热解纤维素菌属F32中乙酰木聚糖酯酶Axe7的表征

Characterization of an acetyl xylan esterase Axe7 from Caldicellulosiruptor sp. F32

摘要:

【目的】从嗜热厌氧微生物热解纤维素菌属F32 (Caldicellulosiruptor sp. F32)菌株中鉴定出可水解木聚糖侧链乙酰基团的脂酶。【方法】通过基因组序列注释、比对以及蛋白结构预测的方法，发现一个潜在的脂酶7家族的(CE-7)乙酰木聚糖脂酶Axe7。利用基因克隆、质粒构建以及在大肠杆菌中表达目标蛋白并纯化等实验方法，获得了该酶的重组蛋白。【结果】以4-甲基乙酸伞形酯(4-Methylumbelliferyl-acetate)作为底物时，该酶的最适反应pH在6.5?7.0之间，最适反应温度为85 °C，在最适的温度和pH条件下，Axe7活性半衰期(Half-life)超过4 h。在不同金属离子(1.5 mmol/L)存在下，Axe7活性可保持为最适反应酶活的(66.3±4.6)%?(95.7±2.3)%之间，说明金属离子对其酶活有一定的影响。通过测定酶动力学发现Axe7的Km和kcat值分别为0.39 mmol/L和66.95 s?1。【结论】从高温厌氧微生物中发现并表征一个热稳定性良好的乙酰木聚糖脂酶，为木质纤维素的高温糖化和生物炼制提供了一个可工业化的潜在选择。

Abstract:

[Objective] A thermostable acetyl xylan esterase (Axe) was identified from a thermophilic bacterium Caldicellulosiruptor sp. F32. [Methods] Based on genome annotation, protein blast, and protein structural prediction, a family-7 carbohydrate esterase (Axe7) was found. After gene cloning, plasmid construction, protein expression and purification in Escherichia coli, the recombinant protein Axe7 was produced. [Results] On the substrate of 4-Methylumbelliferyl-acetate (4-MUA), the optimal pH of Axe7 was between 6.5 and 7.0, the optimal temperature was 85 °C, and the half-life of Axe7 under the optimal condition was more than 4 hours. By incubation with 1.5 mmol/L different metal ions, the retained activities of Axe7 were between (66.3±4.6)% and (95.7±2.3)%, suggesting the effect of metal ions on enzyme activity. The results of enzyme kinetics showed that the Km of Axe7 was 0.39 mmol/L and kcat was 66.95 s?1. [Conclusion] Our findings provide a potential enzyme for the biorefinery industry.