Ammonium Sulfate Fractionation Research Articles

Biochemical properties of β-glucosidase from Turkish Hacıhaliloğlu apricot (Prunus armenica L.) as affected by harvest year

β-glucosidase was extracted and purified from Turkish Hacihaliloğlu apricot variety and some of its biochemical properties were studied in a two-year study (2008 and 2009). Two isoenzymes (isoenzyme A and B) were obtained upon ammonium sulfate fractionation, ion exchange chromatography using DEAE-Toyopearl 650 M and gel filtration chromatography using Sephadex G-100. The pH optimum was found to be 5.0 for both isoenzymes. The optimum temperature for isoenzyme A activity in both years was found to be 50 °C, whereas those for the isoenzyme B in 2008 and 2009 were 50 °C and 55 °C, respectively. Km values of the isoenzyme A and B were found to be 2.49 and 1.12 mM in 2008, and 2.41 and 1.06 mM in 2009, respectively. The Z values for isoenzyme A in 2008 and 2009 were found to be 20.3 °C and 17.9 °C, respectively. Those for isoenzyme B in 2008 and 2009 were 21.1 °C and 23.0 °C, respectively. Ea values for isoenzyme A were 108.1 kj/molK and 122.3 kj/molK, while those for isoenzyme B were found to be 103.5 kj/mol K and 95.1 kj/molK in 2008 and 2009, respectively. Of the inhibitors tested, CaCl2 was the most effective.

Protective effects and plausible mechanisms of antler-velvet polypeptide against hydrogen peroxide induced injury in human umbilical vein endothelial cells.

Antler velvet polypeptide (VAP) is a prominent bioactive component of antler velvet. Whereas uncharacterized crude extracts have typically been used in pharmacological studies, in this study, the velvet polypeptide was isolated and purified by acid water extraction, ethanol precipitation, ammonium sulfate fractionation and precipitation, and chromatography, progressively. Human umbilical vein endothelial cells (HUVECs) were induced with H2O2 followed purified polypeptide treatment. Cell viability was evaluated by MTT assay. The apoptosis of cells was detected by fluorescence microscopy and flow cytometry. A cell analyzer was used to measure the mitochondrial membrane potential. The intracellular reactive oxygen species (ROS) levels were determined by flow cytometry. Oxidative stress related biochemical parameters were detected, and the expression of apoptosis-related proteins was examined by Western blot analysis. The results indicated that a 7.0 kDa polypeptide (VAP II) was isolated from antler velvet. VAP II enhanced cell viability, decreased cell apoptosis, reversed depolarization of mitochondrial membrane potential, decreased ROS levels, inhibited oxidative stress, and regulated the downstream signaling apoptotic cascade expression caused by H2O2. The protective effects of VAP II on HUVECs suggests a potential strategy for the treatment of cardiovascular disease.

Extracellular Alkaline Lipase from a Novel Fungus Curvularia sp. DHE 5: Optimisation of Physicochemical Parameters, Partial Purification and Characterisation.

Thirty isolated fungal strains were screened for lipase production using Phenol Red plates, containing tributyrin as lipidic substrate, and a novel fungus identified genetically as Curvularia sp. DHE 5 was found as the most prominent strain. Various agro-industrial substrates were evaluated as inert supports for lipase production in solid-state fermentation. The highest yield of lipase ((83.4±2.2) U/g on dry mass basis) was reported with wheat bran medium after seven days of fermentation at pH=7.0, temperature of 30 °C, 70% moisture content, inoculum size of 1.27·107 spore/mL and 2% olive oil as an inducer. Supplementation of the medium with 0.05% KCl as an ion source further increased lipase production to (88.9±1.2) U/g on dry mass basis. The enzyme was partially purified through ammonium sulphate fractionation (40%) followed by dialysis, and its optimum pH and temperature were reported at 8.0 and 50 °C, respectively, with remarkable pH and thermal stability.

Cloning and characterization of d-threonine aldolase from the green alga Chlamydomonas reinhardtii

d-Threonine aldolase (DTA) catalyzes the pyridoxal 5’-phosphate (PLP)-dependent interconversion of d-threonine and glycine plus acetaldehyde. The enzyme is a powerful tool for the stereospecific synthesis of various β-hydroxy amino acids in synthetic organic chemistry. In this study, DTA from the green alga Chlamydomonas reinhardtii was discovered and characterized, representing the first report to describe the existence of eukaryotic DTA. DTA was overexpressed in recombinant Escherichia coli BL21 (DE3) cells; the specific activity of the enzyme in the cell-free extract was 0.8 U/mg. The recombinant enzyme was purified to homogeneity by ammonium sulfate fractionation, DEAE-Sepharose, and Mono Q column chromatographies (purified enzyme 7.0 U/mg). For the cleavage reaction, the optimal temperature and pH were 70 °C and pH 8.4, respectively. The enzyme demonstrated 90% of residual activity at 50 °C for 1 h. The enzyme catalyzed the synthesis of d- and d-allo threonine from a mixture of glycine and acetaldehyde (the diastereomer excess of d-threonine was 18%). DTA was activated by several divalent metal ions, including manganese, and was inhibited by PLP enzyme inhibitors and metalloenzyme inhibitors.

Prolonged Production and Aggregation Complexity of Cold-Active Lipase from Pseudomonas proteolytica (GBPI_Hb61) Isolated from Cold Desert Himalaya.

Pseudomonas, being the common inhabitant of colder environments, are suitable for the production of cold-active enzymes. In the present study, a newly isolated strain of Pseudomonas from cold desert site in Indian Himalayan Region, was investigated for the production of cold-active lipase. The bacteria were identified as Pseudomonas proteolytica by 16S rDNA sequencing. Lipase production by bacteria was confirmed by qualitative assay using tributyrin and rhodamine-B agar plate method. The bacterium produced maximum lipase at 25°C followed by production at 15°C while utilizing olive, corn, as well as soybean oil as substrate in lipase production broth. Enzyme produced by bacteria was partially purified using ammonium sulphate fractionation. GBPI_Hb61 showed aggregation behaviour which was confirmed using several techniques including gel filtration chromatography, dynamic light scattering, and native PAGE. Molecular weight determined by SDS-PAGE followed by in-gel activity suggested two lipases of nearly similar molecular weight of ~50kDa. The enzyme showed stability in wide range of pH from 5 to 11 and temperature up to 50°C. The enzyme from GBPI_Hb61 exhibited maximum activity toward p-nitrophenyldecanoate (C10). The stability of enzyme was not affected with methanol while it retained more than 75% activity when incubated with ethanol, acetone, and hexane. The bacterium is likely to be a potential source for production of cold-active lipase with efficient applicability under multiple conditions.

Purification and Characterization of an Extracellular Phosphatase Enzyme From Bacillus spp.

Phosphorus is one of the most important nutrients for plant growth and development. Chemical Pi fertilizer is used to provide the phosphorus for the plants, but it is mostly fixed in the soil into insoluble form and become unavailable to the plants. Phosphate-solubilizing bacteria have lots of application in agriculture as biological fertilizer. Consumption of biofertilizers instead of chemical fertilizers can lead to environmental pollution reduction and crop production enhancement using sustainable farming. In this study, a phosphatase-producing bacterium was isolated from agricultural soil in Kerman. Screening of phosphate solubilizing bacteria was performed on the PVK medium, based on clear area diameter. The best bacterium (AG41) was identified based on 16s rDNA gene. The optimum condition for production of phosphatase was also determined and it was purified and characterized. Sequence alignment and phylogenetic tree results show that AG41 is closely related to Bacillus subtilis, with 98% homology. Phosphatase activity was determined by end point method. The best carbon, nitrogen and phosphate sources for enzyme production were 1.0% glucose, 0.5% ammonium sulfate and (0.25%) sodium phytate +(0.25%) tricalcium phosphate, respectively. Bacterial phosphatase was partially purified using ammonium sulfate fractionation followed by dialysis. Results showed that the optimum temperature for the purified enzyme activity was 40oC and it was stable at temperatures below 60°C. This enzyme was stable between pH 3.0-7.0, and the optimal pH activity was found to 5.0. These results indicated that this strain can be a notable candidate for using as biofertilizers.

Insight into the biochemical, kinetic and spectroscopic characterization of garlic (Allium sativum) phytocystatin: Implication for cardiovascular disease

Phytocystatins are cysteine proteinase inhibitors present in plants. They play crucial role in maintaining protease-anti protease balance and are involved in various endogenous processes. Thus, they are suitable and convenient targets for genetic engineering which makes their isolation and characterisation from different sources the need of the hour. In the present study a phytocystatin has been isolated from garlic (Allium sativum) by a simple two-step process using ammonium sulphate fractionation and gel filtration chromatography on Sephacryl S-100HR with a fold purification of 152.6 and yield 48.9%. A single band on native gel electrophoresis confirms the homogeneity of the purified inhibitor. The molecular weight of the purified inhibitor was found to be 12.5kDa as determined by SDS-PAGE and gel filtration chromatography. The garlic phytocystatin was found to be stable under broad range of pH (6–8) and temperature (30°C–60°C). Kinetic studies suggests that garlic phytocystatins are reversible and non-competitive inhibitors having highest affinity for papain followed by ficin and bromelain. UV and fluorescence spectroscopy revealed significant conformational change upon garlic phytocystatin-papain complex formation. Secondary structure analysis was performed using CD and FTIR. Garlic phytocystatin possesses 33.9% alpha-helical content as assessed by CD spectroscopy.

Purification and characterization of an antiviral protein from prickly pear (Opuntia ficus-indica (L.) Miller) cladode

The infectivity of three RNA plant viruses belonging to the genera Cucumovirus, Potyvirus and Tobamovirus was inhibited by an aqueous extract of prickly pear (Opuntia ficus-indica (L.) Miller) cladode. This antiviral activity was not dialyzable, and not related to isolated plant polysaccharides, but was sensitive to heating and extract treatment with trichloroacetic acid and sodium dodecyl sulfate. An antiviral protein with MW of 28.3 kDa was purified from cactus extract by ammonium sulfate fractionation followed by ion exchange chromatography. The isolated protein inhibited Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus and Tobacco mosaic virus at a concentration of 40 μg mL−1. An IC50 of 2.8 μg mL−1 was calculated for isolated protein for CMV inhibition on faba bean plants. It was demonstrated that the antiviral protein has RNase, but not DNase and protease activity. It interacted with viral nucleic acid in far Western dot blot analysis, was resistant to 2-mercaptoethanol and ethylenediaminetetraacetic acid, and was active in the pH range of 4–10. To our knowledge, this is the first report on inhibitory effect of prickly pear extract on members of Tobamovirus, Potyvirus and Cucumovirus, as well as isolation and characterization of an antiviral protein from prickly pear cladode. This plant can be regarded as an encouraging source of antiviral protein to develop environmentally-friendly compounds for management of plant viral diseases.

Purification and Characterization of Hyaluronate Lyase from Arthrobacter globiformis A152.

A hyaluronate lyase was obtained by cultivating Arthrobacter globiformis strain A152. The enzyme was purified to homogeneity from the supernatant by ammonium sulfate fractionation, Q Sepharose Fast Flow, and Sephadex G-100 chromatography. The purification resulted in a 32.78-fold increase in hyaluronate lyase activity with specific activity of 297.2U/mg. The molecular weight of the enzyme determined by SDS-PAGE was approximately 73.7kDa. Using hyaluronic acid (HA) as a substrate, the maximal reaction rate (Vmax) and the Michaelis-Menten constant (Km) of hyaluronate lyase were found to be 4.76μmol/min/ml and 0.11mg/ml, respectively. The optimum pH and temperature values for hyaluronate lyase activity were pH 6.0 and 42°C, respectively. This enzyme was stable at pH 4-10, 5-7, and 5-7 at 4, 37, and 42°C, respectively. Investigation about temperature effects on hyaluronate lyase displayed that it was stable at 30-37°C and also showed high activity at 37°C. The enzymatic activity was enhanced by Ca2+ and was strongly inhibited by Cu2+ and SDS. These properties suggested that the hyaluronate lyase in this study could bring promising prospects in medical and industry applications.

Allium sativum Protease Inhibitor: A Novel Kunitz Trypsin Inhibitor from Garlic Is a New Comrade of the Serpin Family.

PurposeThis study was aimed to purify and characterize the Protease inhibitor (PI) from a plant Allium sativum (garlic) with strong medicinal properties and to explore its phytodrug potentials.MethodsAllium sativum Protease Inhibitor (ASPI) was purified using ammonium sulphate fractionation and Fast Protein Liquid Chromatography on anion exchanger Hi-Trap DEAE column. The purified protein was analyzed for its purity and molecular weight by SDS PAGE. The confirmation of presence of trypsin inhibiting PI was performed by MALDI TOF-TOF and analyzed by MASCOT database. The ASPI was further investigated for its kinetic properties and stability under extreme conditions of pH, temperature and chemical denaturants. Secondary structure was determined by Circular Dichorism (CD) spectroscopy.ResultsASPI of ~15 kDa inhibited trypsin and matched "truncated kunitz Trypsin Inhibitor (Glycine max)" in MASCOT database. The purified ASPI showed 30376.1371 U/mg specific activity with a fold purity of 159.92 and yield ~93%. ASPI was quite stable in the range of pH 2–12 showing a decline in the activity around pH 4–5 suggesting that the pI value of the protein as ASPI aggregates in this range. ASPI showed stability to a broad range of temperature (10–80°C) but declined beyond 80°C. Further, detergents, oxidizing agents and reducing agents demonstrated change in ASPI activity under varying concentrations. The kinetic analysis revealed sigmoidal relationship of velocity with substrate concentration with Vmax 240.8 (μM/min) and Km value of 0.12 μM. ASPI showed uncompetitive inhibition with a Ki of 0.08±0.01 nM). The Far UV CD depicted 2.0% α -helices and 51% β -sheets at native pH.ConclusionsTo conclude, purified ~15 kDa ASPI exhibited fair stability in wide range of pH and temperature Overall, there was an increase in purification fold with remarkable yield. Chemical modification studies suggested the presence of lysine and tryptophan residues as lead amino acids present in the reactive sites. Therefore, ASPI with trypsin inhibitory property has the potential to be used as a non-cytotoxic clinical agents.

Purification and characterization of 2S albumin from Nelumbo nucifera.

The 2S albumins are a group of seed storage proteins that have recently attracted considerable attention in the field of allergen science due to their allergenic potential. A new 2S albumin from seeds of Nelumbo nucifera (Nn-2S alb) was purified to electrophoretic homogeneity by the combination of ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. The protein has a molecular mass of about 12kDa estimated by SDS-PAGE, in good agreement with 12.5±0.01kDa determined by ESI-MS. Circular dichroism data showed that protein contained about 66% α-helices as estimated by K2D3, indicating that the protein was predominantly helical. The sedimentation coefficient (s°20,w) of the predicted model was 1.72±0.21S. The predicted 3-dimensional structure of the Nn-2S alb revealed that the protein has a region of 12 amino acids which largely corresponds to the conserved immuno-dominant epitope of 2S allergens.

Purification and characterization of chondroitinase ABC from Acinetobacter sp. C26

An extracellular chondroitinase ABC (ChSase ABC, EC 4.2.2.4) produced by cultivating Acinetobacter sp. C26, was purified to homogeneity from the supernatant by ammonium sulfate fractionation, Q-Sepharose Fast Flow and Sephadex G-100 chromatography. The 76kDa enzyme was purified 48.09-fold to homogeneity with specific activity of 348.64U/mg, Using the chondroitin sulfate A (CS-A) as substrate, the maximal reaction rate (Vmax) and Michaelis-Menten constant (Km) of ChSase ABC were found to be 10.471μmol/min/ml and 0.105mg/ml, respectively. The enzyme showed the highest activity at the optimal conditions of pH 6.0 and 42 ∘C, respectively. This enzyme was stable at pH 5–10, 5–9 and 5–7 at 4°C, 37°C and 42°C, respectively. Investigation about thermal stability of ChSase ABC displayed that it was stable at 37°C. ChSase ABC activity was increased in presence of Na+, K+, Mn2+, 1,10-phenanthrolin and strongly inhibited by Cu2+, Hg2+, Al3+and SDS. These properties suggested that ChSase ABC from Acinetobacter sp. C26 bring promising prospects in medical and industry applications.

Characterization of a novel protease from Aeribacillus pallidus strain VP3 with potential biotechnological interest

The present study investigates the purification and physico-chemical characterization of an extracellular protease from the Aeribacillus pallidus strain VP3 previously isolated from a geothermal oil-field (Sfax, Tunisia). The maximum protease activity recorded after 22h of incubation at 45°C was 3000U/ml. Pure enzyme, designated as SPVP, was obtained after ammonium sulfate fractionation (40–60%)-dialysis followed by heat-treatment (70°C for 30min) and UNO Q-6 FPLC anion-exchange chromatography. The purified enzyme is a monomer of molecular mass about 29kDa. The sequence of the 25 NH2-terminal residues of SPVP showed a high homology with those of Bacillus proteases. The almost complete inhibition by PMSF and DIFP confirmed that SPVP is a member of serine protease family. Its optima of pH and temperature were pH 10 and 60°C, respectively. Its half-life times at 70 and 80°C were 8 and 4h, respectively. Its catalytic efficiency was higher than those of SAPCG, Alcalase Ultra 2.5L, and Thermolysin type X. SPVP exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively and high resistance against organic solvents. These properties make SPVP a potential candidate for applications in detergent formulations and non-aqueous peptide biocatalysis.

Partial purification of the molybdenum-reducing enzyme from Bacillus pumilus strain Lbna

Molybdenum is an emerging pollutant. Bioremediation of this heavy metal is possible by the mediation of Mo-reducing bacteria. These bacteria contain the Mo-reducing enzymes that can conver toxic soluble molybdenum into molybdenum blue; a less soluble and less toxic form of the metal. To date only the enzyme has been purified from only one bacterium. The aim of this study is to purify the Mo-reducing enzyme from a previously isolated Mo-reducing bacterium Bacillus pumilus strain Lbna using ammonium sulphate fractionation followed by ion exchange and then gel filtration. Two clear bands were obtained after the gel filtration step with molecular weights of 70 and 100 kDa. This indicates that further additional purification methods need to be used to get a purified fraction. Hence, additional steps of chromatography such as hydroxyapatite or chromatofocusing techniques can be applied in the future.

Partial purification and characterization of the molybdenum-reducing enzyme from the glyphosate-degrading Burkholderia vietnamiensis strain AQ5-12

In this study, a novel glyphosate-degrading shows the ability to reduce molybdenum to molybdenum blue. The enzyme from this bacterium was partially purified and partially characterized to ascertain whether the Mo-reducing enzyme from this bacterium shows better or lower efficiency in reducing molybdenum compared to other Mo-reducing bacterium that only exhibits a single biotransformation activity. The enzyme was partially purified using ammonium sulphate fractionation. The Vmax for the electron donating substrate or NADH was at 1.905 nmole Mo blue/min while the Km was 6.146 mM. The regression coefficient was 0.98. Comparative assessment with the previously characterized Mo-reducing enzyme from various bacteria showed that the Mo-reducing enzyme from Burkholderia vietnamiensis strain AQ5-12 showed a lower enzyme activity.

Plant-produced Crimean-Congo haemorrhagic fever virus nucleoprotein for use in indirect ELISA

Crimean-Congo haemorrhagic fever (CCHF) is a disease of serious public concern caused by the CCHF virus (CCHFV). Anti-CCHFV IgG in humans can be detected using ELISA with native antigen prepared from cell cultures which have been infected with virus or from brain tissue of suckling mice which have been inoculated with virus. However, the preparation of these reagents requires high biosafety levels and is expensive. A safer, more cost-effective recombinantly-produced NP reagent is desirable. Recently, plants have been shown to be a cost-effective and safe system for expression of recombinant proteins. This work describes cloning of the CCHFV NP gene into three different plant expression systems and comparison of expression in Nicotiana benthamiana. The highest expressing construct was selected. Expressed NP was purified by ammonium sulphate fractionation prior to histidine affinity chromatography. Purified NP was tested in an indirect ELISA to determine if the recombinant antigen was able to detect anti-CCHFV IgG in sera from convalescent patients. Plant-produced NP detected IgG antibodies against CCHFV in 13/13 serum samples from convalescent patients and 0/13 samples collected from volunteers with no history of CCHFV infection. Results were compared with commercially available immunofluorescent assays and 100% concordance was obtained between the two assays. This suggests that a full evaluation of the plant produced NP for application as a safe recombinant is warranted.

Structural and functional studies on a variant of cystatin purified from brain of Capra hircus

Cystatins, known for their ubiquitous presence in mammalian system are thiol protease inhibitors serving important physiological functions. Here, we present a variant of cystatin isolated from brain of Capra hircus (goat) which is glycosylated but lacks disulphide bonds. Caprine brain cystatin (CBC) was isolated using alkaline treatment, ammonium sulphate fractionation (40–60%) and gel filtration chromatography on Sephacryl S-100HR column with an overall yield of 26.29% and 322-fold purification. The inhibitor gave a molecular mass of ~44 kDa as determined by SDS-PAGE and gel filtration behaviour. The Stokes radius and diffusion coefficient of CBC were 27.14 Å and 8.18 × 10−7 cm2 s−1, respectively. Kinetic data revealed that CBC inhibited thiol proteases reversibly and competitively, with the highest inhibition towards papain (Ki = 4.10 nM) followed by ficin and bromelain. CBC possessed 34.7% α-helical content as observed by CD spectroscopy. UV, fluorescence, CD and FTIR spectroscopy revealed significant conformational change upon CBC-papain complex formation. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic parameters – ΔH, ΔS, ΔG along with N (binding stoichiometry) for CBC-papain complex formation. Binding stoichiometry (N = .97 ± .07 sites) for the CBC-papain complex indicates that cystatin is surrounded by nearly one papain molecule. Negative ΔH (−5.78 kcal mol−1) and positive ΔS (11.01 cal mol−1 deg−1) values suggest that the interaction between CBC and papain is enthalpically as well as entropically favoured process. The overall negative ΔG (−9.19 kcal mol−1) value implies a spontaneous CBC-papain interaction.

Amylase from bacillus thuringiensis isolated from tapioca waste: isolation, partial purification and characterization

Microorganism enzymes are the most widely used in industrial applications. Tapioca liquid waste could be a great source of amylase-producing bacteria. The aim of this research was to isolate the amylase-producing bacteria form the tapioca waste, to produce amylase and to purify the resulted amylase. The screening, identification, and the optimal production condition of the amylase‒producing bacteria were studied. The optimization of bacteria production growth phase and the amylase production time were investigated. The crude amylase was purified using ammonium sulfate fractionation followed by SDS PAGE electrophoresis to identify the molecular weight and to purity of the amylase. The amylase activity assay used was based on the measuring of resulted reducing sugar by Somogyi-Nelson method. The result showed that the amylase producing bacteria was identified as Bacillus thuringiensis. The exponential phase of the bacteria growth for bacteria adaptation before production was 18 h and the optimal production time of amylase enzyme was 24 h. The highest specific activity of the purified amylase was fraction (FHD) 40% with specific activity of 37.56 ± 0.38U/mg. The SDS PAGE of FHD 40% profile showed two clear bands with molecular weight of 32 kDa and 35 kDa respectively

Study on isolation and determination of glycoprotein from tea plant leaves

Tea is a healthy food, because it has various bioactive components. Recent research indicated that tea glycoprotein might be a bioactive component in tea. In this report, total proteins were extracted from tea fresh leaves, and coarse glycoproteins were isolated through the methods of ammonium sulfate fractions (45%--70%) and Sephadex G-100 chromatography. According to control staining of glycan and polypeptide, glycoproteins were determinated by using Pierce GelCode Glycoprotein Staining Kit and Coomassie Brilliant Blue R250, and three tea glycoproteins were fast purified from the SDS-gels. Sixteen native bioactive glycoproteins were gained from tea total proteins by ConA-Sepharose 4B affinity chromatography.

Purification and Characterization of Hexavalent Chromate Reductase Activity in Cell Free Extract of Bacillus subtilis Strain Isolated From Treated Tannery Effluent

A Bacillus subtilis strain previously isolated from treated tannery effluent was tolerant to a maximum of 1400 mg/L Cr(VI) and reduced 77% toxic Cr(VI) to less toxic Cr(III) within 24 hrs. at pH 8.5 and 35°C temperature under shaking condition (120 rpm). The chromate reductase enzyme was found to be membrane bound and it has been purified using ammonium sulphate fractionation, anionexchange chromatography and gel filtration chromatography on Sephadex G-75 by cell free extract of the bacterium. The enzyme was characterized based on optimal temperature, pH, metal ions and initial Cr(VI) concentration in the reaction mixture. Maximum chromate reductase activity was observed at pH 8.5 and 35ºC temperature. The enzyme was significantly active between pH 6.5-10.0 and was found to be stable between 30ºC-40ºC. The chromate reductase activity was slightly decreased with increasing “the concentration of Cr(VI). The enzymatic activity was completely inhibited by Hg(II) and As(III) while Ni(II), Cd(II), Mn(II), Zn(II), Pb (II) and Co (II) showed moderate effect on chromate reductase activity of Bacillus subtilis and the chromate reduction was enhanced by Cu(II) and Fe(III). Keywords: Bacillus subtilis, chromate reductase, Cr(VI), tannery.