A few of the potential applications of nitrile biotransformations are discussed below.

Acrylamide Acrylamide monomer is an important commodity

chemical used to make synthetic fibres, flocculent agents and polymers for petroleum recovery, with a world-wide

demand of 200,000 tpa. It is conventionally synthesized by the hydrolysis of acrylonitrile using the catalyst, raney copper at about 1 00°C. The production of this com­modity chemical is desirable under mild conditions as the chemical method has several draw-backs like the use of high temperature, etc.52,)3 . Nitto Chemical Indus­try, Japan, started the industrial production of acrylamide through a biotechnological route in 1 985 using Coryne­bacterium sp. N-774 (Rhodococcus sp. N-774), Later, the second generation acrylamide process using a supe­rior bacterium, P chlororaphis B23 with a production capacity of 6,000 tpa has been establ ished in 1 988. The productivity of this process has been further enhanced in 1 99 1 by another more powerfu l b iocatalyst , R. rhodochrous J I in the third generation process, The co­balt nitrile hydratase of this organism is a robust and superior enzyme for acrylamide production as it is more heat stable, up to 50°C and tolerates high concentrations

••

RAMAKRISHNA et at. : MICROBIAL METABOLISM OF NITRILES 94 1

of not only acrylonitrile but also the product, acrylamide up to the concentration of 50% (w/v). As a result of the high productivity of the cel ls, the capacity could be in­creased to 30,000 tpa without any major change in the plan t53. A comparison of the three generations of biocatalysts used for Nitto's acrylamide process clearly shows the superiority of R. rhodochrous J 1 in terms of specific activity, productivity and final concentration of acrylamide achievable, etc.53.K5. 1 34. Overall , the biotech­nological process is much simpler and economical , the recovery ofunreacted acrylonitrile is unnecessary as the conversion is more than 99.99% and a very pure prod­uct is obtained53. The Nitto's acrylamide plant using the biocatalyst continues to be operated successful ly.

Nicotinamide, Nicotinic Acid and p-Amino­benzoic Acid

Nicotinamide is a useful v itamin and is also used as animal feed supplement. Nagasawa and coworkers 1 35 showed 1 00% molar conversion of 3-cyanopyridine to nicotinamide by R. rhodochrous 1 1 nitrile hydratase. Be­sides n icotinamide, other commercially important com­pounds such as nicotinic acid and p-aminobenzoic acid have been shown to be produced by resting cells of No­cardia sp. and R. rhodochrous 1 1 K9, 1 30, As chemical syn­theses of these compounds are very complex and require harsh conditions, their production by biotechnological route that uses only mild conditions is very attractive. Recently, Lonza has announced that a biological pro­cess based on the enzymat ic convers ion of 3 -cyanopyridine by the R. rhodochrous cells with nitrile hydratase wilI be used in a 3000 tpa nicotinamide plant in Guanzhou, South China. This technology has been licensed from Nitto Chemical Industries, Japan, and the plant is due to come on-stream very soon 1 37 •

Acrylic Acid and Methacrylic Acid Acrylic acid and methacrylic acid are commercially

important starting materials for the synthesis of various polymers, They are traditionally manufactured by gas­phase oxidation of propylene and isobutylene in the pres­ence of oxide catalysts at a high temperature l3X , Severe problems with the catalysts such as coking, inactivation, etc. and polymerization of the products at high tempera­tures are some of the draw-backs in the chemical syn­theses. So for the production of these acids the use of nitrile-metabolizing organisms as biocatalysts, which re­quire only mild conditions as opposed to traditional chemical methods, is attractive 13K. Nocardia rhodochrous

LL l OO-2 1 has been found to convert acrylonitrile to acrylic acid but is unable to metabolize the acrylic acid further9. S imilarly, R. rhodochrous 1 1 n i trilase induced by E-caprolactam is also shown to be a very powerful biocatalyst of value for the industrial scale production of acryl ic acid and methacrylic acid w ith a h igh molar conversion and without any by-product formation 1 3K. Processes for the preparation of ammonium acrylate and ammon ium methacryl ate from acry lon i t r i le or methacrylonitri1e, respectively, using R. rhodochrous nitrilase with low Km for acrylonitrile have been pat­ented '39, '40.

Hydroxamic Acids Hydroxamic acids (HA), which form very stable che­

lates with a number of metal ions, are also constituents of growth factors, food additives, antibiotics, antibiotic antagonists, tumor inhibitors, antifungal agents, cell di­vision factors and enzyme inhibitors and are used as re­agents in analytical chemistry for metal determinations73. u-Aminohydroxamic acid derivatives have medical ap­plications, since they are potent inhibitors of several matrix metalloproteases, the zinc endopeptidase enzymes involved in the t issue remodel l ing73 , Some of the polymerizable unsaturated HA and mid-chain or long­chain HA are used for wastewater treatment and nuclear technology to eliminate metal ions73, ' 4 1 . '43. The long­chain HA are also efficient surfactants '44 . As the chemi­cal methods for the manufacture of these highly useful compounds have the drawbacks of requiring many sol­vents, sometimes high temperatures, n itrogen atmosphere and cumbersome steps, use of biocatalysts for produc­ing these molecules facil i tates some otherwise difficult reactions under mild conditions73, '44 . Recently, the ami­dases from the organism, Rhodococcus sp. R3 1 2 have been reported to have the exceptional capability of cata­lyzing the biosynthesis of the HA compounds under mild conditions in reaction media devoid of organic sol­vents 73. 1 44 . The Rhodococcus sp. R3 1 2 wide-spectrum amidase can synthesize short-chain C2-C

3 HA, while the

Rhodococcus sp. R3 1 2 adipamidase catalyzes the syn­thesis of C

4-CK HA and the Candida parapsilosis l ipase/

acyl transferase produces the hydrophobic long-chain HA144.

Optically Active Compounds Use of nitrile-hydrolyzing enzymes offers a potential

method for enantioselective conversion of racemic u-

942 J SCI IND RES VOL.58 DECEMBER 1 999

hydroxy or a-amino ni triles to stereospecific (0- or L­isomer) a-hydroxy or a-amino acids (or amides) under mild conditions. Using these organisms, lactic acid has been produced from lactonitrile which can be easily and economically made by reacting acetaldehyde and hydro­gen cyanidel,�. Nitto Chemical Industry patented a pro­cess for microbial manufacture of optically active (0- or L-) lactic acid (e.e., 1 00%) from racemic lactonitri le by several microorganisms including Enterobacter Sp. 145. Lactic acid has the potential of becoming a very large volume commodity chemical intermediate for use as feedstock for b iodegradable polymers, oxygenated chemicals, plant growth regulators, environmentally friendly 'green ' solvents and speciality chemical inter­mediates l 4n. Of even greater commercial interest is the synthesis of other expensive, optically pure D- or L-acids from corresponding racemic ni triles. Rhodococcus sp. R3 1 2 has converted OL-aminonitriles to L-amino acids in 50% yield, the remainder being o-aminoamidel . Aminopropionitrile has been stereospecifically converted to L-a lan i n e by u s i ng immob i l ized ce l l s of an Acinetobacter sp. 102. Optically active aminoacids have been produced from a-aminonitriles by the cells of R. rhodochrous PA-34 having n itrilase2�. The non-steroi­dal, anti- inflammatory compounds such as (S)-( +)­ibuprofen, naproxen and ketoprofen can be made from corresponding ni triles by enantiomer selective transfor­mation by Acinetobacter sp. AK226, Agrobacterium tumefaciens d3, Rhodococcus sp. C3II, R. erythropolis MP50, R. equi A4, respectively l 03, 1 1 1 . l l i . A process us­ing ultrafiltration membrane bioreactor or immobilzation on porus cellulose beads has been described for the pro­duction of (S)-( + )-ibuprofen 147, 14X . Simi larly, optically pure 2-ary lhydroxy propionic acids having herbicidal activity have been prepared from the corresponding ra­cemic nitriles by Brevibacterium imperiale CBS49874)(�).

Adipic Acid and Caprolactam Interestingly, Novo Industri AlS, Denmark, using

n itrilase, nitrile hydratase and amidase prepared precur­sors of nylon-6 and nylon-6,6, adipic acid and caprolac­tam by converting adiponitrile into either the correspond­ing cyanoamides or cyanocarboxylic acids, etc . , that can be further converted to precursors by chemical synthe­sis�5. Another enzymatic process for adipic acid produc­tion from adiponitrile using mutants of Rhodococcus sp. R3 I 2, which have h igher adiponitrile-metabolizing ca­pabi lity has also been reportedn7, 14�- 1 5 1 . Novo Industri AIS h as been cons ider ing the b i ocon vers i on of

adiponitrile to produce adipic acid and caprolactam on a commercial scale for nylon manufacture.

Biodegradation of Nitrile-containing Industrial Effluents

A very important aspect of biotechnological poten­tial of nitrile metabol ism is the biodegradation of ni­trile-containing industrial effluents. Manufacturing of n itriles on a large scale world-wide is usual ly associ­ated with the formation of a large amount of toxic efflu­ents. These effluents pose a great danger to the environ­ment and microbial degradation is the most convenient and cost-effective technology for their detoxification.

A) Acrylonitrile Plant Wastewater Acrylon itrile (ACN) is manufactured conventional ly

by ammoxidation process where propylene is oxidized in the presence of ammonia and air (oxygen). This gen­erates two major ACN effluent streams: I ) wastewa­ter-I in the quench column after stripping volatile or­ganics, designated as K01 1 (EPA hazardous waste code) wastewater, contains very high amounts of organics, cya­n ides, total dissolved solids (TDS) mostly as ammonium sulphate l 52, 2) wastewater-II, the stripper column bot­tom after recovery of crude acetonitrile, designated as KO 1 3, is relatively dilute with lower amount of organics and cyanideJ52. The ACN wastewater typical ly contains cyanides, acrolein, acrylonitrile, acetonitri le, ammonia and other reaction products along with heavy organic material 1 53 . Since the ACN wastewater contains cyanide mostly in the form of inorganic cyanide, many chemical oxidation methods such as alkaline chlorination, etc. can be used for the removal 154 , 1 55. Due to its high toxicity, acclimation of biological systems to degrade ACN waste­water has general ly been only partially successful . Kato and Yamamural56 failed to adapt an ordinary activated sludge system to degrade even dilute concentrat ions of ACN wastewater. However, by augmentation of a ni­trile-degrading Nocardia to the activated sludge, the system degraded 1 0-50 mg/L cyanide. Similarly, Fuj i i and Oshimil57 also treated the wastewater by activated sludge having Alcaligenes or Achromobacter that de­grade nitriles and cyanide. Mimura and coworkers l 5x h ave reported the appl ication of Corynebacterium nitrophilus having the abi lity to degrade acetonitrile, hydrocarbons, carboxylic acids, alcohols and ketones, for biotreatment. Ramakrishna and coworkersl 5� have studied the activated carbon powder-activated sludge (PAC-AS) process for the treatment of ACN plant waste-

I

RAMAKRISHNA et al. : MICROBIAL METABOLISM OF NITRILES 943

water, which has shown advantageous effects such as enhanced COD reduction and specific respiration rate of the activated sludge. However, with prolonged opera­tion the advantages diminished as irreversible adsorp­tion of non-biodegradable components inhibited the in situ regenerati on of carbon surface . Knowles and Wyattl60- 162 developed and patented a novel biological process for the degradation of ACN wastewater using a mixed cul ture of Alcaligenes, Pseudomonas, Flavobac­terium, Acinetobacter spp and Bacillus megaterium ca­pable of degrading the main wastewater constituents. An integrated process for the treatment of KO I I and KO 1 3 ACN wastewaters i s proposed by Zimpro Passavant En­vironmental Systems, Inc. USA'52. The treatment in­cludes, first step pretreatment by wet air oxidation, then a second step passage through evaporator/crystall izer to remove ammonium sulphate and a third step biophysi­cal powdered activated carbon treatment (PACT). Mishra and coworkers 163 have also successfully treated the ACN wastewater by a wet air oxidation process followed by biotreatment. Indian Petrochemicals Corporation Ltd (IPCL) have studied various processes and found a physi­cochemical treatment fol lowed by b iotreatment to be commercially feasible 'M, ' 65 . For biotreatment, a mixed microbial culture containing Acetobacter, Acinetobacter, Arthrobacter, Aeromonas, Bacillus, Flavobacterium and Pseudomonas has been developed with advantageous properties l ike better COD and cyanide degradation, settleabil ity, shock resistance, growth and higher spe­cific respiration ratel64. The efficiency of this process improved significantly by using polypropylene pads or active carbon powder as biomass support material re­sulting in a very stable performance under shock load­ing conditions '64 and the know-how is currently being translated in to a commercial scale biotreatment plant.

B) Wastewater from A diponitrile and Nylon Manufacture

Adiponitrile is manufactured commerc ially by sev­eral processes, such as the DuPont Process based on cata­lytic addition of HCN to butadiene and the Monsanto process based on electrodimerization of acrylonitrile'. Though not much information is avai lable on the biotreatment of adiponitrile and £-caprolactam manufac­ture effluents, biodegradation of adiponitrile and acry­lonitrile by organisms l ike Rhodococcus sp. R3 1 2 1 , Arthrobacter Sp. 166, 1 67, Klebsiella pneumoniae '6x, etc . is well documented. B iodegradation of £-caprolactam and related compounds 'fi9 and nylon-6 wastewater contain-

ing £-caprolactam by Pseudomonas aeruginosa MCM B-407 170 has been reported.

Miscellaneous Uses The crude oi l produced from shale deposits contains

h igh levels of undesirable n itrogen and sulphur com­pounds. As a result, the upgradation of shale oil is gen­erally carried out by costly hydrotreatment at h igh tem­peratures and pressures. A b iotechnological process us­ing P aeruginosa and P fluorescence, which selectively degraded the shale oil n i triles, has been described for the upgradation of shale oi ls '7 1 . Such biotech process potentially reduces the cost of shale oi l upgradation .

The nitrile-metabolizing organi sms can also be used for the detoxification of food products containing toxic cyanoglucosides such as cassava roots, a staple food in developing countries 1 72 . Rhodococcus sp. R3 1 2 with an endocellular �-glucosidase, a nitrile hydratase and an amidase ha s been u sed to remove the tox i c cyanoglucosides and a-hydroxy n i triles in cassava pulpl72 .

Another interesting appl ication of the n i trile-metabo­l izing organisms is the enzymatic decontamination of aqueous polymer emulsions (latexes) of n i trile rubbers containing acrylonitri le ' 7, by means of whole cells and cellular lysates of Brevibacterium imperiale CBS 49874, Corynebacterium nitrophilus ATCC 2 1 4 1 9 or Novo's biocatalyst SP 409.

A significant use of n i trile metabolizing organisms has been the development of herbicide resistant plants for the crop i mprovement. Genes from Klebsiella ozaenae or Myrothecium verruca ria that degrade the herbicides, dichlorobenil, bromoxynil and cyanamide, respectively, have been used to develop transgenic to­bacco or tomato plants resistant to the herbicides75,Kl . These transgenic plants may soon be available in the market.

Conclusions A wide variety of microorganisms are reported to have

the capabil i ty to metabolize different types of n i triles. The microbial degradation of nitriles proceeds through two distinct enzymatic pathways: n itrilase catalyzes the direct hydrolysis of n i triles to carboxylic acids and am­monia, while n itrile hydratase catalyzes the hydration of n itriles to amides fol lowed by their conversion to the carboxylic acids plus ammonia by amidase.

Nitrilases are multimeric, sulphydryl enzymes that do not have any metal cofactors or prosthetic groups, while

944 J SCI IND RES VOL.58 DECEMBER 1 999

nitrile hydratases, the most studied among the nitrile hy­drolyzing enzymes, are metal loenzymes having iron or cobalt. Recent studies have shown novel ligand struc­tures of metal binding sites, the unique photoactivation of ferric nitrile hydratase and the mechanism of hyper induction of these enzymes. The versati le biocatalytic nature and applications of nitri le converting enzymes are now increasingly recognised for the production of sev­eral pharmaceuticals and fine chemicals . Also, micro­bial nitrile hydrolysis has been shown as a potential method for the preparation of optically active nitriles, amides and carboxylic acids, not generally feasible by chemical routes . A commercial process involving the mult i-ki loton scale synthesis of acry lamide using Rhodococcus rhodochrous Jl nitrile hydratase is the best example of a ful ly developed industrial application of this biotechnology. Similarly, for nicotinamide also commercial scale biotechnological process is being es­tablished.

The nitrile metabolizing microorganisms by virtue of their capability to eliminate the highly toxic nitrile compounds play a very significant role in al leviating en­vironmental pollution by way of bioremediation of en­vironmental matrices contaminated with toxic nitrile compounds . The genes from the nitrile herbicide degrad­ing organisms have been used to develop transgenic to­bacco or tomato plants with herbicide resistance. Though recent developments broadened the scope of potential application of these versatile biocatalysts in chemical synthesis and bioremediation, further application-ori­ented studies are required to ful ly harness their biotech­nological potential .

Acknowledgements The authors thank the management of Indian Petro­

chemicals Corporation Ltd, for their continued support and permission for publication of this article.

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