西黑冠长臂猿粪便微生物宏基因组来源的高比活α-l-阿拉伯呋喃糖苷酶的重组表达及酶学性质

Recombinant expression and characterization of high-specific activity α-l-arabinofuranosidase from Nomascus concolor fecal microbial metagenome

摘要:

【背景】α-l-阿拉伯呋喃糖苷酶是一类重要的半纤维素酶，能协同其他半纤维素酶降解木聚糖，在食品、医药、生物质能转化中具有应用价值。【目的】挖掘新型α-l-阿拉伯呋喃糖苷酶基因，对其进行异源表达、纯化并研究其酶学性质。【方法】从西黑冠长臂猿粪便微生物宏基因组中扩增α-l-阿拉伯呋喃糖苷酶基因，在大肠杆菌BL21(DE3)中进行异源表达，并进行酶学性质研究。【结果】从粪便微生物宏基因组中扩增得到α-l-阿拉伯呋喃糖苷酶基因AbfNC2b_38，并获得重组α-l-阿拉伯呋喃糖苷酶AbfNC2b_38，其分子量为57.04 kDa。AbfNC2b_38的最适作用条件为55 ℃、pH 6.0，K_m和V_max分别为(6.48±0.73) mmol/L和(1 248.0±114.6) U/mg，与其他宏基因组来源的α-l-阿拉伯呋喃糖苷酶相比具有最高比活300.81 U/mg。AbfNC2b_38具有较好的乙醇和NaCl耐受性，30%乙醇下耐受1 h保持68%的活性；25% NaCl中耐受1 h，相对酶活仍保持在约70%。与木聚糖酶协同降解山毛榉木聚糖时，协同率最高为1.21。【结论】从西黑冠长臂猿粪便微生物宏基因组中获得新型α-l-阿拉伯呋喃糖苷酶基因AbfNC2b_38并成功异源表达。AbfNC2b_38具有较好的乙醇和NaCl耐受性，能与木聚糖酶协同作用提高木聚糖的降解效率，在饲料、食品加工等领域具有潜在的应用价值。

关键词:粪便微生物宏基因组;α-l-阿拉伯呋喃糖苷酶;协同作用

Abstract:

[Background] As a valuable hemicellulase, α-l-arabinofuranosidase (EC 3.2.1.55) can degrade xylan with other hemicellulases and is applied in food, medicine, and biomass energy conversion. [Objective] This study aimed to clone a novel α-l-arabinofuranosidase gene and analyze the heteroexpression, purification, and enzymatic properties of the protein. [Methods] The α-l-arabinofuranosidase gene was amplified from the fecal microbial metagenome of Nomascus concolor and heterologously expressed in Escherichia coli BL21(DE3), and then the enzymatic properties of the protein were examined. [Results] After α-l-arabinofuranosidase gene AbfNC2b_38 was amplified from the fecal microbial metagenome, a recombinant protein AbfNC2b_38 with a molecular weight of 57.04 kDa was expressed. At the optimum conditions of 55 °C and pH 6.0, the enzyme showed the K_m of (6.48±0.73) mmol/L, the V_max of (1 248.0±114.6) U/mg, and the highest specific activity of 300.81 U/mg compared with the α-l-arabinofuranosidases of other metagenomic sources. This enzyme was highly tolerant to ethanol and NaCl. It showed the relative activity of 68% after being treated with 30% ethanol for 1 h and relative activity of approximately 70% after being exposed to 25% NaCl for 1 h. Moreover, it had the highest synergistic rate of 1.21 when synergistically degrading beech xylan with xylanase. [Conclusion] A novel α-l-arabinofuranosidase gene AbfNC2b_38 was cloned from the fecal microbial metagenome of N. concolor, which was successfully heterologously expressed. AbfNC2b_38 was highly tolerant to both ethanol and NaCl, and can cooperate with xylanase to improve the degradation efficiency of xylan. Hence, this enzyme is of significant application potential in feed and food processing.

Key words:fecal microbial metagenome;α-l-arabinofuranosidase;synergy