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 acidophilicβ-1,4-xylanase and its Ca^(2+)-dependent carbohydrate-binding module

作　　者：葛慧华[1] 刘婷 杨纯 张光亚[1] GE Huihua;LIU Ting;YANG Chun;ZHANG Guangya(Department of Bioengineering and Biotechnology,Huaqiao University,Xiamen 361021,Fujian,China)

机构地区：[1]华侨大学生物工程与技术系,福建厦门361021

出　　处：《微生物学报》2023年第8期3252-3263,共12页Acta Microbiologica Sinica

基　　金：福建省自然科学基金(2020J01079)。

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(kcat/Km)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。Ca^(2+)、Mg2+和Ni2+对酶催化活性均有明显促进作用,其中Ca^(2+)效果更为明显。仅当含有Ca^(2+)时,CBM4513才对β-1,4-木聚糖具有特异性结合能力,属于Ca^(2+)依赖型CBM,其最大结合量为9.13μmol/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Ca^(2+)依赖型CBM,进一步丰富了它们的基因和蛋白资源。[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 verifiedβ-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-lengthβ-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℃and pH 3.0,being a psychrophilic and acidophilicβ-1,4-xylanase.The truncatedβ-1,4-xylanase(Xy14513-T)demonstrated the highest activity at 20°C and pH 4.0 and the catalytic efficiency(kcat/Km)20%lower than that of Xyl4513,indicating the positive effects of CBM on the stability and catalytic performance ofβ-1,4-xylanase.In addition,Ca^(2+),Mg2+,and Ni2+improved the catalytic activities,and Ca^(2+)showed the best performance.Only in the presence of Ca^(2+),CBM4513 had the specific binding ability toβ-1,4-xylan,demonstrating a Ca^(2+)-dependent CBM,and the maximum binding amount was 9.13μmol/g.[Conclusion]We obtained a novel psychrophilic and acidophilicβ-1,4-xylanase with a Ca^(2+)-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.

关 键 词：木聚糖酶 Ca^(2+)依赖型碳水化合物结合模块(CBM) 嗜冷嗜酸酶 低聚木糖 

分 类 号：Q936[生物学—微生物学]