Purification and characterization of arylacetonitrile-specific nitrilase of Alcaligenes sp. MTCC 10675.

Arylacetonitrile-hydrolyzing nitrilase (E.C. 3.5.5.5) of Alcaligenes sp. MTCC 10675 has been purified by up to 46-fold to homogeneity and 32% yield using ammonium sulfate fractionation, Sephacryl S-300 gel permeation, and anion exchange chromatography. The molecular weight of the native enzyme was estimated to be 520 ± 60 kDa. The subunit has a molecular weight of 60 ± 14 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of the purified enzyme were 6.5 and 50 °C, respectively. The purified arylacetonitrilase has a half-life of 3 H 20 Min at its optimum temperature. The value for Vmax, Km , kcat , and ki of enzyme for mandelonitrile as a substrate was 50 ± 05 µmol/Min/mg, 13 ± 02 mM, 26 ± 03 Sec(-) , and 32.4 ± 03 mM, respectively. Alcaligenes sp. MTCC 10675 arylacetonitrilase amino acid sequence has variations from other reported arylacetonitrilase, namely, A11G, N21H, D149N, S170T, P171R, S179A, Q180N, and S191A, and it has a high thermal stability and catalytic rate as compared with the already purified arylacetonitrilase.

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. Hg(2+), Ag(+), Pb(3+), and Co(2+) 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.).

Simultaneous purification of nitrile hydratase and amidase of Alcaligenes sp. MTCC 10674.

Alcaligenes sp. MTCC 10674 has a bienzymatic system for the hydrolysis of nitriles. The nitrile hydratase and amidase have been purified simultaneously to homogeneity using a combination of (NH)4SO4 precipitation, ion exchange chromatography and gel permeation chromatography. Nitrile hydratase and amidase have molecular weight of 47 and 114 kDa, respectively and exist as heterodimer. Optimum temperatures for maximum activity of nitrile hydratase and amidase were 15 °C (2.4 U/mg protein) and 45 °C (2.3 U/mg protein), respectively. Nitrile hydratase showed maximum 7.8 U/mg protein at 50 mM acrylonitrile and amidase has 9.2 U/mg protein at 25 mM propionamide. Nitrile hydratase has Vmax 10 µmol/min/mg and Km 40 mM, while amidase has Vmax 12.5 µmol/min/mg and Km 45.5 mM, respectively. Heavy metal ions Hg2+, Ag+, Pb2+ and Cu2+ were strong inhibitors of nitrile hydratase and amidase activity.

An isobutyronitrile-induced bienzymatic system of Alcaligenes sp. MTCC 10674 and its application in the synthesis of α-hydroxyisobutyric acid.

Alcaligenes sp. MTCC 10674 was isolated as acetone cyanohydrin hydrolyzing bacterium from soil of orchid gardens of Himachal Pradesh. Acetone cyanohydrin hydrolyzing activity of this organism comprised nitrile hydratase and amidase activities. It exhibited higher substrate specificity towards aliphatic hydroxynitrile (acetone cyanohydrin) in comparison to arylaliphatic hydroxynitrile. Isobutyronitrile (40 mM) acted as a carbon source as well as inducer for growth of Alcaligenes sp. MTCC 10674 and expression of acetone cyanohydrin hydrolyzing activity. Optimization of culture condition using response surface methodology increased acetone cyanohydrin hydrolyzing activity by 1.3-fold, while inducer mediation approach increased the activity by 1.2-fold. The half life of this enzyme was 25 h at 15 °C. V max and K m value for acetone cyanohydrin hydrolyzing enzyme was 0.71 µmol mg(-1) min(-1) and 14.3 mM, when acetone cyanohydrin was used as substrate. Acetone cyanohydrin hydrolyzing enzyme encountered product inhibition and IC50 and K i value were calculated to be 28 and 10.2 mM, respectively, when product α-hydroxyisobutyric acid was added in the reaction. Under optimized reaction conditions at 40 ml fed batch scale, 3 mg dcw ml (-) resting cells of Alcaligenes sp. MTCC 10674 fully converted 0.33 M acetone cyanohydrin into α-hydroxyisobutyric acid (1.02 g) in 6 h 40 min. The characterization of acetone cyanohydrins hydrolyzing activity revealed that it comprises bienzymatic nitrile hydrolyzing system, i.e. nitrile hydratase and amidase for the production of α-hydroxyisobutyric acid from acetone cyanohydrin and maximum 70 % yield is being reported for the first time.

Characterization of bhatooru, a traditional fermented food of Himachal Pradesh: microbiological and biochemical aspects.

A number of traditional fermented products are prepared and consumed in Himachal and the types of traditional fermented products of Himachal are unique and different from other areas. Bhatooru is an indigenous leavened bread or roti and constitutes the staple diet of rural population of Himachal. The microbiological analysis of the inoculums (malera) revealed that it composed of a consortium of microorganisms. Population of Lactobacillus, Leuconostoc and Saccharomyces cerevisiae increased from 4.77 to 8.0 log cfu/g of dry matter in 10 h of fermentation. The amount of total proteins increased from 13.6 to 18.4 % (w/w). The total sugars during fermentation decreased from 74.1 to 50.1 % (w/w) on dry weight basis. However, the reducing sugar level of the fermenting samples increased significantly from 7.8 to 16.5 mg/g dry matter in the first 4 h and thereafter, it gradually decreased to 10.0 mg/g dry matter. Similarly starch content decreased from 70.2 to 48.3 % (w/w) on dry weight basis by 10 h of fermentation. In fermented samples protease activity increased from 0.48 U/g dry matter to 11.5 U/g in 6 h and then decreased to 3.21 U/g on dry weight basis at 10 h. Amylase activity initially increased from 65.0 to 79.4 U to 6 h and then declined to 69.9 U/g of dry matter. Fermentation in bhatooru significantly enhanced the B vitamin levels especially thiamine, riboflavin and nicotinic acid and essential amino acids viz methionine, phenylalanine, threonine, lysine and leucine.

Cloning, sequencing, and expression of nitrile hydratase gene of mutant 4D strain of Rhodococcus rhodochrous PA 34 in E. coli.

The NHase encoding gene of mutant 4D was isolated by PCR amplification. The NHase gene of mutant 4D was successfully cloned and expressed in Escherichia coli by using Ek/LIC Duet cloning kits (Novagen). For the active expression of the NHase gene, the co-expression of small cobalt transporter gene (P-protein gene) has also been co-expressed with NHase gene E. coli. The nucleotide sequence of this NHase gene revealed high homology with the H-NHase of Rhodococcus rhodochrous J1. The recombinant E. coli cells showed higher NHase activity (5.9 U/mg dcw) as compared to the wild (4.1 U/mg dcw) whereas it is less than the mutant strain (8.4 U/mg dcw). Addition of cobalt ion in Luria-Bertani medium is needed up to a very small concentration (0.4 mM) for NHase activity. The recombinant E. coli exhibited maximum NHase activity at 6 h of incubation and was purified with a yield of 56 % with specific activity of 37.1 U/mg protein.

Bioconversion of acrylonitrile to acrylamide using polyacrylamide entrapped cells of Rhodococcus rhodochrous PA-34.

The nitrile hydratase (NHase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The resting cells (having NHase activity) (8 %; 1 mL corresponds to 22 mg dry cell mass, DCM) were immobilized in polyacrylamide gel containing 12.5 % acrylamide, 0.6 % bisacrylamide, 0.2 % diammonium persulfate and 0.4 % TEMED. The polyacrylamide entrapped cells (1.12 mg DCM/mL) completely converted acrylonitrile in 3 h at 10 °C, using 0.1 mol/L potassium phosphate buffer. In a partitioned fed batch reactor, 432 g/L acrylamide was accumulated after 1 d. The polyacrylamide discs were recycled up to 3×; 405, 210 and 170 g/L acrylamide was produced in 1st, 2nd and 3rd recycling reactions. In four cycles, a total of 1217 g acrylamide was produced by recycling the same mass of entrapped cells.

Hydrolysis of benzonitrile herbicides by soil actinobacteria and metabolite toxicity.

The soil actinobacteria Rhodococcus rhodochrous PA-34, Rhodococcus sp. NDB 1165 and Nocardia globerula NHB-2 grown in the presence of isobutyronitrile exhibited nitrilase activities towards benzonitrile (approx. 1.1-1.9 U mg(-1) dry cell weight). The resting cell suspensions eliminated benzonitrile and the benzonitrile analogues chloroxynil (3,5-dichloro-4-hydroxybenzonitrile), bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodo-4-hydroxybenzonitrile) (0.5 mM each) from reaction mixtures at 30 degrees C and pH 8.0. The products were isolated and identified as the corresponding substituted benzoic acids. The reaction rates decreased in the order benzonitrile >> chloroxynil > bromoxynil > ioxynil in all strains. Depending on the strain, 92-100, 70-90 and 30-51% of chloroxynil, bromoxynil and ioxynil, respectively, was hydrolyzed after 5 h. After a 20-h incubation, almost full conversion of chloroxynil and bromoxynil was observed in all strains, while only about 60% of the added ioxynil was converted into carboxylic acid. The product of ioxynil was not metabolized any further, and those of the other two herbicides very slowly. None of the nitrilase-producing strains hydrolyzed dichlobenil (2,6-dichlorobenzonitrile). 3,5-Dibromo-4-hydroxybenzoic acid exhibited less inhibitory effect than bromoxynil both on luminescent bacteria and germinating seeds of Lactuca sativa. 3,5-Diiodo-4-hydroxybenzoic acid only exhibited lower toxicity than ioxynil in the latter test.

Citric acid production by Aspergillus niger van. Tieghem MTCC 281 using waste apple pomace as a substrate.

A solid state fermentation process was tried for the production of citric acid from apple pomace left after juice extraction using Aspergillus niger van. Tieghem MTCC 281 spores as inoculum (36.8 × 10(4) spores/100 g of pomace). The yield of citric acid was optimized by varying the amount of methanol (1-5% v/w), temperature (25-35°C) and time of incubation (1-7 days) for fermentation process. Optimum yield of citric acid (4.6 g/100 g of pomace) was recorded with 4% (v/w) methanol after 5 days of incubation at 30°C.

Purification of a hyperactive nitrile hydratase from resting cells of Rhodococcus rhodochrous PA-34.

A propionitrile-induced nitrile hydratase (NHase), a promising biocatalyst for synthesis of organic amides has been purified from cell-free extract of Rhodococcus rhodochrous PA-34. About 11-fold purification of NHase was achieved with 52% yield. The SDS-PAGE of the purified enzyme revealed that it consisted of two subunits of 25.04 kD and 30.6 kD. However, the molecular weight of holoenzyme was speculated to be 86 kD by native-PAGE. This NHase exhibited maximum activity at pH 8.0 and temperature 40°C. Half-life was 2 h at 40°C and 0.5 h at 50°C. The Km and Vmax were 167 mM and 250 µmole/min/mg using 25 mM 3-cyanopyridine as substrate. AgNO(3), Pb(CH(3)COO)(2) and HgCl(2) inhibited the NHase to extent of 89-100%.

Bioconversion of benzonitrile to benzoic acid using free and agar entrapped cells of Nocardia globerula NHB-2.

The free and agar immobilized cells of Nocardia globerula NHB-2 having nitrilase (EC 3.5.5.1) activity were used to catalyse the transformation of benzonitrile to benzoic acid. The whole cells of N. globerula NHB-2 were immobilized in agar which exhibited maximum conversion of benzonitrile to benzoic acid in 0.1 M potassium phosphate buffer pH 7.5 (free cells) 8.0 (immobilized cells), temperature 40 degrees C, cells 2 mg dcm ml(-1) reaction mixture and benzonitrile (4% v/v) in 4 h (free cells). The effect of temperature on the stability of nitrilase was studied and cells retained 100% activity at 30 degrees C and lost 50% activity at 40 degrees C. In a fed batch mode of reaction 108 and 84 gl(-1) benzoic acid was produced using free and agar entrapped cells (2 g dcm). The agar immobilized cells were recycled up to three times and 80, 62, 20 gl(-1) benzoic acid was again produced respectively in each of three cycles and a total 244 g benzoic acid was produced by recycling the same mass of immobilized biocatalyst.

Bioconversion of butyronitrile to butyramide using whole cells of Rhodococcus rhodochrous PA-34.

Butyramide is an important chemical commodity, which is used for the synthesis of hydroxamic acids and electrorheological fluids and for the preparation of beta-amodoorganotin compounds. The nitrile hydratase (Nhase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of butyronitrile to butyramide. The maximum Nhase activity [18 U/mg dry cell weight (dcw)] of whole cells of R. rhodochrous PA-34 was observed at pH 7.0 with 10% (v/v) butyronitrile and 1 mg cells (dcw)/ml reaction mixture at 10 degrees C. The cells of R. rhodochrous PA-34 retained almost 50% activity when incubated for 1 h in the presence of 85% (v/v) butyronitrile. A yield of 597 g of butyramide (6.8 M) was obtained using 60% (v/v) butyronitrile, 1 g cells (dry weight) in a 1-l batch reaction at 10 degrees C for 6 h.

Bench scale conversion of 3-cyanopyidine to nicotinamide using resting cells of Rhodococcus rhodochrous PA-34.

The nitrile hydratase (NHase, EC 3.5.5.1) activity of Rhodococcus rhodochrous PA-34 was explored for the conversion of 3-cyanopyridine to nicotinamide. The NHase activity (∼18 U/mg dry cell weight, dcw) was observed in 0.1 M phosphate buffer, pH 8.0 containing 1M 3-cyanopyridine as substrate, and 0.75 mg of resting cells (dry cell weight) per ml reaction mixture at 40°C. However, 25°C was more suitable for prolonged batch reaction at high substrate (3-cyanopyridine) concentration. In a batch reaction (1 liter), 7M 3-cyanopyridine (729 g) was completely converted to nicotinamide (855 g) in 12h at 25°C using 9.0 g resting cells (dry cell weight) of R. rhodochrous PA-34.

Constitutive acetonitrile hydrolysing activity of Nocardia globerula NHB-2: Optimization of production and reaction conditions.

Nocardia globerula NHB-2 exhibited an intracellular acetonitrile hydrolysing activity (AHA) when cultivated in nutrient broth supplemented with glucose (10.0 g/l) and yeast extract (1.0 g/l), at pH 8.0, 30 degrees C for 21 hr. Maximum AHA was recorded in the culture containing 0.1 M of sodium phosphate buffer, (pH 8.8) at 45 degrees C for 15 min with 600 micromol of acetonitrile and resting cells of N. globerula NHB-2 equivalent to 1.0 ml culture broth. This activity was stable up to 40 degrees C and was completely inactivated at or above 60 degrees C. About five-fold increase in AHA was observed after optimization of culture and reaction conditions. Under the optimized conditions, this organism hydrolyzed various nitriles and amides such as propionitrile, benzonitrile. acetamide, and acrylamide to corresponding acids. This nitrile/amide hydrolysing activity of N. globerula NHB-2 has potential applications in enzymatic synthesis of organic acids and bioremediation of nitriles and amides contaminated soil and water system.

Purification and characterization of a small size protease from Bacillus sp. APR-4.

A thermostable extracellular protease of Bacillus sp. APR-4 was purified by size-exclusion and ion-exchange chromatographic methods and its properties were studied. The purified enzyme had a specific activity of 21,000 U/mg of protein and gave single band on SDS/PAGE with a molecular mass of 16.9 KDa. This protease had an optimal pH of 9 and exhibited its highest activity at 60 degrees C. The enzyme activity was inhibited by EDTA, suggesting the presence of metal residue at the active site. Ca2+ (5 mM) had stabilising effect on the activity of protease, but Cu2+ (5 mM) had inhibitory effect. The enzyme exhibited highest specificity towards casein (1%) and had a Km of 26.3 mg/ml and a Vmax of 47.6 U/mg with casein as a substrate. The stability of this enzyme was evaluated in the presence of some organic solvents and the enzyme was stable in methanol, petroleum ether and ethanol. Detergents (Wheel, Farishta) had stimulatory effect on the activity of this enzyme.

Protein enrichment of apple pomace by co-culture of cellulolytic moulds and yeasts.

The co-culture of cellulolytic moulds and yeasts on apple pomace in solid-state fermentation (SSF) and liquid-state fermentation (LSF) increased the protein content of apple pomace. The co-culture of Candida utilis and Aspergillus niger was the best among several combinations and increased the protein content of dried and pectin-extracted apple pomace to 20% and 17%, respectively, under SSF conditions.

Effect of fatty acids on aflatoxin production by Aspergillus parasiticus.

The effect of saturated and unsaturated fatty acids on aflatoxin production was studied in a synthetic medium. The aflatoxin production decreased (10-75%) in the presence of lauric acid and palmitic acid but the addition of behenic and sebacic acid stimulated aflatoxin production by 125-541%. Linolenic and linoleic acids effected aflatoxin production and mycelium growth. An 34-fold increase in aflatoxin production was observed with 50 mM linoleic acid. An inverse relationship was observed between aflatoxin production and mycelium mass, irrespective of the nature of the fatty acid.

Effect of metal ions on aflatoxin production by Aspergillus parasiticus.

The effect of iron, copper, cobalt, cadmium, zinc, molybdenum, magnesium and manganese salts was studied on aflatoxin production in relation to mycelial mass. Iron, copper and cadmium salts decreased the aflatoxin production to different levels but a mixed trend was observed depending on salt concentration, with molybdenum, magnesium and manganese. Cobalt and zinc salts stimulated aflatoxin production at all concentrations studied. The maximum increase in aflatoxin production, 655% and 519% was observed in the presence of zinc sulfate and sodium molybdate, respectively. A negative correlation was observed between aflatoxin production and vegetative growth of fungus.

Mechanism of action of aflatoxin B1 in Bacillus megaterium.

Bacillus megaterium cells from various growth phases were equally susceptible to the lethal effects of aflatoxin B1. Known surfactants (EDTA and Tween-80) accentuated the effects of aflatoxin B1. Viability and inulin uptake in aflatoxin B1-exposed cells decreased considerably. The effect was concentration dependent. A straight-line relationship observed in the death curve indicated a single target for aflatoxin B1 action in B. megaterium. Leakage of intracellular constituents in B. megaterium was also concentration dependent, and this can be related to the extent of cell membrane damage.