基于密码子优化的FAD依赖葡萄糖脱氢酶在毕赤酵母中的高效表达及酶学性质
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摘要
旨在获得表达量高的FAD依赖的葡萄糖脱氢酶。通过FAD依赖的葡萄糖脱氢酶密码子优化,人工合成基因片段,构建重组表达载体pMD-GDH,转化毕赤酵母X33菌株后利用甲醇诱导培养实现分泌表达。结果显示,经试管水平筛选阳性转化子获得一株酶活高且稳定的重组菌株,在10 L发酵罐培养时经过136 h诱导培养,酶活达到257 600 U/L。酶学性质分析表明,以葡萄糖为底物时最适温度和pH分别为55℃和7.0,在50℃下处理150 min仍有70%的活性,在pH 4-7范围内,37℃保温4 h,FAD-GDH仍能保持50%以上的活性。金属离子Cu~(2+)对酶活抑制作用比较大。FAD-GDH的底物专一性较好,以葡萄糖为最适底物。经毕赤酵母密码子偏好性优化实现了FAD依赖的葡萄糖脱氢酶在毕赤酵母中的高效表达,为应用于血糖检测提供理论依据。
The objective of this work is to obtain high-yield FAD-dependent glucose dehydrogenase(FAD-GDH). First,by optimizing the codons of FAD-GDH gene according to the codon preference of Pichia pastoris,the gene fragment was artificially synthesized,then the recombinant vector pMD-GDH containing FAD-GDH e gene was constructed and transferred into P. pastoris(X33),and the secretory expression by methanol induction was achieved. Results showed that a stable recombinant strain with high enzymatic activity was obtained by screening positive transformants at test-tube level. In the 10 L fermenter after 136 h induction culture,enzyme activity reached 257 600 U/L. The analysis of enzymatic characterization demonstrated that the optimal pH and temperature while using glucose as substrate were 7.0 and55℃,respectively. The initial enzyme activity still remained 70% after 150 min treatment at 50℃. In the range of pH 4-7,FAD-GDH still retained over 50% activity after incubated at 37℃ for 4 h. Cu~(2+) presented relatively large inhibition to enzyme activity. FAD-GDH harbored a relative high substrate specificity and D-glucose was the optimal substrate. The efficient expression of FAD-GDH in P. pastoris is achieved by optimizing the codon preference of P. pastoris,which provides a theoretical basis for the detection of blood glucose.
The objective of this work is to obtain high-yield FAD-dependent glucose dehydrogenase(FAD-GDH). First,by optimizing the codons of FAD-GDH gene according to the codon preference of Pichia pastoris,the gene fragment was artificially synthesized,then the recombinant vector pMD-GDH containing FAD-GDH e gene was constructed and transferred into P. pastoris(X33),and the secretory expression by methanol induction was achieved. Results showed that a stable recombinant strain with high enzymatic activity was obtained by screening positive transformants at test-tube level. In the 10 L fermenter after 136 h induction culture,enzyme activity reached 257 600 U/L. The analysis of enzymatic characterization demonstrated that the optimal pH and temperature while using glucose as substrate were 7.0 and55℃,respectively. The initial enzyme activity still remained 70% after 150 min treatment at 50℃. In the range of pH 4-7,FAD-GDH still retained over 50% activity after incubated at 37℃ for 4 h. Cu~(2+) presented relatively large inhibition to enzyme activity. FAD-GDH harbored a relative high substrate specificity and D-glucose was the optimal substrate. The efficient expression of FAD-GDH in P. pastoris is achieved by optimizing the codon preference of P. pastoris,which provides a theoretical basis for the detection of blood glucose.
引文
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[15]高庆华,胡美荣,吴芳彤,等.点青霉葡萄糖氧化酶基因的克隆及其酶学性质研究[J].生物技术通报, 2016, 32(7):152-159.
[16]Tsuya T, Ferri S, Fujikawa M, et al, Cloning and functional expression of glucose dehydrogenase complex of Burkholderia cepacia in Escherichia coli[J]. Journal of Biotechnology, 2006,123:127-136.
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[19]罗超.产葡萄糖脱氢酶工程菌发酵及重组葡萄糖脱氢酶酶学性质研究[D].杭州:浙江大学, 2015.
[20]杨愈丰. FAD依赖的葡萄糖脱氢酶(FAD-GDH)的重组表达、结构与功能研究[D].广州:华南理工大学, 2014.
[2]Fapyane D, Lee SJ, Kang SH, et al, High performance enzyme fuel cells using a genetically expressed FAD-dependent glucose dehydrogenaseα-subunit of Burkholderia cepacia immobilized in a carbon nanotube electrode for low glucose conditions[J]. Physical Chemistry Chemical Physics, 2013, 15:9508-9512.
[3]Southcott M, Mac Vittie K, Halámek J, et al, A pacemaker powered by an implantable biofuel cell operating under conditions mimicking the human blood circulatory system-battery not included[J].Physical Chemistry Chemical Physics, 2013, 15:6278-6283.
[4]Sygmund C, Staudigl P, Klausberger M, et al, Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris[J].Microbial cell factories, 2011, 10:1-9.
[5]Gu L, Zhang J, Liu B, et al. High-Level extracellular production of glucose oxidase by recombinant Pichia pastoris using a combined strategy[J]. Applied Biochemistry and Biotechnology, 2015, 175(3):1429-1447.
[6]朱泰承,李寅.毕赤酵母表达系统发展概况及趋势[J].生物工程学报, 2015, 31(6):929-938.
[7]Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli:advances and challenges[J]. Frontiers in Microbiology, 2014, 5(172):172-184.
[8]Yang J, Liu L. Codon optimization through a two-step gene synthesis leads to a high-level expression of Aspergillus niger lip2 gene in Pichia pastoris[J]. Journal of Molecular Catalysis B:Enzymatic,2010, 63(3):164-169.
[9]Al-Hawash AB, Zhang X, Ma F. Strategies of codon optimization for high-level heterologous protein expression in microbial expression systems[J]. Gene Reports, 2017:S2452014417300614.
[10]Wu AB, Chen HD, Tang ZZ, et al. Synthesis of Drosophila melanogaster acetylcholinesterase gene using yeast preferred codons and its expression in Pichia pastoris[J]. ChemicoBiological Interactions, 2008, 175(1):403-405.
[11]陈惠,赵海霞,王红宁,等.植酸酶基因中稀有密码子的改造提高其在毕赤酵母中的表达量[J].中国生物化学与分子生物学报, 2005, 21(2):171-175.
[12]孙风敏,韩焱,李文利.基于密码子优化的蛋白酶K在毕赤酵母中的表达及分离纯化[J].微生物学通报, 2014, 41(11):2198-2207.
[13]Gao Z, Li Z, Zhang Y, et al. High-level expression of the Penicillium notatum glucose oxidase gene in Pichia pastoris using codon optimization[J]. Biotechnology Letters, 2012, 34(3):507-514.
[14]周利伟.青霉来源葡萄糖脱氢酶的克隆、表达及其酶学性质研究[D].北京:中国农业科学院, 2012.
[15]高庆华,胡美荣,吴芳彤,等.点青霉葡萄糖氧化酶基因的克隆及其酶学性质研究[J].生物技术通报, 2016, 32(7):152-159.
[16]Tsuya T, Ferri S, Fujikawa M, et al, Cloning and functional expression of glucose dehydrogenase complex of Burkholderia cepacia in Escherichia coli[J]. Journal of Biotechnology, 2006,123:127-136.
[18]Omura H, Sanada H, Yada T, et al, Coenzyme-linked glucose dehydrogenase and polynucleotide encoding the same[P].Europe:EP2380980, Oct. 26, 2011.
[17]Krainer FW, Dietzsch C, et al. Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway[J]. Microbial Cell Factories, 2012, 11(1):22-36.
[19]罗超.产葡萄糖脱氢酶工程菌发酵及重组葡萄糖脱氢酶酶学性质研究[D].杭州:浙江大学, 2015.
[20]杨愈丰. FAD依赖的葡萄糖脱氢酶(FAD-GDH)的重组表达、结构与功能研究[D].广州:华南理工大学, 2014.