Piwu Li1,2, Kang Li2, Xu Li2, Fei Zhao2, Ruiming Wang1,2, Junqing Wang3,4. 1. State Key Laboratory of Biobased Material and Green Papermaking (LBMP) (Qilu University of Technology), Jinan, 250353, Shandong, People's Republic of China. 2. Key Laboratory of Shandong Microbial Engineering, QILU University of Technology (Shandong Academy of Sciences), Jinan, 250353, Shandong, People's Republic of China. 3. State Key Laboratory of Biobased Material and Green Papermaking (LBMP) (Qilu University of Technology), Jinan, 250353, Shandong, People's Republic of China. wjqtt.6082@163.com. 4. Key Laboratory of Shandong Microbial Engineering, QILU University of Technology (Shandong Academy of Sciences), Jinan, 250353, Shandong, People's Republic of China. wjqtt.6082@163.com.
Abstract
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.
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.
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