Engineering Carboxylic Acid Reductases Enzyme through Rational Protein Designing

Kulkarni, Adwait (2023) Engineering Carboxylic Acid Reductases Enzyme through Rational Protein Designing. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS dissertation of Adwait Kulkarni (18MS005)) Thesis_18MS005.pdf - Submitted Version Restricted to Repository staff only Download (2MB)

Abstract

Enzymes are biological catalysts that can carry out complex biochemical reactions in ambient temperature and solvent conditions. Traditional industrial catalytic processes have often had a severe impact on the environment. Biocatalysts have shown to be an excellent alternative to traditional catalysis and a promising solution to the problems related to sustainability. However, certain limiting factors for enzymatic catalysis, such as temperature sensitivity and solvent ionic strength, etc., prevent it from being widely adopted as the primary catalyst in the industry. These issues can be solved by employing different protein engineering strategies. By making targeted changes in the DNA sequence that encodes for the enzyme of interest, the amino acids in those positions and, consequently, properties of the enzymes can be altered. This work uses such protein engineering approaches on Carboxylic Acid Reductases, a class of enzymes that convert carboxylic acids into aldehydes. This project aims to make three essential mutations in the adenylation domain of a MmCAR (Mycobacterium marinum), which has been shown to improve enzyme activity. Then the PR di-domain - which is responsible for thermostability- of this mutated CAR will be swapped with that from an Ancient CAR (AncCAR-A) - which is constructed through Ancestral Sequence Reconstruction (ASR) - thus making the hybrid enzyme highly active while being thermostable at the same time.

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