D-甘露糖异构酶的克隆表达及酶法制备D-甘露糖
|
摘要
将大肠杆菌BL21中的D-甘露糖异构酶(MIase)基因yihS克隆到表达载体pET-28a(+)中,并转入大肠杆菌BL21(DE3)感受态细胞中表达。重组菌株经IPTG诱导培养6 h后MIase发酵酶活可达4.2 U/mL。重组MIase生产D-甘露糖时不需要金属离子的参与。该酶在40℃和pH 7.5条件下表现出催化D-果糖的最高活性,转化率为25%左右;通过研究底物浓度对酶活性的影响,得出该酶的活性不受底物浓度的抑制,以D-果糖为底物时,动力学参数Km为123.32mmol/L,Vmax为113.64μmol/(min·mg),催化效率kcat/Km为0.691 (s·mmol/L)。
The gene encoding D-mannose isomerase(MIase) from Escherichia coli(E. coli) BL21 was cloned into expression vector pET-28 a(+),and then the recombinant plasmid was transformed into the strain E. coli BL21(DE3). Efficient MIase intracellular expression by the recombinant E.coli BL21 was achieved with an activity of 4.2 U/mL(D-mannose forming) after IPTG induction for 6 h. The enzyme was identified as a metal-independent protein. The enzyme activity reached the maximum with a conversion rate of 25% at 40 ℃ and pH 7.5. Using D-fructose as the substrate,a Km value of 123.32 mmol/L,Vmaxvalue of 113.64 μmol/(min·mg) and catalytic efficiency kcat/Kmvalue of 0.691 s·mmol/L were estimated,respectively.
The gene encoding D-mannose isomerase(MIase) from Escherichia coli(E. coli) BL21 was cloned into expression vector pET-28 a(+),and then the recombinant plasmid was transformed into the strain E. coli BL21(DE3). Efficient MIase intracellular expression by the recombinant E.coli BL21 was achieved with an activity of 4.2 U/mL(D-mannose forming) after IPTG induction for 6 h. The enzyme was identified as a metal-independent protein. The enzyme activity reached the maximum with a conversion rate of 25% at 40 ℃ and pH 7.5. Using D-fructose as the substrate,a Km value of 123.32 mmol/L,Vmaxvalue of 113.64 μmol/(min·mg) and catalytic efficiency kcat/Kmvalue of 0.691 s·mmol/L were estimated,respectively.
引文
[1]TONNESEN A S.Clinical pharmacology and use of diuretics[M].ASA Refresher Courses Anesthesiol,1983,11:217-236.
[2]HU X,ZHANG P,MIAO M,et al.D-mannose:properties,production,and applications:an overview[J].Comprehensive Reviews in Food Science and Food Safety,2016,15(4):773-785.
[3]SHARMA A,JEN R,BUTLER A M,et al.The developing human preterm neonatal immune system:A case for more research in this area[J].Clinical Immunology,2012,145(1):61-68.
[4]DIELUBANZA E J,SCHAEFFER A J.Urinary tract infections in women[J].Medical Clinics of North America,2011,95:27-41.
[5]OYOFO B A,DELOACH J R,CORRIER D E,et al.Prevention of Salmonella typhimurium colonization of broilers with D-mannose[J].Poultry Science,1989,68:1357-1360.
[6]RANTA K,NIEMINEN K,EKHOLM F S,et al.Evaluation of immunostimulatory activities of synthetic mannose-containing structures mimicking theβ-(1→2)-linked cell wall mannans of Candida albicans[J].Clinical and Vaccine Immunology,2012,19:1889-1893.
[7]MENDONCA R T,AGUAYO M G,RODRIGUEZ J,et al.Extraction and characterization of hemicelluloses from Pinus radiata and its feasibility for bioethanol production[J].Revista Arvore,2013,37(1):175-180.
[8]ZHAO Guanghui,WANG Guanbin,LI Junping,et al.The orthogonal experiment analysis on the producing mannose from dextrose by catalyzing effect of ammonium molybdate[J].Contemporary Chemical Industry,2005,34:39-41.(in Chinese)
[9]TAKASAKI Y,HINOKI K,KATAOKA Y,et al.Enzymatic production of d-mannose from d-fructose by mannose isomerase[J].Journal of Fermentation and Bioengineering,1993,76(3):237-239.
[10]JIA Ming,MU Wangmeng,ZHANG Tao,et al.Expression of D-psicose 3-epimerase in Bacillus subtilis[J].Journal of Food Science and Biotechnology,2014,33(11):1129-1135.(in Chinese)
[11]BICAS J,SILVA J,DIONISIO A.Biotechnological production of bio flavors and functional sugars[J].Food Science and Technology International,2010,30:7-18.
[12]PALLERONI N J,DOUDOROFF M.Mannose isomerase of Pseudomonas saccharophila[J].Journal of Biological Chemistry,1956,218(1):535-548.
[13]SAMBROOK J,FRITSCH E F,MANIATIS T.Molecular cloning:a Laboratory Manual[M].2nd ed.Cold Spring Harbor:Cold Spring Harbor Laboratory Press,1989.
[14]HE Feng,LIU Lijian,ZHUO Renxi,et al.Immobilization of papain on porous silica beads:preparation and characterization[J].Ion Exchange and Adsorption,1997,13(3):290-294.(in Chinese)
[15]HANG Hua,ZHAO Meng,ZHOU Liuming,et al.Research advances on inulin fructotransferase[J].Food and Fermentation Industries,2011,37(1):117-121.(in Chinese)
[16]TANG Qunfang,LI Jianghua,LIU Long,et al.Enhanced production of solvent-stable alpha glycosidase from bacillus licheniformis jxc-1 by an optimal feeding strategy[J].Journal of Food Science and Biotechnology,2016,1:12-18.(in Chinese)
[17]程丽芳.解纤维热酸菌L-阿拉伯糖异构酶的克隆表达、性质研究及分子改造[D].无锡:江南大学,2010.
[18]WANG Jiangbing,DENG Yong.Hydrolysis of soybean protein/solate with alcalase[J].Science and Technology of Food Industry,2002,23(1):61-63.(in Chinese)
[19]ZHOU Rong,YANG Jiguo,WANG Yonghua,et al.Research on enzymatic and kinetic characteristics of a new lipase[J].Modern Food Science and Technology,2007,23(9):8-11.(in Chinese)
[2]HU X,ZHANG P,MIAO M,et al.D-mannose:properties,production,and applications:an overview[J].Comprehensive Reviews in Food Science and Food Safety,2016,15(4):773-785.
[3]SHARMA A,JEN R,BUTLER A M,et al.The developing human preterm neonatal immune system:A case for more research in this area[J].Clinical Immunology,2012,145(1):61-68.
[4]DIELUBANZA E J,SCHAEFFER A J.Urinary tract infections in women[J].Medical Clinics of North America,2011,95:27-41.
[5]OYOFO B A,DELOACH J R,CORRIER D E,et al.Prevention of Salmonella typhimurium colonization of broilers with D-mannose[J].Poultry Science,1989,68:1357-1360.
[6]RANTA K,NIEMINEN K,EKHOLM F S,et al.Evaluation of immunostimulatory activities of synthetic mannose-containing structures mimicking theβ-(1→2)-linked cell wall mannans of Candida albicans[J].Clinical and Vaccine Immunology,2012,19:1889-1893.
[7]MENDONCA R T,AGUAYO M G,RODRIGUEZ J,et al.Extraction and characterization of hemicelluloses from Pinus radiata and its feasibility for bioethanol production[J].Revista Arvore,2013,37(1):175-180.
[8]ZHAO Guanghui,WANG Guanbin,LI Junping,et al.The orthogonal experiment analysis on the producing mannose from dextrose by catalyzing effect of ammonium molybdate[J].Contemporary Chemical Industry,2005,34:39-41.(in Chinese)
[9]TAKASAKI Y,HINOKI K,KATAOKA Y,et al.Enzymatic production of d-mannose from d-fructose by mannose isomerase[J].Journal of Fermentation and Bioengineering,1993,76(3):237-239.
[10]JIA Ming,MU Wangmeng,ZHANG Tao,et al.Expression of D-psicose 3-epimerase in Bacillus subtilis[J].Journal of Food Science and Biotechnology,2014,33(11):1129-1135.(in Chinese)
[11]BICAS J,SILVA J,DIONISIO A.Biotechnological production of bio flavors and functional sugars[J].Food Science and Technology International,2010,30:7-18.
[12]PALLERONI N J,DOUDOROFF M.Mannose isomerase of Pseudomonas saccharophila[J].Journal of Biological Chemistry,1956,218(1):535-548.
[13]SAMBROOK J,FRITSCH E F,MANIATIS T.Molecular cloning:a Laboratory Manual[M].2nd ed.Cold Spring Harbor:Cold Spring Harbor Laboratory Press,1989.
[14]HE Feng,LIU Lijian,ZHUO Renxi,et al.Immobilization of papain on porous silica beads:preparation and characterization[J].Ion Exchange and Adsorption,1997,13(3):290-294.(in Chinese)
[15]HANG Hua,ZHAO Meng,ZHOU Liuming,et al.Research advances on inulin fructotransferase[J].Food and Fermentation Industries,2011,37(1):117-121.(in Chinese)
[16]TANG Qunfang,LI Jianghua,LIU Long,et al.Enhanced production of solvent-stable alpha glycosidase from bacillus licheniformis jxc-1 by an optimal feeding strategy[J].Journal of Food Science and Biotechnology,2016,1:12-18.(in Chinese)
[17]程丽芳.解纤维热酸菌L-阿拉伯糖异构酶的克隆表达、性质研究及分子改造[D].无锡:江南大学,2010.
[18]WANG Jiangbing,DENG Yong.Hydrolysis of soybean protein/solate with alcalase[J].Science and Technology of Food Industry,2002,23(1):61-63.(in Chinese)
[19]ZHOU Rong,YANG Jiguo,WANG Yonghua,et al.Research on enzymatic and kinetic characteristics of a new lipase[J].Modern Food Science and Technology,2007,23(9):8-11.(in Chinese)