Biotransformation and Mutational Studies on Amidase enzyme of microbial isolate NIT 36

Abstract

newline ABSTRACT newlineMicroorganisms particularly bacteria are a unique source of a variety of enzymes. In this study, amidase (EC 3.5.1.4) producing microbes were screened from the soil of thermal sites such as Tattapani of Himachal Pradesh. The single isolate which showed maximum amidase activity was named as NIT-36. It was isolated from Tattapani hot water springs located in Mandi district of Himachal Pradesh. Though isolated from a thermal site this isolate does not show activity at high temperatures therefore it is categorized as a mesophile. Isolate NIT-36 was confirmed as Rhodococcus pyridinivornas NIT-36 on the basis of colony morphology, stained preparation, biochemical tests as well as 16S rRNA sequencing and showed 99% similarity to Rhodococcus pyridinivornas. Response Surface Methodology was used to generate a process model for obtaining optimal conditions for important parameters influencing amidase activity as well as acyl transfer activity of amidase enzyme. Further scale up studies for acetohydroxamic acid production was done by both batch and fed-batch methods. newlineThis study also incorporates mutational studies. A mutant was generated by deleting a few sequences of amidase enzyme. Wild type amidase and mutated amidase were cloned in E. coli and sequencing was performed. Both the wild and mutant strains show appreciable amidase activity towards a variety of substrates. The amidase gene from wild and mutated strain was cloned by using pET vector. The individual changes at the gene level were also recorded and a correlation was established between corresponding genotypic and phenotypic changes by employing various bioinformatics tools. The conserved residues were identified and comparative analysis with previously reported amidases was undertaken. Bioinformatic analysis revealed the unique composition of amidase protein of R. pyridinivorans NIT-36.