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

【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18.8g/L和82.6μg/min。1mmol/L和5mmol/L的Mg^(2+)、Zn^(2+)、Cu^(2+)、Ni^(2+)、EDTA和SDS,5 mmol/L的Ca^(2+)和β-巯基乙醇对rRuXyn024具有明显的抑制作用,其中5 mmol/L的Cu^(2+)、β-巯基乙醇使rRuXyn024接近失活。Mn^(2+)对rRuXyn024具有明显促进作用,1 mmol/L、5 mmol/L的Mn^(2+)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。

[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°C,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,rRuXyn024showcased the K_(m)and V_(max)of 18.8 g/L and 82.6μg/min,respectively.The activity of rRuXyn024 was inhibited by Mg^(2+),Zn^(2+),Cu^(2+),Ni^(2+),EDTA,and SDS at 1 mmol/L and 5 mmol/L,as well as 5 mmol/L Ca^(2+)andβ-mercaptoethanol.Cu^(2+)andβ-mercaptoethanol at 5 mmol/L nearly inactivated the enzyme.Mn^(2+)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.