参与脂肪酸和糖类合成的两个酶的克隆、表达纯化及晶体学研究
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摘要
微生物中的各种酶类具有巨大的潜在应用价值。它的开发和利用,对于提高人们生活质量有重要意义。对于这些蛋白酶的理化性质、生物活性和催化机理等方面的基础研究有助于发现它更广阔的应用前景。如今,酶类在食品发酵工业生产,生物制剂等方面已得到广泛的应用。
本文借助结构生物学中晶体学的方法,通过获悉蛋白酶的空间结构来推测酶的功能,探讨催化机理。研究中以激烈火球菌(Pyrococcus furiosus)中潜在的参与脂肪酸合成酶类PFS(PF2046)和脆弱拟杆菌(Bacteroides.fragilis 9343)中的糖合成酶FKP为研究对象,将其基因构建至原核表达载体pMCSG7中,使用大肠杆菌E.coli BL21(DE3)大量表达出带有His标签的可溶性融合蛋白,经镍离子柱亲和层析,离子交换及分子筛层析纯化后,得到纯度达95%以上蛋白。使用商品化试剂盒进行晶体生长条件筛选,解析了PFS的三维结构,并对其功能进行初步研究。同时得到了FKP蛋白的微晶,经过结晶条件优化获得分辨率3.5 ?的晶体,为FKP蛋白的结构解析奠定基础。对这两个酶的性质和结构的认识,为深度利用这两种酶开发食品制剂或在食品及相关工业上的广泛应用提供详了理论切入点。
Many microbiology enzymes have great potential in industrial application and have played very important roles in human life. Studies on the Physiochemical Characteristics、biological activity and catalytic mechanism of enzymes are important for their possible wider applications. Nowadays, enzymes have been commonly applied in food fermentation and biomedical regent development.
In this thesis, two enzymes were selected to probe the crystal structures in order to provide clue to understand their function and catalytic mechanism, including a putative fatty acid synthetase PFS (PF2046) from Pyrococcus furio sus and a pyrophosphorylase FKP from Bacteroides.fragilis 9343. Both of these genes were cloned into the expression vector pMCSG7 and used E.coli BL21 (DE3) as a host for large-scale protein expression. The bacteria were broken and the soluble portion was purified by nickel affinity column, followed by ion-exchange and size-exclusion chromatography. The purity of the two enzymes was above 95%. Protein crystallization was performed by using commercial screening kits. PFS’s structure was solved through the B-factor sharpening method when protein crystal was big enough for data collection. FKP's small crystal was diffracted to 3.5 ? and was very close to structure solving. Structure and biochemistry characterization of these two enzymes will provide people more information to develop new food additives or their extensive application in food and related industry.
本文借助结构生物学中晶体学的方法,通过获悉蛋白酶的空间结构来推测酶的功能,探讨催化机理。研究中以激烈火球菌(Pyrococcus furiosus)中潜在的参与脂肪酸合成酶类PFS(PF2046)和脆弱拟杆菌(Bacteroides.fragilis 9343)中的糖合成酶FKP为研究对象,将其基因构建至原核表达载体pMCSG7中,使用大肠杆菌E.coli BL21(DE3)大量表达出带有His标签的可溶性融合蛋白,经镍离子柱亲和层析,离子交换及分子筛层析纯化后,得到纯度达95%以上蛋白。使用商品化试剂盒进行晶体生长条件筛选,解析了PFS的三维结构,并对其功能进行初步研究。同时得到了FKP蛋白的微晶,经过结晶条件优化获得分辨率3.5 ?的晶体,为FKP蛋白的结构解析奠定基础。对这两个酶的性质和结构的认识,为深度利用这两种酶开发食品制剂或在食品及相关工业上的广泛应用提供详了理论切入点。
Many microbiology enzymes have great potential in industrial application and have played very important roles in human life. Studies on the Physiochemical Characteristics、biological activity and catalytic mechanism of enzymes are important for their possible wider applications. Nowadays, enzymes have been commonly applied in food fermentation and biomedical regent development.
In this thesis, two enzymes were selected to probe the crystal structures in order to provide clue to understand their function and catalytic mechanism, including a putative fatty acid synthetase PFS (PF2046) from Pyrococcus furio sus and a pyrophosphorylase FKP from Bacteroides.fragilis 9343. Both of these genes were cloned into the expression vector pMCSG7 and used E.coli BL21 (DE3) as a host for large-scale protein expression. The bacteria were broken and the soluble portion was purified by nickel affinity column, followed by ion-exchange and size-exclusion chromatography. The purity of the two enzymes was above 95%. Protein crystallization was performed by using commercial screening kits. PFS’s structure was solved through the B-factor sharpening method when protein crystal was big enough for data collection. FKP's small crystal was diffracted to 3.5 ? and was very close to structure solving. Structure and biochemistry characterization of these two enzymes will provide people more information to develop new food additives or their extensive application in food and related industry.
引文
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2大岛太郎.好热性细菌[M].北京:科学出版社,1983.55-60
3周得庆.微生物学教程[M].北京:高等教育出版社,1993.111-113
4 Woese C,Fox GE.Phylogenetic structure of the prokaryotie domain:the primary kingdoms[J].Proe Natl Acad Sci,1977,74:5088-5090
5 Woses C.R.,Kandler O et al.Toward a natural system of organisms:proposal for the domains archaea,bacteria and eucaryai[J].Proe Natl Acad Sci,1990,81:4576-4579.
6 Wiegel J,Canganella F.Extreme Thermophiles,Encyclopedia of Life Science [M].Nature Publishing Group,2001.33-36
7沈萍.微生物学[M].北京:高等教育出版社,2000.205-213
8 Melchior D.L.Lipid phase transitions and regulation of membrane fluidity in prokaryotes [J].CurrTop Membranes Transp,1982,17:263-316
9 Yokoyama A,Shizuri Y, Hoshino T, et al.Arch Microbiol[M].1996,165,342-345
10 Jack L.C.M, Van de Vossenberg.The essence of being extremophilic:the role of the unique archaeal membrane lipids Extremophiles[J]. J Mol Biol,1998, 2:163-170
11 Brown J.W,Haas E,et al. Characterization of ribonuclease P RNAs from the rmophilic bacteria[J].Nucleic Acids Research,1993,2l(3):671—679
12郦惠燕,邵靖字.嗜热菌的耐热分子机制[J].生命科学,2000,12(1):30-33
13 Sandman IL Reeve J.N.Structure and functional relationship O arehaeal and eukaryal histones and nucleosomes[J].Arch Microbiol,2000,173:165-169
14 Julie Hollien,Susan Marqusee.Structural distribution of stability in a thermophilic enzyme[J].PNAS,1999, 96(24): 13674-13678
15卢柏松,王国力等.嗜热与嗜常温微生物的蛋白质氨基酸组成比较[J].微生物学报,1998,38(1):20-25
16 Jaenicki R . Stability of proteins in extreme environments : erystallins and enz3anes[J].FASEB,1996,10:84-92
17 Rothschild L.J.,Mancinelli R.L.Life in extreme environment[J].Nature,2001,409:1092-1101
18钟旭美,张宝善等.耐热性酶在食品中的应用研究进展[J].食品研究与开发,2005,26(2):30-33
19 Hanzawa S,Hoaki T et al.An extremelythermostable serine protease from a hyperthermophilic archaeon desulfurococcus strain SY,isolated from a deepsea hydrothermal vent[J].J Mar Biotechnol,1996,4:121-126
20 Mayr J,Lupas A, et al. A hyperthermostable protease of the subtilisin family bound to the surface of the layer of the archaeonStaphylothermus marinus[J].Curr Biol, 1996,6:739-749
21 Klingeberg M, Galunsky B, et al. Purification and properties of highly thermostable,SDS resistant and stereospecific proteinase from the extremether mophilic archaeon Thermococcus stetteri[J]. Appl Environ Microbiol.1995, 61:3098-3104
22 Hyeung jang,Byoung Kim, et al. A novel subtilisin like serine protease fromThermoanaerobacter yonseiensisKB-1:its cloning, expression, and biochemical properties[J]. Extre mephiles, 2002,6(3):233-243
23 Koch R, Canganella F et al. Purification and properties of a thermostable pullulanase from a newly isolated thermophilic anaerobic bacterium[J] Appl.Environ Microbiol,1997,63(5):1088-1094
24 Brown S H,Sjholm C et al.Purification and characterization of a highly thermostable glucose isomerase produced by the extremely thermophilic eubacterium Thermotoga maritime[J]. Biotechnol Bioeng, 1993,41:878-886
25 Schuliger J W,Brown S H et al.Purification and characterization of a novel amylolytic enzyme from ES4,a marine hyperthermophilic archaeum[J].Mol.Mar. Biol.Biotechnol,1993,2:76-78
26 Dong G,Vieille C et al.Cloning, sequencing, and expression of the gene encoding extracellularɑ-amylase form Pyrococcus furiosus and biochemical characterization of the recombinant enzyme[J].Appl Environ Microbiol,1997b,63:3577-3584
27陈卫,张灏.一种高温乳糖酶的酶学性质及对牛乳中乳糖的水解[J].中国乳品工业2002,30(6):15-18
28 Fiala,G,Steen, K O et al. Pyrococcus furiosus sp.nov represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100℃[J].Arch. Microbiol, 1986, 145:56-61
29 Peeples, T. L., Kelly et al. Bioenergetic response of the extreme thermoacidophile Metallosphaera sedula to thermal and nutritional stress[J]. Appl.Environ. Microbiol,1995,61: 2314-2321
30和致中.超高温古生菌强烈炽热球菌(Pyrococcus furiosus)的生理代谢[J].生命科学,2000,12(4): 189-193
31 Schafer T, Schonheit P. Pyruvate metabolism of the hyperthermophilic archaebacterium Pyrococcus furiosus: Acetate formation from acetyl-CoA and ATP synthesis arecatalyzed by an acetyl-CoA synthetase (ADP-forming)[J].Arch Microbiol, 1991,155:366-377
32 Bryant F O,Adams M W W. Characterization of hydrogenase from the hyperthermophilic archaebacterium, Pyrococcus furiosus[J]. J Biol Chem,1989,264:5070-5079
33 Schafer T , Schonheit P.Maltose fermentation to acetate、CO2 and H2 in the anaerobic hyperthermophilic archaeon Pyrococcus fuciosus:evidence for the operation of a novel sugar fermentation pathway[J].Arch Microbiol,1992,158:188-202
34 Mukund S, Adams M W . Characterization of a tungsten-iron-sulfur protein exhibiting novel spectroscopic and redox properties from the hyperthermophilic archaebacterium Pyrococcus furiosus[J]. J Biol Chem, 1990,265: 11508-11516
35 Mukund S, Adams M W . The novel tungsten-iron-sulfur protein of the hyperthermophilic archaebacterium, Pyrococcus furiosus, is an aldehyde ferredoxin oxidoreductase. Evidence for its participation in a unique glycolytic pathway[J]. J Biol Chem, 1991,266: 14208-14216
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