Abstract
Chitosanase plays an important role in enzymatic production of chitosan oligosaccharides (COSs). The present study describes the gene cloning and high-level expression of a high-efficiency chitosanase from Bacillus mojavensis SY1 (CsnBm). The gene encoding CsnBm was obtained by homologous cloning, ligated to pPICZαA, and transformed into Pichia pastoris X33. A recombinant strain designated X33-C3 with the highest activity was isolated from 120 recombinant colonies. The maximum activity and total protein concentration of recombinant strain X33-C3 were 6,052 U/ml and 3.75 g/l, respectively, which were obtained in fed-batch cultivation in a 50-l bioreactor. The optimal temperature and pH of purified CsnBm were 55°C and 5.5, respectively. Meanwhile, CsnBm was stable from pH 4.0 to 9.0 and 40 to 55°C. The purified CsnBm exhibited high activity toward colloidal chitosan with degrees of deacetylation from 85 to 95%. Furthermore, CsnBm exhibited high efficiency to hydrolyze different concentration of colloidal chitosan to produce COSs. The result of this study not only identifies a high-efficiency chitosanase for preparation of COSs, but also casts some insight into the high-level production of chitosanase in heterologous systems.
Highlights
As the second abundant polysaccharide on earth, chitin is composed of N-acetylglucosamine linked by β-1,4 glycoside bonds, and the lack of solubility limits its industrial application (Kaczmarek et al, 2019; Schmitz et al, 2019)
Sequence analysis showed that the open reading frame of chitosanase from Bacillus mojavensis SY1 (CsnBm) was 837 bp (Accession No, OK172330), which encoded 278 amino acid residues
The results of National Center for Biotechnology Information (NCBI) blastp revealed that CsnBm shared 98.1% identity to chitosanase from B. mojavensis UCMB5075 (Accession No WP_168747460.1), followed by chitosanase Bacillus halotolerans (96.3% identity, Accession No WP_059336147.1)
Summary
As the second abundant polysaccharide on earth, chitin is composed of N-acetylglucosamine linked by β-1,4 glycoside bonds, and the lack of solubility limits its industrial application (Kaczmarek et al, 2019; Schmitz et al, 2019). High Efficiency Chitosanase chitin or chitosan, COSs have many advantages such as excellent water solubility, low viscosity, and biodegradability (Li et al, 2016; Yuan et al, 2019). The preparation methods of COSs mainly include chemical, physical, and enzymatic method. Among these three methods, the enzymatic production of COSs has received more attention due to its environmental friendliness, high identification of hydrolysis sites, non-toxicity, and controllability (Yuan et al, 2019). The high production cost limits the enzymatic method as the main choice for industrialscale preparation of COSs (Yuan et al, 2019).
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