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

Abstract

Due to the various biological activities of chitosan oligosaccharides (COSs), they have great potential value for use in many areas. Chitosanase plays an important role in enzymatic preparation of COSs. Herein, a gene encoding a chitosanase (ShCsn46) from marine Streptomyces hygroscopicus R1 was cloned and the sequences encoding ShCsn46 without signal peptide were optimized based on the codon usage of Pichia pastoris (P. pastoris). In addition, the optimized gene was ligated to pPICZαA and transformed to P. pastoris X33. After screening, a recombinant strain named X33-Sh33 with the highest activity was isolated from 96 recombinant colonies. The maximum activity and total protein concentration of the recombinant strain ShCsn46 were 2250 U/ml and 3.98 g/l, respectively. The optimal pH and temperature of purified ShCsn46 were 5.5 and 55°C, respectively. Meanwhile, ShCsn46 was stable from pH 5.0 to 10.0 and 40 to 55°C, respectively. The purified ShCsn46 was activated by Mn2+ and inhibited by Cu2+, Fe2+, and Al3+. In addition, substrate specificity of the purified ShCsn46 showed highest activity toward colloidal chitosan with 95% degree of deacetylation. Furthermore, the purified ShCsn46 exhibited high efficiency to hydrolyze 4% colloidal chitosan to prepare COSs. COSs with degree of polymerization of 2–6, 2–5, and 2–4 were controllably produced by adjusting the reaction time. This study provides an excellent chitosanase for the controllable preparation of COSs with a desirable degree of polymerization.

Controllable preparation of chitosan oligosaccharides via a recombinant chitosanase from marine Streptomyces lydicus S1 and its potential application on preservation of pre-packaged tofu

“…According to the previous studies, we found that the pH stability of chitosanases from GH46 family varies greatly in accordance with their sources. The chitosanases from S. avermitilis , B. mojavensis SY1, and S. hygroscopicus R1 were stable from pH 4.0 to 8.0 ( Guo et al, 2021 ; Wang et al, 2021a , b ). Additionally, the residual activity of chitosanases from Bacillus sp.…”

“…For instance, the cold adaptive chitosanase from G. sunshinyii is only stable at the temperature from 5 to 35°C ( Qin et al, 2018a ). In addition, the chitosanases from S. hygroscopicus R1and B. amyloliquefaciens are mesophilic enzyme, which are stable at the temperature in the range from 35 to 55°C ( Luo et al, 2020 ; Wang et al, 2021b ). However, the thermal stable chitosanase from Streptomyces roseolus displays higher residual activity even though the incubating temperature reached 65°C ( Jiang et al, 2012 ).…”

“…As the value of OD 600 reached 0.6, 1 mM Isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to culture solution and cultivated at 16°C and 200 rpm for 2 h. Finally, the recombinant strains was collected by centrifuging at 8,000 × g and 6°C for 10 min and disrupted by sonication. The method for purification of recombinant SlCsn46 was similar to previous studies ( Qin et al, 2018b ; Wang et al, 2021b ) using Ni-IDA sefinose resin chromatography (Sangon Biotech, Shanghai, China). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analysis the purified recombinant SlCsn46.…”

“…It has been reported that the reaction time for the chitosanase CsnBm from Bacillus mojavensis SY1 decomposes 92.3% of 4% colloidal chitosan into COSs with DP from 2 to 6 is only 15 min ( Wang et al, 2021a ). Similarly, 91.2% of 4% colloidal chitosan could be hydrolyzed and converted into COSs at 20 min by a GH46 chitosanase from Streptomyces hygroscopicus R1 ( Wang et al, 2021b ). According to the previous studies, the chitosanases from GH46 are mainly from bacteria.…”

“…Among different bacteria, the genus Streptomyces is an important producer for preparation of different chitosanases, some of which have been characterized such as ShCsn46, SaCsn46A, Sn1-CSN, CsnN174, and Csn21c from S. hygroscopicus R1, Streptomyces avermitilis , Streptomyces niveus , Streptomyces sp. N174, and Streptomyces albolongus ATCC 27414, respectively ( Guo et al, 2019 , 2021 ; Ding et al, 2020 ; Chen et al, 2021 ; Wang et al, 2021b ). However, there are few reports about the bioinformatics analysis, heterologous expression, and characterization of chitosanase from Streptomyces lydicus .…”