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.     

Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326

Bacillus cereus MCM B-326, isolated from buffalo hide, produced an extracellular protease. Maximum protease production occurred (126.87+/-1.32 U ml(-1)) in starch soybean meal medium of pH 9.0, at 30 degrees C, under shake culture condition, with 2.8 x 10(8) cells ml(-1) as initial inoculum density, at 36 h. Ammonium sulphate precipitate of the enzyme was stable over a temperature range of 25-65 degrees C and pH 6-12, with maximum activity at 55 degrees C and pH 9.0. The enzyme required Ca(2+) ions for its production but not for activity and/or stability. The partially purified enzyme exhibited multiple proteases of molecular weight 45 kDa and 36 kDa. The enzyme could be effectively used to remove hair from buffalo hide indicating its potential in leather processing industry.     

Enzymatic depilation of animal hide: identification of elastase (LasB) from Pseudomonas aeruginosa MCM B-327 as a depilating protease

Conventional leather processing involving depilation of animal hide by lime and sulphide treatment generates considerable amounts of chemical waste causing severe environmental pollution. Enzymatic depilation is an environmentally friendly process and has been considered to be a viable alternative to the chemical depilation process. We isolated an extracellular protease from Pseudomonas aeruginosa strain MCM B-327 with high depilation activity using buffalo hide as a substrate. This 33 kDa protease generated a peptide mass fingerprint and de novo sequence that matched perfectly with LasB (elastase), of Pseudomonas aeruginosa. In support of this data a lasB mutant of MCM B-327 strain lacked depilatory activity and failed to produce LasB. LasB heterologously over-produced and purified from Escherichia coli also exhibited high depilating activity. Moreover, reintroduction of the lasB gene to the P. aeruginosa lasB mutant via a knock-in strategy also successfully restored depilation activity thus confirming the role of LasB as the depilating enzyme.     

Statistical optimization of medium components for production of extracellular chitinase by Basidiobolus ranarum: a novel biocontrol agent against plant pathogenic fungi

The influence of concentration of medium components such as colloidal chitin, lactose, malt extract, yeast extract, and peptone on the chitinase production from Basidiobolous ranarum at the flask level were studied by using statistical tool Central Composite Design (CCD) and analysed by Response Surface Methodology (RSM). The results revealed that colloidal chitin, malt extract and peptone had significant effect (P < 0.01) on the chitinase production at their individual levels. The polynomial equation of the model developed incorporates 3 linear, 3 quadratic and 5 interactive terms. Maximum chitinase production of 3.47 U ml(-1) was achieved with 1.5% colloidal chitin, 0.125% lactose, 0.025% malt extract and 0.075% peptone. After optimization, chitinase activity was increased by 7.71 fold. A second order polynomial equation was found to be useful for the development of efficient bioprocess for chitinase production. To screen the biotechnological potential of this enzyme, degradation of fungal mycelia by ammonium sulphate precipitate of the same was studied for several pathogenic fungi-in vitro which showed promising results particularly against Rhizoctonia solani and Fusarium solani. This study provides the first evidence showing the effectiveness of RSM for the development of a robust statistical model for the chitinase production by Basidiobolus and for its application in the biocontrol of phytopathogenic fungi. (? 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).     

Optimization of protease activity of alkaliphilic bacteria isolated from an alkaline lake in India

The effect of pH on the conversion of glucose to hydrogen by a mixed culture of fermentative bacteria was evaluated. At 36 degrees C, six hours hydraulic retention, over 90% of glucose was degraded at pH ranging 4.0-7.0, producing biogas and an effluent comprising mostly fatty acids. At the optimal pH of 5.5, the biogas comprised 64 +/- 2% of hydrogen with a yield of 2.1 +/- 0.1 mol-H2/mol-glucose and a specific production rate of 4.6 +/- 0.4 l-H2/(g-VSS day). The effluent was composed of acetate (15.3-34.1%) and butyrate (31.2-45.6%), plus smaller quantities of other volatile fatty acids and alcohols. The diversity of microbial communities increased with pH, based on 16S rDNA analysis by denaturing gradient gel electrophoresis (DGGE).     

Production of 2,3-butanediol from glucose byBacillus licheniformis

Bacillus licheniformis produced 2,3-butanediol from glucose with an optimum yield of 47 g/100 g glucose after 72 h of growth on a peptone/beef extract medium containing 2% (w/v) glucose at pH 6.0 and 37°C. This yield of 2,3-butanediol was higher than those previously reported forKlebsiella oxytoca (37 g/100 g glucose) andBacillus polymyxa (24 g/100 glucose).     

Effect of supplementation of micronutrients and phytochemicals to fructooligosaccharides on growth response of probiotics and E. coli

Probiotics and prebiotics, which can change the colonic microenvironment, are the areas of current interest. Unutilizable fractions of the foods and fortificants, which reach the colon can affect the profile of probiotics. Effects of eight such factors viz. zinc sulphate, zinc carbonate, ferrous sulphate, ferric citrate, quercetin, gallic acid, phytic acid, and oxalic acid were, therefore, investigated on 24 H growth of Lactobacillus acidophilus (L1) and Lactobacillus plantarum (L2), two isolates of bifidobacteria (longum (L3) and bifidum (L4)) and a marketed consortium (L5) of eight probiotic cultures. MRS medium with marketed fructooligosaccharide as the only source of carbon was used for study of dose response curves. Quercetin and zinc sulphate showed significant positive effect for L1 and L5 (P < 0.01), whereas there was slight positive effect or no effect on growth of other probiotics. Phytic acid showed a significant inhibitory effect for L2 and a slight inhibitory effect on L3 and L4 whereas L5 were able to tolerate phytic acid. Oxalic acid had slight positive effect for L1 (P < 0.05) and L5 and no effect on growth of other probiotics (P > 0.05). Further, zinc sulphate, ferrous sulphate, quercetin, and oxalic acid significantly inhibited growth of E. coli (P < 0.05)     

Characterisation of copolymer, poly (hydroxybutyrate-co-hydroxyvalerate) (PHB-co-PHV) produced by Halomonas campisalis (MCM B-1027), its biodegradability and potential application

Characterisation of polyhydroxyalkanoate (PHA) film produced by haloalkalitolerant Halomonascampisalis (MCM B-1027) in 14 L SS fermenter revealed it to have composition of monomer units, HB:HV as 96:4 as analysed by ¹H NMR indicating the PHA as a co-polymer of PHB-co-PHV, molecular weight by gel permeation chromatography as 2.08 × 10⁶, melting temperature 166.51 °C, tensile strength 18.8 MPa; two relaxations namely beta transition corresponding to the glass rubber transition and alpha transition corresponding to crystalline relaxation by Dynamic Mechanical Thermal analysis and only one relaxation corresponding to MWS interfacial polarisation with activation energy of 129 kJ/mol by broadband dielectric spectroscopy. Optical microscopic studies showed typical Maltese-cross pattern of spherulites. The PHA film was found to be biodegradable by standard ASTM method as well as by soil burial method. The leak proof polymer bags prepared from the film could be used as a packaging material.     

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.     

Extracellular chitinase production by some members of the saprophytic Entomophthorales group

Thirteen strains were isolated from different habitats, belonging to two genera, namely Conidiobolus and Basidiobolus, related to saprophytic Entomophthorales. Chitin flake colonization and agar-well diffusion tests were used to screen potential extracellular chitinase-producing strains in plate assays. Preliminary screening resulted in five chitinase producers that were further studied quantitatively. Results indicated that studied isolates of this group produced chitinase at different levels in chitin-containing as well as non-chitin-containing medium. Conidiobolus coronatus was found to be the most significant chitinase producer, giving 0.261 U/ml using colloidal chitin as a carbon source, among the isolates under study. This communication also reports the chitinolytic activity of Basidiobolus haptoporus, the effect of environmental and nutritional parameters on chitinase production, and utilization of fungal biomass as a carbon source, which hitherto had not been elaborated from this genus.