Muhammad Mohsin Javed

Detection of Four Novel Polymorphisms in PrP gene of Pakistani sheep (Damani and Hashtnagri) and goats (Kamori and Local Hairy) breeds

Scrapie is a fatal neurodegenerative disorder of sheep and goats caused by post-translational conformational change in the host-encoded prion protein (PrPC). Susceptibility or resistance to scrapie has been associated with the presence of polymorphisms in the prion protein (PrP) gene. In the present study, we analyzed the PrP gene sequence to determine the frequency of polymorphisms in 56 sheep (28 each from Damani and Hashtnagri breeds) and 56 goats (28 each from Kamori and Local Hairy breeds). A total of 7 amino acid polymorphisms were detected in the PrP gene for sheep and 4 for goats. These amino acid polymorphisms were combined in 13 alleles and 15 genotypes in sheep and 5 alleles and 6 genotypes in goats. The overall frequency of the most sheep scrapie-resistant polymorphism (Q171R) was calculated to be 0.107. The most scrapie-susceptible polymorphism (A136V) was not detected in any of the studied sheep. The overall frequency of scrapie-associated polymorphism (H143R) in goats was found to be 0.152. Along with already known amino acid polymorphisms, two novel polymorphisms were also detected for each of sheep (Q171N and T191I) and goats (G22C and P63L). However, the overall frequency of these polymorphisms was extremely low.

Cloning and expression of β-glucosidase gene from Bacillus licheniformis into E. coli BL 21 (DE3)

A 1.4 Kb fragment of Bacillus licheniformis ATCC 14580 encoding β-glucosidase was cloned and expressed in Escherichia coli. β-Glucosidase expressed by E. coli harboring cloned gene was located entirely in the intracellular fraction. Recombinant β-glucosidase protein was purified to homogeneity level and the molecular weight was found to be 53 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. It gave maximum activity at 50°C and pH 6. Km and Vmax were 0.206 mM and 1.26 U/mg, respectively, with p-nitrophenyl-β-D-glucopyranoside, while activation energy Ea, enthalpy of activation ?H and entropy of activation ΔS were found to be 66.31 kJ/mol, 64.04 kJ/mol and 48.28 J/mol/K, respectively. The pKa1 and pKa2 of the ionizable groups of active site residues involved in Vmax were found to be 5.5 and 7.0, respectively. When the recombinant β-glucosidase protein was used as a member of consortium with endoglucanase and exoglucanase for the saccharification of wheat straw, 123% increase in saccharification was observed.

Metabolic engineering and thermodynamic characterization of an extracellular β-glucosidase produced by Aspergillus niger

The production of an extracellular β-glucosidase by Aspergillus niger NRRL 599 was optimized using submerged fermentation technique. Effect of different media, different carbon sources, initial pH of the fermentation medium, temperature, incubation period and inoculum size on the production of β- glucosidase enzyme was investigated. A. niger NRRL 599 produced maximum extracellular β- glucosidase (4.48 U/mg) in Eggins and Pugh medium with 1% wheat bran (w/v) at pH 5.5 inoculated with 4% conidial suspension after 96 h of incubation at 30°C. Purified β-glucosidase gave K m and V max values of 3.11 mM and 20.83 U/mg respectively for pNPG hydrolysis. The enzyme was optimally active at pH 4.8 and at temperature of 60°C. Thermodynamic parameters, E a , ΔH and ΔS were found to be 52.17 KJ/mol, 49.90 J/mol.K and -71.69 KJ/mol, respectively. The pKa1 and pKa 2 of ionizable groups of active site residues involved in V max were calculated to be 4.1 and 6.0 respectively. Key words : β-Glucosidase, Aspergillus niger, kinetics, thermodynamics.

Biosorption of mercury from industrial effluent by fungal consortia

ABSTRACT Mercury is present in different types of industrial effluents that cause environmental pollution. Conventional methods such as precipitation, oxidation/reduction, ion exchange, filtration, membranes, and evaporation are extremely expensive or inefficient for the removal of mercury from diluted solutions. In this context, the biosorption process has recently been shown to be an effective and economical method. The present work describes the mercury biosorption ability of three fungi, i.e., Aspergillus niger, Trichoderma viride, and Humicola insolens. Monocultures of these strains and 10 different combinations were investigated. The consortium of 24-h-old H. insolens and 48-h-old of A. niger and T. viride in equal ratio was found to be compatible. This consortium decreased the residual mercury from 2.02 to 0.001 μ g/L after 7 days of incubation, and caused a significant reduction in chemical oxygen demand (COD) (92.6%) from an initial level of 21 mg/L.

Characterization Of A Novel Hydrolytic Enzyme Producing Thermophilic Bacterium Isolated From The Hot Spring Of Azad Kashmir-Pakistan

A thermophilic bacterium (TP-2) was isolated from the Tatta Pani hot spring in Azad Kashmir and was characterized using phenotypic and genotypic characters. The strain developed cream colored, round, smooth, flat and slimy colonies while the cells were Gram positive rods that ranged in size from about 2.1-3.6 μm to 0.2-0.3 μm in width. Sequence analysis of its 16S rRNA gene showed that isolate TP-2 had 89% homology with Geobacillus debilis. It grew within pH range of 5.5 to 8.5 with optimum growth at pH 7.0. The isolate showed optimum growth at 65oC and gave positive results for gelatin hydrolysis (GEL), ortho nitrophenyl-β-D-galactopyranosidase (ONPG), and nitrate production and produced acid from sucrose, glucose and maltose. It utilized glucose, fructose, maltose, lactose, sucrose, xylan, starch, filter paper and carboxymethylcellulose as sole carbon source. Isolate TP-2 produced significant amount of industrially important enzymes i.e. extracellular α-amylase, CMCase, FPase, Xylanase, Protease and Lipase and intracellular CMCase and FPase.

Optimization of Culture Conditions for the Production of Lovastatin by Aspergillus Terreus in Submerged Fermentation

The production of lovastatin by Aspergillus terreus PU-PCSIR-1 was studied using submerged fermentation technique. Effects of pH, incubation temperature, incubation time, and the media components on the production of lovastatin were investigated. Maximum lovastatin yield (198.90 mg/L) was achieved after 14 days of incubation at temperature 28°C and pH 7.4. Peptonized milk, linoleic acid, glucose, yeast extract, and trace elements had a positive impact on the production of lovastatin by A. terreus.