| 库巴德巴利酵母FBKL2.0130 D-阿拉伯糖醇脱氢酶基因的克隆表达及酶学性质研究 |
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| 引用本文： | 邱韵,刘逸寒,王晓丹.库巴德巴利酵母FBKL2.0130 D-阿拉伯糖醇脱氢酶基因的克隆表达及酶学性质研究[J].中国酿造,2020,39(9):163. |
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| 作者姓名： | 邱韵 刘逸寒 王晓丹 |
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| 作者单位： | （1.贵州大学 酿酒与食品工程学院，贵州 贵阳 550025；2.贵州大学 贵州省发酵工程与生物制药重点实验室，贵州 贵阳 550025；3.天津科技大学 生物工程学院 天津市工业微生物重点实验室 工业发酵微生物教育部重点实验室，天津 300457） |
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| 基金项目： | 贵州省工业公关项目（黔科合支撑[2016]2340）；贵州省科技计划项目（黔科合平台人才[2019]5645）；贵州省科技计划项目（黔科合支撑[2018]2314）；贵州省科技计划项目（黔科合支撑[2018]2834） |
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| 摘 要： | 该研究从库巴德巴利酵母FBKL2.0130中克隆D-阿拉伯糖醇脱氢酶基因ARD，利用闪电克隆技术构建重组质粒Yep-PAK，并将重组质粒导入酿酒酵母（Saccharomyces cerevisiae）W303-1A中，得到重组菌株W303-ARD并成功表达。并对重组菌株和亲本菌株的生长性能进行测定，并对重组D-阿拉伯糖醇脱氢酶酶学性质进行研究。结果表明，与亲本菌株相比，重组菌株的D-阿拉伯糖醇脱氢酶的还原活力从（32.7±0.803） U/mL提高至（122.4±1.012） U/mL，氧化活力从（18.46±0.105） U/mL提高至（97.30±0.826）U/mL。重组D-阿拉伯糖醇脱氢酶的最适反应温度为30 ℃，在25～40 ℃范围内氧化、还原活力均保持稳定。还原反应的最适pH值为9.0，在pH 9.0～11.0范围内比较稳定；氧化反应的最适pH值为7.0，在pH 7.0～8.0的范围内能保持较高的活性。Cu2+、Ba2+、Zn2+均能抑制重组酶活力；Mg2+对重组酶活力无明显影响；Ca2+、Mn2+、Fe3+均能提高重组酶活力。
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| 关 键 词： | D-阿拉伯糖醇脱氢酶 库巴德巴利酵母FBKL2.0130 异源表达 酶学性质 |
Clone and expression of D-arabinitol dehydrogenase gene from Debaryomyces coudertii FBKL2.0130 and its enzymatic property |
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| QIU Yun,LIU Yihan,WANG Xiaodan.Clone and expression of D-arabinitol dehydrogenase gene from Debaryomyces coudertii FBKL2.0130 and its enzymatic property[J].China Brewing,2020,39(9):163. |
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| Authors: | QIU Yun LIU Yihan WANG Xiaodan |
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| Affiliation: | (1.School of Liquor and Food Engineering, Guizhou University, Guiyang 550025, China; 2.Guizhou Province Key Laboratory of Fermentation Engineering and Biopharmacy, Guizhou University, Guiyang 550025, China; 3.Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China) |
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| Abstract: | The D-arabitol dehydrogenase gene ARD was cloned from Debaryomyces coudertii FBKL2.0130, and the recombinant plasmid Yep-PAK was constructed by flash clone technology and imported into Saccharomyces cerevisiae W303-1A, thus the recombinant strain W303-ARD was obtained and successfully expressed. The growth performances of both recombinant strain and parent strain were tested, and the enzymatic properties of recombinant D-arabinol dehydrogenase were studied. The results showed that compared with the parent strain, the reduction activity of D-arabitol dehydrogenase of the recombinant strain increased from (32.7±0.803) U/ml to (122.4±1.012) U/ml, and the oxidation activity increased from (18.46±0.105) U/ml to (97.30±0.826) U/ml. The optimal reaction temperature for the recombinant D-arabinitol dehydrogenase was 30 ℃, and the oxidation and reduction activity could maintain good stability between 25 ℃ and 40 ℃. The optimal pH for the recombinant D-arabinitol dehydrogenase was 9.0 in reduction reaction and 7.0 in oxidation reaction. The reduction activity of recombinant D-arabinitol dehydrogenase was relatively stable between pH 9.0 to 11.0, and its oxidation activity could be maintained at a relatively high level in the range of pH 7.0 to 8.0. Cu2+, Ba2+, Zn2+ could all inhibit the activity of the recombinant D-arabinitol dehydrogenase, Mg2+ had no significant effect, and Ca2+, Mn2+, Fe3+ could all improve the activity. |
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| Keywords: | D-arabinitol dehydrogenase Debaryomyces coudertii FBKL2 0130 heterologous expression enzymatic property |
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