Production of an alkaline protease by Bacillus cereus MCM B-326 and its application as a dehairing agent

The present investigation describes microbial production of an alkaline protease and its use in dehairing of buffalo hide. Bacillus cereus produced extracellular protease when grown on a medium containing starch, wheat bran and soya flour (SWS). The ammonium sulphate precipitated (ASP) enzyme was applied for dehairing of buffalo hide. Microscopic observation of longitudinal section of buffalo hide revealed that the epidermis was completely removed and hair was uprooted leaving empty follicles in the hide. The ASP enzyme was stable for one month at ambient temperature between 25–35 °C. Enzymatic dehairing may be a promising shift towards an environment-friendly leather processing method.     

A novel extracellular protease from Pseudomonas aeruginosa MCM B-327: enzyme production and its partial characterization

The focus of this study was on production, purification and characterization of dehairing protease from Pseudomonas aeruginosa MCM B-327, isolated from vermicompost pit soil. Optimum protease activity, 395 U mL(-1), was observed in the medium containing soybean meal and tryptone, at pH 7 and 30 °C. The crude enzyme exhibited dehairing activity. As compared to chemical method, enzymatic method of dehairing showed reduction in COD, TDS and TSS by 34.28%, 37.32% and 51.58%, respectively. Zymogram of crude enzyme on native-PAGE presented two bands with protease activity of molecular weights of 56 and 67 kDa. Both proteases showed dehairing activity. Out of these, 56kDa protease (PA02) was purified 3.05-folds with 2.71% recovery. The enzyme was active in pH range 7-9 and temperature 20-50 °C with optimum pH of 8 and temperature 35°C. Moreover, the enzyme activity of PA02 protease was not strongly inhibited by specific inhibitor showing the novel nature of enzyme compared to serine, cysteine, aspartyl and metalloproteases. Kinetic studies indicated that substrate specificity of PA02 protease was towards various natural and synthetic proteolytic substrates but inactive against collagen and keratin. These findings suggest protease secreted by P. aeruginosa MCM B-327 may have application in dehairing for environment-friendly leather processing.     

Purification and characterization of an extracellular alkaline serine protease with dehairing function from Bacillus pumilus

An extracellular alkaline serine protease (called DHAP), produced by a Bacillus pumilus strain, demonstrates significant dehairing function. This protease is purified by hydrophobic interaction chromatography, ion exchange, and gel filtration. DHAP had a pI of 9.0 and a molecular weight of approximately 32,000 Dalton. It shows maximal activity at pH 10 and with a temperature of 55 degrees C; the enzyme activity can be completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP). The first 20 amino acid residues of the purified DHAP have been determined with a sequence of AQTVPYGIPQIKAPAVHAQG. Alignment of this sequence with other alkaline protease demonstrates its high homology with protease from another B. pumilus strain.     

Production and characterization of tannase from Bacillus cereus KBR9

A tannase-producing soil bacteria has been isolated and identified as Bacillus cereus. It can degrade tannic acid and produce maximum tannase (0.22 U/ml) at stationary phases of growth (24 h). Maximum growth and enzyme production occurred with initial medium pH of 4.5-5.0. Partial purified tannase showed optimum activity at pH 4.5 and 40 degrees C. It remains stable up to 30 degrees C and pH 4.5 to 5.0. The enzyme is salt tolerant, stable up to 2 m of NaCl and retains 82% original activity in 3 m.     

Production and purification of a calcium-dependent protease from Bacillus cereus BG1

The production and purification of a calcium-dependent protease by Bacillus cereus BG1 were studied. The production of the protease was found to depend specifically on the calcium concentration in the culture medium. This suggests that this metal ion is essential for the induction of protease production and/or stabilisation of the enzyme after synthesis. The calcium requirement is highly specific since other metal ions (such as Mg²⁺ and Ba²⁺, which both activate the enzyme) are not able to induce protease production. The most appropriate medium for growth and protease production comprises (g L^–1) starch 5, CaCl₂ 2, yeast extract 2, K₂HPO₄ 0.2 and KH₂PO₄ 0.2. The protease of BG1 strain was purified to homogeneity by ultrafiltration, heat treatment, gel filtration on Sephacryl S-200, ion exchange chromatography on DEAE-cellulose and, finally, a second gel filtration on Sephacryl S-200, with a 39-fold increase in specific activity and 23% recovery. The molecular weight was estimated to be 34 kDa on SDS-PAGE. The optimum temperature and pH of the purified enzyme were determined to be 60°C and 8.0, respectively, in 100 mM Tris-HCl buffer + 2 mM CaCl₂.     

Purification and characterization of a solvent and detergent-stable novel protease from Bacillus cereus

A protease-producing bacterium was isolated from slaughterhouse waste samples, Hyderabad, India. It was related to Bacillus cereus on the basis of 16S rRNA gene sequencing and biochemical properties. The protease was purified to homogeneity using ammonium sulfate precipitation, and ion exchange chromatography with a fold purification of 1.8 and a recovery of 49%. The enzyme had a relative molecular weight of 28 kDa, pH and temperature optima for this protease were 10 and 60 °C. The activity was stable between a pH range of 7.0 and 12.0. The activity was inhibited by EDTA and enhanced (four-fold) by Cu²⁺ ions indicating the presence of metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents and anionic surfactants. The enzyme also showed stability in the presence of organic solvents.     

Purification and characterization of a novel extracellular protease from Bacillus cereus KCTC 3674

Bacillus cereus KCTC 3674 excretes several kinds of extracellular proteases into the growth medium. Two proteases with molecular masses of approximately 36-kDa and 38-kDa, as shown by SDS-PAGE, were purified from the culture broth. The 38-kDa protease was purified from B. cereus cultivated at 37 degrees C, and the 36-kDa protease was obtained from the B. cereus cultivated at 20 degrees C. The 38-kDa protease was identified as an extracellular neutral (metallo-) protease and was further characterized. The 36-kDa protease was shown to be a novel enzyme based on its N-terminal amino acid sequence, its identification as a metallo-enzyme that was strongly inhibited by EDTA and o-phenanthroline, its hemolysis properties, and its optimal pH and temperature for activity of 8.0 and 70 degrees C, respectively.     

Production and characterization of collagenase from a new Amazonian Bacillus cereus strain

Abstract

A new collagenase producing a strain of Bacillus cereus, isolated from the pollen of a bee of Amazon Region (Brazil), had its enzyme characterized and the production medium composition and culture conditions enhanced. A two-level design on three factors, namely initial medium pH, the substrate (gelatin) concentration and agitation intensity, allowed identifying the first two variables as the most significant ones, while a central composite design (CCD) was subsequently used to identify their optimal levels. Statistics highlighted maximized collagenolytic activity when substrate concentration and initial medium pH were selected at their highest levels (positive effects), whereas agitation intensity at the lowest (negative effect). Triplicate runs performed under predicted optimal conditions (pH 7.8 and 1.7% gelatin concentration) yielded a collagenolytic activity (305.39?±?5.15?U) 4.6- to 15-fold those obtained with the preliminary design. The enzyme displayed optimum activity at 45?°C and pH 7.2, was stable over wide ranges of pH values and temperatures (7.2–11.0 and 25–50?°C, respectively) and was strongly inhibited by 10?mM phenylmethylsulphonyl fluoride. The zymogram showed two prominent bands at 50 and 76?kDa. These results are a first attempt to elucidate the features of this new collagenase, its production conditions, and possible scale-up.     

Production and characterization of two hemolysins of Bacillus cereus.

Bacillus cereus strain B-48 produced two hemolysins with molecular weights of 52,000 (H-I) and 31,000 (H-II). A mutant was isolated that produced only H-II but was identical with the wild type in all other respects. We exploited this mutant to produce H-II for study that was free of contamination by H-I. By manipulation of media composition, we produced H-I in the absence of H-II. The hemolysins were precipitated differently by ammonium sulfate, and both exhibited the Arrhenius effect when heated. Both hemolysins attached rapidly to erythrocytes; however, lysis by H-I was immediate, while lysis by H-II followed after a lag. Hemolysis by H-I and H-II increased in rate with increasing temperature and was absent at 0 degrees C. Only H-I was inhibited by cholesterol. The hemolysins of B. cereus appeared similar to the hemolysins of B. thuringiensis. H-I probably is identical with cereolysin.     

Purification and characterization of alkaline protease with novel properties from Bacillus cereus strain S8

Proteases are the hydrolytic enzymes which hydrolyzes peptide bond between proteins with paramount applications in pharmaceutical and industrial sector. Therefore production of proteases with efficient characteristics of biotechnological interest from novel strain is significant. Hence, in this study, an alkaline serine protease produced by Bacillus cereus strain S8 (MTCC NO 11901) was purified and characterized. The alkaline protease was purified by ammonium sulfate precipitation (50%), ion exchange (DEAE-Cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. As a result of this purification, a protein with specific activity of 300U/mg protein was obtained with purification fold 17.04 and recovery percentage of 34.6%. The molecular weight of the purified protease was determined using SDS-PAGE under non-reducing (71?kDa) and reducing conditions (35?kDa and 22?kDa). Zymogram analysis revealed that proteolytic activity was only associated with 22?kDa. These results indicate that existence of the enzyme as dimer in its native state. The molecular weight of the protease (22?kDa) was also determined by gel filtration (Sephadex G-200) chromatography and it was calculated as 21.8?kDa. The optimum activity of the protease was observed at pH 10.0 and temperature 70?°C with great stability towards pH and temperature with casein as a specific substrate. The enzyme was completely inhibited by PMSF and TLCK indicating that it is a serine protease of trypsin type. The enzyme exhibits a great stability towards organic solvents, oxidizing and bleaching agents and it is negatively influenced by Li²⁺ and Co²⁺ metal ions. The purified protein was further characterized by Matrix Assisted Laser Desorption Ionization/Mass Spectroscopy (MALDI/MS) analysis which reveals that total number of amino acids is 208 with isoelectric point 9.52.     

Laundry detergent compatibility of the alkaline protease from Bacillus cereus

The endogenous protease activity in various commercially available laundry detergents of international companies was studied. The maximum protease activity was found at 50 degrees C in pH range 10.5-11.0 in all the tested laundry detergents. The endogenous protease activity in the tested detergents retained up to 70% on incubation at 40 degrees C for 1 h, whereas less than 30% activity was only found on incubation at 50 degrees C for 1 h. The alkaline protease from an alkalophilic strain of Bacillus cereus was studied for its compatibility in commercial detergents. The cell free fermented broth from shake flask culture of the organism showed maximum activity at pH 10.5 and 50 degrees C. The protease from B. cereus showed much higher residual activity (more than 80%) on incubation with laundry detergents at 50 degrees C for 1 h or longer. The protease enzyme from B. cereus was found to be superior over the endogenous proteases present in the tested commercial laundry detergents in comparison to the enzyme stability during the washing at higher temperature, e.g., 40-50 degrees C.     

地衣芽孢杆菌TG116胞外蛋白酶产酶条件与酶学性质

【背景】从独角莲中分离得到的地衣芽孢杆菌TG116是一株对植物病原菌具有广谱抗性作用的生防菌株。【目的】优化TG116的产酶条件并探索其酶学性质,进一步了解其抗菌机制。【方法】采用Folin-Phenol显色法与响应曲面法,优化菌株TG116的产酶条件并研究其蛋白酶的酶学性质。【结果】菌株TG116产酶最适条件为:温度40.83°C,p H 8.01,发酵时间53.74 h,增加通气量可以显著提高酶活力。按照优化后的条件培养48 h后,上清液蛋白酶活力从57.46 U/mL达到了254.07 U/mL。酶学性质研究表明:该酶为碱性蛋白酶,最适反应pH为8.5,最适反应温度为50°C,具有良好的温度和pH稳定性,EDTA对酶活具有强烈的抑制作用,金属离子Mg~(2+)、Ca~(2+)、Na~+、Co~(2+)、K~+等对酶活也具有一定的抑制作用。【结论】菌株TG116具有良好的p H与温度稳定性,在实际应用中蛋白酶不易失活,可以分解真菌的细胞壁蛋白成分,破坏细胞壁结构,从而抑制甚至杀死病原菌,达到抗菌作用。     

Preliminary characterization of adenosine deaminase from Bacillus cereus

Adenosine deaminase from Bacillus cereus is quite unstable, similarly to other bacterial deaminases, but it shows a peculiar stabilizing effect by some monovalent cations. These include K+, Li+, NH4+ and to a lesser extent Cs+. Maximal stabilization of the deaminase is exerted by K+ at concentrations higher than 20 mM. The enzyme can be rapidly inactivated by sulphydryl reagents such as p-hydroxymercuribenzoate. Since adenosine deaminase from B. cereus, in addition to monovalent cations, is stabilized also by dithiothreitol, a possible influence of monovalent cations on the reactivity of some sulphydryl groups on the enzyme has been suggested.     

Phenotypic characterization of Bacillus thuringiensis and Bacillus cereus

One hundred and thirty-seven strains of Bacillus thuringiensis and 35 strains of Bacillus cereus were tested for the presence or absence of 99 traits. An analysis of these data indicated that strains of B. thuringiensis were indistinguishable from B. cereus, except for their ability to produce parasporal crystals. This conclusion was based on a comparison of the phenotypic properties of B. thuringiensis and B. cereus, as well as on the results of numerical analyses of the data which grouped strains into clusters on the basis of phenotypic similarity. In the resulting dendrograms, strains of B. thuringiensis and B. cereus were interspersed, exhibiting no tendency to segregate. In addition, with the exception of serovar israelensis, strains on B. thuringiensis belonging to the same flagellar serovar showed little or no tendency to group in different clusters. A comparison of the phenotypic differences between serovars indicated that the greater the number of strains in the serovars, the fewer, if any, phenotypic traits separating them. This suggests that the properties reported to differentiate serovars can be attributed to the internal phenotypic diversity of the species. Characterization of 10 mosquitocidal strains of Bacillus sphaericus indicated that the traits employed in this study readily distinguished these highly related organisms from strains of B. thuringiensis and B. cereus.     

Production and characterization of monoclonal antibodies against vegetative cells of Bacillus cereus

Two monoclonal antibodies (MAbs) against Bacillus cereus were produced. The MAbs (8D3 and 9B7) were selected by enzyme-linked immunosorbent assay for their reactivity with B. cereus vegetative cells. They reacted with B. cereus vegetative cells while failing to recognize B. cereus spores. Immunoblotting revealed that MAb 8D3 recognized a 22-kDa antigen, while MAb 9B7 recognized two antigens with molecular masses of approximately 58 and 62 kDa. The use of MAbs 8D3 and 9B7 in combination to develop an immunological method for the detection of B. cereus vegetative cells in foods was investigated.     

Production,isolation and characterization of extracellular protease of an alkaliphilic strain of Arthrobacter ramosus,MCM B-351 isolated from the alkaline lake of Lonar,India

An extracellular protease was produced by Arthrobacter ramosus isolated from the alkaline lake of Lonar, Buldhana District of Maharashtra, India when grown on a synthetic medium of pH 10 containing casein. The optimum conditions for production were 3.0% initial casein concentration, 2% inoculum of 1 × 10⁸ cells/ml, pH 9.0, temperature 30 °C and shaken culture conditions. The protease was purified by ammonium sulphate precipitation followed by Sephadex G-100 chromatography. Two proteases viz. Arthro I and Arthro II, having molecular weights 21 and 11.4 kDa respectively were isolated. The Arthro II fraction had K _m 395 g/ml and V _max 10.55 g/min for azocasein. The maximum activity of enzyme was at 55 °C and pH 8. It was thermostable (up to 80 °C), alkali stable (pH 12) and stable in commercial detergent. The enzyme may contain a thiol group at the active site.     

A novel serine alkaline protease from Bacillus altitudinis GVC11 and its application as a dehairing agent

A serine alkaline protease from a newly isolated alkaliphilic Bacillus altitudinis GVC11 was purified and characterized. The enzyme was purified to homogeneity by acetone precipitation, DEAE-cellulose anion exchange chromatography with 7.03-fold increase in specific activity and 15.25% recovery. The molecular weight of alkaline protease was estimated to be 28 kDa by SDS PAGE and activity was further assessed by zymogram analysis. The enzyme was highly active over a wide range of pH 8.5 to 12.5 with an optimum pH of 9.5. The optimum temperature of purified enzyme was 45 °C and Ca²⁺ further increased the thermal stability of the enzyme. The enzyme activity was enhanced by Ca²⁺ and Mg²⁺ and inhibited by Hg²⁺. The present study is the first report to examine and describe production of highly alkaline protease from Bacillus altitudinis and also its remarkable dehairing ability of goat hide in 18 h without disturbing the collagen and hair integrity.     

Purification and characterization of a DNA-dependent ATPase from Bacillus cereus

A DNA-dependent ATPase (molecular weight 71 000) free of nuclease activity has been purified from Bacillus cereus. The enzyme shows similar characteristics as the enzyme isolated from Escherichia coli and Bacillus subtilis. Heat denatured DNA stimulates the rate of ATP hydrolysis to ADP and Pi to an extent about tenfold higher than the native DNA. Double stranded DNA without single stranded regions is not a suitable cofactor for the enzyme. The ATPase is inhibited by adenosine 5'-(beta, gamma-imino)-diphosphate, while another ATP analogue, adenosine 5'-(beta, gamma-methylene)-diphosphate has no effect on ATPase activity. KM for ATP is 0.38 mM, the apparent KM for nucleotide equivalent DNA is 1.2 microM. Evidence of the unwinding function of the enzyme is presented.     

Separation and characterization of an autolytic endo-beta-glucosaminidase from Bacillus cereus

1. An autolytic endo-beta-glucosaminidase, capable of cleaving the glycoside linkages of N-unsubstituted glucosamine in the glycan moiety of cell wall peptidoglycan, was purified 470-fold from a salt extract of the 2,000 x g precipitate fraction obtained after sonication of a lysozyme-resistant strain of Bacillus cereus. The properties of this enzyme were studied. 2. The purified enzyme preparation was also active towards the glycan chain of fully N-acetylated cell wall peptidoglycan. 3. The endo-beta-glucosaminidase was inactive towards the cell wall peptidoglycan unless the peptide portion of this polymer was removed either by the action of N-acetylmuramyl-L-alanine amidase or by the treatment with alkali in aqueous dimethyl sulfoxide. 4. Studies on the action of this enzyme towards chemically modified glycans revealed that the carboxyl groups of muramic acid residues are indispensable to a substrate for this enzyme.