Promote the expression and corrected folding of an extremely stable N-demethylase by promoter reconstruction, native environment simulation and surface design

Thermomicrobiumroseumsarcosine oxidase (TrSOX) was a N-demethylasewith specific substrate chiral selectivity, outstanding thermostability and environmental resistance. To promote the expression of TrSOX in Bacillus subtilisW600, the HpaII promoter ofpMA5 plasmidwas replaced by constitutive or inducible promoters. Through orthogonal experiment, the expression process was optimized, B. subtilis W600 cells containing pMA5-PxyltrSOX plasmid were cultivated until OD600nm reached 2.0 and were then induced with 1.6% xylose at 37 degrees C for 2 h, and the native environment of T. roseum was simulated by heating at 80 degrees C, with the productivity of TrSOX increased from similar to 8.3 to similar to 66.7 mu g/g wet cells; and the simulated high temperature was the key switch for the final folding. To reduce the surface hydrophobicity, a S320R mutantwas built to forma hydrophilic lid around the entrance of the substrate pocket, and the yield of TrSOX (S320R) was similar to 163.0 mu g/gwet cells, approximately 20 folds as that in the initial expression system. This mutant revealed the similar secondary structure, stability, resistance, chiral substrate selectivity and optimal reaction environment with wild type TrSOX; however, the N-demethylation activities for amino acid derivative substrates were dramatically increased, while those for hydrophobic non-amino acid compounds were repressed. (c) 2021 Elsevier B.V. All rights reserved.