Transformation of aliphatic and aromatic nitrilesby a nitrilase from Pseudomonas sp.

Jasvinder Dhillon, Suneel Chhatre, Rishi Shanker, and N. Shivaraman

Abstract: A Pseudomonassp. (S1) isolated from a garden soil possessed a unique nitrilase, which is capable ofcatalyzing the direct hydrolysis of both potassium and organic cyanides to their corresponding carboxylic acids andammonia, without the formation of amide as an intermediate. The nitrilase was purified with 4.8% recovery in threesteps from a cell extract of the strain. The relative mobility of the homogenous enzyme preparation in SDS and nativepolyacrylamide gels indicated molecular weight of 41 kDa, approximately.Pseudomonassp. (S1) utilized all thenitriles as carbon and nitrogen sources. The enzyme was induced by both aliphatic and aromatic nitriles, while thealiphatic olefinic nitrile – acrylonitrile was the most suitable substrate. The nitrilase also catalyzed the hydrolysis ofacetonitrile, adiponitrile, benzonitrile, butyronitrile, glutaronitrile, phenylacetonitrile, succinodinitrile, and potassiumcyanide, with the formation of ammonia and the corresponding carboxylic acids. The Michaelis–Menten constant, Km,of the partially purified nitrilase for acetonitrile, acrylonitrile, adiponitrile, benzonitrile, and potassium cyanidepresented values of 11.26, 5.88, 10.28, 12.27, and 0.75 mM, respectively.

Key words: nitriles, enzyme kinetics, nitrilase, partial purification,Pseudomonassp.

Résumé: Un Pseudomonassp. (S1), isolé d’une terre à jardinage, possédait une nitrilase particulière capable decatalyser l’hydrolyse directe à la fois du potassium et des cyanures organiques en leurs acides carboxyliquescorrespondants et en ammoniaque sans formation d’amide comme intermédiaire. La nitrilase a été purifiée en troisétapes à partir d’un extrait brut de la souche avec un pourcentage de récupération de 4.8%. La mobilité relative d’unepréparation enzymatique homogène en gel de polyacrylamide de base et de SDS a suggéré un poids moléculaired’environ 41 kDa. LePseudomonassp. (S1) utilisait tous les nitriles comme sources de carbone et d’azote. L’enzymepouvait être induite par les nitriles aromatiques et aliphatiques et le meilleur substrat était l’acrylonitrile – nitrileoléfinique aliphatique. La nitrilase pouvait aussi catalyser l’hydrolyse de l’acétonitrile, de l’adiponitrile, du benzonitrile,du butyronitrile, du glutaronitrile, du phénylacétonitrile, du succinonitrile et du cyanure de potassium avec formationd’ammoniaque et des acides carboxyliques correspondants. Les constantes Km de Michaelis–Menten pour la nitrilasepartiellement purifiée étaient respectivement 11.26, 5.88, 10.28, 12.27, et 0.75 mM pour l’acétonitrile, l’acrylonitrile,l’adiponitrile, le benzonitrile, et le cyanure de potassium.

Mots clés: nitriles, cinétique enzymatique,Pseudomonassp., purification partielle.

Dhillon et al. 815

Introduction

Organic cyanide compounds and their derivatives are usedextensively in many industrial operations. Nitriles are syn-thesized on a large scale as solvents, plastics, synthetic rub-ber, pharmaceuticals, herbicides, and starting material forother industrially important chemicals. Nitriles are alsowidely used in organic synthesis as precursors for com-pounds such as amides and organic acids (DiGeronimo andAntoine 1976). Enzymes converting nitriles to higher valueacids or amides have attracted increasing attention as bio-catalysts for processing organic chemicals. The microbialdegradation of nitriles has been found to proceed through

two enzymatic pathways: (i) aromatic nitriles, heterocyclicand certain unsaturated aliphatic nitriles are catabolized di-rectly to the corresponding acids and ammonia by nitrilaseenzyme; (ii ) saturated aliphatic nitriles are catabolized intwo stages: first by conversion to the corresponding amideby the enzyme nitrile hydratase, and then to the correspond-ing carboxylic acid and ammonia by the enzyme amidase(Asano et al. 1982).

Nitrilases that utilize benzonitrile and related aromaticnitriles as substrates have been purified and characterized byvarious workers (Harper 1977; Bandyopadhyay et al. 1986;Stalker et al. 1988; Kobayashi et al. 1990; Kobayashi et al.1998; Nagasawa and Yamada 1989). However, aliphaticnitriles cannot act as substrates for these nitrilases.Kobayashi et al. (1990) have described the occurrence of anovel nitrilase inRhodococcus rhodochrousK22 that actspreferentially on aliphatic nitriles. Nitrile hydratases knownto act on aliphatic nitriles have been purified and character-ized from Pseudomonas chlororaphisB23, Brevibacteriumsp. strain R312 andRhodococcus rhodochrousJ1 (Nagasawaand Yamada 1989).

Can. J. Microbiol.45: 811–815 (1999) © 1999 NRC Canada

811

Received April 23, 1999. Revision received July 6, 1999.Accepted July 27, 1999.

J. Dhillon, S. Chhatre, R. Shanker, and N. Shivaraman.1

National Environmental Engineering Research Institute(NEERI), Nehru Marg, Nagpur 440 020, India.

1Author to whom all correspondence should be addressed(e-mail: jkdhillon@hotmail.com)

In all the enzymatic studies reported so far, specific en-zymes for the degradation/transformation of either alkali ororganic cyanides, namely sodium cyanide, acetonitrile,acrylonitrile, and crotononitrile, have been studied in organ-isms such as Pseudomonas putidaand RhodococcusrhodochrousK22 (Kobayashi et al. 1990; Babu et al. 1994).There is only one report on the conversion of both alkali cy-anide and aliphatic nitriles by a single bacterial species, butunfortunately it does not specify the enzyme responsible forthis transformation (Babu et al. 1994). In the present paper,we report the partial purification of an acetonitrile-induciblenitrilase from Pseudomonassp. (S1) that can transformaliphatic and aromatic nitriles as well as alkali cyanides(Dhillon and Shivaraman 1999).

Materials and methods

ChemicalsChemicals used in the study were of the highest purity available

commercially, while nitriles and cyanide used were as follows:acetonitrile and acrylonitrile (Sisco Research Laboratories Pvt.Ltd., India), adiponitrile (Aldrich Chemical Company Inc.,U.S.A.), phenylacetonitrile (S.D. Fine Chem. Ltd., India),butyronitrile, benzonitrile, glutaronitrile, succinodinitrile (MerckSchuchardt, Germany), and potassium cyanide (FERAK, Ger-many). Sephadex G–200 superfine was obtained from Pharmacia(U.S.A.). Marker proteins for molecular mass determination andCoommassie Brilliant Blue R-250 and G-250 were purchased fromBioRad Laboratories (Richmond, Calif.).

Enzyme assay and definition of unitsNitrilase activity was assayed by the method described by

Kobayashi et al. (1990) except that the substrate used wasacrylonitrile (94.34 mM) instead of crotononitrile. The amount ofcarboxylic acid formed in the reaction mixture and the ammoniareleased were estimated as end products of the enzymatic reaction.One unit of enzyme is defined as the amount required to catalyzethe formation of 1 mM of ammonia per min from the organic/alkalicyanide under the conditions described above.

Enzyme activity was assayed in terms of ammonia formed, esti-mated colorimetrically by Nesslerization method (APHA, AWWA,WPCF 1992). The protein content in the enzyme fractions was de-termined by the Coommassie Brilliant Blue G-250 dye-bindingmethod of Bradford (1976).

Substrate SpecificityTo check the substrate specificity of the enzyme, the enzyme as-

say was carried out as mentioned above. A comparatively low con-centration of nitriles was used because of the low solubility ofaromatic nitriles in water. The amount of ammonia produced after30 min, in the reaction mixture, was estimated colorimetrically.

The following cyanide compounds were tested for substratespecificity: acrylonitrile, acetonitrile, butyronitrile, glutaronitrile,adiponitrile, succinonitrile, benzonitrile, phenyl acetonitrile, andpotassium cyanide.

Enzyme PurificationAll purification steps were performed at 0 to 4°C, unless other-

wise specified. For purification, 100 mM Tris buffer containing50 mM KCl, 1 mM EDTA, and 1 mMβ-mercaptoethanol wasused, unless specified otherwise. Washed cells (5 g wet weight)from 2.5 L of culture broth were suspended in 100 mM Tris buffer(pH 7; 50 mL) and sonicated at 19 kHz for 10 min with 30 s burstsand intervals, with an ultrasonic oscillator (Heat Systems, U.S.A.).

The cell debris was removed by centrifugation at 25 000 ×g. Theresulting supernatant solution was subjected to ultracentrifugationat 120 000 ×g for 2 h in aBeckman OptimaTM TLX, at 4°C. Solidammonium sulphate was added to the cytosolic supernatant to give40% saturation. After slow stirring for 30 min, the precipitate wasremoved by centrifugation at 6500 ×g and ammonium sulphatewas added to obtain 65% saturation. The suspension was then cen-trifuged and the pellet dissolved in 100 mM Tris buffer, followedby dialysis for 4 h against two changes of 50 mM Tris buffer. Af-ter dialysis, the enzyme was concentrated and placed on aSephadex-75 column (1.5 × 23 cm), equilibrated with 0.1 M phos-phate buffer containing 0.1 M KCl, 1 mMβ-mercaptoethanol, and1 mM EDTA. The enzyme was eluted in the same buffer, activefractions combined, and finally concentrated. The enzyme solutionobtained was passed to a Sephadex G-200 superfine column (1.5 ×23 cm) equilibrated with 0.1 M phosphate buffer. The rate of col-umn elution was maintained at 1 mL/h. The active fractions werecombined and concentrated using a Centricon concentrator (MWcut off 30 kDa), and stored at –70°C after addition of 20% glyc-erol.

The fraction obtained from the Sephadex 200 superfine columnwas concentrated and loaded on a native polyacrylamide gel,where one lane was loaded with standard protein marker and allthe other lanes were loaded with the enzyme fraction. The laneloaded with the standard protein marker and its adjacent lane werecut from the native gel and stained with Coommassie BrilliantBlue R-250. This stained piece of gel was placed adjacent to theunstained portion and the pieces of gel showing prominent bandswere sliced off from the gel, crushed, and suspended in 0.1 Mphosphate buffer (pH 7.0) separately, further re-concentrated usingCentricon concentrator, and then tested for enzyme activity. Thefraction showing the enzyme activity was once again subjected tosodium dodecyl sulphate – polyacrylamide gel electrophoresis(SDS–PAGE) along with standard protein markers to confirm thepurification of the enzyme.

Analytical methodsSDS–PAGE was performed in 10% polyacrylamide slab gels,

using Tris-glycine buffer system (Sambrook et al. 1989). Proteinswere stained with Coommassie Brilliant Blue R-250 and destainedin methanol : acetic acid : water mixture (9:2:9 v/v). The relativemolecular weight of the subunits of the enzyme was determinedfrom the relative mobilities of standard proteins. Cyanide was esti-mated by the silver nitrate titration method and ammonia by theNesslerization method (APHA, AWWA, WPCF 1992).

The nitriles and carboxylic acids were estimated by capillarygas chromatography using a 15 m CP-SIL 8B column with FID ina Perkin Elmer Autosystem Gas Chromatograph withLC1022software. The conditions were: oven temperature 45°C to 270°C in15 min at the rate of 20°C/min with steady temperature for 1 minat 45°C and 3 min at 270°C. Both the injector and detector temper-atures were 300°C.

Determination of kinetic constants of the partiallypurified nitrilase enzyme of Pseudomonassp. (S1) onorganic and alkali cyanides

The Michaelis–Menten constant (Km) and maximum rate of re-action (Vmax) for the enzyme were determined using acetonitrile,acrylonitrile, adiponitrile, benzonitrile, and potassium cyanide assubstrates. The fraction eluted from the Sephadex G-200 superfinecolumn after concentration was used as partially purified enzymefor the kinetic studies. A typical assay mixture consisted of 20 mgenzyme protein and 20–600 mM acetonitrile or acrylonitrile; 10–250 mM adiponitrile or benzonitrile; or 1–10 mM potassium cya-nide made up in 1 mL of 0.1 M phosphate buffer. Incubations werecarried out at 37°C in a shaker water bath for 5–240 min and the

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812 Can. J. Microbiol. Vol. 45, 1999

reaction terminated by addition of HCl (100 mL × 0.1 M), fol-lowed by development of color for ammonia estimation by Ness-lerization.

Results

Enzyme purificationThe enzyme was purified 10.9-fold with a yield of 4.76%

from the cell-free extract ofPseudomonassp. (S1), usingacrylonitrile as substrate, and by adopting the purificationprocedures described in the text. The enzyme catalyzed thehydrolysis of acrylonitrile to acrylic acid at 193 mmol/min/mg of protein under the standard reaction conditions.The various fractions collected after each step of the purifi-cation process were subjected to SDS–PAGE (Table 1),which showed one major band (Fig. 1, Lane 2) running closeto the standard ovalbumin of 43 kDa. Thus, the molecularweight of the enzyme from thePseudomonassp. (S1) couldbe deduced to be approximately 41 kDa, which is similar tothat reported for nitrilase in literature (Kobayashi et al.1990; Kobayashi et al. 1998).

Determination of kinetic constants of the partiallypurified nitrilase enzyme of Pseudomonassp. (S1) onorganic and alkali cyanides

The nitrilase ofPseudomonassp. (S1) showed a broadsubstrate specificity for cyanide compounds including satu-rated (acetonitrile, butyronitrile, adiponitrile, glutaronitrile,succinonitrile) and olefinic (acrylonitrile) aliphatic nitriles,aromatic (benzonitrile) and aralkyl (phenylacetonitrile)nitriles, and alkali cyanide (KCN). The Michaelis–Mentenconstants (Km and Vmax) for the aliphatic nitriles (saturatedand olefinic), aromatic nitrile, aralkyl nitrile, and alkali cya-nide were computed from Lineweaver–Burke plots (Ta-ble 2). The maximum rate of reaction was highest for thealiphatic olefinic nitrile acrylonitrile (276 mM/mg/min) andlowest for the alkali cyanide KCN (8.8 mM/mg/min), in theorder acrylonitrile > acetonitrile > adiponitrile > benzonitrile> KCN, while the respective Km values for transformation ofacetonitrile, acrylonitrile, adiponitrile, benzonitrile, and po-tassium cyanide were found to be 11.26, 5.88, 10.28, 12.27,and 0.75 mM.

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Purification stepTotal protein(mg)

Total activity(units)

Specific activity(units/mg protein) Purification Yield (%)

Crude extract 49.2 32.23 1.61 1.00 10060% Ammonium sulphate fraction 30.72 31.82 3.18 1.98 62.44Sephadex G-200 (superfine) fraction 5.51 29.85 5.97 3.71 11.21Fraction eluted from native gel 2.33 52.59 17.53 10.90 4.76

Table 1. Purification of nitrilase enzyme fromPseudomonassp. (S1).

Fig. 1. Molecular weight determination of the partially purified nitrilase using standard protein markers of high and low molecularweight. Lane 1, standard marker of high molecular weight proteins. Lane 2, nitrilase enzyme isolated fromPseudomonassp. (S1).Lane 3, standard marker of low molecular weight proteins.

Discussion

It was originally assumed that nitriles, in which the cyanogroup is conjugated with a double bond such as benzonitrile,were directly hydrolyzed to the corresponding acid and am-monia by nitrilase (Harper 1977; Bandyopadhyay et al.1986; Stalker et al. 1988). However, aliphatic nitriles werecatabolized in two stages, via conversion to the correspond-ing amides and then to acids and ammonia by nitrilehydratase and amidase, respectively (Asano et al. 1982;Nagasawa and Yamada 1989). In contrast to these early ob-servations, Kobayashi et al. (1990) reported the occurrenceof a novel nitrilase, which preferentially attacked aliphaticnitriles. In the former respect, the nitrilase extracted fromisolate S1 not only exhibits activity similar to knownbenzonitrile-specific nitrilases but also transforms saturatedaliphatic nitriles. Our findings are not in agreement with thehypothesis that nitrilases hydrolyze only aromatic nitriles.The enzyme resembles the novel nitrilase fromRhodococcusrhodochrousK22, which is known to act on aliphatic nitriles(Kobayashi et al. 1990), and is also similar to the nitrilasefrom Rhodococcus rhodochrousJ1, reported recently byKobayashi et al. (1998), which catalyses the hydrolyticcleavage of the C–N triple bond in benzonitrile as well ashydrolysis of benzamide to benzoic acid and ammonium,stoichiometrically.

In the present study, the partially purified nitrilase ofPseudomonassp. (S1) catalyzes the direct hydrolysis of cya-nide compounds to the corresponding carboxylic acids andammonia, without the formation of amides as intermediates,which is characteristic of nitrilases and not of nitrile hydra-tases. The yield and the degree of purification obtained withthe three-step purification procedure, 4.76-fold purificationwith 10.9% yield, is comparable with the 3.09-fold purifica-tion and 10.4% yield reported by Kobayashi et al. (1990),for the nitrilase from Rhodococcus rhodochrousK22. Amore than 100% increase (from 32 units to 53 units) in theyield of enzyme activity may be due to the activation of en-zyme and the removal of inhibitors during the purification ofthe enzyme. Near-homogeneity after a 10.9-fold purificationmay be possible because the enzyme is inducible, and wheninduced represents almost 5% of the total cell protein.

Further evidence that this enzyme is a nitrilase has beenprovided by the SDS–PAGE and native gel electrophoresis,by yielding a single prominent band of MW of 41 kDa(Kobayashi et al. 1990). This corresponds to the molecularweight of nitrilases reported by Kobayashi et al. (1990) andNagasawa and Yamada (1989) fromRhodococcusisolates,and not of nitrile hydratase (27 kDa) (Bauer et al. 1998).Dhillon and Shivaraman (1999) have shown during experi-ments with growing cells as well as resting cells ofPseudo-

monassp. (S1) that the cells directly transformed the nitrilesto corresponding carboxylic acid without formation of amideas an intermediate. This direct conversion is known to becarried out by nitrilase (Kobayashi et al. 1990; Nagasawaand Yamada 1989).

The aliphatic olefinic nitrile, acrylonitrile, known to be aneffective nucleophilic agent for the modification of proteinsulphydryl groups (Nagasawa et al. 1990) appears to be themost suitable substrate for this enzyme, despite its alkylatingability. The transformation rate of this aliphatic olefinicnitrile was the highest, followed by saturated aliphaticmononitrile, and acetonitrile. Similar observations on sub-strate toxicity and specificity have been made with anacrylamide-attacking amidase from a soilPseudomonassp.,perhaps indicative of the evolutionary relationship betweenbacterial nitrilases, amidases, and nitrile hydratases (Shankeret al. 1991). The slowest rate of transformation was ob-served with the alkali cyanide. The increase in the rate oftransformation of the enzyme from benzonitrile to acrylo-nitrile, except KCN, may be due to the increase in the num-ber of hydrogen-bonding positions for the enzyme, with theincrease in number of unsaturated carbon atoms in the sub-strate (Bui et al. 1984). The kinetic constants obtained withthe aliphatic nitriles recorded in this study are very close tothe values reported by Kobayashi et al. (1990) for the nitri-lase ofRhodococcus rhodochrousK22.

The observation of the transformation of KCN to formicacid by the nitrilase ofPseudomonassp. (S1) is unique. Al-though the transformation of KCN to formamide by cyanidehydratase (Cluness et al. 1993) or formic acid by cyanidase(Basheer et al. 1992) was reported earlier, there is no suchreport available in the literature about the ability of anitrilase enzyme to transform KCN. Bui et al. (1984) havereported the kinetics of the conversion of KCN to formicacid by nitrile hydratase fromBrevibacteriumspecies. Babuet al. (1994) have studied the kinetics of degradation ofacetonitrile and sodium cyanide to CO2 and NH3 but did notspecify the enzyme responsible for the conversion. It isprobably the first observation where a single nitrilase is ca-pable of catalyzing conversions of both aliphatic and aro-matic nitriles as well as alkali cyanide. Such organisms andtheir enzymes are potential biocatalysts in biotreatment ofhazardous industrial wastes.

Acknowledgement

The authors thank Dr. P. Khanna, Director, NEERI for hisinterest and encouragement.

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814 Can. J. Microbiol. Vol. 45, 1999

Substrate Km (mM) Vmax Vmax/Km Substrate concentration (mM)

Acrylonitrile 5.88 276 46.77 20–500Adiponitrile 10.28 21.8 2.12 10–250Acetonitrile 11.26 87 7.72 20–500Benzonitrile 12.27 10.6 0.86 10–250Potassium cyanide 0.75 8.8 11.73 1–10

Note: Vmax is expressed as mM of ammonia/mg of protein/min.

Table 2. Kinetic constants for the partially purified nitrilase on various substrates.

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