来源于Alicyclobacillus sp.A4葡萄糖异构酶的克隆表达及转化应用研究

doi:10.13560/j.cnki.biotech.bull.1985.2018-0408

摘要/Abstract

摘要： 葡萄糖异构酶在体外可以将D-葡萄糖异构化为D-果糖,是商业制备高果糖浆的关键酶。本研究从嗜热菌Alicyclobacillus sp.A4中克隆了葡萄糖异构酶（A4GI）基因,并在大肠杆菌BL21中成功进行了表达。利用His-tag蛋白纯化磁珠对粗酶液进行纯化,并对已纯化的重组A4GI进行详细的酶学性质及转化率的测定。结果表明：A4GI的最适温度和pH分别为65℃和pH 7.5,该酶在pH 6.0-11.0之间很稳定,在pH 6.0-11.0缓冲液中37℃处理1 h后,剩余酶活99% 以上。最适反应条件下,重组酶对D-葡萄糖的K_m与V_max值为99.8 mM与3.75 µmol/min/mg。在不同浓度的葡萄糖转化实验中,A4GI的转化率在一定底物浓度范围内随底物浓度增加呈现升高的趋势,在底物葡萄糖浓度为 3 M时达到52.7% 的最高转化率,因此可以实现在高浓度葡萄糖下进行高效转化,降低后期浓缩的生产成本,具有较好的工业应用潜力。

关键词: 葡萄糖异构酶, 高果糖浆, 转化率, Alicyclobacillus sp.A4

Abstract: Glucose isomerase is a key enzyme for the commercial preparation of high fructose corn syrup by isomerizing D-glucose to D-fructose in vitro. The glucose isomerase（A4GI）gene from the thermophilic Alicyclobacillus sp. A4 strain was cloned and successfully expressed in Escherichia coli BL21. His-tag protein-purified magnetic beads were used to purify the crude enzyme solution,and the detailed enzymatic properties and conversion rate of the purified recombinant A4GI were determined. The results showed that the optimal temperature and pH of A4GI were 65℃ and pH 7.5,respectively. The enzyme was stable over a broad pH range of 6.0-11.0. After incubation at 37℃ for 1 h,the enzyme retained over 99% of its initial activity at pH 6.0-11.0. Under optimal reaction conditions,the K_m and V_max values of the recombinant enzyme for D-glucose were 99.8 mmol/L and 3.75 µmol/min/mg. In the experiment of glucose conversion at different concentrations,the conversion rate of A4GI tended to increase along with the increased concentration of D-glucose,and the conversion rate reached the peak of 52.7% at the substrate concentration of 3 mol/L. Thus,high-efficiency conversion under high concentration of glucose by A4GI may be achieved,resulting in the reduced cost of post-concentration,and there is great potential for it to be applied in industry.

Key words: glucose isomerase, high fructose corn syrup, conversion rate, Alicyclobacillus sp. A4

戴晨霞, 曹慧方, 罗会颖, 姚斌, 柏映国, 徐波. 来源于Alicyclobacillus sp.A4葡萄糖异构酶的克隆表达及转化应用研究[J]. 生物技术通报, 2018, 34(11): 144-151.

DAI Chen-xia, CAO Hui-fang, LUO Hui-ying, YAO Bin, BAI Ying-guo, XU Bo. Cloning,Expression and Conversion Application of Glucose Isomerase from Alicyclobacillus sp.A4[J]. Biotechnology Bulletin, 2018, 34(11): 144-151.

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