蜡样芽孢杆菌W-3木聚糖酶xynA基因原核表达及其酶学性质研究

doi:10.16431/j.cnki.1671-7236.2023.10.025

Abstract

Abstract: 【Objective】 The objective of this study was to clone the xynA gene of Bacillus cereus W-3 and analyze its bioinformatically.The allogeneic expression and enzymatic characteristics were studied.【Method】 xynA gene was cloned by homologous amplification,prokaryotic expression vector pCola-xynA was constructed,and heterologous expression was performed using Escherichia coli BL21 (DE3).The recombinant protein was isolated and purified by nickel column affinity chromatography and identified by SDS-PAGE. The bioinformatic analysis of xynA protein was carried out by online software.The enzymatic properties of xynA at different temperature and pH were studied using beech xylanan as substrate.【Result】 xynA gene was 642 bp in length and contained a complete open reading frame,encoding 213 amino acids.Amino acid sequence comparison showed that the similarity between xynA and Bacillus methylotrophicus subspecies FZB42 xylanase (AJD80562.1) was 96%,the similarity of NBRC15307 xylanase with Paenibacillus macerans (AAZ17386.1) and Bacillus altitudinis with the family xylanase (WP-007407578.1) was 94%.The N-terminal of xynA protein contained a signal peptide with a theoretical isoelectric point of 9.42 and a molecular weight of 23.3 ku.The protein was a hydrophilic protein with stable structure,5 potential glycosylation sites and 38 phosphorylation sites,and belongs to the glycoside hydrolase 11 family.The enzyme activity of xynA was 733.826 U/mg,the optimum temperature was 70 ℃,and the optimum pH was 9.0.It had good stability at 60-80 ℃ and pH 8.0-11.0,50.3% enzyme activity could be retained at 80 ℃ for 1 h,and 94.63% enzyme activity could be retained at 70 ℃ and pH 9.0 for 1 h.【Conclusion】 Xylanase xynA gene was successfully cloned from the natural microorganism Bacillus cereus W-3 strain.xynA protein was a stable hydrophilic protein,belonging to the glycoside hydrolase 11 family.xynA showed the characteristics of heat resistance,alkali resistance,high enzyme activity,and wide adaptability to temperature and pH.The results laid a theoretical foundation for the development and utilization of xynA.

Key words: Bacillus cereus; xylanase; prokaryotic expression; bioinformatics; enzymatic properties

CLC Number:

Q814.9

YAN Lihuan, XIAO Yao, ZHAO Jian, ZHAO Jinpeng, XUE Zhiquan, JING Xiaoyuan, FENG Yan. Study on Prokaryotic Expression and Enzymatic Properties of Xylanase xynA Gene in Bacillus cereus W-3[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(10): 4114-4124.

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Study on Prokaryotic Expression and Enzymatic Properties of Xylanase xynA Gene in Bacillus cereus W-3

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