普通生酮基古龙酸菌中山梨糖脱氢酶和山梨酮脱氢酶的纯化及酶学性质分析
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摘要
【目的】对源于普通生酮基古龙酸菌(Ketogulonicigenium vulgare WSH-001)的山梨糖脱氢酶(Sorbose dehydrogenase,SDH)和山梨酮脱氢酶(Sorbosone dehydrogenase,SNDH)的酶学性质进行分析。【方法】以K.vulgare WSH-001基因组DNA为模板,PCR扩增得到山梨糖脱氢酶基因(sdh)和山梨酮脱氢酶基因(sndh),构建重组表达质粒p ET28a-sdh、p ET28a-sndh,并分别转入大肠杆菌BL21(DE3)中。利用镍柱亲和层析和凝胶过滤层析得到纯化的SDH和SNDH。【结果】成功构建产SDH和SNDH的大肠杆菌BL21(DE3)并对目的酶进行纯化。SDS-PAGE分析结果表明,SDH和SNDH的大小分别为64 k Da和48 k Da,与理论预测值一致。显色法测得SDH酶活为3.15 U/mg,最适反应温度为30°C,最适反应pH为8.0左右;SNDH酶活为6.12 U/mg,最适反应温度为35°C,最适反应pH为8.0左右。在pH 3.0、4.0、5.0的偏酸性条件下,2个酶的酶活受到显著影响。【结论】表达并纯化了来源于普通生酮基古龙酸菌来源的SDH、SNDH,并进行了酶学性质分析,为利用SDH、SNDH实现维生素C前体2-酮基-L-古龙酸的一步法发酵生产提供了必要的参考。
[Objective] We purified and characterized L-sorbose dehydrogenase and L-sorbosone dehydrogenase from Ketogulonicigenium vulgare WSH-001. [Methods] L-sorbose dehydrogenase gene(sdh) and L-sorbosone dehydrogenase gene(sndh) from K. vulgare WSH-001 were amplified by PCR. The amplified fragments were inserted into p ET-28a(+) to obtain expression plasmids,namely p ET28a-sdh and p ET28a-sndh. Escherichia coli BL21(DE3) harboring the above plasmids was used to express SDH and SNDH. Purified SDH and SNDH were obtained by using His TrapTM affinity chromatography and gel filtration chromatography. [Results] SDH and SNDH from K. vulgare WSH-001 were expressed in E. coli BL21(DE3) and purified. The molecular weight of SDH and SNDH were 64 k Da and 48 k Da on SDS-PAGE,respectively. Colorimetric assay showed that the enzyme activity of SDH and SNDH was 3.15 U/mg and 6.12 U/mg,respectively. The optimum temperature and pH of the purified SDH were 30 °C and 8.0,respectively,whereas the optimum temperature and pH of the purified SNDH were 35 °C and 8.0,respectively. The enzyme activity of SDH and SNDH was extremely low at pH 3.0,4.0 and 5.0. [Conclusion] SDH and SNDH from K. vulgare WSH-001 were expressed in E. coli BL21(DE3) and characterized. The results could provide essential reference for the achievement of one-step fermentation of 2-KLG.
[Objective] We purified and characterized L-sorbose dehydrogenase and L-sorbosone dehydrogenase from Ketogulonicigenium vulgare WSH-001. [Methods] L-sorbose dehydrogenase gene(sdh) and L-sorbosone dehydrogenase gene(sndh) from K. vulgare WSH-001 were amplified by PCR. The amplified fragments were inserted into p ET-28a(+) to obtain expression plasmids,namely p ET28a-sdh and p ET28a-sndh. Escherichia coli BL21(DE3) harboring the above plasmids was used to express SDH and SNDH. Purified SDH and SNDH were obtained by using His TrapTM affinity chromatography and gel filtration chromatography. [Results] SDH and SNDH from K. vulgare WSH-001 were expressed in E. coli BL21(DE3) and purified. The molecular weight of SDH and SNDH were 64 k Da and 48 k Da on SDS-PAGE,respectively. Colorimetric assay showed that the enzyme activity of SDH and SNDH was 3.15 U/mg and 6.12 U/mg,respectively. The optimum temperature and pH of the purified SDH were 30 °C and 8.0,respectively,whereas the optimum temperature and pH of the purified SNDH were 35 °C and 8.0,respectively. The enzyme activity of SDH and SNDH was extremely low at pH 3.0,4.0 and 5.0. [Conclusion] SDH and SNDH from K. vulgare WSH-001 were expressed in E. coli BL21(DE3) and characterized. The results could provide essential reference for the achievement of one-step fermentation of 2-KLG.
引文
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[3]Zhu YB,Liu J,Du GC,Zhou JW,Chen J.Sporulation and spore stability of Bacillus megaterium enhance Ketogulonigenium vulgare propagation and 2-keto-L-gulonic acid biosynthesis.Bioresource Technology,2012,107:399–404.
[4]Bremus C,Herrmann U,Bringer-Meyer S,Sahm H.The use of microorganisms in L-ascorbic acid production.Journal of Biotechnology,2006,124(1):196–205.
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[7]Wang XB,Liu J,Du GC,Zhou JW,Chen J.Efficient production of L-sorbose from D-sorbitol by whole cell immobilization of Gluconobacter oxydans WSH-003.Biochemical Engineering Journal,2013,77:171–176.
[8]Urbance JW,Bratina BJ,Stoddard SF,Schmidt TM.Taxonomic characterization of Ketogulonigenium vulgare gen.nov.,sp.nov.and Ketogulonigenium robustum sp.nov.,which oxidize L-sorbose to 2-keto-L-gulonic acid.International Journal of Systematic and Evolutionary Microbiology,2001,51(3):1059–1070.
[9]Asakura A,Hoshino T.Isolation and characterization of a new quinoprotein dehydrogenase,L-sorbose/L-sorbosone dehydrogenase.Bioscience,Biotechnology,and Biochemistry,1999,63(1):46–53.
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[11]Gao LL,Du GC,Zhou JW,Chen J,Liu J.Characterization of a group of pyrroloquinoline quinone-dependent dehydrogenases that are involved in the conversion of L-Sorbose to2-Keto-L-gulonic acid in Ketogulonicigenium vulgare WSH-001.Biotechnology Progress,2013,29(6):1398–1404.
[12]Gao LL,Hu YD,Liu J,Du GC,Zhou JW,Chen J.Stepwise metabolic engineering of Gluconobacter oxydans WSH-003for the direct production of 2-keto-L-gulonic acid from D-sorbitol.Metabolic Engineering,2014,24:30–37.
[13]Xu A,Yao J,Yu L,Lv S,Wang J,Yan B,Yu Z.Mutation of Gluconobacter oxydans and Bacillus megaterium in a two-step process of L-ascorbic acid manufacture by ion beam.Journal of Applied Microbiology,2004,96(6):1317–1323.
[14]Wei DZ,Yuan WK,Yin GL,Yuan ZY,Chen MH.Studies on kinetic model of vitamin C two-step fermentation process.Chinese Journal of Biotechnology,1992,8(3):277–282.(in Chinese)魏东芝,袁渭康,尹光琳,袁中一,陈敏恒.维生素C二步发酵过程动力学模型的研究.生物工程学报,1992,8(3):277–283.
[15]Xue ZY,Yin GL.Study on the purification of L-sorbose dehydrogenase and it’s physical chemical and enzyme properties.Microbiology,2000,27(2):89–92.(in Chinese)薛震役,尹光琳.L-山梨糖脱氢酶的纯化及性质的研究.微生物学通报,2000,27(2):89–92.
[16]Hao AY,Jia Q,Wu HT,Zhou HR,Geng WF,Gao WL,Zhao JQ,Jia JG.Isolation and characteristics research of L-sorbose dehydrogenase in Ketogulonigenium sp.WB0104.Industrial Microbiology,2008,38(1):10–14.(in Chinese)郝爱鱼,贾茜,吴洪涛,周华芮,耿文飞,高文利,赵军强,贺建功.酮古龙酸菌WB0104中L-山梨糖脱氢酶的分离和性质研究.工业微生物,2008,38(1):10–14.
[17]Araki T,Nakatsuka T,Kobayashi F,Watanabe-Ishimaru E,Sanada H,Tamura T,Inagaki K.Reactivity of sorbose dehydrogenase from Sinorhizobium sp.97507 for1,5-anhydro-d-glucitol.Bioscience,Biotechnology,and Biochemistry,2015,79(7):1130–1132.
[18]Zhao SG,Yao LM,Su CX,Wang T,Wang J,Tang ML,Yu ZL.Purification and properties of a new L-sorbose dehydrogenase accelerative protein from bacillus megaterium bred by ion-beam implantation.Plasma Science and Technology,2008,10(3):398–402.
[19]Gao SY,Zhang WC,Wang JH,Guo AG.Integration and expression of sdh gene in Escherichia coli.Acta Microbiologica Sinica,2005,45(1):139–141.(in Chinese)高书颖,张惟材,汪建华,郭蔼光.山梨糖脱氢酶基因在大肠杆菌染色体上整合及表达.微生物学报,2005,45(1):139–141.
[20]Zhang GQ,Chen WW,Xiong XH,Wang JH,You S,Zhang WC.Prokaryotic expression and enzyme activity identification of L-sorbone dehydrogenase of Ketogulonigenium vulgare.Letters in Biotechnology,2012,23(4):511–514.(in Chinese)张谷青,陈微微,熊向华,汪建华,游松,张惟材.酮古龙酸菌山梨酮脱氢酶的原核表达及活性鉴定.生物技术通报,2012,23(4):511–514.
[21]Wang PP,Xia Y,Li JH,Kang Z,Zhou JW,Chen J.Overexpression of pyrroloquinoline quinone biosynthetic genes affects L-sorbose production in Gluconobacter oxydans WSH-003.Biochemical Engineering Journal,2016,112:70–77.
[22]Hoshino T,Sugisawa T,Fujiwara A.Isolation and characterization of NAD(P)-dependent L-sorbosone dehydrogenase from Gluconobacter melanogenus UV10.Agricultural and Biological Chemistry,1991,55(3):665–670.
[23]Huang WS,Zhang R.Microbial fermentation of vitamin C.Xinjiang Agricultural Sciences,2004,41(S):38–39.(in Chinese)黄文书,张瑞.维生素C生物发酵.新疆农业科学,2004,41(S):38–39.
[24]Zhang W,Yan B,Wang J,Yao J,Yu Z.Purification and characterization of membrane-bound L-sorbose dehydrogenase from Gluconobacter oxydans GO112.Enzyme and microbial technology,2006,38(5):643–648.
[25]Miyazaki T,Sugisawa T,Hoshino T.Pyrroloquinoline quinone-dependent dehydrogenases from Ketogulonicigenium vulgare catalyze the direct conversion of L-sorbosone to L-ascorbic acid.Applied and Environmental Microbiology,2006,72(2):1487–1495.
[26]Yang YH,LüYK,Chen JM,Zhou JW.Bioprocess optimization for the production of 2-keto-L-gulonic acid by a one-step fermentation strain.Food and Fermentation Industries,2016,42(7):60–64.(in Chinese)杨燕花,吕永坤,陈吉铭,周景文.2-酮基-L-古龙酸一步发酵生产菌株发酵过程优化.食品与发酵工业,2016,42(7):60–64.
[2]Jacob RA,Sotoudeh G.Vitamin C function and status in chronic disease.Nutrition in Clinical Care,2002,5(2):66–74.
[3]Zhu YB,Liu J,Du GC,Zhou JW,Chen J.Sporulation and spore stability of Bacillus megaterium enhance Ketogulonigenium vulgare propagation and 2-keto-L-gulonic acid biosynthesis.Bioresource Technology,2012,107:399–404.
[4]Bremus C,Herrmann U,Bringer-Meyer S,Sahm H.The use of microorganisms in L-ascorbic acid production.Journal of Biotechnology,2006,124(1):196–205.
[5]Hancock RD,Viola R.Biotechnological approaches for L-ascorbic acid production.Trends in Biotechnology,2002,20(7):299–305.
[6]Hancock RD,Viola R.The use of micro-organisms for L-ascorbic acid production:current status and future perspectives.Applied Microbiology and Biotechnology,2001,56(5/6):567–576.
[7]Wang XB,Liu J,Du GC,Zhou JW,Chen J.Efficient production of L-sorbose from D-sorbitol by whole cell immobilization of Gluconobacter oxydans WSH-003.Biochemical Engineering Journal,2013,77:171–176.
[8]Urbance JW,Bratina BJ,Stoddard SF,Schmidt TM.Taxonomic characterization of Ketogulonigenium vulgare gen.nov.,sp.nov.and Ketogulonigenium robustum sp.nov.,which oxidize L-sorbose to 2-keto-L-gulonic acid.International Journal of Systematic and Evolutionary Microbiology,2001,51(3):1059–1070.
[9]Asakura A,Hoshino T.Isolation and characterization of a new quinoprotein dehydrogenase,L-sorbose/L-sorbosone dehydrogenase.Bioscience,Biotechnology,and Biochemistry,1999,63(1):46–53.
[10]Liu LM,Li Y,Zhang J,Zhou ZM,Liu J,Li XM,Zhou JW,Du GC,Wang L,Chen J.Complete genome sequence of the industrial strain Ketogulonicigenium vulgare WSH-001.Journal of Bacteriology,2011,193(21):6108–6109.
[11]Gao LL,Du GC,Zhou JW,Chen J,Liu J.Characterization of a group of pyrroloquinoline quinone-dependent dehydrogenases that are involved in the conversion of L-Sorbose to2-Keto-L-gulonic acid in Ketogulonicigenium vulgare WSH-001.Biotechnology Progress,2013,29(6):1398–1404.
[12]Gao LL,Hu YD,Liu J,Du GC,Zhou JW,Chen J.Stepwise metabolic engineering of Gluconobacter oxydans WSH-003for the direct production of 2-keto-L-gulonic acid from D-sorbitol.Metabolic Engineering,2014,24:30–37.
[13]Xu A,Yao J,Yu L,Lv S,Wang J,Yan B,Yu Z.Mutation of Gluconobacter oxydans and Bacillus megaterium in a two-step process of L-ascorbic acid manufacture by ion beam.Journal of Applied Microbiology,2004,96(6):1317–1323.
[14]Wei DZ,Yuan WK,Yin GL,Yuan ZY,Chen MH.Studies on kinetic model of vitamin C two-step fermentation process.Chinese Journal of Biotechnology,1992,8(3):277–282.(in Chinese)魏东芝,袁渭康,尹光琳,袁中一,陈敏恒.维生素C二步发酵过程动力学模型的研究.生物工程学报,1992,8(3):277–283.
[15]Xue ZY,Yin GL.Study on the purification of L-sorbose dehydrogenase and it’s physical chemical and enzyme properties.Microbiology,2000,27(2):89–92.(in Chinese)薛震役,尹光琳.L-山梨糖脱氢酶的纯化及性质的研究.微生物学通报,2000,27(2):89–92.
[16]Hao AY,Jia Q,Wu HT,Zhou HR,Geng WF,Gao WL,Zhao JQ,Jia JG.Isolation and characteristics research of L-sorbose dehydrogenase in Ketogulonigenium sp.WB0104.Industrial Microbiology,2008,38(1):10–14.(in Chinese)郝爱鱼,贾茜,吴洪涛,周华芮,耿文飞,高文利,赵军强,贺建功.酮古龙酸菌WB0104中L-山梨糖脱氢酶的分离和性质研究.工业微生物,2008,38(1):10–14.
[17]Araki T,Nakatsuka T,Kobayashi F,Watanabe-Ishimaru E,Sanada H,Tamura T,Inagaki K.Reactivity of sorbose dehydrogenase from Sinorhizobium sp.97507 for1,5-anhydro-d-glucitol.Bioscience,Biotechnology,and Biochemistry,2015,79(7):1130–1132.
[18]Zhao SG,Yao LM,Su CX,Wang T,Wang J,Tang ML,Yu ZL.Purification and properties of a new L-sorbose dehydrogenase accelerative protein from bacillus megaterium bred by ion-beam implantation.Plasma Science and Technology,2008,10(3):398–402.
[19]Gao SY,Zhang WC,Wang JH,Guo AG.Integration and expression of sdh gene in Escherichia coli.Acta Microbiologica Sinica,2005,45(1):139–141.(in Chinese)高书颖,张惟材,汪建华,郭蔼光.山梨糖脱氢酶基因在大肠杆菌染色体上整合及表达.微生物学报,2005,45(1):139–141.
[20]Zhang GQ,Chen WW,Xiong XH,Wang JH,You S,Zhang WC.Prokaryotic expression and enzyme activity identification of L-sorbone dehydrogenase of Ketogulonigenium vulgare.Letters in Biotechnology,2012,23(4):511–514.(in Chinese)张谷青,陈微微,熊向华,汪建华,游松,张惟材.酮古龙酸菌山梨酮脱氢酶的原核表达及活性鉴定.生物技术通报,2012,23(4):511–514.
[21]Wang PP,Xia Y,Li JH,Kang Z,Zhou JW,Chen J.Overexpression of pyrroloquinoline quinone biosynthetic genes affects L-sorbose production in Gluconobacter oxydans WSH-003.Biochemical Engineering Journal,2016,112:70–77.
[22]Hoshino T,Sugisawa T,Fujiwara A.Isolation and characterization of NAD(P)-dependent L-sorbosone dehydrogenase from Gluconobacter melanogenus UV10.Agricultural and Biological Chemistry,1991,55(3):665–670.
[23]Huang WS,Zhang R.Microbial fermentation of vitamin C.Xinjiang Agricultural Sciences,2004,41(S):38–39.(in Chinese)黄文书,张瑞.维生素C生物发酵.新疆农业科学,2004,41(S):38–39.
[24]Zhang W,Yan B,Wang J,Yao J,Yu Z.Purification and characterization of membrane-bound L-sorbose dehydrogenase from Gluconobacter oxydans GO112.Enzyme and microbial technology,2006,38(5):643–648.
[25]Miyazaki T,Sugisawa T,Hoshino T.Pyrroloquinoline quinone-dependent dehydrogenases from Ketogulonicigenium vulgare catalyze the direct conversion of L-sorbosone to L-ascorbic acid.Applied and Environmental Microbiology,2006,72(2):1487–1495.
[26]Yang YH,LüYK,Chen JM,Zhou JW.Bioprocess optimization for the production of 2-keto-L-gulonic acid by a one-step fermentation strain.Food and Fermentation Industries,2016,42(7):60–64.(in Chinese)杨燕花,吕永坤,陈吉铭,周景文.2-酮基-L-古龙酸一步发酵生产菌株发酵过程优化.食品与发酵工业,2016,42(7):60–64.