Cloning, Expression and Characterisation of Newly Mined Ketoreductase in Komagataella phaffii

Optically active alcohol synthesis has been widely investigated, as they represent an important category of precursors for fine chemicals and pharmaceuticals. This study focused on the expression, purification, and characterization of the gene encoding 2,4-dienoyl-CoA reductase mined from the genome of Komagataella phaffii, here used as a ketoreductase. The impact of the promoter on the expression level of the secreted ketoreductase (kpKRED) was investigated. The kpKRED gene was inserted into an expression vector that had either an inducible alcohol oxidase 1 promoter or a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. The expressed kpKRED was purified by anion exchange chromatography and the specific activity was found to be 2902 U/mg. A catalytic efficiency K_cat/K_M of 3.80 was observed for ethyl acetoacetate, with K_M value of 4.68 for p-methoxy acetophenone. The purified kpKRED reduced the ketones with optimal activity at temperature 35°C and pH 6.5. Moreover, pure kpKRED exhibited broad substrate specificity towards ketones and ketoesters, with the maximum activity observed for ethyl acetoacetate. It was found that kpKRED, is capable of utilizing both NADH and NADPH as cofactors. In addition, purified kpKRED exhibits efficient solvent tolerance towards 5 organic solvents, such as ethyl alcohol and iso-propylalcohol. This enzyme retained catalytic activity in 10% organic solvents, indicating its strong resilience to organic solvents highlighting its robustness and versatility. This extensive investigation emphasizes the capability of kpKRED as a reliable and adaptable biocatalyst for industrial applications.