Identification of deleterious single nucleotide polymorphism (SNP)s in the human TBX5 gene & prediction of their structural & functional consequences: An in silico approach.

T-box transcription factor 5 gene (TBX5) encodes the transcription factor TBX5, which plays a crucial role in the development of heart and upper limbs. Damaging single nucleotide variants in this gene alter the protein structure, disturb the functions of TBX5, and ultimately cause Holt-Oram Syndrome (HOS). By analyzing the available single nucleotide polymorphism information in the dbSNP database, this study was designed to identify the most deleterious TBX5 SNPs through in silico approaches and predict their structural and functional consequences. Fifty-eight missense substitutions were found damaging by sequence homology-based tools: SIFT and PROVEAN, and structure homology-based tool PolyPhen-2. Various disease association meta-predictors further scrutinized these SNPs. Additionally, conservation profile of the amino acid residues, their surface accessibility, stability, and structural integrity of the native protein upon mutations were assessed. From these analyses, finally 5 SNPs were detected as the most damaging ones: [rs1565941579 (P85S), rs1269970792 (W121R), rs772248871 (V153D), rs769113870 (E208D), and rs1318021626 (I222N)]. Analyses of stop-lost, nonsense, UTR, and splice site SNPs were also conducted. Through integrative bioinformatics analyses, this study has identified the SNPs that are deleterious to the TBX5 protein structure and have the potential to cause HOS. Further wet-lab experiments can validate these findings.

Optimization and partial characterization of culture conditions for the production of alkaline protease from Bacillus licheniformis P003.

Proteolytic enzymes have occupied a pivotal position for their practical applications. The present study was carried out under shake flask conditions for the production of alkaline protease from Bacillus licheniformis P003 in basal medium containing glucose, peptone, K2HPO4, MgSO4 and Na2CO3 at pH 10. The effect of culture conditions and medium components for maximum production of alkaline protease was investigated using one factor constant at a time method along with its characterization. Maximum level of enzyme production was obtained after 48h of incubation with 2% inoculum size at 42°C, under continuous agitation at 150 rpm, in growth medium of pH 9. Highest enzyme production was obtained using 1% rice flour as carbon source and 0.8% beef extract as organic nitrogen source. Results indicated that single organic nitrogen source alone was more suitable than using in combinations and there was no significant positive effect of adding inorganic nitrogen sources in basal medium. After optimization of the parameters, enzyme production was increased about 20 fold than that of in basal medium. The crude enzyme was highly active at pH 10 and stable from pH 7-11. The enzyme showed highest activity (100%) at 50°C, and retained 78% relative activity at 70°C. Stability studies showed that the enzyme retained 75% of its initial activity after heating at 60°C for 1h. The enzyme retained about 66% and 46% of its initial activity after 28 days of storage at 4°C and room temperature (25°C) respectively. Mn(2+) and Mg(2+) increased the residual activity of the enzyme, whereas Fe(2+) moderately inhibited its residual activity. When pre-incubated with Tween-20, Tween-80, SDS and H2O2, each at 0.5% concentration, the enzyme showed increased residual activity. These characteristics may make the enzyme suitable for several industrial applications, especially in leather industries.

Production and partial characterization of extracellular amylase enzyme from Bacillus amyloliquefaciens P-001.

Amylases are one of the most important enzymes in present-day biotechnology. The present study was concerned with the production and partial characterization of extracellular amylase from Bacillus amyloliquefaciens P-001. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Enzyme production was induced by a variety of starchy substrate but corn flour was found to be a suitable natural source for maximum production. Tryptone and ammonium nitrate (0.2%) as nitrogen sources gave higher yield compared to other nitrogen sources. Maximum enzyme production was obtained after 48 hrs of incubation in a fermentation medium with initial pH 9.0 at 42°C under continuous agitation at 150 rpm. The size of inoculum was also optimized which was found to be 1% (v/v). Enzyme production was 2.43 times higher after optimizing the production conditions as compared to the basal media. Studies on crude amylase revealed that optimum pH, temperature and reaction time of enzyme activity was 6.5, 60°C and 40 minutes respectively. About 73% of the activity retained after heating the crude enzyme solution at 50°C for 30 min. The enzyme was activated by Ca(2+) (relative activity 146.25%). It was strongly inhibited by Mn(2+), Zn(2+) and Cu(2+), but less affected by Mg(2+) and Fe(2+).