一种瘤胃木聚糖酶的酶学特性分析及其对农作物秸秆的水解作用

Enzymatic properties and straw-degrading effect of a xylanase derived from rumen

摘要:

【目的】研究重组木聚糖酶rRuXyn024的酶学特性及其对农作物秸秆的水解作用。【方法】从肉牛瘤胃中克隆RuXyn024基因，运用生物信息学工具对该基因序列进行详细分析；构建表达载体pET-RuXyn024，并将其转化入大肠杆菌BL21(DE3)菌株中，实现RuXyn024的异源表达；对rRuXyn024的酶学特性进行深入分析，并研究其对农作物秸秆的降解能力。【结果】RuXyn024编码358个氨基酸，大小约为40 kDa，属于GH 10家族。rRuXyn024的最适pH和温度分别为7.0和40℃，在pH 6.0−9.0和30−70℃下能分别保持最大活性的60%和70%以上。以麦秸木聚糖为底物时，rRuXyn024的K_m值和V_max值分别为18.8 g/L和82.6µg/min。1 mmol/L和5 mmol/L的Mg²⁺、Zn²⁺、Cu²⁺、Ni²⁺、EDTA和SDS，5 mmol/L的Ca²⁺和β-巯基乙醇对rRuXyn024具有明显的抑制作用，其中5 mmol/L的Cu²⁺、β-巯基乙醇使rRuXyn024接近失活。Mn²⁺对rRuXyn024具有明显促进作用，1 mmol/L、5 mmol/L的Mn²⁺使rRuXyn024的活性分别提高了46.9%和35.8%。rRuXyn024水解麦秸木聚糖产生了以木三糖和木二糖为主的寡糖产物。rRuXyn024能够有效水解玉米秸、稻秸、豆秸和油菜秸，其中对玉米秸的水解作用最强。【结论】rRuXyn024具有宽泛的pH和温度适应性，在改善农作物秸秆的反刍动物利用方面具有较大的应用潜力。

Abstract:

[Objective] To investigate the enzymatic properties and straw-degrading effect of a recombinant xylanase rRuXyn024.[Methods] We cloned RuXyn024 from the rumen of beef cattle and used bioinformatics tools for detailed sequence analysis.The expression vector pET-RuXyn024 was constructed and transformed into Escherichia coli BL21(DE3) for heterologous expression of RuXyn024.Furthermore,the enzymatic properties and straw-degrading effect of rRuXyn024 were examined.[Results] RuXyn024 was composed of 358 amino acid residues and had a molecular weight of approximately 40 kDa,belonging to the GH 10 family.The optimal pH and temperature of rRuXyn024 were pH 7.0 and 40℃,respectively.The relative activity of RuXyn024 at pH 6.0−9.0 and 30−70℃ remained above 60% and 70%,respectively.With xylan from wheat straw as the substrate,rRuXyn024 showcased the K_m and V_max of 18.8 g/L and 82.6µg/min,respectively.The activity of rRuXyn024 was inhibited by Mg²⁺,Zn²⁺,Cu²⁺,Ni²⁺,EDTA,and SDS at 1 mmol/L and 5 mmol/L,as well as 5 mmol/L Ca²⁺ and β-mercaptoethanol.Cu²⁺ and β-mercaptoethanol at 5 mmol/L nearly inactivated the enzyme.Mn²⁺ at 1 mmol/L and 5 mmol/L increased the activity of rRuXyn024 by 46.9% and 35.8%,respectively.The degradation of xylan from wheat straw by rRuXyn024 produced oligosaccharides,including xylotriose and xylobiose.rRuXyn024 could degrade maize straw,rice straw,soybean straw,and rapeseed straw,with the strongest degrading effect on maize straw.[Conclusion] rRuXyn024 exhibits tolerance to broad scopes of pH and temperature and significant potential for improving the utilization of straw by ruminants.