Characterization of recombinant endo-1,4-ß-xylanase of Bacillus halodurans C-125 and rational identification of hot spot amino acid residues responsible for enhancing thermostability by an in-silico approach.

Increased demand of enzymes for industrial use has led the scientists towards protein engineering techniques. In different protein engineering strategies, rational approach has emerged as the most efficient method utilizing bioinformatics tools to produce enzymes with desired reaction kinetics; physiochemical (temperature, pH, half life, etc) and biological (selectivity, specificity, etc.) characteristics. Xylanase is one of the widely used enzymes in paper and food industry to degrade xylan component present in plant pulp. In this study endo 1,4-ß-xylanase (Xyl-11A) from Bacillus halodurans C-125 was cloned in pET-22b (+) vector and expressed in Escherichia coli BL21 (DE3) expression strain. The enzyme had Michaelis constant Km of 1.32 mg ml-1 birchwoodxylan (soluble form) and maximum reaction velocity (Vmax) 73.53 mmol min-1 mg-1 with an optimum temperature of 75 °C and pH 9.0. The thermostability analysis showed that enzyme retained more than 80% of its residual activity when incubated at 75 °C for 2 h. In addition, to increase Xyl-11A thermostability, an in-silico analysis was performedto identify the hot spot amino acid residues. Consensus-based amino acid substitution was applied to evaluate multiple sequence alignment of homologs and identified 20 amino acids positions by following Jensen-Shnnon Divergence method. 3D models of 20 selected mutants were analyzed for conformational transition in protein structures by using NMSim server. Two selected mutants T6K and I17M of Xyl-11A retained 40, 60% residual activity respectively, at 85 °C for 120 min as compared to wild type enzyme which retained 37% initial activity under same conditions, confirming the enhanced thermostability of mutants. The present study showed a good approach for the identification of promising amino acid residues responsible for enhancing the thermostability of enzymes of industrial importance.

Prediction of epitopes of Neisseria Gonorrhoeae against USA human leukocyte antigen background: An immunoinformatic approach towards development of future vaccines for USA population.

Gonorrheal infections are the second most prevalent sexually transmitted diseases STDs in the USA populations after Chlamydia. These infections have now become an urgent problem to address because Neisseria gonorrhoeae is capable of developing resistance to multiple antibiotic classes. Infection with these antibiotic-resistant strains has become the major public health concern. Although extensive researches are ongoing to control its transmission and to develop the productive treatments against this pathogen, no effective vaccine could develop till now. The present study will effectively contribute to the future designing of USA specific epitope-based vaccines. Through computational approaches, this study has highlighted putative epitopes from Neisseria gonorrhoeae which restrict maximally to the frequent HLA alleles in the USA populations. Antigenic and non-allergenic nature of predicted epitopes was verified using vexijen and AllerTOP tools respectively. Total seven epitopes, four from class-I and three from class-II were antigenic as well as non-allergenic. These epitopes showed USA population coverage of 99.3% with no allergenic response. Still, additional studies are needed to validate the immunogenic properties of the predicted epitopes which are likely vaccine target for gonorrhoea in the USA populations.

Effects of black cumin seed (Nigella sativa L.) on performance and immune system in newly evolved crossbred laying hens.

BACKGROUND: A limited number of studies have tested the effect of diets supplemented with black cumin seed (BCS) on the performance of laying hen. OBJECTIVE: To determine the effects of various levels of dietary BCS on the performance of laying hen. BIRDS AND METHODS: Three hundred 40-week-old rural Leghorn (White Leghorn males X F1 females of Fayoumi male X Rhode Island Red female) crossbred hens were randomly assigned to four groups with five replicates of 15 birds each (75 laying hens per group), and fed diets supplemented with 0%, 3%, 4% or 5% BCS for 60 days. Statistical analysis was performed by means of ANOVA. RESULTS: BCS at all levels revealed greater egg production, egg weight and egg mass than the control group. Similarly, feed conversion was improved by 0.50, 0.55 and 0.36 points compared to controls with the addition of 3%, 4% or 5% levels of BCS, respectively. Haugh units and yolk weights of eggs from hens that were fed diets containing 3%, 4% and 5% BCS were significantly greater than those from the control group. Supplementation of 4% or 5% BCS significantly increased shell thickness and decreased serum LDL cholesterol and egg yolk cholesterol concentration as compared to other groups. Mean hemagglutination inhibition titers against Newcastle disease virus were higher than in controls. CONCLUSION: BCS at levels of 4% or 5% positively influenced egg production, egg weight and shell quality. Furthermore, it decreased the concentration of serum LDL cholesterol and egg yolk cholesterol concentrations and enhanced immunity against Newcastle disease virus.