Nitrile biotransformation by Aspergillus niger

A nitrile-converting enzyme activity was induced in Aspergillus niger K10 by 3-cyanopyridine. The whole cell biocatalyst was active at pH 3–11 and hydrolyzed the cyano group into acid and/or amide functions in benzonitrile as well as in its meta- and para-substituted derivatives, cyanopyridines, 2-phenylacetonitrile and thiophen-2-acetonitrile. Amides constituted a significant part of the total biotransformation products of 2- and 4-cyanopyridine, 4-chlorobenzonitrile, 4-tolunitrile and 1,4-dicyanobenzene, while -substituted acrylonitriles gave amides as the sole products.     

Hyperinduction of nitrilases in filamentous fungi

2-Cyanopyridine proved to act as a powerful nitrilase inducer in Aspergillus niger K10, Fusarium solani O1, Fusarium oxysporum CCF 1414, Fusarium oxysporum CCF 483 and Penicillium multicolor CCF 2244. Valeronitrile also enhanced the nitrilase activity in most of the strains. The highest nitrilase activities were produced by fungi cultivated in a Czapek-Dox medium with both 2-cyanopyridine and valeronitrile. The specific nitrilase activities of these cultures were two to three orders of magnitude higher than those of cultures grown on other nitriles such as 3-cyanopyridine or 4-cyanopyridine.     

Immobilization of fungal nitrilase and bacterial amidase – two enzymes working in accord

A nitrilase from Aspergillus niger and an amidase from Rhodococcus erythropolis co-immobilized on a 1-mL Butyl Sepharose column were used for the hydrolysis of 4-cyanopyridine into isonicotinic acid. The former enzyme converted the nitrile into the acid:amide mixture (molar ratio ca. 3:1), while the latter enzyme hydrolyzed the amide by-product. Therefore, the ratio of amide in the total product decreased to about 5%. Sodium sulfate was used as a component of the elution buffer, as the commonly used ammonium sulfate (0.8 M) acted as an amidase inhibitor. The hydrolysis of 4-cyanopyridine by a nitrilase from F. solani gave isonicotinic acid and isonicotinamide at a molar ratio of about 98:2. When using this enzyme and the amidase immobilized on two columns operated in tandem, the percentage of isonicotinamide in total product decreased to <0.2%.     

Characterization and functional cloning of an aromatic nitrilase from Pseudomonas putida CGMCC3830 with high conversion efficiency toward cyanopyridine

Nitrilases have long been considered as an attractive alternative to chemical catalyst in carboxylic acids biosynthesis due to their green characteristics and the catalytic potential in nitrile hydrolysis. A novel nitrilase from Pseudomonas putida CGMCC3830 was purified to homogeneity. pI value was estimated to be 5.2 through two-dimensional electrophoresis. The amino acid sequence of NH₂ terminus was determined. Nitrilase gene was cloned through CODEHOP PCR, Degenerate PCR and TAIL-PCR. The open reading frame consisted of 1113 bp encoding a protein of 370 amino acids. The predicted amino acid sequence showed the highest identity (61.6%) to nitrilase from Rhodococcus rhodochrous J1. The enzyme was highly specific toward aromatic nitriles such as 3-cyanopyridine, 4-cyanopyridine, and 2-chloro-4-cyanopyridine. It was classified as aromatic nitrilase. The nitrilase activity could reach up to 71.8 U/mg with 3-cyanopyridine as substrate, which was a prominent level among identified cyanopyridine converting enzymes. The kinetic parameters K_m and V_max for 3-cyanopyridine were 27.9 mM and 84.0 U/mg, respectively. These data would warrant it as a novel and potential candidate for creating effective nitrilases in catalytic applications of carboxylic acids synthesis through further protein engineering.     

A propionitrile-induced nitrilase of <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. NDB 1165 and its application in nicotinic acid synthesis

Rhodococcus sp. NDB 1165, a nitrile-transforming organism was isolated from temperate forest soil of Himalayas. The nitrilase (EC 3.5.5.2) activity of this organism had higher substrate specificity toward aromatic nitriles (benzonitrile, 3-cyanopyridine and 4-cyanopyridine) and unsaturated aliphatic nitrile (acrylonitrile) in comparison to saturated aliphatic nitriles (acetonitrile, propionitrile, butyronitrile and isobutyronitrile) nitrile and arylacetonitrile (phenylacetonitrile and indole-3-acetonitrile). The nitrilase of Rhodococcus sp. NDB 1165 was inducible in nature and propionitrile proved to be an efficient inducer. However, the salts of ferrous and cobalt ions had an inhibitory effect. Under optimized reaction conditions (pH 8.0 and temperature 45°C) the nitrilase activity of this organism was 2.39 ± 0.07 U/mg dry cell mass (dcm). The half-life of this enzyme was 150 min and 40 min at 45°C and 50°C respectively. However, it was quite stable at 40°C and around 58 % activity was retained even after 6 h at this temperature. The V _max and K _m value of this nitrilase were 1.67 μmol/ml min and 0.1 M respectively using 3-cyanopyridine as substrate. However, the decrease in V _max and K _m values (0.56 μmol/ml min and 0.02 M, respectively) were ␣observed at >0.05 M 3-cyanopyridine which revealed that this enzyme experienced uncompetitive inhibition at higher substrate concentrations. Under optimized reaction conditions, 1.6 M 3-cyanopyridine was successfully converted in to nicotinic acid using 2.0 mg resting cells (dcm)/ml reaction mixture in 11 h. This is the highest production of nicotinic acid i.e. 8.95 mg/mg resting cells (dcm)/h as compared to nitrilase systems reported hitherto.     

Continuous hydrolysis of 4-cyanopyridine by nitrilases from <Emphasis Type="Italic">Fusarium solani</Emphasis> O1 and <Emphasis Type="Italic">Aspergillus niger</Emphasis> K10

The operational stabilities of nitrilases from Aspergillus niger K10 and Fusarium solani O1 were examined with 4-cyanopyridine as the substrate in continuous-stirred membrane reactors (CSMRs). The former enzyme was fairly stable at 30 °C with a deactivation constant (k _d) and enzyme half-life of 0.014 h⁻¹ and 50 h, respectively, but the latter exhibited an even higher stability characterized by k _d = 0.008 h⁻¹ and half-life of 87 h at 40 °C. Another advantage of this enzyme was its high chemoselectivity, i.e., selective transformation of nitriles into carboxylic acids, while the amide formed a high ratio of A. niger K10 nitrilase product. High conversion rates (>90%) were maintained for about 52 h using the nitrilase from F. solani O1 immobilized in cross-linked enzyme aggregates (CLEAs). The purity of isonicotinic acid was increased from 98% to >99.9% by using two CSMRs connected in series, the first one containing the F. solani O1 nitrilase and the second the amidase from Rhodococcus erythropolis A4 (both enzymes as CLEAs), the amidase hydrolyzing the by-product isonicotinamide.     

Purification and characterization of a nitrilase from Fusarium solani O1

An intracellular nitrilase was purified from a Fusarium solani O1 culture, in which the enzyme (up to 3000 U L⁻¹) was induced by 2-cyanopyridine. SDS-PAGE revealed one major band corresponding to a molecular weight of approximately 40 kDa. Peptide mass fingerprinting suggested a high similarity of the protein with the putative nitrilase from Gibberella moniliformis. Electron microscopy revealed that the enzyme molecules associated into extended rods. The enzyme showed high specific activities towards benzonitrile (156 U mg⁻¹) and 4-cyanopyridine (203 U mg⁻¹). Other aromatic nitriles (3-chlorobenzonitrile, 3-hydroxybenzonitrile) also served as good substrates for the enzyme. The rates of hydrolysis of aliphatic nitriles (methacrylonitrile, propionitrile, butyronitrile, valeronitrile) were 14–26% of that of benzonitrile. The nitrilase was active within pH 5–10 and at up to 50 °C with optima at pH 8.0 and 40–45 °C. Its activity was strongly inhibited by Hg²⁺ and Ag⁺ ions. More than half of the enzyme activity was preserved at up to 50% of n-hexane or n-heptane or at up to 15% of xylene or ethanol. Operational stability of the enzyme was examined by the conversion of 45 mM 4-cyanopyridine in a continuous and stirred ultrafiltration-membrane reactor. The nitrilase half-life was 277 and 10.5 h at 35 and 45 °C, respectively.     

Cyanide Metabolism in Higher Plants: Cyanoalanine Hydratase is a NIT4 Homolog

Cyanoalanine hydratase (E.C. 4.2.1.65) is an enzyme involved in the cyanide detoxification pathway of higher plants and catalyzes the hydrolysis of β-cyano-l-alanine to asparagine. We have isolated the enzyme from seedlings of blue lupine (Lupinus angustifolius) to obtain protein sequence information for molecular cloning. In contrast to earlier reports, extracts of blue lupine cotyledons were found also to contain cyanoalanine-nitrilase (E.C. 3.5.5.4) activity, resulting in aspartic acid production. Both activities co-elute during isolation of cyanoalanine hydratase and are co-precipitated by an antibody directed against Arabidopsis thaliana nitrilase 4 (NIT4). The isolated cyanoalanine hydratase was sequenced by nanospray-MS/MS and shown to be a homolog of Arabidopsis thaliana and Nicotiana tabacum NIT4. Full-length cDNA sequences for two NIT4 homologs from blue lupine were obtained by PCR using degenerate primers and RACE-experiments. The recombinant LaNIT4 enzymes, like Arabidopsis NIT4, hydrolyze cyanoalanine to asparagine and aspartic acid but show a much higher cyanoalanine-hydratase activity. The two nitrilase genes displayed differential but overlapping expression. Taken together these data show that the so-called ‘cyanoalanine hydratase’ of plants is not a bacterial type nitrile hydratase enzyme but a nitrilase enzyme which can have a remarkably high nitrile-hydratase activity.     

Purification,characterization, and substrate specificity of a glucoamylase with steroidal saponin-rhamnosidase activity from <Emphasis Type="Italic">Curvularia lunata</Emphasis>

It has been previously reported that a glucoamylase from Curvularia lunata is able to hydrolyze the terminal 1,2-linked rhamnosyl residues of sugar chains at C-3 position of steroidal saponins. In this work, the enzyme was isolated and identified after isolation and purification by column chromatography including gel filtration and ion-exchange chromatography. Analysis of protein fragments by MALDI-TOF/TOF™ proteomics Analyzer indicated the enzyme to be 1,4-alpha-D-glucan glucohydrolase EC 3.2.1.3, GA and had considerable homology with the glucoamylase from Aspergillus oryzae. We first found that the glucoamylase was produced from C. lunata and was able to hydrolyze the terminal rhamnosyl of steroidal saponins. The enzyme had the general character of glucoamylase, which hydrolyze starch. It had a molecular mass of 66 kDa and was optimally active at 50°C, pH 4, and specific activity of 12.34 U mg of total protein⁻¹ under the conditions, using diosgenin-3-O-α-L-rhamnopyranosyl(1→4)-[α-L-rhamnopyranosyl (1→2)]-β-D-glucopyranoside (compound II) as the substrate. Furthermore, four kinds of commercial glucoamylases from Aspergillus niger were investigated in this work, and they had the similar activity in hydrolyzing terminal rhamnosyl residues of steroidal saponin. This project was supported by the National Natural Science Foundation of China (NSFC; 30572333).     

A novel nitrilase from Rhodobacter sphaeroides LHS-305: cloning, heterologous expression and biochemical characterization

In this study, a novel nitrilase gene from Rhodobacter sphaeroides was cloned and overexpressed in Escherichia coli. The open reading frame of the nitrilase gene includes 969 base pairs, which encodes a putative polypeptide of 322 amino acid residues. The molecular weight of the purified native nitrilase was about 560 kDa determined by size exclusion chromatography. This nitrilase showed one single band on SDS-PAGE with a molecular weight of 40 kDa. This suggested that the native nitrilase consisted of 14 subunits with identical size. The optimal pH and temperature of the purified enzyme were 7.0 and 40 °C, respectively. The kinetic parameters V _max and K _m toward 3-cyanopyridine were 77.5 μmol min^?1 mg^?1 and 73.1 mmol/l, respectively. The enzyme can easily convert aliphatic nitrile and aromatic nitriles to their corresponding acids. Furthermore, this enzyme demonstrated regioselectivity in hydrolysis of aliphatic dinitriles. This specific characteristic makes this nitrilase have a great potential for commercial production of various cyanocarboxylic acids by hydrolyzing readily available dinitriles.     

<Emphasis Type="Bold">Characterization of nitrile hydratation catalysed by </Emphasis><Emphasis Type="BoldItalic">Nocardia</Emphasis><Emphasis Type="Bold"> sp. 108</Emphasis>

Abstract Nocardia sp. 108 exhibited strong acrylonitrile-hydrating activity and its nitrile hydratase was Co²⁺-dependent. Nocardia sp. 108 was active within a broad pH range from 6.0 to 10.0 at 30°C and thermostable at temperatures below 35°C, but became unstable at temperatures above 45°C. Furthermore, it was found that Nocardia sp. 108 can hydrate indole-3-acetonitrile, p-chlorobenzonitrile, p-hydroxybenzylcyanide, 3,4,5-trimethoxybenzonitrile, p-aminobenzonitrile, 3-cyanopyridine, o-chlorobenzonitrile to the corresponding amides and hence displayed a broad substrate specificity. The temperature and pH optima for these hydrations were 28°C and pH 7.0–7.5, respectively. At the observed concentrations, acrylonitrile was completely converted within 5 min, while 3,4,5-trimethoxybenzonitrile, p-aminobenzonitrile, indole-3-acetonitrile, p-chlorobenzonitrile were approximately 21.71, 8.98, 34.44, 93.10% hydrated. p-Chlorobenzonitrile appeared to be the preferred aromatic nitrile for Nocardia sp. 108.     

Molecular and biochemical characterization of an extracellular serine-protease from <Emphasis Type="Italic">Vibrio metschnikovii</Emphasis> J1

A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases when it was grown at 30°C in media containing casein as carbon source (14,000 U ml⁻¹). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60°C. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic constants K _m and K _cat of the purified enzyme using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide were 0.158 mM and 1.14 × 10⁵ min⁻¹, respectively. The catalytic efficiency (K _cat /K _m) was 7.23 × 10⁸ min⁻¹ M⁻¹. The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme.     

Chemotaxonomic studies ofAsplenium sect.Hymenasplenium (Aspleniaceae)

We previously isolated and characterized a new free amino acid withd-configuration at the α-carbon,trans-3, 4-dehydro-d-2-aminopimelic acid and its related amino acids,d-2-aminopimelic acid and 4-hydroxy-l-2-aminopimelic acid fromAsplenium unilaterale. In this paper, we report that the biosynthetic relationshps among these three amino acids were studied using¹⁴C-and³H-labeled compounds as tracers. Glutamate and aspartate were shown to be good precursors and it was suggested that 4-hydroxy-l-2-aminopimelic acid is biosynthesized first and the twod-amino acids are derived from it. Furthermore, the distribution patterns of these non-protein amino acids inAsplenium sect.Hymenasplenium were examined in detail and they were evaluated by their biosynthetic pathway. Morphological characters especially on their rhizomes were also examined and their character phylogeny was determined by outgroup comparison. Taking all the characters available into account, the phylogenetic relationship among 7 species ofAsplenium sect.Hymenasplenium in Japan and Taiwan is discussed by the transformed cladistic method.     

Characterization of salt-tolerant glutaminase from Stenotrophomonas maltophilia NYW-81 and its application in Japanese soy sauce fermentation

Glutaminase from Stenotrophomonas maltophilia NYW-81 was purified to homogeneity with a final specific activity of 325 U/mg. The molecular mass of the native enzyme was estimated to be 41 kDa by gel filtration. A subunit molecular mass of 36 kDa was measured with SDS-PAGE, thus indicating that the native enzyme is a monomer. The N-terminal amino acid sequence of the enzyme was determined to be KEAETQQKLANVVILATGGTIA. Besides l-glutamine, which was hydrolyzed with the highest specific activity (100%), l-asparagine (74%), d-glutamine (75%), and d-asparagine (67%) were also hydrolyzed. The pH and temperature optima were 9.0 and approximately 60°C, respectively. The enzyme was most stable at pH 8.0 and was highly stable (relative activities from 60 to 80%) over a wide pH range (5.0–10.0). About 70 and 50% of enzyme activity was retained even after treatment at 60 and 70°C, respectively, for 10 min. The enzyme showed high activity (86% of the original activity) in the presence of 16% NaCl. These results indicate that this enzyme has a higher salt tolerance and thermal stability than bacterial glutaminases that have been reported so far. In a model reaction of Japanese soy sauce fermentation, glutaminase from S. maltophilia exhibited high ability in the production of glutamic acid compared with glutaminases from Aspergillus oryzae, Escherichia coli, Pseudomonas citronellolis, and Micrococcus luteus, indicating that this enzyme is suitable for application in Japanese soy sauce fermentation.     

Characterization of a family 54 α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase from <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

A glycosyl hydrolase family 54 (GH54) α-l-arabinofuranosidase gene (abfA) of Aureobasidium pullulans was amplified by polymerase chain reaction from genomic DNA and a 498-amino-acid open reading frame deduced from the DNA sequence. Modeling of the highly conserved A. pullulans AbfA protein sequence on the crystal structure of Aspergillus kawachii AkabfB showed that the catalytic amino acid arrangement and overall structure were highly similar including the N-terminal catalytic and C-terminal arabinose binding domains. The abfA gene was expressed in Saccharomyces cerevisiae, and the heterologous enzyme was purified. The protein was monomeric, migrating at 49 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and eluting at 36 kDa upon gel filtration. AbfA showed maximal activity at 55°C and between pH 3.5 and pH 4. The enzyme had a K _m value for p-nitrophenyl-α-l-arabinofuranoside of 3.7 mM and a V _max of 34.8 μmol min⁻¹ mg protein⁻¹. Arabinose acted as a noncompetitive inhibitor with a K _i of 38.4 mM. The enzyme released arabinose from maize fiber, oat spelt arabinoxylan, and wheat arabinoxylan, but not from larch wood arabinogalactan or α-1,5-debranched arabinan. AbfA displayed low activity against α-1,5-l-arabino-oligosaccharides. The enzyme acted synergistically with endo-β-1,4-xylanase in the breakdown of wheat arabinoxylan. Binding of AbfA to xylan from several sources confirmed the presence of a functional carbohydrate-binding module. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The α-d-mannan core of a complex cell-wall heteroglycan of Trichoderma reesei is responsible for β-glucosidase activation

A heteroglycan responsible for the binding of the enzyme β-1,4-d-glucosidase (EC 3.2.1.21) to fungal cell walls was isolated from cell walls of the filamentous fungusTrichoderma reesei. The heteroglycan, composed of mannose, galactose, glucose, and glucuronic acid, also activated β-1,4-d-glucosidase, β-1,4-d-xylosidase andN-acetyl-β-1,4-d-glucosaminidase activity in vitro. The structural backbone of this heteroglycan was prepared by acid hydrolysis and gel filtration. The molecular structure of the core of the heteroglycan was determined by NMR studies as a linear α-1,6-d-mannan. The mannan core obtained by acid degradation stimulated the β-glucosidase activity by 90%. Several glycosidases fromAspergillus niger were also activated by theT. reesei heteroglycan. The β-glucosidase ofTrichoderma was activated by mannan fromSaccharomyces cerevisiae to a comparable extent.     

Purification and characterization of threonine dehydrogenase from Clostridium sticklandii

Threonine dehydrogenase from Clostridium sticklandii has been purified 76-fold from cells grown in a defined medium to a homogeneous preparation of 234 units · mg^-1 protein. Purification was obtained by chromatography on Q-Sepharose fast flow and Reactive green 19-Agarose. The native enzyme had a molecular mass of 67 kDa and consisted of two identical subunits (33 kDa each). The optimum pH for catalytic activity was 9.0. Only l-threo-threo-nine, dl--hydroxynorvaline and acetoin were substrates; only NAD was used as the natural electron acceptor. The apparent K _m values for l-threonine and NAD were 18 mM and 0.1 mM, respectively. Zn²⁺, Co²⁺ and Cu²⁺ ions (0.9 mM) inhibited enzyme activity. The N-terminal amino acid sequence revealed similarities to the class of non-metal short-chain alcohol dehydrogenases, whereas the threonine dehydrogenase from Escherichia coli belongs to the class of medium chain, zinc-containing alcohol dehydrogenases.Abbreviations PMSF phenylmethylsulfonyl fluoride - Dea diethanolamine - Tris tris-(hydroxy-methyl)-aminomethane - Nbs ₂ 5,5-dithiobis-(2-nitrobenzoic acid) - ApADN 3-acetylpyridine adenine diucleotide - thio-NAD thionicotinamide adenine dinucleotide - NBT nitro blue tetrazolium chloride     

Dimethylformamide is a novel nitrilase inducer in Rhodococcus rhodochrous

Nitrilases are of commercial interest in the selective synthesis of carboxylic acids from nitriles. Nitrilase induction was achieved here in three bacterial strains through the incorporation of a previously unrecognised and inexpensive nitrilase inducer, dimethylformamide (DMF), during cultivation of two Rhodococcus rhodochrous strains (ATCC BAA-870 and PPPPB BD-1780), as well as a closely related organism (Pimelobacter simplex PPPPB BD-1781). Benzonitrile, a known nitrilase inducer, was ineffective in these strains. Biocatalytic product profiling, enzyme inhibition studies and protein sequencing were performed to distinguish the nitrilase activity from that of sequential nitrile hydratase-amidase activity. The expressed enzyme, a 40-kDa protein with high sequence similarity to nitrilase protein Uniprot Q-03217, hydrolyzed 3-cyanopyridine to produce nicotinic acid exclusively in strains BD-1780 and BD-1781. These strains were capable of synthesising both the vitamin nicotinic acid as well as β-amino acids, a compound class of pharmaceutical interest. The induced nitrilase demonstrated high enantioselectivity (> 99%) in the hydrolysis of 3-amino-3-phenylpropanenitrile to the corresponding carboxylic acid.

     

Purification and biochemical characterization of a broad substrate specificity thermostable alkaline protease from Aspergillus nidulans

Aspergillus nidulans PW1 produces an extracellular carboxylesterase activity that acts on several lipid esters when cultured in liquid media containing olive oil as a carbon source. The enzyme was purified by gel filtration and ion exchange chromatography. It has an apparent MW and pI of 37 kDa and 4.5, respectively. The enzyme efficiently hydrolyzed all assayed glycerides, but showed preference toward short- and medium-length chain fatty acid esters. Maximum activity was obtained at pH 8.5 at 40°C. The enzyme retained activity after incubation at pHs ranging from 8 to11 for 12 h at 37°C and 6 to 8 for 24 h at 37°C. It retained 80% of its activity after incubation at 30 to 70°C for 30 min and lost 50% of its activity after incubation for 15 min at 80°C. Noticeable activation of the enzyme is observed when Fe²⁺ ion is present at a concentration of 1 mM. Inhibition of the enzyme is observed in the presence of Cu²⁺, Fe³⁺, Hg²⁺, and Zn²⁺ ions. Even though the enzyme showed strong carboxylesterase activity, the deduced N-terminal amino acid sequence of the purified protein corresponded to the protease encoded by prtA gene.