苏云金芽胞杆菌几丁质酶BtCHI1的原核表达与性质分析

doi:10.3969/j.issn.1674-7968.2022.01.014

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Abstract Chitinases (CHI) are a series of glycoside hydrolases (GHs) that break chitin into chitooligosaccharides ((GlcNAc)_n, 10≥n≥2) or N-acetylglucosamine (GlcNAc). In this study, a chitinase gene derived from Bacillus thuringensis, BtCHI1, was cloned and heterologously expressed in Escherichia coli BL21(DE3)plysS. The enzymatic properties, kinetic parameters, and substrate hydrolytic patterns recombinant enzyme BtCHI1 were characterized. The results showed that the molecular mass of BtCHI1 was approximate 75 kD. Its optimum temperature and pH were 40 ℃ and 7.0, respectively. The enzyme was unstable after heat-challenge at 50 ℃ for 1 h, retaining only 20.11% residual activity. However, the enzyme was considerably stable in broad pH buffers ranging from 3.0 to 10.0, retained over 80% residual activities after incubation for 1 h, respectively. Substrate kinetics assays indicated the recombinant BtCHI1 was active against chitin, colloidal chitin and chitosan, with V_max values of (4.62±0.46), (0.52±0.03) and (0.22±0.02) μmol/(min·mg), respectively. Nevertheless, the enzyme was inactive towards hydroxypropyl chitosan or glycol chitosan. Meanwhile, the enzyme was considerably resilient to 0.5~10 mmol/L Na⁺, Al³⁺ or EDTA (P>0.05). However, after treatment with 0.5~10 mmol/L Ca²⁺ or 5~10 mmol/L SDS for 1 h, the activities of BtCHI1 decline significantly (P<0.01). Thin-layer chromatography and high-performance liquid chromatography analysis revealed that BtCHI1 releases chitobiose from chitin and colloidal chitin, and further converted it into GlcNAc. Additionally, BtCHI1 (0.28 U) was observed to liberate (1.00±0.04) mg/mL and (1.71±0.11) mg/mL from natural shrimp and crab shell substrates. These results provide new insights into chitooligosaccharides/GlcNAc development and natural chitin substrate utilization.

Key words： Bacillus thuringensis Chitinase Enzymatic properties Hydrolytic products Shrimp and crab shell

Received: 07 April 2021

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Corresponding Authors: *Emirate14@zju.edu.cn;zhanghuien@zwu.edu.cn

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	WU Feng
	ZHOU Ye-Bo
	JIANG Lu-Xin
	SUN Xiao-Bao
	YIN Shang-Jun
	QIAN Guo-Ying
	WANG Jia-Kun
	WANG Zheng-Dong
	ZHANG Hui-En
	WANG Qian

Cite this article:

WU Feng,ZHOU Ye-Bo,JIANG Lu-Xin, et al. Prokaryotic Expression and Characterization Analysis of Chitinase BtCHI1 from Bacillus thuringensis[J]. 农业生物技术学报, 2022, 30(1): 151-163.

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http://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2022.01.014 OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2022/V30/I1/151

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