Objective: This study focused on improving the production of chitosan oligosaccharides by chitinase degradation of chitin. Methods: Using genetic engineering methods to clone, prokaryotic expressing, and investigating enzymatic properties of the chitinase from Streptomyces diastaticus, and the chitinase activity was explored by homology modeling, amino acid comparison of the catalytic domain, and site-directed mutagenesis. Results: After cloning and prokaryotic expression, the enzyme production cycle was shortened from 7 d to 24 h, and the enzyme activity reached 132 U/L, which was 32% higher than the original bacterial enzyme activity (100 U/L). The amino acids that determine the activity of chitinase was Asp at position 128, 130, and Glu at position 132 of the catalytic domain. Conclusion: Clonal expression of the chitinase gene resulted in a shorter enzyme production cycle and increased enzyme activity.

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