Attenuated substrate inhibition of a haloketone reductase via structure-guided loop engineering.

Substrate inhibition of enzymes is one of the main obstacles encountered frequently in industrial biocatalysis. Haloketone reductase SsCR was seriously inhibited by substrate 2,2',4'-trichloroacetophenone. In this study, two essential loops were found that have a relationship with substrate binding by conducting X-ray crystal structure analysis. Three key residues were selected from the tips of the loops and substituted with amino acids with lower hydrophobicity to weaken the hydrophobic interactions that bridge the two loops, resulting in a remarkable reduction of substrate inhibition. Among these variants, L211H showed a significant attenuation of substrate inhibition, with a Ki of 16 mM, which was 16 times that of the native enzyme. The kinetic parameter kcat/Km of L211H was 3.1 × 103 s-1 mM-1, showing the comparable catalytic efficiency to that of the wild-type enzyme (WT). At the substrate loading of 100 mM, the space time yield of variant L211H in asymmetric reduction of the haloketone was 3-fold higher than that of the WT.

Enhancing transglutaminase production of Streptomyces mobaraensis by iterative mutagenesis breeding with atmospheric and room-temperature plasma (ARTP).

OBJECTIVES: To improve the fermentation production of transglutaminase (TGase) from Streptomyces mobaraensis for applications in the food industry, the atmospheric and room-temperature plasma (ARTP) mutagenesis was applied to breed S. mobaraensis mutants with increased TGase production. RESULTS: After eight rounds of iterative ARTP mutagenesis, four genetically stable mutants, Sm5-V1, Sm6-V13, Sm2-V10, and Sm7-V12, were identified, which showed increased TGase production by 27, 24, 24, and 19%, respectively. The best mutant Sm5-V1 exhibited a maximum TGase activity of 5.85 U/mL during flask fermentation. Compared to the wild-type strain, the transcription levels of the zymogen TGase genes in the mutants increased significantly as indicated by quantitative real-time PCR, while the gene nucleotide sequences of the mutants did not change at all. It was shown that the overexpression of TGase zymogen gene in the mutants contributes to the increase in TGase production. CONCLUSIONS: ARTP is a potentially efficient tool for microbial mutation breeding to bring some significant changes required for the industrial applications.

Identification of key residues in Debaryomyces hansenii carbonyl reductase for highly productive preparation of (S)-aryl halohydrins.

Key residues of Debaryomyces hansenii carbonyl reductase in the determination of the reducing activity towards aryl haloketones were identified through combinatorial mutation of conserved residues. This study provides a green and efficient biocatalyst for the synthesis of (S)-aryl halohydrins.