Significant enhancement of the thermal stability and catalytic efficiency of transglutaminase in Streptomyces mobaraensis engineered through the novel S. mobaraensis genomic mutant library construction method GHR/Sml

Abstract

Herein, we developed a novel Streptomyces mobaraensis genomic mutant library construction method, GHR/Sml, to directly and significantly enhance the thermal stability and catalytic efficiency of TGase in the genome of S. mobaraensis. First, 13 key amino acid residues and their mutations for enhanced thermal stability were identified using error-prone PCR and site-directed mutagenesis. Then, the GHR/Sml method was developed to construct a TGase genomic mutant library with 13 mutations. Positive mutants S23Y/Y24N/S250R, S23Y/Y24N/S303K, S23Y/Y24N/K294L, S23Y/Y24N/S199A/R208L, S23Y/Y24N, and S250R were obtained from 1500 total mutants; their half-life values at 50 °C were increased by 9.3-, 9.5-, 8.7-, 9.0-, 6.9-, and 4.8-fold compared with that of TG_LD, respectively. Furthermore, the k_cat/K_m of mutant S23Y/Y24N/S250R increased by 1.25-fold over that of TG_LD. The activity of S23Y/Y24N/S250R reached 65.34 U/mL in a 1000-L fermenter, which was the highest activity reported. This novel GHR/Sml method is of great significance for systematically improving properties of additional enzymes in the genome of S. mobaraensis.