Objective: To improve enzyme activity of Glucosamine-6-phosphate synthase (Glms) of Bacillus subtilis by site saturation mutagenesis at Leu593, Ala594, Lys595, Ser596 and Val597 based on computer-aided semi-rational design.
Results: The results indicated that L593S had the greatest effect on the activity of BsGlms and the enzyme activity increased from 5 to 48 U/mL. The mutation of L593S increased the yield of glucosamine by 1.6 times that of the original strain. The binding energy of the mutant with substrate was reduced from - 743.864 to - 768.246 kcal/mol. Molecular dynamics simulation results showed that Ser593 enhanced the flexibility of the protein, which ultimately led to increased enzyme activity.
Conclusion: We successfully improved BsGlms activity through computer simulation and site saturation mutagenesis. This combination of methodologies may fit into an efficient workflow for improving Glms and other proteins activity.
Keywords: Computer simulation; Enzyme activity; Glucosamine-6-phosphate synthase; Molecular dynamics simulation; Site saturation mutagenesis.