Engineering of L-glutamate oxidase as the whole-cell biocatalyst for the improvement of α-ketoglutarate production

In this study, error-prone PCR mutagenesis of Streptomyces mobaraensis LGOX followed by high-throughput screening was performed to yield four single point mutants with improved enzymatic activity, termed F94L, S280T, I282M and H533R. Moreover, site-saturation mutagenesis at these four residues was employed, yielding two additionally improved mutants, termed I282L and H533L. Subsequently, we employed combinatorial mutagenesis of two, three and four point mutants, and the best mutant S280TH533L showed 90% higher enzymatic activity than the wild-type control. The data also showed that the presence of these point mutations greatly enhanced enzymatic activity, but did not alter its optimum temperature and pH. Furthermore, the S280TH533L mutant had the maximal velocity (Vmax) of 231.3 μmol/mg/min and the Michaelis-Menten constant (KM) of 2.7 mM, which were the highest Vmax and lowest KM values of LGOX reported so far. Finally, we developed a whole-cell biocatalyst for α-KG production by co-expression of both S280TH533L mutant and KatE catalase. Randomized ribosome binding site (RBS) sequences were introduced to generate vectors with varying expression levels of S280TH533L and KatE, and two optimized co-expression strains were obtained after screening. The α-KG production reached a maximum titer of 181.9 g/L after 12 h conversation using the optimized whole-cell biocatalyst, with a molar conversion rate of substrate higher than 86.3% in the absence of exogenous catalase, while th...

Source: Enzyme and Microbial Technology - February 1, 2020 Category: Biotechnology Source Type: research