新型耐热β-1,4-木糖苷酶的重组表达及酶学性质

Biochemical characterization of a novel thermostable β-1,4-xylosidase from Caldicellulosiruptor owensensis OL

摘要:

[目的]拟对来源于热解纤维素果汁杆菌的新型β-木糖苷酶基因（CoXyl B）进行重组表达和酶学性质研究。[方法]在大肠杆菌系统中成功表达CoXyl B基因，并通过镍柱亲和层析、强阴离子交换和凝胶层析等纯化方法获得纯酶。[结果]对CoXyl B酶学性质的研究结果显示，在以4-对硝基苯酚-β-D-木糖苷为底物时，该酶的最适反应温度为90℃，最适反应pH为6.0。在40-70℃范围内CoXyl B酶活较高且比较稳定。在pH 5.0-6.0之间，70℃孵育1 h后，CoXyl B的相对酶活仍保留80%以上。Ag⁺、高浓度的SDS和PMSF对酶活力的抑制作用较显著，而高浓度Mg²⁺、Li⁺和EDTA对酶活力的激活作用较为明显。CoXyl B的k_cat和K_m值分别为5.0×10^-3 s^-1和1.9 mmol/L。薄层层析色谱显示CoXyl B具有降解木二糖、木三糖和木四糖的能力。[结论]本研究鉴定出CoXyl B为一种新型的极端耐热木糖苷酶，CoXyl B的酶学性质研究将为其在食品热加工以及生物降解领域中的应用提供参考。

Abstract:

[Objective] A thermostable β-xylosidase from the thermophile Caldicellulosiruptor owensensis OL (CoXyl B) was characterized. [Methods] Recombinant CoXyl B was heterogeneously expressed in Escherichia coli and then purified using Ni-NTA, anion exchange and Superdex 200 chromatography. Further, the hydrolysis activity of CoXyl B was studied using p-nitrophenyl-D-xyloside (pNPX) and xylooligosaccharide as substrates. [Results] The optimum temperature and pH of CoXyl B was 90℃ and pH 6.0, respectively. CoXyl B maintained stable at temperature between 40℃ and 70℃. After incubation at 70℃ for 1 h, CoXyl B retained more than 80% of its initial activity at both pH 5.0 and 6.0. Significantly, Ag⁺, SDS and PMSF exhibited negative effect on the activity of CoXyl B, whereas Mg²⁺, Li⁺ and EDTA significantly enhanced the activity of CoXyl B. The kinetic parameters of CoXyl B towards pNPX were k_cat of 5.0×10^-3 s^-1 and K_m of 1.9 mmol/L. Moreover, CoXyl B exhibited efficient hydrolysis activity towards xylobiose, xylotriose and xylotetraose. [Conclusion] Our work suggested the application potential of a new thermostable β-xylosidase (CoXyl B) in the area of xylan degradation at high temperature.