嗜冷嗜酸β-1,4-木聚糖酶及其钙离子依赖型碳水化合物结合模块

Psychrophilic and acidophilic β-1,4-xylanase and its Ca2+-dependent carbohydrate-binding module

摘要:

【目的】β-1,4-木聚糖酶是木聚糖降解的关键酶之一，嗜冷嗜酸木聚糖酶在功能性低聚木糖的制备中具有重要作用，但相关报道较少。【方法】从太平洋火色杆菌(Flammeovirga pacifica)菌株WPAGA1基因组发掘到一条新型的木聚糖酶序列，经基因合成、质粒构建和表达，并对其进行分离纯化及酶学性质研究。【结果】该木聚糖酶(Xyl4513)具有2个保守结构域，一个属于糖苷水解酶11家族(glycoside hydrolase family 11, GH11)催化模块(Xyl4513-T)，另一个属于碳水化合物结合模块(carbohydrate-binding module, CBM) 60家族(CBM4513)，这是一种非常罕见的GH11家族木聚糖酶含有CBM的现象。纯化后的Xyl4513最适反应温度和pH值分别为30 ℃、3.0，这一特性说明Xyl4513为嗜冷嗜酸β-1,4-木聚糖酶；而截短的木聚糖酶Xyl4513-T最适反应温度和pH值分别为20 ℃、4.0，且催化效率(k_cat/K_m)较前者下降了20%，说明CBM4513对酶稳定性和催化效率有较大影响。Ca²⁺、Mg²⁺和Ni²⁺对酶催化活性均有明显促进作用，其中Ca²⁺效果更为明显。仅当含有Ca²⁺时，CBM4513才对β-1,4-木聚糖具有特异性结合能力，属于Ca²⁺依赖型CBM，其最大结合量为9.13 µmol/g。【结论】本文获得了一种新型的嗜冷嗜酸木聚糖酶和相应的Ca²⁺依赖型CBM，进一步丰富了它们的基因和蛋白资源。

Abstract:

[Objective] β-1,4-xylanase is one of the key enzymes in the biodegradation of xylan. Psychrophilic and acidophilic xylanases play an important role in preparing functional xylooligosaccharides, whereas little is known about these enzymes. [Methods] We discovered a novel xylanase gene by functional annotation of the genome of the deep-sea bacterium Flammeovirga pacifica strain WPAGA1. The sequence alignment suggested 60% identity of this sequence with the verified β-1,4-xylanase from Clostridium saccharobutylicum (ID: P17137). Then, we constructed the recombinant plasmid and transformed it into the host cells for expression. After purifying the enzyme by nickel column, we examined the enzyme properties. [Results] The full-length β-1,4-xylanase (Xyl4513) had two conserved domains: a catalytic module belonging to the glycoside hydrolase family 11 (Xy14513-T) and a carbohydrate-binding module (CBM) belonging to family 60 (CBM4513). This was a rare phenomenon that the GH11 xylanase contained CBM. The purified Xyl4513 showed the highest activity at 30 °C and pH 3.0, being a psychrophilic and acidophilic β-1,4-xylanase. The truncated β-1,4-xylanase (Xy14513-T) demonstrated the highest activity at 20 °C and pH 4.0 and the catalytic efficiency (k_cat/K_m) 20% lower than that of Xyl4513, indicating the positive effects of CBM on the stability and catalytic performance of β-1,4-xylanase. In addition, Ca²⁺, Mg²⁺, and Ni²⁺ improved the catalytic activities, and Ca²⁺ showed the best performance. Only in the presence of Ca²⁺, CBM4513 had the specific binding ability to β-1,4-xylan, demonstrating a Ca²⁺-dependent CBM, and the maximum binding amount was 9.13 μmol/g. [Conclusion] We obtained a novel psychrophilic and acidophilic β-1,4-xylanase with a Ca²⁺-dependent CBM, which enriched the related gene and protein resources. The findings of this study will provide valuable information for exploring the stability, catalytic mechanism, and engineering of xylanases and CBMs.