Heterologous Expression and Characterization of a High-Efficiency Chitosanase From Bacillus mojavensis SY1 Suitable for Production of Chitosan Oligosaccharides

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.

Overexpression and Biochemical Properties of a GH46 Chitosanase From Marine Streptomyces hygroscopicus R1 Suitable for Chitosan Oligosaccharides Preparation

“…The expression level of chitosanases from Mitsuaria sp., Streptomyces sp. N174, B. amyloliquefaciens, A. fumigatus CJ22-326, and Bacillus mojavensis SY1 (B. mojavensis SY1) are almost 1.6, 8.5, 4.5, 3.1, and 3.8 g/l, respectively (Peng et al, 2013;Ding et al, 2019;Luo et al, 2020;Zhou et al, 2020;Wang J. R. et al, 2021). Similar with previous works, the production of ShCsn46 was 3.91 g/l and the recombinant ShCsn46 was the main protein of supernatant (Figure 3B).…”

“…Meanwhile, the hydrolytic pattern of ShCsn46 is similar to many previous reported chitosanases. Most of the chitosanases from GH46 display endo-type property and without activity toward (GlcN) 2 and (GlcN) 3 (Jiang et al, 2012;Luo et al, 2020;Sun et al, 2020;Zhou et al, 2020;Wang J. R. et al, 2021). Furthermore, the hydrolytic process of (GlcN) 6 revealed that ShCsn46 may have two binding and cutting modes, which are "4 + 2" and "3 + 3" splitting mode, respectively (Figure 5F).…”

“…As a member of the GH75 family, the chitosanase from A. fumigatus CJ22-326 could hydrolyze 2% colloidal chitosan to produce COSs with different DP (2-6) and the hydrolysis rate is 97.29% after 8 h of reaction in the presence of 30 U/ml of this chitosanase (Zhou et al, 2020). As shown in Table 4, the GH46 family chitosanases BaCsn46A, Csn-SH, and CsnBm from B. amyloliquefaciens, B. atrophaeus BSS, and B. mojavensis SY1 exhibit higher efficiency toward colloidal than non-specific enzymes and chitosanase from GH8 and GH75 family (Qin et al, 2018;Cui et al, 2021;Wang J. R. et al, 2021). In this study, after 20 min of reaction in the presence of 10 U/ml ShCsn46, the hydrolysis rate of 4% colloidal chitosan reached 91.2%, which reveals that ShCsn46 has great potential and competitiveness for preparation of COSs.…”

“…Therefore, improvement of the production of Streptomyces chitosanases is a key factor for further industrial application. Heterelogous expression of chitosanases in Pichia pastoris is an effective way to enhance the production of target chitosanases (Peng et al, 2013;Ding et al, 2019;Luo et al, 2020;Wang J. R. et al, 2021). As a mature host, P. pastoris is widely used to produce recombinant protein due to its advantages such as extracellular secretion of recombinant protein, low secretion level of endogenous protein, high expression level of target recombinant protein, and mature fermentation process (Ahmad et al, 2014).…”

Overexpression of Bacillus circulans alkaline protease in Bacillus subtilis and its potential application for recovery of protein from soybean dregs

“…As a mature expression host, B. subtilis is widely used to produce a variety of enzymes and proteins due to its extracellular secretion of target recombinant protein (Song et al, 2015;Cui et al, 2018). Compared with E. coli and Pichia pastoris, B. subtilis exhibits several advantages such as ease of genetic manipulation, being classified as generally recognized as safe (GRAS) and easy cultivation, and short fermentation time (Gu et al, 2018;Wang et al, 2021b). Recently, several proteases are heterologously expressed in B. subtilis including alkaline and neutral proteases from Bacillus clausii TCCC1004, Geobacillus stearothermophilus (G. stearothermophilus), Bacillus licheniformis, and Idiomarina sp.…”