乳糖诱导E.coli发酵生产FAD为辅基的葡萄糖脱氢酶
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摘要
黄素腺嘌呤二核苷酸(FAD)为辅基的葡萄糖脱氢酶(FAD-GDH,EC1.1.5.9),具有辅基结合紧密、催化效率高的优点,可替代目前诊断用葡萄糖氧化酶应用于血糖指标的临床生化检测。本文选取Burkholderia cepacia的葡萄糖脱氢酶基因(gdh)构建表达质粒pTrc99a-gdh,转化E. coli BL21(DE3)。异丙基硫代半乳糖苷(IPTG)诱导发酵,通过酶活测定及聚丙烯酰胺凝胶电泳分析,获得可溶性表达的FAD-GDH,分子量约为60000。利用乳糖替代IPTG作为诱导剂,摇瓶水平初步探索诱导条件,酶活达到994U/L。7.5L发酵罐放大培养该菌,采用梯度流加补料策略,分阶段温度控制,并采用不同的乳糖流加速率进行诱导。当采用0.3mL/min的乳糖流加速率时,酶活达22200U/L,菌体量达69.48g/L。经过镍柱层析,最终获得纯酶的比酶活104.5U/mg。为医用诊断原料用酶葡萄糖脱氢酶新酶种的工业化生产提供借鉴。
FAD-conjugated glucose dehydrogenase(FAD-GDH, EC1.1.5.9) has the advantages of tightly binding of prosthetic groups and high catalytic efficiency. The glucose dehydrogenase gene(gdh) of the bacterial source Burkholderia cepacia was selected to construct the expression plasmid pTrc99 a-gdh. In order to obtain high-yield FAD-GDH, the recombinant E. coli BL21(DE3) was induced using IPTG. The supernatant was analyzed by enzyme activity and SDS-PAGE electrophoretogram, which indicated` that soluble expression was obtained. Using lactose instead of IPTG as the inducer, the shake flask level was initially explored for inducing conditions, and the enzyme activity reached 994 U/L. The gradient feed fermentation of E. coli was carried out to produce FAD-GDH in 7.5 L fermenter under stepped temperature control strategy. Under the condition of lactose addition rate of 0.3 mL/min, enzyme activity and dry cell weight reached 22200 U/L and 69.48 g/L, respectively. After nickel column chromatography, it was found that the enzyme had a specific activity of 104.5 U/mg. The study provides a certain reference for industrial production of the new glucose dehydrogenase.
FAD-conjugated glucose dehydrogenase(FAD-GDH, EC1.1.5.9) has the advantages of tightly binding of prosthetic groups and high catalytic efficiency. The glucose dehydrogenase gene(gdh) of the bacterial source Burkholderia cepacia was selected to construct the expression plasmid pTrc99 a-gdh. In order to obtain high-yield FAD-GDH, the recombinant E. coli BL21(DE3) was induced using IPTG. The supernatant was analyzed by enzyme activity and SDS-PAGE electrophoretogram, which indicated` that soluble expression was obtained. Using lactose instead of IPTG as the inducer, the shake flask level was initially explored for inducing conditions, and the enzyme activity reached 994 U/L. The gradient feed fermentation of E. coli was carried out to produce FAD-GDH in 7.5 L fermenter under stepped temperature control strategy. Under the condition of lactose addition rate of 0.3 mL/min, enzyme activity and dry cell weight reached 22200 U/L and 69.48 g/L, respectively. After nickel column chromatography, it was found that the enzyme had a specific activity of 104.5 U/mg. The study provides a certain reference for industrial production of the new glucose dehydrogenase.
引文
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[15] FRUCHTL M, SAKON J, BEITLE R. Alternate carbohydrate and nontraditional inducer leads to increased productivity of a collagen binding domain fusion protein via fed-batch fermentation[J]. Journal of Biotechnology, 2016, 226:65-73.
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[17]田中宪彰,结城健介.黄素腺嘌呤双核苷酸结合型葡萄糖脱氢酶:CN103981157A[P]. 2014-08-13.TANAKA M, YUKI K. Flavin adenine dinucleotide binding glucose dehydrogenase:CN103981157A[P].. 2014-08-13.
[18]田岛辽子,一柳敦,市川惠一,等.黄素结合型葡萄糖脱氢酶:CN102292439A[P].. 2010-04-19.TIDAO L, YILIU D, ICHIKAWA K, et al. Flavin-binding glucose dehydrogenase:CN102292439[P]. 2010-04-19.
[19] YANG Y, HUANG L, WANG J, et al. Expression, characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus[J]. Enzyme and Microbial Technology, 2015, 68:43-49.
[20] SYGMUND C, STAUDIGL P, KLAUSBERGER M. Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris[J]. Microbial Cell Factories, 2011, 10:106-115.
[2]徐娴,谢承佳,何冰芳.枯草芽孢杆菌葡萄糖脱氢酶基因的克隆及高效表达[J].食品与生物技术学报, 2007, 26(5):75-78.XU X, XIE C J, HE B F. Cloning and high-level expression of glucose dehydrogenase gene from Bacillus subtilis[J]. Journal of Foods and Biotechnology, 2007, 26(5):75-78.
[3] IGARASHI S, OKUDA J, IKEBUKURO K, et al. Molecular engineering of PQQGDH and its applications[J]. Archives of Biochemistry and Biophysics, 2004, 428(1):52-63.
[4] MORI K, NAKAJIMA M, KOJIMA K, et al. Screening of Aspergillusderived FAD-glucose dehydrogenases from fungal genome database[J]. Biotechnology Letters, 2011, 33(11):2255-2263.
[5] YANG Y F, HUANG L, WANG J F, et al. Efficient expression,purification, and characterization of a novel FAD-dependent glucose dehydrogenase from Aspergillus terreus in Pichia pastoris[J]. Journal of Microbiology and Biotechnology, 2014, 24(11):1516-1524.
[6] CHRISTOPH S, PETRA S, MIRIAM K, et al. Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris[J]. Microbial Cell Factories, 2011, 10:106.
[7] MURATA K, AKATSUKA W, SADAKANE T, et al. Glucose oxidation catalyzed by FAD-dependent glucose dehydrogenase within Os complex-tethered redox polymer hydrogel[J]. Electrochimica Acta,2014, 136:537-541.
[8] SU L Q, HONG R Y, WU J. Enhanced extracellular expression of gene-optimized Thermobifida fusca cutinase in Escherichia coli by optimization of induction strategy[J]. Process Biochemistry, 2015, 50(7):1039-1046.
[9]陈亮,任随周,许玫英.乳糖替代IPTG诱导脱色酶TpmD基因在大肠杆菌中的高效表达[J].微生物学通报, 2009, 36(4):551-556.CHEN L, REN S Z,XU M Y. Expression of Tpm D gene in lactase induced by lactose instead of IPTG in E.coli[J]. Microbiology, 2009, 36(4):551-556.
[10]杨海麟,王长城,张玲.高密度培养重组E.coli产胆固醇氧化酶的乳糖诱导策略[J].食品工业科技, 2011, 32(2):162-165.YANG H L,WANG C C, ZHANG L. High-density culture of lactose induced cholesterol oxidase in recombinant E. coli[J]. Food Industry Science and Technology, 2011, 32(2):162-165.
[11]张翔,张彦昊,刘孝永.产壳聚糖酶菌株ncps116发酵条件优化及其酶学性质[J].化工进展, 2018, 37(6):2354-2363.ZHANG X, ZHANG Y H, LIU X Y. Optimization of fermentation conditions and enzymatic properties of the chitosan producing strain ncps116[J]. Chemical Industry and Engineering Progress, 2018, 37(6):2354-2363.
[12]孙艳,王长城,张玲.重组大肠杆菌产胆固醇氧化酶的指数流加策略[J].化工进展, 2010, 29(1):130-133.SUN Y, WANG C C, ZHANG L. Indexed federation strategies for cholesterol oxidase production from recombinant E. coli[J]. Chemical Industry and Engineering Progress, 2010, 29(1):130-133.
[13] SU L Q, HUANG Y, WU J. Enhanced production of recombinant Escherichia coli glutamate decarboxylase through optimization of induction strategy and addition of pyridoxine[J]. Bioresource Technology, 2015, 198:63-69.
[14] CHEN X, ZHOU L, TIAN K, et al. Metabolic engineering of Escherichia coli:a sustainable industrial platform for bio-based chemical production[J]. Biotechnology Advances, 2013, 31(8):1200-1223.
[15] FRUCHTL M, SAKON J, BEITLE R. Alternate carbohydrate and nontraditional inducer leads to increased productivity of a collagen binding domain fusion protein via fed-batch fermentation[J]. Journal of Biotechnology, 2016, 226:65-73.
[16]相场洋志.新型葡萄糖脱氢酶:CN1962859[P]. 2006-03-13.YOSHIHIKO F. New glucose dehydrogenase:CN1962859[P]. 2006-03-13.
[17]田中宪彰,结城健介.黄素腺嘌呤双核苷酸结合型葡萄糖脱氢酶:CN103981157A[P]. 2014-08-13.TANAKA M, YUKI K. Flavin adenine dinucleotide binding glucose dehydrogenase:CN103981157A[P].. 2014-08-13.
[18]田岛辽子,一柳敦,市川惠一,等.黄素结合型葡萄糖脱氢酶:CN102292439A[P].. 2010-04-19.TIDAO L, YILIU D, ICHIKAWA K, et al. Flavin-binding glucose dehydrogenase:CN102292439[P]. 2010-04-19.
[19] YANG Y, HUANG L, WANG J, et al. Expression, characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus[J]. Enzyme and Microbial Technology, 2015, 68:43-49.
[20] SYGMUND C, STAUDIGL P, KLAUSBERGER M. Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris[J]. Microbial Cell Factories, 2011, 10:106-115.
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