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-tolerant β-xylosidase from Leifsonia sp.ZF2019

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Cu²⁺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。

Abstract:

[Objective] We cloned and expressed a novel β-xylosidase Xyl4900 from Leifsonia sp.ZF2019 isolated from Artipe eryx larvae and then investigated its enzymatic properties,aiming to provide foundations for developing β-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 the β-glucosidase of GH3 family while had a domain of β-xylosidase.It was a novel β-xylosidase that could specifically hydrolyze 4-nitrophenyl β-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 Cu²⁺ 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.