黑曲霉耐热型葡聚糖酶纤维素结合结构域的替换与融合体酶性质

Replacement of cellulose binding domain of Aspergillus niger thermostable glucanase and properties of fusion enzymes

该文旨在通过更换纤维素结合结构域(cellulose binding domain, CBD)的方式探索获得具有纤维素水解能力的耐热型葡聚糖酶的方法。黑曲霉(Aspergillus niger)来源的葡聚糖酶AnEglA6具有优越的耐热性能,在禽类饲料生产中具有应用前景。通过更换不同家族来源的碳水化合物结合结构域(carbohydrate binding module, CBM)编码区,构建了融合体葡聚糖酶基因,分别在巴斯德毕赤酵母(Pichia pastoris)中进行异源表达。AnEglA6催化结构域与分别来源于嗜热菌(Caldicellulosiruptor kristjanssonii)和荧光假单胞菌(Pseudomonas fluorescens)的木聚糖酶的底物结合结构域CBM9、CBM10结合获得的融合体酶AnEg-CBM9和AnEg-CBM10均具有较高的纤维素水解能力,并且底物范围进一步扩大,对微晶纤维素也具有一定的水解能力。AnEg-CBM10热稳定性有显著的提高,在85℃下酶活力半衰期达到40 min左右,能短时耐受90℃高温。融合体酶AnEg-CBM10是一种热稳定性提高、具有纤维素水解能力的新型葡聚糖酶。

This work aimed to investigate a novel approach to obtain thermostable glucanases capable of cellulose hydrolysis by repla-cing the cellulose binding domain(CBD)with carbohydrate binding domain(CBM)from various families.The focus of this study was the AnEglA6 enzyme obtained from Aspergillus niger and has excellent thermostability and potential application in poultry feed production.Fu-sion genes were constructed by replacing the CBD-encoding region of eglA6 with CBMs and then expressed in Pichia pastoris.The fusion enzyme AnEg-CBM9 was obtained by combining the catalytic domain of AnEglA6 with the CBM of xylanase from the hyperthermophilic bacterium Caldicellulosiruptor kristjansonii.AnEg-CBM10 was composed of eglA6 and CBM of xylanase from Pseudomonas fluorescens.Both fusion enzymes exhibited high cellulose hydrolysis activity and expanded their substrate range by demonstrating certain hydrolysis activity on microcrystalline cellulose.AnEg-CBM10 showed remarkable improvement in thermal stability,with a half-life of approximately 40 min at 85℃and the ability to withstand high temperatures of 90℃for a brief period.Therefore,AnEg-CBM10 is a novel glucanase with im-proved thermal stability and cellulose hydrolysis activity.