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农业生物技术学报  2022, Vol. 30 Issue (1): 151-163    DOI: 10.3969/j.issn.1674-7968.2022.01.014
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Prokaryotic Expression and Characterization Analysis of Chitinase BtCHI1 from Bacillus thuringensis
WU Feng1, ZHOU Ye-Bo1, JIANG Lu-Xin2, SUN Xiao-Bao3, YIN Shang-Jun1, QIAN Guo-Ying1, WANG Jia-Kun3, WANG Zheng-Dong4, ZHANG Hui-En1,*, WANG Qian1,3,*
1 College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China;
2 Hangzhou Centre for Agricultural Technology Extension, Hangzhou 310019, China;
3 College of Animal Sciences, Zhejiang University, Hangzhou 310058, China;
4 Ningbo Yutian Marine Biological Technology Co., LTD., Ningbo 315729, China
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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 Vmax 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 wordsBacillus thuringensis      Chitinase      Enzymatic properties      Hydrolytic products      Shrimp and crab shell     
Received: 07 April 2021     
ZTFLH:  S182  
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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