CLONING, EXPRESSION, PURIFICATION AND BIOINFORMATICS ANALYSIS OF ARSENITE OXIDASE (homologue) FROM Comamonas testosteroni KF1

Abstract

Arsenic pollution in water is a major problem that has been observed in many underdeveloped nations across the world. Arsenic (As) contamination has recently drawn major attention to the environmental destiny and behavior of As in Southeast Asian nations including Bangladesh, India, China, Chile, and Japan. Water contamination with As is brought on by a number of natural and anthropogenic processes, both direct and indirect. Because it causes As poisoning in humans, arsenic-contaminated water poses a serious hazard to humanity. Therefore, it is essential to handle polluted water as such and provide clean water to the public at this time. Different approaches have been suggested for treating As-contaminated water. Bioremediation is one such strategy to reduce environmental contamination. Despite the fact that heavy metals kill microorganisms, some bacteria, such as arsenic-tolerant bacteria, are resistant to heavy metals. Bacteria limit the toxicity of arsenite and arsenate by either reducing ingestion, actively exporting the arsenicals, or by chemically altering them to produce versions that are relatively less dangerous. As (V) is a hundred times less poisonous than As (III), as is well known. As a result, the arsenic bioremediation techniques involve a two-step process: first, As (III) is biologically oxidized by arsenite oxidizing bacterial strains, and subsequently the As (V) that results is eliminated. It has been documented that a broad variety of bacteria isolated from diverse polluted environments had the capacity to manufacture Arsenite oxidase, that then catalyzes different enzymatic oxidations of As3+ to As5+. It has been discovered that one of the Alcaligenes faecalis bacterial strains has the enzyme Arsenite Oxidase. This Arsenite Oxidase enzyme underwent sequence alignment analysis as a result of which As3+ to As5+ enzymatic oxidation has been described. It has been discovered that one of the Alcaligenes faecalis bacterial strains has the enzyme Arsenite Oxidase. We used Uniprot to perform sequence alignment analysis on the Arsenite vi Oxidase enzyme sequence of A. faecalis against the bacterial strains present in the Macromolecular Characterization Unit Laboratory, Dept. of Biosciences and Bioengineering, IIT Roorkee, and we discovered homologous protein sequences in the various strains. In the project a gene which was found to be homologous to arsenite oxidase was amplified from Comamonas testosteroni KF1 bacterial strains and clones into cloning vectors E. coli DH5α. Successful cloning was obtained in E. coli DH5α host cells. However, protein of 104 kDa could not be confirmed by SDS PAGE analysis and hence, the protein may not have purified due to various reasons. 3D structure of arsenite oxidase was predicted from AlphaFol2 and analyzed on Pymol.