Heterologous Expression and Characterization Analysis of a High-Efficiency Chitosanase from Bacillus halotolerans R1

Abstract:

The present study employed homologous cloning to obtain the chitosanase gene from Bacillus subtilis, and prepared recombinant Bacillus subtilis chitosanase(CsnBh46) through heterologous expression. The recombinant CsnBh46 enzyme was characterized to determine its enzymatic properties. The full-length recombinant CsnBh46 consists of 278 amino acids, and its three-dimensional structure reveals it is divided into upper and lower domains, each containing nine α-helices and two β-strands. The maximum enzymatic activity of recombinant CsnBh46 expressed in Pichia pastoris X33 was 6 375 U/mL, with a total protein mass concentration of 4.3 g/L. The optimal reaction pH and temperature for purified recombinant CsnBh46 were 6.0 and 60 ℃, respectively. Meanwhile, CsnBh46 was activated by Mn²⁺, Ca2+ ^{and Mg2+. The favorite substrate for recombinant CsnBh46 was colloidal chitosan with 95% degree of deacetylation. Furthermore, recombinant CsnBh46 efficiently hydrolyzed colloidal chitosan and allowed for the targeted preparation of chitosan oligosaccharides with different degrees of polymerization based on reaction time. This study provides a foundation for the industrial application of CsnBh46.}