Geobacillus thermodenitrificans α-半乳糖苷酶原核表达及其酶学性质
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摘要
本文利用大肠杆菌原核表达系统对来源于Geobacillus thermodenitrificans的α-半乳糖苷酶编码基因进行了重组表达,并对该酶的酶学性质进行了研究。结果表明,该α-半乳糖苷酶的分子量为100～110 kDa,以p NPG为底物时,该酶的最适反应温度为70℃,最适pH6.0,且该酶具有较好的温度稳定性和pH稳定性; Hg~+、Ag~+、Cu~(2+)离子能完全抑制α-半乳糖苷酶的活性,Fe~(3+)、Mn~(2+)、Zn~(2+)等离子对α-半乳糖苷酶的活性具有不同程度的激活作用;以pNPG为底物测得该酶的米氏常数K_m=10.04 mmol/L,最大反应速度V_(max)=18.25μmol/min。
In this study,the gene encoding α-galactosidase from Geobacillus thermodenitrificans was expressed recombinantly using the prokaryotic expression system of Escherichia coli,and the enzymatic properties of the enzyme were studied.The results showed that,the molecular weight of the α-galactosidase was 100 ～ 110 kDa. When p NPG was used as the substrate,the optimum reaction temperature of the enzyme was 70 ℃,the optimum pH was 6.0,and the enzyme had good temperature stability and pH stability.Hg~+,Ag~+and Cu~(2+) ions could completely inhibit its enzyme activity,Fe~(3+),Mn~(2+),Zn~(2+) and other ions had different degrees of activation of α-galactosidase activity.The Michaelis constant was measured using pNPG as a substrate,K_m= 10.04 mmol/L,the maximum reaction rate V_(max)= 18.25 μmol/min.
In this study,the gene encoding α-galactosidase from Geobacillus thermodenitrificans was expressed recombinantly using the prokaryotic expression system of Escherichia coli,and the enzymatic properties of the enzyme were studied.The results showed that,the molecular weight of the α-galactosidase was 100 ～ 110 kDa. When p NPG was used as the substrate,the optimum reaction temperature of the enzyme was 70 ℃,the optimum pH was 6.0,and the enzyme had good temperature stability and pH stability.Hg~+,Ag~+and Cu~(2+) ions could completely inhibit its enzyme activity,Fe~(3+),Mn~(2+),Zn~(2+) and other ions had different degrees of activation of α-galactosidase activity.The Michaelis constant was measured using pNPG as a substrate,K_m= 10.04 mmol/L,the maximum reaction rate V_(max)= 18.25 μmol/min.
引文
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[12]Wang H,Shi P,Luo H,et al.A thermophilicα-galactosidase from Neosartorya fischeri P1 with high specific activity,broad substrate specificity and significant hydrolysis ability of soymilk[J].Bioresource Technology,2014,153:361-364.
[13]王柏婧,冯雁,王师钰,等.嗜热酶的特性及其应用[J].微生物学报,2002(2):259-262.
[14]密士军,柏映国,孟昆,等.一种来源于青霉的新的α-半乳糖苷酶的分离纯化及其酶学性质[J].微生物学报,2007(1):156-160.
[15]Nazina T N,Tourova T P,Poltaraus A B,et al. Taxonomic study of aerobic thermophilic bacilli:Descriptions of Geobacillus subterraneus[J]. International Journal of Systematic&Evolutionary Microbiology,2001,51:433-446.
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[17]周峻沛,潘璐,李俊俊,等.黑颈鹤粪便分离菌Arthrobacter sp.GN14的α-半乳糖苷酶基因克隆、表达与酶学特性[J].微生物学报,2012,52(5):611-619.
[18]Zapater I G,Ullah A H J,Wodzinski R J. Extracellularα-galactosidase(EC 3.2.1.22)from Aspergillus ficuum NRRL3135 purification and characterization[J]. Preparative Biochemistry,1990,20(3-4):263-296.
[19] Karshikoff A, Ladenstein R. Ion pairs and the thermotolerance of proteins from hyperthermophiles:A ‘traffic rule’for hot roads[J]. Trends in Biochemical Sciences,2001,26(9):550-557.
[2]李苏红,朱旻鹏,李拖平.重组水稻α-半乳糖苷酶的分离纯化及酶学性质研究[J].食品科学,2010,31(21):304-307.
[3]张宗东.黄瓜(Cucumis sativus L.)酸性α-半乳糖苷酶的离体表达与酶学性质研究[D].扬州:扬州大学,2008.
[4]沈汪洋,金征宇.发芽咖啡豆α-半乳糖苷酶的性质研究[J].安徽农业科学,2011,39(19):11958-11960.
[5]沈仁权,陈中孚,顾其敏,等.嗜热脂肪芽孢杆菌中一株对分解代谢物不敏感突变型的α-半乳糖苷酶的纯化及其性质[J].复旦学报:自然科学版,1981(2):60-68.
[6]陈俊亮,田芬,霍贵成,等.长双歧杆菌α-半乳糖苷酶的分离纯化及酶学性质[J].食品科学,2014,35(7):118-122.
[7]刘彩琴,何国庆.臭曲霉ZU-G1α-半乳糖苷酶的分离纯化及酶学性质[J].中国食品学报,2009,9(4):70-75.
[8]郝桂娟,张凯,王学智,等.α-半乳糖苷酶的研究进展[J].中国畜牧兽医,2013,40(3):149-154.
[9]Zhao H,Lu L,Xiao M,et al.Cloning and characterization of a novel alpha-galactosidase from Bifidobacterium breve 203 capable of synthesizing gal-alpha-1,4 linkage[J]. Fems Microbiology Letters,2008,285(2):278-283.
[10]Slominski B A,Campbell L D,Guenter W.Oligosaccharides in canola meal and their effect on nonstarch polysaccharide digestibility and true metabolizable energy in poultry[J]. Poultry Science,1994,73(1):156-162.
[11]Coon C N,Leske K L,Akavanichan O,et al. Effect of oligosaccharide-free soybean meal on true metabolizable energy and fiber digestion in adult roosters[J].Poultry Science,1990,69(5):787-793.
[12]Wang H,Shi P,Luo H,et al.A thermophilicα-galactosidase from Neosartorya fischeri P1 with high specific activity,broad substrate specificity and significant hydrolysis ability of soymilk[J].Bioresource Technology,2014,153:361-364.
[13]王柏婧,冯雁,王师钰,等.嗜热酶的特性及其应用[J].微生物学报,2002(2):259-262.
[14]密士军,柏映国,孟昆,等.一种来源于青霉的新的α-半乳糖苷酶的分离纯化及其酶学性质[J].微生物学报,2007(1):156-160.
[15]Nazina T N,Tourova T P,Poltaraus A B,et al. Taxonomic study of aerobic thermophilic bacilli:Descriptions of Geobacillus subterraneus[J]. International Journal of Systematic&Evolutionary Microbiology,2001,51:433-446.
[16]Fridjonsson O,Watzlawick H,Gehweiler A,et al.Cloning of the gene encoding a novel thermostableα-galactosidase from Thermus brockianus ITI360[J]. Applied and Environmental Microbiology,1999,65(9):3955-3963.
[17]周峻沛,潘璐,李俊俊,等.黑颈鹤粪便分离菌Arthrobacter sp.GN14的α-半乳糖苷酶基因克隆、表达与酶学特性[J].微生物学报,2012,52(5):611-619.
[18]Zapater I G,Ullah A H J,Wodzinski R J. Extracellularα-galactosidase(EC 3.2.1.22)from Aspergillus ficuum NRRL3135 purification and characterization[J]. Preparative Biochemistry,1990,20(3-4):263-296.
[19] Karshikoff A, Ladenstein R. Ion pairs and the thermotolerance of proteins from hyperthermophiles:A ‘traffic rule’for hot roads[J]. Trends in Biochemical Sciences,2001,26(9):550-557.