Jitender Sharma

Chymosin and other milk coagulants: sources and biotechnological interventions

Calf rennet, which consists of over 90% chymosin, is commonly used in cheese industries for the curdling of milk. Various animal, plant and microbial sources have been exploited as possible alternatives to calf rennet. The coagulating properties of the enzymatic preparations (coagulants) from these sources differ in terms of their physicochemical factors. The cheese industry has always sought out novel and stable enzyme sources, and recombinant chymosin has been found to be an effective alternative since it possesses several advantages over plant and microbial milk-clotting enzymes. This paper reviews the use of various milk coagulants, especially animal coagulants, for cheese making. Advancements in genetic and protein engineering to produce recombinant chymosin are discussed in addition to evaluating its identity to the rennet available from natural sources.

Application of cellulase-free xylano-pectinolytic enzymes from the same bacterial isolate in biobleaching of kraft pulp.

A synergistic action of xylano-pectinolytic enzymes from the same bacterial isolate Bacillus pumilus was evaluated for the prebleaching of kraft pulp. The enzymatic prebleaching of kraft pulp resulted in 8.5% reduction in kappa number of the pulp, showing remarkable delignification with the enzyme treatment. This approach resulted in 25% reduction in active chlorine consumption in subsequent bleaching stages without any decrease in brightness. Increase in Burst factor (9%), Tear factor (4.6%), Breaking length (4.4%), Double fold number (12.5%), Gurley porosity (4%) and Viscosity (11.8%) of enzyme treated pulp reflected the significant improvement in pulp properties. This is the first report describing the use of xylanase and pectinase produced simultaneously in the same production medium from the same bacterial isolate for effective biobleaching of kraft pulp. Use of this xylano-pectinolytic synergism in paper and pulp industry will ultimately help in making the process not only economically feasible but also eco-friendly.

Effect of nutrients supplementation on anaerobic sludge development and activity for treating distillery effluent

Startup of laboratory anaerobic reactors and treatment efficiency were investigated by supplementing the distillery effluent feed with macronutrients (Ca, P) and micronutrients (Ni, Fe and Co) under mesophilic conditions. Calcium and phosphate were deterimental to the treatment efficiency and sludge granulation. Traces of salts of iron, nickel and cobalt, individually and in combinations improved the COD removal efficiency and sludge granulation process.

Characterization of statistically produced xylanase for enrichment of fruit juice clarification process

Critical factors for xylanase production of Bacillus stearothermophilus under batch fermentation and for clarification of citrus fruit juice using this xylanase were optimized through central composite design of response surface methodology. Statistical approach resulted in an increase of 1.19-fold in xylanase yield over conventional method. Model equation for juice clarification included independent variables viz. temperature, incubation time and enzyme dose to study the dependent variables such as yield, acidic neutrality and filterability etc. Coefficient of determination, R(2) for enzyme production model and for different juice properties were in accordance with the linearity of the model. On the basis of the contour plots the optimum enzyme dose was 12.5 IU/g of xylanase. Enzymatic treatment has resulted in the improvement of twofold in the release of reducing sugars and 52.97% in juice yield, whereas 35.34% reduction in turbidity was observed.

Short communicationOrganoiodine (III)-mediated synthesis of 3-aryl/heteroaryl-5,7-dimethyl-1,2,4-triazolo[4,3-c]pyrimidines as antibacterial agents

Synthesis of 12 new 3-aryl/heteroaryl-5,7-dimethyl-1,2,4-triazolo[4,3-c]pyrimidines (3a-l) has been accomplished by the oxidation of pyrimidinylhydrazones (2a-l) of various aryl/heteroaryl aldehydes using 1.1equiv. of iodobenzene diacetate (IBD) in dichloromethane. All the compounds 3a-l tested in vitro for their antibacterial activity against two Gram-positive bacteria namely, Bacillus subtilis, Bacillus stearothermophilus and two Gram-negative bacteria Pseudomonas putida, Escherichia coli. Two compounds, namely 3-(2,4-dichlorophenyl)-5,7-dimethyl-1,2,4-triazolo [4,3-c]pyrimidine (3j) and 3-(4-hydroxy-2-methoxyphenyl)-5,7-dimethyl-1,2,4-triazolo[4,3-c]pyrimidine (3l) were found to be equipotent or more potent than the commercially available antibiotics (chloramphenicol and streptomycin).

Bleach-boosting effect of crude xylanase from Bacillus stearothermophilus SDX on wheat straw pulp

Pretreatment of wheat straw pulp using cellulase-free xylanase produced from Bacillus stearothermophilus SDX at 60°C for 120min resulted in 4.75% and 22.31% increase in brightness and whiteness, respectively. Enzyme dose of 10U/g of oven dried pulp at pH 9 decreased the kappa number and permanganate number by 7.14% and 5.31%, respectively. Further chlorine dioxide and alkaline bleaching sequences (CDED(1)D(2)) resulted in 1.76% and 3.63% increase in brightness and whiteness, respectively. Enzymatic prebleaching of pulp decreased 20% of chlorine consumption without any decrease in brightness. Improvement in various pulp properties like viscosity, burst factor, burstness, breaking length, double fold, gurley porosity, tear factor, and tearness were also observed after bleaching of xylanase treated wheat straw pulp.

Identification, Characterization, and Regulation of a Novel Antifungal Chitosanase Gene (cho) in Anabaena spp.

ABSTRACT Two contrasting cyanobacterial species (Anabaena fertilissima and Anabaena sphaerica) were selected based on differences in antifungal behavior in order to study the mechanism for production of an antifungal enzyme and the genes responsible for this production. In A. fertilissima, chitosanase and antifungal activities were increased significantly under of growth-limiting conditions (8 of light and 16 h of darkness). The lack of such activities in A. sphaerica was associated with high levels of protein that accumulated during the stationary phase (at 28 days) under the same light conditions. The gene putatively responsible for chitosanase and antifungal activities was amplified using specific primers, and sequence analysis of the amplified products (1.086 and 1.101 kb in A. sphaerica and A. fertilissima, respectively) showed that they belong to the glycoside hydrolase 3 (GH3)-like family of Anabaena variabilis ATCC 29413. Pairwise alignment of the corresponding protein sequences identified a putative signal peptide (amino acids 1 to 23) and some amino acid changes in the sequence of A. fertilissima which may be responsible for functioning of the chitosanase and the observed antifungal activity. Hydrolysis of the chitosan oligosaccharide (GlcN)5 to (GlcN)2 and (GlcN)3 confirmed the presence of chitosanase activity in A. fertilissima. Site-directed mutagenesis of the A. fertilissima chitosanase-encoding gene (cho) led to identification of catalytic residues (Glu-121 and Glu-141) important for the antifungal effect of the cho product. The level of expression of cho was monitored by quantitative real-time reverse transcription-PCR, which indicated that transcription of this gene is significantly enhanced under conditions that retard growth, such as a long dark period.

Comparison and suitability of gel matrix for entrapping higher content of enzymes for commercial applications

To check the suitability of enzyme entrapped beads for use in pharmaceutical industry, amylase enzyme was entrapped in agar/agarose, polyacrylamide gels and calcium alginate beads. Sodium alginate of 1% concentration was found to be best with respect to immobilization efficiency and calcium alginate beads so obtained were not much susceptible to breakage. When sodium alginate- amylase mixture was added from a height of about 20-30 cm. into CaCl2 solution, size of beads was large at higher alginate concentration due to the increase in the size of droplet formation before entering into CaCl2 solution. Enzyme entrapped polyacrylamide and agar/agarose gels were fragile and could not withstand repeated use whereas enzyme entrapped in large calcium alginate beads was used successfully for 50 cycles for the conversion of starch into product without much damage to the beads under stirring conditions. Amylase preparation was also mixed with urease, lysozyme and coimmobilized in large sized calcium alginate beads. These beads were used for 10 repeated cycles to check the conversion of substrates into their products by their respective enzymes and we concluded that an enzyme or mixture of two or three enzymes can be immobilized in the same large sized calcium alginate beads. This will save the additional cost of bioreactor, manpower, maintenance conditions required for the conversion of one drug into another using enzyme/s entrapped in large sized beads.

Xylanase production using agro-residue in solid-state fermentation from Bacillus pumilus ASH for biodelignification of wheat straw pulp

Two stage statistical design was used to optimize xylanase production from Bacillus pumilus ASH under solid-state fermentation. Initially, Plackett–Burman designing (PB) was used for the selection of crucial production parameters. Peptone, yeast extract, incubation time, moisture level and pH were found to be the crucial factors for the xylanase production. Crucial variables were further processed through central composite designing (CCD) of response surface methodology (RSM) to maximize the xylanase yield. Each significant factor was investigated at five different levels to study their influence on enzyme production. Statistical approach resulted in 2.19-fold increase in xylanase yield over conventional strategy. The determination coefficient (R2) as shown by analysis of variance (ANOVA) was 0.9992, which shows the adequate credibility of the model. Potential of cellulase-free xylanase was further investigated for biobleaching of wheat straw pulp. Xylanase aided bleaching through XCDED1D2 sequence resulted in 20 and 17% reduction in chlorine and chlorine dioxide consumption as compared to control. Significant increase in pulp brightness (%ISO), whiteness and improvement in various pulp properties was also observed.

Enrichment and isolation of endosulfan-degrading microorganism from tropical acid soil

Endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,3,4-benzo-dioxathiepin-3-oxide) is a cyclodiene organochlorine currently used as an insecticide all over the world and its residues are posing a serious environmental threat. This study reports the enrichment and isolation of a microbial culture capable of degrading endosulfan with minimal production of endosulfan sulfate, the toxic metabolite of endosulfan, from tropical acid soil. Enrichment was achieved by using the insecticide as sole sulfur source. The enriched microbial culture, SKL-1, later identified as Pseudomonas aeruginosa, degraded up to 50.25 and 69.77 % of α and β endosulfan, respectively in 20 days. Percentage of bioformation of endosulfan sulfate to total formation was 2.12% by the 20th day of incubation. Degradation of the insecticide was concomitant with bacterial growth reaching up to an optical density of 600 nm (OD600) 2.34 and aryl sulfatase activity of the broth reaching up to 23.93 μg pNP/mL/hr. The results of this study suggest that this novel strain is a valuable source of potent endosulfan–degrading enzymes for use in enzymatic bioremediation. Further, the increase in aryl sulfatase activity of the broth with the increase in degradation of endosulfan suggests the probable involvement of the enzyme in the transformation of endosulfan to its metabolites.