Influence of metal ions on bioremediation activity of protocatechuate 3,4-dioxygenase from Stenotrophomonas maltophilia KB2

The aim of this paper was to describe the effect of various metal ions on the activity of protocatechuate 3,4-dioxygenase from Stenotrophomonas maltophilia KB2. We also compared activity of different dioxygenases isolated from this strain, in the presence of metal ions, after induction by various aromatic compounds. S. maltophilia KB2 degraded 13 mM 3,4-dihydroxybenzoate, 10 mM benzoic acid and 12 mM phenol within 24 h of incubation. In the presence of dihydroxybenzoate and benzoate, the activity of protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase was observed. Although Fe³⁺, Cu²⁺, Zn²⁺, Co²⁺, Al³⁺, Cd²⁺, Ni²⁺ and Mn²⁺ ions caused 20–80 % inhibition of protocatechuate 3,4-dioxygenase activity, the above-mentioned metal ions (with the exception of Ni²⁺) inhibited catechol 1,2-dioxygenase to a lesser extent or even activate the enzyme. Retaining activity of at least one of three dioxygenases from strain KB2 in the presence of metal ions makes it an ideal bacterium for bioremediation of contaminated areas.     

Crucial factors affecting the nitrile hydratase production of Mesorhizobium sp. F28

Mesorhizobium sp. F28 contains cobalt-NHase, which effectively converts acrylonitrile into acrylamide. When urea was added to the culture medium, the NHase activity was 62.3 U ml^?1 (R2A–R2A/urea) after 22.5 h of cultivation, which was similar to that in the medium without addition (R2A–R2A, 70.0 U ml^?1). The relative activity of the purified NHase was 100%, 92%, 94%, and 92% in the medium containing, respectively, 0 mM, 2 mM, 5 mM, and 10 mM of urea. Urea had no significant effect on the purified NHase activity of Mesorhizobium sp. F28. This research did not observe the NHase production by Mesorhizobium sp. F28 when acrylonitrile was supplemented in the culture medium except that cobalt ions existed. The highest enzyme activity was 328.5 U ml^?1 as cobalt ions were added in the pre-culture and culture medium after 22.5 h of cultivation (R2A/Co-R2A/Co); compared to media without cobalt ions (R2A–R2A, 22.5 h, 70.5 U ml^?1) this is an almost five-fold enhancement. It can be concluded that culture media containing cobalt ions was beneficial for the formation of active NHase of Mesorhizobium sp. F28.     

Expression,purification, and characterization of recombinant mangrove glutamine synthetase

To expand our knowledge about the relationship of nitrogen use efficiency and glutamine synthetase (GS) activity in the mangrove plant, a cytosolic GS gene from Avicennia marina has been heterologously expressed in and purified from Escherichia coli. Synthesis of the mangrove GS enzyme in E. coli was demonstrated by functional genetic complementation of a GS deficient mutant. The subunit molecular mass of GSI was ~40 kDa. Optimal conditions for biosynthetic activity were found to be 35 °C at pH 7.5. The Mg²⁺-dependent biosynthetic activity was strongly inhibited by Ni²⁺, Zn²⁺, and Al³⁺, whereas was enhanced by Co²⁺. The apparent K _m values of AmGLN1 for the substrates in the biosynthetic assay were 3.15 mM for glutamate, and 2.54 mM for ATP, 2.80 mM for NH₄ ⁺ respectively. The low affinity kinetics of AmGLN1 apparently participates in glutamine synthesis under the ammonium excess conditions.     

Some properties of a deoxyribonuclease isolated from Aspergillus niger

A deoxyribonuclease, isolated from the mycelia of Aspergillus niger, grown as surface cultures on a liquid medium, was partially purified and had an optimum pH of 9.5 and an optimum temperature of 37°C. The enzyme was strongly activated by Mg²⁺ while Mn²⁺, Ca²⁺ or Co²⁺ activated the enzyme to lesser extents. Thiol reagents, mercaptoethanol and dithiothreitol (DTT) activated the enzyme. S-Adenosylmethionine at low concentration (2.8?5.0 × 10^?2 mM) activated the enzyme but at a higher concentration (11.5 × 10^?2 mM) and above it inhibited the enzyme. The effect of EDTA on the enzyme was variable. The enzyme had both ATP-dependent and independent activities, the former usually being higher. ATP could be replaced by CTP or GTP. The nucleoside diphosphates ADP, CDP and GDP could replace ATP but they were not as effective as the triphosphates.     

Expression, purification and characterization of flavin reductase from Citrobacter freundii A1

Flavin reductase plays an important biological role in catalyzing the reduction of flavin by NAD(P)H oxidation. The gene that codes for flavin reductase from Citrobacter freundii A1 was cloned and expressed in Escherichia coli BL21(DE3)pLysS. In this study, we aimed to characterize the purified recombinant flavin reductase of C. freundii A1. The recombinant enzyme was purified to homogeneity and the biochemical profiles, including the effect of pH, temperature, metal ions and anions on flavin reductase activity and stability, were determined. This enzyme exhibited optimum activity at 45 °C in a 10-min reaction at pH 7.5 and was stable at temperatures up to 30 °C. At 0.1 mM concentration of metal ions, flavin reductase activity was stimulated by divalent cations including Mn²⁺, Sr²⁺, Ni²⁺, Sn²⁺, Ba²⁺, Co²⁺, Mg²⁺, Ca²⁺ and Pb²⁺. Ag⁺ was noticeably the strongest inhibitor of recombinant flavin reductase of C. freundii A1. This enzyme should not be defined as a standard flavoprotein. This is the first attempt to characterize flavin reductase of C. freundii origin.     

Cloning, expression and characterization of a novel esterase from Bacillus pumilus

The gene encoding esterase (CE1) from Bacillus pumilus ARA with a calculated molecular weight of 28.4 kDa was cloned, sequenced and efficiently expressed in Escherichia coli. The open reading frame of 747 nucleotides encoded a protein, which was classified as a carboxylesterase with an identity of 87 % to esterase from Bacillus subtilis 168. Recombinant CE1 was purified in a single step to electrophoretic homogeneity by IMAC (Ni²⁺). The enzyme displayed maximum activity toward p-nitrophenyl (pNP) acetate at 37–40 °C and pH?6.5–7.0. It was stable in the pH range from 6.5 to 8.0, and at temperature from 25 to 40 °C. Among four p-nitrophenyl esters tested, the best substrate was pNP acetate with K _m and k _cat values of 0.33 mM and 4.07 s^?1, respectively. Amounts of 2 mM Ca²⁺ and Co²⁺ significantly increased the esterase activity to 190 and 121 %, respectively. These results suggest that CE1 has very attractive applications of increasing feed digestibility in animal nutrition in this moderate temperature range.     

Characterization and application of a newly synthesized 2-deoxyribose-5-phosphate aldolase

A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 °C, respectively. The recombinant DERA was stable at pH 4.0–7.0 and at temperatures below 50 °C. The enzyme activity was inhibited by 1 mM of Ni²⁺, Ba²⁺ and Fe²⁺. The apparent K _m and V _max values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 μmol min^?1 mg^?1, for 2-deoxyribose were 0.033 mM and 2.59 μmol min^?1 mg^?1, respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose.     

Role of Divalent Metal Ions on Activity and Stability of Thermostable Dipeptidase from Bacillus stearothermophilus

Thermostable dipeptidase from Bacillus stearothermophilus, a typical metalloenzyme containing 1.0g atom of Zn per mole of subunit of the dimeric enzyme was markedly activated by exogenous divalent metal ions such as Mn²⁺, Co²⁺, and Cd²⁺ . In contrast, several others including Ba²⁺, Hg²⁺, and Cu²⁺ considerably inhibited the enzyme, even the inherent metal, Zn²⁺, being slightly inhibitory. To study the metal-binding properties of this dipeptidase, the enzyme was completely resolved to the inactive, Zn-free apoenzyme by treatment with EDTA in the presence of guanidine hydrochloride in a weakly acidic buffer. The apoenzyme was readily reconstituted by incubation with either Zn²⁺, Mn²⁺, or Co²⁺, restoring the catalytic activity. The Mn-reconstituted enzyme had nearly twice the activity of the original Zn-enzyme. Combined with kinetic analyses of reconstitution of the apoenzyme with metal ions, these results show that the enzyme has two non-identical metal-binding sites, each with a different property. Furthermore, substitution of Mn²⁺ or Co²⁺ for Zn²⁺ considerably lowered the thermostability of the enzyme without affecting the overall conformation of the enzyme protein, suggesting that the prosthetic Zn is playing dual roles in conformational stability and catalysis of the thermostable dipeptidase.     

Fermentative production of extracellular amylase from novel amylase producer,Tuber maculatum mycelium,and its characterization

Abstract

Truffles are symbiotic hypogeous edible fungi (form of mushroom) that form filamentous mycelia in their initial phase of the growth cycle as well as a symbiotic association with host plant roots. In the present study, Tuber maculatum mycelia were isolated and tested for extracellular amylase production at different pH on solid agar medium. Furthermore, the mycelium was subjected to submerged fermentation for amylase production under different culture conditions such as variable carbon sources and their concentrations, initial medium pH, and incubation time. The optimized conditions after the experiments included soluble starch (0.5% w/v), initial medium pH of 7.0, and incubation time of 7 days, at room temperature (22?±?2?°C) under static conditions which resulted in 1.41?U/mL of amylase. The amylase thus obtained was further characterized for its biocatalytic properties and found to have an optimum activity at pH 5.0 and a temperature of 50?°C. The enzyme showed good thermostability at 50?°C by retaining 98% of the maximal activity after 100?min of incubation. The amylase activity was marginally enhanced in presence of Cu²⁺ and Na⁺ and slightly reduced by K⁺, Ca²⁺, Fe²⁺, Mg²⁺, Co²⁺, Zn²⁺, and Mn²⁺ ions at 1?mM concentration.     

Enhancement of production and activity of alkaline zinc metalloprotease from <Emphasis Type="Italic">Salinivibrio proteolyticus</Emphasis> using low intensity direct electric current and zinc nanoparticles

Objectives

To improve the production and activity of an alkaline zinc metalloprotease from Salinivibrio proteolyticus in response to ZnSO₄ (ionic and nanoparticle forms) and low intensity direct electric current (LIDC).

Results

A DC of 50 µA for 10 min increased enzyme production from 35 to 53 U ml^?1 when applied to the stationary phase bacterial cells. Zn²⁺ improved enzyme production better than zinc nanoparticles (52 vs. 43.5 U ml^?1). Zinc nanoparticles (0.5 mM) added to an enzyme reaction mixture containing casein (0.65 %) and 20 mM Tris/HCl buffer (pH 8) improved enzyme activity more than Zn²⁺ (42 vs. 36 U ml^?1).

Conclusion

LIDC exposure (50 µA, 10 min) to the stationary phase bacterial cells increases metalloprotease production in Salinivibrio. A low concentration of zinc nanoparticles (0.5 mM) increases maximum enzyme activity.

     

Production and characterization of a solvent-tolerant protease from a novel marine isolate Bacillus tequilensis P15

Thirty-six proteolytic bacteria were isolated from the Jakhau coast, Kutch, India, amongst which isolate P15 identified as Bacillus tequilensis (JQ904626) was found to produce an extracellular solvent-- and detergent-tolerant protease (116.69?±?0.48 U/ml) and was selected for further investigation. Deoiled Jatropha seedcake (JSC) was found to be a suitable substrate for protease production under submerged condition. Upon optimization of process parameters following one-factor-at-a-time approach, an overall 6.4-fold (860.27?±?18.48 U/ml) increase in protease production was achieved. The maximum protease yield was obtained using a medium containing 2 % (w/v) deoiled JSC as substrate (pH of 8.0) upon 36 h of fermentation at 30 °C. The optimum temperature and pH for activity of B. tequilensis P15 protease was found to be 50 °C and 8.0, respectively. The enzyme exhibited a half-life of 190 min at 50 °C, which was enhanced to 270 min in presence of 5 mM Ca²⁺. The enzyme exhibited significant stability in almost all the solvents tested in the range of log P _ow varying from 8.8 to ?0.76. The enzyme activity was strongly inhibited by PMSF at 5 mM concentration, whereas the presence of EDTA (5 mM) and pCMB (5 mM) enhanced enzyme activity by 20.9 and 13.7 %, respectively. The enzyme was also found to be stable in the presence of surfactants, commercial detergents and bleach-oxidant (H₂O₂). This protease was demonstrated to be effective in removal of blood stains from fabrics, dehairing of hide, and stripping off the gelatin from used photographic films.     

Molecular Cloning,Expression and Characterization of Pectin Methylesterase (<Emphasis Type="Italic">Ct</Emphasis>PME) from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0–9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca²⁺ or Mg²⁺ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca²⁺ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.     

Purification and characterization of a novel thermo-active amidase from Geobacillus subterraneus RL-2a

A thermostable amidase produced by Geobacillus subterraneus RL-2a was purified to homogeneity, with a yield of 9.54 % and a specific activity of 48.66 U mg^?1. The molecular weight of the native enzyme was estimated to be 111 kDa. The amidase of G. subterraneus RL-2a is constitutive in nature, active at a broad range of pH (4.5–11.5) and temperature (40–90 °C) and has a half-life of 5 h and 54 min at 70 °C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co²⁺, Hg²⁺, Cu²⁺, Ni²⁺, and thiol reagents. The presence of mid-chain aliphatic and amino acid amides enhances the enzymatic activity. The acyl transferase activity was detected with propionamide, butyramide and nicotinamide. The enzyme showed moderate stability toward toluene, carbon tetrachloride, benzene, ethylene glycol except acetone, ethanol, butanol, propanol and dimethyl sulfoxide. The K _m and V _max of the purified amidase with nicotinamide were 6.02 ± 0.56 mM and 132.6 ± 4.4 μmol min^?1 mg^?1 protein by analyzing Michaelis–Menten kinetics. The results of MALDI-TOF analysis indicated that this amidase has homology with the amidase of Geobacillus sp. C56-T3 (gi|297530427). It is the first reported wide-spectrum thermostable amidase from a thermophilic G. subterraneus.     

Effect of the metals iron, copper and silver on fluorobenzene biodegradation by Labrys portucalensis

Organic and metallic pollutants are ubiquitous in the environment. Many metals are reported to be toxic to microorganisms and to inhibit biodegradation. The effect of the metals iron, copper and silver on the metabolism of Labrys portucalensis F11 and on fluorobenzene (FB) biodegradation was examined. The results indicate that the addition of 1 mM of Fe²⁺ to the culture medium has a positive effect on bacterial growth and has no impact in the biodegradation of 1 and 2 mM of FB. The presence of 1 mM of Cu²⁺ was found to strongly inhibit the growth of F11 cultures and to reduce the biodegradation of 1 and 2 mM of FB to ca. 50 %, with 80 % of stoichiometrically expected fluoride released. In the experiments with resting cells, the FB degraded (from 2 mM supplied) was reduced ca. 20 % whereas the fluoride released was reduced to 45 % of that stoichiometrically expected. Ag⁺ was the most potent inhibitor of FB degradation. In experiments with growing cells, the addition of 1 mM of Ag⁺ to the culture medium containing 1 and 2 mM of FB resulted in no fluoride release, whereas FB degradation was only one third of that observed in control cultures. In the experiments with resting cells, the addition of Ag⁺ resulted in 25 % reduction in substrate degradation and fluoride release was only 20 % of that stoichiometrically expected. The accumulation of catechol and 4-fluorocatechol in cultures supplemented with Cu²⁺ or Ag⁺ suggest inhibition of the key enzyme of FB metabolism—catechol 1,2-dioxygenase.     

Heterologous overexpression of Vigna radiata epoxide hydrolase in Escherichia coli and its catalytic performance in enantioconvergent hydrolysis of p-nitrostyrene oxide into (R)-p-nitrophenyl glycol

Two native epoxide hydrolases (EHs) were previously discovered from mung bean powder (Vigna radiata), both of which can catalyze the enantioconvergent hydrolysis of p-nitrostyrene oxide (pNSO). In this study, the encoding gene of VrEH1 was successfully cloned from the cDNA of V. radiata by RT-PCR and rapid amplification of cDNA ends (RACE) technologies. High homologies were found to two putative EHs originated from Glycine max (80 %) and Medicago truncatula (79 %). The vreh1 gene constructed in pET28a(+) vector was then heterologously overexpressed in Escherichia coli BL21(DE3), and the encoded protein was purified to homogeneity by nickel affinity chromatography. It was shown that VrEH1 has an optimum activity at 45 °C and is very thermostable with an inactivation energy of 468 kJ mol^-1. The enzyme has no apparent requirement of metal ions for activity, and its activity was strongly inhibited by 1 mM of Ni²⁺, Cu²⁺, Fe²⁺, or Co²⁺. By adding 0.1 % Triton X-100, the enzyme activity could be significantly increased up to 340 %. VrEH1 shows an unusual ability of enantioconvergent catalysis for the hydrolysis of racemic pNSO, affording (R)-p-nitrophenyl glycol (pNPG). It displays opposite regioselectivity toward (S)-pNSO (83 % to C_α) in contrast to (R)-pNSO (87 % to C_β). The K _M and k _cat of VrEH1 were determined to be 1.4 mM and 0.42 s^-1 for (R)-pNSO and 5.5 mM and 6.2 s^-1 for (S)-pNSO. This thermostable recombinant VrEH1 with enantioconvergency is considered to be a promising biocatalyst for the highly productive preparation of enantiopure vicinal diols and also a good model for understanding the mechanism of EH stereoselectivity.     

Manganese dependent production of 5′-nucleotidase by alkalophilic Bacillus No. C-3

Alkalophilic Bacillus no. C-3 isolated from soil produced 5′-nucleotidase (EC 3.1.3.5) extracellularly when cultured in a medium containing Mn²⁺. The unique point of enzyme production is that the enzyme was produced well in the medium containing a rather high concentration of Mn²⁺, in spite of a small difference in growth. The optimum concentration of Mn²⁺ for the enzyme production was 10 mM and over. Mn²⁺ could not be replaced by other divalent cations when added singly. In the presence of 10 mM Mn²⁺, the enzyme production was repressed by the addition of 0.5 mM phosphate to the medium. The course of the enzyme production closely paralleled the increase in growth. The optimum pH for the enzyme activity was 9.2–9.5, and KHCO₃-K₂CO₃ buffer was suitable for the enzyme.     

Production,Purification, and Characterization of α-Amylase from Solventogenic Clostridium sp. BOH3

A solventogenic strain of Clostridium sp. BOH3 produces extracellular α-amylase (7.15 U/mg protein) in reinforced clostridial medium supplemented with sugarcane bagasse hydrolysate (1 % w/v) and a small amount of starch (0.1 % w/v), which is essential for the expression of α-amylase. In the presence of α-amylase, BOH3 utilizes starch directly without any pretreatment and produces butanol almost equivalent (～90 %) to the production of butanol from glucose. α-Amylase can be purified from culture supernatant by using one-step weak anion exchange chromatography with a yield of 43 %. In peptide fingerprinting analysis, this enzyme shows homology with α-amylase produced by Clostridium acetobutylicum ATCC824. However, the molecular weight is 54 kDa, which is smaller than α-amylase of ATCC824 (84 kDa). This enzyme has optimum temperature at 45–50 °C and optimum pH at 4.5–5.5. Under this condition, the enzyme activity is 91.32 U/mg protein, and its K _m and V _max values are 1.71?±?0.02 mg/ml and 96.13?±?0.15 μmol/min/mg protein, respectively. Activity of this α-amylase can be enhanced (>1.5 times) by addition of Ca²⁺ and Co²⁺ and its activity can be maintained at an acidic pH (pH 3–5) for about 24 h. These unique characteristics suggest that this enzyme can be used for saccharification of starch for production of biofuel in one pot.     

The inhibitory effect of Ca2+ on the flavonoid production of Tetrastigma hemsleyanum suspension cells induced by metal elicitors

Tetrastigma hemsleyanum suspension cells were treated with four metal salts to screen suitable elicitors for the promotion of plant cell biomass and flavonoid production. The effects of calcium ions (Ca²⁺) on induction were also studied. It was found that the most effective elicitors were 50 μM of the heavy metal ion copper (Cu²⁺) and 100 μM of the rare earth element cerium (Ce³⁺). The maximal biomass levels under respective treatments over a 16-d culture period increased by 1.3- and 1.6-fold, and the total flavonoid content was 1.8- and 1.6-fold greater than the control, respectively. Reducing the exogenous Ca²⁺ concentration or adding Ca²⁺ antagonists (1 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N-tetraacetc acid (EGTA) or 1 mM verapamil) strengthened inductive effects of metal elicitors and enhanced flavonoid production. However, 0.5 μM of the calcium ionophore A23187 showed contrary results. The increase in exogenous Ca²⁺ concentration in the presence of A23187 suppressed H₂O₂ bursts and peroxidase activity caused by metal elicitors. The results suggest that Ca²⁺ plays an inhibitory role in the plant cell response to metal elicitors. This suppression could have been caused by Ca²⁺ preventing the cells from absorbing metal ions and then easing the induction, or because the decrease of Ca²⁺ concentration worked as an induction signal. Therefore, reducing the Ca²⁺ concentration in culture medium, or adding Ca²⁺ antagonists could be used to improve flavonoid production and cell growth in combination with induction by metal elicitors during in vitro culture of T. hemsleyanum suspension cells.     

Optimization of arylacetonitrilase production from Alcaligenes sp. MTCC 10675 and its application in mandelic acid synthesis

Alcaligenes sp. MTCC 10675 has been isolated from soil sample using enrichment method and has nitrilase catalytic system which is highly specific for the hydrolysis of arylaliphatic nitriles. Optimization of culture conditions using response surface methodology and inducer-mediated approach enhanced arylacetonitrilase production significantly (2.4-fold). Isobutyronitrile acted as an effective inducer for the induction of arylacetonitrilase, and it is highly specific for arylacetonitriles (phenyl acetonitrile and mandelonitrile). Arylacetonitrilase has no effect on its relative velocity (V _r) up to 20 mM substrate (mandelonitrile) concentration and at 30 mM mandelonitrile, 23.4 % degree of inhibition (I _d) was recorded. Half life of arylacetonitrilase of Alcaligenes sp. MTCC 10675 was 27.5 h at 25 °C. Hg²⁺, Ag⁺, Pb³⁺, and Co²⁺ were strong inhibitor of arylacetonitrilase activity which resulted into 100 %, 91 %, 84 %, and 83 % inhibition, respectively. Polar protic solvent (dichloromethane, dimethylsulphooxide, and n-butanol) reduce arylacetonitrilase activity up to 80–94 % at 10 % concentration. Alcaligenes sp. MTCC 10675 has higher biocatalytic activity, i.e., 3.9 gg^-1 dcw, which is highest in comparison to till reported organism. Arylacetonitrilase-mediated hydrolysis of racemic mandelonitrile resulted into R-(-) mandelic acid with 99.0 % enantiomeric excess (e.e.)