[Analysis of immobilized L-glutamate oxidase fused with cellulose binding domain on microcrystalline cellulose]

Immobilization of enzymes is important and widely applied in biocatalysis. Streptomyces platensis gene gox, encoding an extracellular L-glutamate oxidase (Gox), was fused to cellulose binding domain (CBDcex) from Cellulomonas fimi and the recombinant protein Gox-CBD was expressed in Escherichia coli. The fusion protein (Gox-CBD) was immobilized onto microcrystalline cellulose. The preparation conditions, binding capacity, properties and stability of the immobilized enzyme were studied. Under the condition of 4 ℃, for 1 hour, the fusion protein Gox-CBD was able to bind microcrystalline cellulose at a ratio of 9.0 mg of protein per gram of microcrystalline cellulose. Enzymatic properties of free and immobilized L-glutamic oxidase (Gox-CBD) were compared. The specific activity of the immobilized enzyme decreased, but its thermal stability increased a lot compared with that of the free Gox-CBD. After incubation at 60 ℃ for 30 min, 70% of the total activity remained whereas the free recombinant Gox completely lost its activity. The immobilized protein was tightly bound to microcrystalline cellulose at pH below 10 or more than 5 mmol/L NaCl. The fusion protein of Gox-CBD can be specifically immobilized on the microcrystalline cellulose on a single step. Therefore, our findings can provide a novel strategy for protein purification and enzyme immobilization.酶的固定化作为一种重要的技术，已在生物催化领域得到了广泛的应用。现将来源于普拉特链霉菌3304 (Streptomyces platensis NTU3304) 产生的胞外L-谷氨酸氧化酶 (L-glutamate oxidase，Gox) 基因gox 融合到来源于粪碱纤维单胞菌Cellulomonas fimi 的纤维素结合域 (CBDcex) 的基因上， 构建表达载体pETM10-Gox-CBD，并在大肠杆菌中表达。通过蛋白纯化获得融合蛋白，并命名为Gox-CBD。利用CBD 对微晶纤维素特异性吸附的特性将其固定在微晶纤维素上，并对固定化酶的制备条件、结合量、酶学性质及其微晶纤维素结合稳定性等进行了研究。在4 ℃条件下结合约1 h，融合蛋白Gox-CBD 结合在纤维素上的结合量即可达到9.0 mg/g。通过对重组型、融合表达游离的以及固定化在微晶纤维素上的谷氨酸氧化酶的酶学性质进行比较发现，固定化酶的比酶活有所降低；但固定化酶的热稳定性相对于游离酶有了很大的提高，在60 ℃孵育30 min 后还保留有约70%的活性，而游离的重组Gox 在相同条件下几乎完全失去活性。当固定化结合蛋白在pH10 或者盐浓度5 mmol/L 的NaCl 条件下可以牢固结合。并且可以通过一步纯化方法固定化融合蛋白Gox-CBD 于微晶纤维素上。因此，L-谷氨酸氧化酶与纤维素结合域融合表达的研究为蛋白的纯化及酶的固定化提供了一种新策略。.