Kalibulla Syed Ibrahim

Methods for Microbiome Analysis

Metagenomics is gaining importance as an invaluable tool as it attempts to determine directly the whole collection of genes and analyze from microbes in a particular environment where they interact with each other by exchanging nutrients, metabolites, and signaling molecules. The development of affordable next-generation sequencers has led to democratization of sequencing, but their ever-growing throughput is making data analysis increasingly complex. This has introduced a plethora of challenges with respect to design of experiments, bioinformatics, and downstream processing. This chapter aims to provide an overview of the currently available methodologies and tools for performing every individual step of a typical metagenomic data set analysis and expected to serve as a useful resource for microbial ecologists and bioinformaticians.

Molecular Cloning, Sequence and Structural Analysis of Dehairing Mn2+ Dependent Alkaline Serine Protease (MASPT) of Bacillus pumilus TMS55

Leather industries release a large amount of pollution-causing chemicals which creates one of the major industrial pollutions. The development of enzyme based processes as a potent alternative to pollution-causing chemicals is useful to overcome this issue. Proteases are enzymes which have extensive applications in leather processing and in several bioremediation processes due to their high alkaline protease activity and dehairing efficacy. In the present study, we report cloning, characterization of a Mn2+ dependent alkaline serine protease gene (MASPT) of Bacillus pumilus TMS55. The gene encoding the protease from B. pumilus TMS55 was cloned and its nucleotide sequence was determined. This gene has an open reading frame (ORF) of 1,149 bp that encodes a polypeptide of 383 amino acid residues. Our analysis showed that this polypeptide is composed of 29 residues N-terminal signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids. We performed bioinformatics analysis to compare MASPT enzyme with other proteases. Homology modeling was employed to model three dimensional structure for MASPT. Structural analysis showed that MASPT structure is composed of nine α-helices and nine β-strands. It has 3 catalytic residues and 14 metal binding residues. Docking analysis showed that residues S223, A260, N263, T328 and S329 interact with Mn2+. This study allows initial inferences about the structure of the protease and will allow the rational design of its derivatives for structure-function studies and also for further improvement of the enzyme.

Protein–Ligand Interactions

Protein–ligand docking is a structural biology tool that predicts the possible binding modes of a ligand with protein.

DNA Marker Analysis

Genetic markers are polymorphic genetic sequences, like RFLPs or microsatellites, that differ within chromosomal alleles. Rather than analysing the sequence directly, this gene is inferred through analysis of a genetic marker. Marker analysis approach is quite helpful in population biology and ecology studies that can be trace patterns in populations like plants, animals, humans, etc.

Protein Sequence Analysis

ExPASy (Expert Protein Analysis System) is a Bioinformatics Resource Portal from Swiss Institute of Bioinformatics that offers Bioinformatics support like accessing scientific databases and software tools for the research in life sciences.

Protein Structure Analysis

Amino acids have been grouped into different categories like polar, nonpolar, acidic, basic, large, small, aliphatic and aromatic, and each group has its own functional features (Table 5.1).

AP-APSE dpol intein: A novel family A DNA polymerase intein domain.

Inteins are “protein introns” that remove themselves from their host proteins through an autocatalytic protein-splicing. After their discovery, inteins have been quickly identified in organisms from all three kingdoms of life - eucarya, bacteria and archaea, but their distribution is sporadic. Here we report the identification and bioinformatics characterization of intein in DNA polymerase A gene of bacteriophage APSE (Acyrthosiphon pisum Secondary Endosymbiont bacteriophage) infecting the Aphid secondary endosymbionts of eukaryotic insects such as Acyrthosiphon pisum, Uroleucon rudbeckiae. The insertion site of intein within APSE family A DNA polymerase extein was identified to be dpola. Hence we propose this as a unique intein of family A DNA polymerase (dpola insertion site) and only reported intein in podoviridae family.