Protein engineering is an important tool which helps in not only designing new enzymes but also enhances enzymatic functions or design proteins with more desirable functions. The project primarily focuses on mutating the engineered enzyme, diol dehydratase, obtained from Klebsiella oxytoca, ppdABC. This enzyme catalyzes the 1,2-diols to produce their respective aldehydes. Diol dehydratase has been previously been engineered to ppdA-C-B to promote catalysis. In order to eliminate the interaction of the substrate with undesired residues to prevent the formation of undesirable hydrogen bonds, the amino acids, primarily at the active site, of Ser202(Ala), Met373(Ala), and Ser149(Ala), were mutated. In order to test if the mutations did, indeed, help in enhancing the catalytic ability of the enzyme, 2,3-butanediol was used as the substrate with the engineered and mutated diol dehydratase. If the mutations had been successfully able to catalyze 2,3-butanediol, the product, 2-butanone, would have been detected. However, no quantity of 2-butanone was detected.
