Song Hui, Zhang Wenyu, Wang Pengju, Tan Huanbo, Su Wencheng, Zhao Shuxin, Zou Peijian
College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, China.
National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Sheng Wu Gong Cheng Xue Bao. 2016 Oct 25;32(10):1348-1361. doi: 10.13345/j.cjb.160084.
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.
酶的固定化很重要且在生物催化中广泛应用。编码胞外L - 谷氨酸氧化酶（Gox）的天蓝色链霉菌基因gox与来自纤维单胞菌的纤维素结合结构域（CBDcex）融合，重组蛋白Gox - CBD在大肠杆菌中表达。融合蛋白（Gox - CBD）被固定在微晶纤维素上。研究了固定化酶的制备条件、结合能力、性质和稳定性。在4℃条件下，保温1小时，融合蛋白Gox - CBD能够以每克微晶纤维素结合9.0毫克蛋白的比例与微晶纤维素结合。比较了游离和固定化L - 谷氨酸氧化酶（Gox - CBD）的酶学性质。固定化酶的比活性降低，但其热稳定性与游离的Gox - CBD相比有很大提高。在60℃孵育30分钟后，仍保留70%的总活性，而游离的重组Gox完全失去活性。在pH低于10或NaCl浓度高于5 mmol/L时，固定化蛋白与微晶纤维素紧密结合。Gox - CBD融合蛋白可以一步特异性地固定在微晶纤维素上。因此，我们的研究结果可为蛋白质纯化和酶固定化提供一种新策略。
