Leifsonia sp.ZF2019中一种新型耐木糖β-木糖苷酶的表达与特征  

Expression and characterization of a novel xylose-tolerantβ-xylosidase from Leifsonia sp.ZF2019

作　　者：何易 焦镕虎 王晨曦 王茜[1] 许光治[1] HE Yi;JIAO Ronghu;WANG Chenxi;WANG Xi;XU Guangzhi(College of Food and Health,Zhejiang Agriculture and Forestry University,Lin’an 311300,Zhejiang,China)

机构地区：[1]浙江农林大学食品与健康学院,浙江临安311300

出　　处：《微生物学报》2022年第4期1452-1463,共12页Acta Microbiologica Sinica

基　　金：国家大学生创新创业训练计划(202010341004);浙江省自然科学基金(LY17C200019)。

摘　　要：【目的】从栀子灰蝶幼虫分离的Leifsonia sp.ZF2019菌株中克隆表达出一种新型β-木糖苷酶Xyl4900,并研究其酶学性质,以期为开发适用于工业生产的β-木糖苷酶提供参考。【方法】采用生物信息学分析技术分析Leifsonia sp.ZF2019菌株的β-木糖苷酶Xyl4900基因并在大肠杆菌中表达了该基因,纯化并研究了其酶学性质。【结果】Xyl4900与GH3家族的β-葡萄糖苷酶同源性高,但带有β-木糖苷酶结构域,可特异性水解对硝基苯基β-D-吡喃木糖苷(pNPX),是一种新型β-木糖苷酶。酶学特性分析显示,Xyl4900在45℃和pH 7.0的条件下酶活性最高,且在pH 6.0–9.0的范围内孵育14 h,仍保持80%以上的酶活力。除Cu^(2+)外,其他金属离子(2.5 mmol/L)对Xyl4900酶活力无明显影响,且对低浓度有机溶剂(5%V/V)有较强耐受性。此外,在20%(W/V)NaCl或100 mmol/L木糖溶液中Xyl4900的酶活性仍高于50%,表现出较好的盐和木糖耐受性。动力学参数分析显示,Xyl4900的K_(m)值、V_(max)值和木糖抑制常数K_(i)值分别为0.80 mmol/L、36.10 U/mg和150.12 mmol/L。特别地,Xyl4900能够很好地降解木二糖、木三糖。【结论】Xyl4900具有良好的pH稳定性、木糖及盐耐受性,能够较好地应用到半纤维素降解和其他工业生产中。[Objective]We cloned and expressed a novelβ-xylosidase Xyl4900 from Leifsonia sp.ZF2019 isolated from Artipe eryx larvae and then investigated its enzymatic properties,aiming to provide foundations for developingβ-xylosidase suitable for industrial application.[Methods]We used bioinformatics tools to analyze the gene of Xyl4900,expressed it in Escherichia coli,and investigated the enzymatic properties of the expressed protein.[Results]Xyl4900 had high homology with theβ-glucosidase of GH3 family while had a domain ofβ-xylosidase.It was a novelβ-xylosidase that could specifically hydrolyze 4-nitrophenylβ-D-xylopyranoside.Xyl4900 had the highest activity at 45℃and pH 7.0,and it still maintained more than 80%of activity after being incubated at pH 6.0–9.0 for 14 h.This enzyme was barely affected by other metal ions(2.5 mmol/L)except Cu^(2+) and had strong tolerance to low-concentration organic solvents(5%,V/V).In addition,the Xyl4900 in 20%(W/V)NaCl or 100 mmol/L xylose solution showed the activity higher than 50%,demonstrating good salt or xylose tolerance.The K_(m),V_(max),and xylose inhibition constant K_(i) of Xyl4900 were 0.80 mmol/L,36.10 U/mg,and 150.12 mmol/L,respectively.In particular,we found that Xyl4900 could well degrade xylobiose and xylotriose.[Conclusion]Xyl4900 showed good pH stability and tolerance to xylose and salt,and thus could be used in hemicellulose degradation and other industrial production practices.

关 键 词：Leifsonia sp.ZF2019 GH3糖苷水解酶 Β-木糖苷酶 盐耐受性 木糖耐受性 

分 类 号：TQ925[轻工技术与工程—发酵工程]