轮枝链霉菌转谷氨酰胺酶异源表达研究
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摘要
转谷氨酰胺酶(Transglutaminase，EC2.3.2.13，TGase)是一种广泛存在于哺乳动物、鱼类、植物、细菌、真菌和绿藻等生物中的一类酶。它可以催化蛋白质分子内及分子之间的共价交联聚合，因此能够直接改变蛋白质本身以及蛋白质所附着的细胞、组织、甚至器官的特性，与血液凝固、伤口愈合、表皮角质化、红细胞膜硬化等生物现象有关，并参与调节细胞生长、分化、增生等生物学功能。它能够提高或改变许多蛋白的成胶性能，在食品、生物医药等方面有着广泛的应用前景。
我们采用PCR技术克隆了轮枝链霉菌(Streptomyces mobaraensis)的转谷氨酰胺酶(MTG)基因，并将其装载入pBV220，pET22b，pET30a等高效表达质粒，转化相应的大肠杆菌宿主菌，研究其表达特性。根据酶蛋白表达量和酶活性对重组菌进行筛选，得到MTG的最适异源表达重组菌pET30a-MTG／BL21(DE3)。在37℃，1mM的IPTG诱导5小时的条件下，MTG表达量最大，达到0.44U／ml发酵液。用商业TGase通过皮下注射兔制备得到抗血清，并对重组MTG进行了Dot blotting和Westen blotting鉴定。
Ni离子金属螯合层析纯化MTG的研究表明，以50mM的咪唑缓冲溶液(20mMNa_3PO_4，0.5MNaCl，pH7.4)洗脱，MTG的回收率较高，以总蛋白计回收率可达到69.2％。每升发酵液可制备约13mg纯MTG，经化学方法测定其酶活约为15.1U／mg蛋白，是目前所报道的基因工程MTG中最高的。酶的稳定性酶学性质研究表明：MTG的最适保存缓冲液为：25mMTris-HCl(pH6.0)，内含0.5mM EDTA，10mM DTT，-80℃保存。重组MTG的最适作用pH6.5，最适作用温度40℃。用SDS-PAGE对其交联BSA蛋白的效果进行的功能鉴定表明：重组MTG与天然或商业化MTG具有同样的蛋白交联活性。
Transglutaminases (protein-glutamine: amine y-glutamyltransferase, EC 2.3.2.13, TGase) widely distributed in most tissues, body fluids including the liver, hair follicles, epidermis, prostate, and blood. TGase is thought to be involved in diverse biological functions, such as maintenance of gross forms of structures and limited degrees of extensibility. TGase is also capable to enhance or change a wide variety of protein's binding properties, which has extensive potential applications in food industry and medical treatment.
TGase gene from Streptoverticillium mobaraense (MTG )is cloned by PCR method. The sequence of MTG is the same as the literature. The expression vectors pET30a, pET22b, and pBV220 were used for the heterologous expression of MTG in E. colt. The highest expression level of MTG was obtained in the E.coli BL21 (DE3)/pET30a-MTG with a yield of 0.44 U/ml fermentation medium after 5 hours induction of ImM IPTG at 37℃. The recombinant MTG was confirmed by dot blotting and Western blotting. Investigation on the purification of MTG by Ni-affinity chromatography showed that 13mg purified MTG could be obtained from 1 liter fermentation medium with 69.2% recovery yield based on the the total protein. The specific activity of the purified MTG reached to 15.1U/mg MTG, which is the highest level among the reported genetic engineered strains. Experimental results on the stability and properties of MTG showed that under -80 癈 the optimized storage buffer, for the recombinant MTG is 25mM Tris-HCl (pH6.0) containing 0.5mM EDTA
and lOmM DTT; the suitable pH and temperature were 6.5 and 40℃ respectively. The biological activity of recombinant MTG was verified by BSA conjunction reaction that was assayed by SDS-PAGE electrophoresis. The results showed that the recombinat MTG possessed the same activity
as the native or commercialized MTG.
我们采用PCR技术克隆了轮枝链霉菌(Streptomyces mobaraensis)的转谷氨酰胺酶(MTG)基因，并将其装载入pBV220，pET22b，pET30a等高效表达质粒，转化相应的大肠杆菌宿主菌，研究其表达特性。根据酶蛋白表达量和酶活性对重组菌进行筛选，得到MTG的最适异源表达重组菌pET30a-MTG／BL21(DE3)。在37℃，1mM的IPTG诱导5小时的条件下，MTG表达量最大，达到0.44U／ml发酵液。用商业TGase通过皮下注射兔制备得到抗血清，并对重组MTG进行了Dot blotting和Westen blotting鉴定。
Ni离子金属螯合层析纯化MTG的研究表明，以50mM的咪唑缓冲溶液(20mMNa_3PO_4，0.5MNaCl，pH7.4)洗脱，MTG的回收率较高，以总蛋白计回收率可达到69.2％。每升发酵液可制备约13mg纯MTG，经化学方法测定其酶活约为15.1U／mg蛋白，是目前所报道的基因工程MTG中最高的。酶的稳定性酶学性质研究表明：MTG的最适保存缓冲液为：25mMTris-HCl(pH6.0)，内含0.5mM EDTA，10mM DTT，-80℃保存。重组MTG的最适作用pH6.5，最适作用温度40℃。用SDS-PAGE对其交联BSA蛋白的效果进行的功能鉴定表明：重组MTG与天然或商业化MTG具有同样的蛋白交联活性。
Transglutaminases (protein-glutamine: amine y-glutamyltransferase, EC 2.3.2.13, TGase) widely distributed in most tissues, body fluids including the liver, hair follicles, epidermis, prostate, and blood. TGase is thought to be involved in diverse biological functions, such as maintenance of gross forms of structures and limited degrees of extensibility. TGase is also capable to enhance or change a wide variety of protein's binding properties, which has extensive potential applications in food industry and medical treatment.
TGase gene from Streptoverticillium mobaraense (MTG )is cloned by PCR method. The sequence of MTG is the same as the literature. The expression vectors pET30a, pET22b, and pBV220 were used for the heterologous expression of MTG in E. colt. The highest expression level of MTG was obtained in the E.coli BL21 (DE3)/pET30a-MTG with a yield of 0.44 U/ml fermentation medium after 5 hours induction of ImM IPTG at 37℃. The recombinant MTG was confirmed by dot blotting and Western blotting. Investigation on the purification of MTG by Ni-affinity chromatography showed that 13mg purified MTG could be obtained from 1 liter fermentation medium with 69.2% recovery yield based on the the total protein. The specific activity of the purified MTG reached to 15.1U/mg MTG, which is the highest level among the reported genetic engineered strains. Experimental results on the stability and properties of MTG showed that under -80 癈 the optimized storage buffer, for the recombinant MTG is 25mM Tris-HCl (pH6.0) containing 0.5mM EDTA
and lOmM DTT; the suitable pH and temperature were 6.5 and 40℃ respectively. The biological activity of recombinant MTG was verified by BSA conjunction reaction that was assayed by SDS-PAGE electrophoresis. The results showed that the recombinat MTG possessed the same activity
as the native or commercialized MTG.
引文
1. Laszlo, L; Sylvia, M.Transglutaminase.Moland Cell Biotech. 1984, 58, 9-35
2. Ando.H; Adachi.M; Umeda.K.etal.Purification and characteristics of a novel transglutamianse derived from microorganisms. Agric. Biol. Chem. 1989.53.2613-2617
3. Klein.D; Gozman.E; Koehn.D. Purification and partial characteristics of transglutamianse Physarum polycephalum. J Bacteriol. 1992, 174, 2599-2605
4. Ando.H; Matsura, A; Susumu.H. Manufacture of transglutaminase with Streptomyces Jpn Kokai Tokkyo Koho JP02286031.1992
5. Bech; Lisbeth.Microbial transglutimanase, their production and use WO96/06931.1996
6．小林克德等 WO96／41866．1996
7．组织型转谷氨酰胺酶的研究进展 李平 朱运松 国外医学分子生物学分册 2002
年第24卷第5期
8. Norlund MA etal. Brain Res, 1999; 851: 154
9．张学兵，岳振峰，徐建祥等 转谷氨酰胺酶的功能特性及其在乳品中的应用中国乳品工业 Vol28，No6
10. NONAKAM, MATSAURAY, MOTOKIM. Incorporation of lys and lysine dipeptides intoα_(sl)-Casein by Ca2+-indenpent MTGase.[J].Bioscience Biotechnology and Biochemistry, 1996, 60 (1): 131
11. SOEDAT, SAKAIT, TOGUCHIS, etal. Studies on the functionalities of microbial ltransglutaminase for food processing Ⅱ: effects of microbial transglutaminase on the texture of gel prepared from skuetouderasurimi. [J]. J of Japanese Society of Food Science and Technology, 1997, 43 (12): 561-567;
12. Nielsen P M. Food Biotechnology, 1995, 3:115～156。
13．廖祥儒等，植物转谷氨酰胺酶，生命的化学 1996，16(5)：31～33
14. Zhu Y, Rinzema A and Tramper J. Applied Microbiology and Biotechnology, 1995, 44: 277～282
15. Kanaji T, Ozaki H Takao T etal. Primary Structure of Microbial Transglutaminase from Streptovertillium sp. strain S-8112 [J], J Biolo Chem, 1993, 268(16): 11565～11572
16. Kashiwagi T, Yokoyama K, Ishikawa K et al. Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense. J Biolo Chem, 2002, 277(46): 44252～60.
17．添田孝彦．食品开发，1993，10：42～45(日)
18．张红城等：转谷氨酰胺酶在食品中的应用 食品与发酵工业 Vol．24 No．3
19. YASHIM, KALYMINS, MUMAZAWAT, etal. Contribution of transglutaminase to the setting of fishpastesatvarious temperature[J]. Fisheries Science, 1996, 62(1): 94 97,
20. KURAISHIC, SAKAMOTOJ, YAMAZAKIK, etal. Production of restructured meat using MTGase without salt or cooking. [J]. J of Food Science, 1997, 62(3): 488-490,
21. HANXQ, STINVASAND. Thermodynamic compatibility of substrate proteins affects their cross linking by TGase [J]. J of Agricultural and Food Chemistry, 1996,
44(5): 1211-1217.
22. JOSEPHD, LANIERTC, HAMANNDD. Temperature and pH effect transglutimanase catalyzed setting of crude fish actomyosin. [J]. J of Food Science, 1994, 59(5): 1018-1023
23. SEGUROK, FAGRAMDMY. MTGase and ε-(γ-glutamyl) lysine crosslink effects on lastic properties of leamaboleogels. [J]. J of Food Science, 1995, 60 (2): 305-311
24. SOEDAT, TOGUCHIS, MUMAZAWAT, etal. Studies on the functionalities of microbial ltransglutarninase for food processing Ⅰ: effects of microbial transglutaminase on the texture of gel prepared from skuetouderasurimi. [J].J of Japanese Society of Food Science and Technology, 1997, 43 (11):345-350
25. SOEDAT, LSHIIT, YAMAZUKIK, etal.The functionalities of MTGase for food application. Ⅰ: effect of transglutimanase on texture of tofu [J].J of Japanese Society of Food Science and Technology, 1997, 43 (2):453-458
26. ELFADIL, BABIKER. Polymerization of soy protein digests by MTGase for improvement of the functional properties [J] Food Research international, 1996, 29(7): 627-634.
27. MOTOKIM, NION, TAKINAMIK. Functional properties of food protein polymerized by transglutaminase [J] Food Research international, 1984, 48: 1257-1261
28. TANIMOTOSY, KINSELLAJE.Enzymatic modification of proteins: effects of transglutimanase cross linking of some physical properties of 13-1acto globulin. [J].J of Agricultural and Food Chemistry, 1988, 28: 281-285
29. Zhu Y, Rinzema A and Tramper J. Applied Microbiology and Biotchnology, 1995, 44: 277～282
30．特许公报．JP03168059，1991(日)
31．特许公报．JP043 60641，1991(日)特许公报．JP02286031，1990(日)
32. AtanabeM, SuzukiT, IkezawaZ, etal. Controlled Enzymatic Treatment of Wheat Proteins for Production of Hyper allergenic Flour[J]Biosci Biotech Biochem, 1994, 58(2): 388～390.
33. Neilson PM. Reaction sand Potential Industrial Applications of Transglutaminase.
[J]Food Biotech, 1995, 9(3): 199～156
34. ZhuY, RinzemaA, Tramper J, etal. Microbial transglutaminase a review of its production and application in food processing [J]. Appl.Microbiol Biotechnol, 1995, 44: 277～282
35．特许公报．JP02171160，1990(日)
36．常忠义，江波，王璋．碳源对轮枝链霉菌SK-2产谷氨酰胺转胺酶的影响生物技术，2000, 10(5)：20～22
37．吴介文；蔡国珍；江善宗．谷氨酰胺转胺酶生产菌株之筛选及影响产量之因子之探讨．中国农业化学会志(台湾)．1996，53，2613-2617
2. Ando.H; Adachi.M; Umeda.K.etal.Purification and characteristics of a novel transglutamianse derived from microorganisms. Agric. Biol. Chem. 1989.53.2613-2617
3. Klein.D; Gozman.E; Koehn.D. Purification and partial characteristics of transglutamianse Physarum polycephalum. J Bacteriol. 1992, 174, 2599-2605
4. Ando.H; Matsura, A; Susumu.H. Manufacture of transglutaminase with Streptomyces Jpn Kokai Tokkyo Koho JP02286031.1992
5. Bech; Lisbeth.Microbial transglutimanase, their production and use WO96/06931.1996
6．小林克德等 WO96／41866．1996
7．组织型转谷氨酰胺酶的研究进展 李平 朱运松 国外医学分子生物学分册 2002
年第24卷第5期
8. Norlund MA etal. Brain Res, 1999; 851: 154
9．张学兵，岳振峰，徐建祥等 转谷氨酰胺酶的功能特性及其在乳品中的应用中国乳品工业 Vol28，No6
10. NONAKAM, MATSAURAY, MOTOKIM. Incorporation of lys and lysine dipeptides intoα_(sl)-Casein by Ca2+-indenpent MTGase.[J].Bioscience Biotechnology and Biochemistry, 1996, 60 (1): 131
11. SOEDAT, SAKAIT, TOGUCHIS, etal. Studies on the functionalities of microbial ltransglutaminase for food processing Ⅱ: effects of microbial transglutaminase on the texture of gel prepared from skuetouderasurimi. [J]. J of Japanese Society of Food Science and Technology, 1997, 43 (12): 561-567;
12. Nielsen P M. Food Biotechnology, 1995, 3:115～156。
13．廖祥儒等，植物转谷氨酰胺酶，生命的化学 1996，16(5)：31～33
14. Zhu Y, Rinzema A and Tramper J. Applied Microbiology and Biotechnology, 1995, 44: 277～282
15. Kanaji T, Ozaki H Takao T etal. Primary Structure of Microbial Transglutaminase from Streptovertillium sp. strain S-8112 [J], J Biolo Chem, 1993, 268(16): 11565～11572
16. Kashiwagi T, Yokoyama K, Ishikawa K et al. Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense. J Biolo Chem, 2002, 277(46): 44252～60.
17．添田孝彦．食品开发，1993，10：42～45(日)
18．张红城等：转谷氨酰胺酶在食品中的应用 食品与发酵工业 Vol．24 No．3
19. YASHIM, KALYMINS, MUMAZAWAT, etal. Contribution of transglutaminase to the setting of fishpastesatvarious temperature[J]. Fisheries Science, 1996, 62(1): 94 97,
20. KURAISHIC, SAKAMOTOJ, YAMAZAKIK, etal. Production of restructured meat using MTGase without salt or cooking. [J]. J of Food Science, 1997, 62(3): 488-490,
21. HANXQ, STINVASAND. Thermodynamic compatibility of substrate proteins affects their cross linking by TGase [J]. J of Agricultural and Food Chemistry, 1996,
44(5): 1211-1217.
22. JOSEPHD, LANIERTC, HAMANNDD. Temperature and pH effect transglutimanase catalyzed setting of crude fish actomyosin. [J]. J of Food Science, 1994, 59(5): 1018-1023
23. SEGUROK, FAGRAMDMY. MTGase and ε-(γ-glutamyl) lysine crosslink effects on lastic properties of leamaboleogels. [J]. J of Food Science, 1995, 60 (2): 305-311
24. SOEDAT, TOGUCHIS, MUMAZAWAT, etal. Studies on the functionalities of microbial ltransglutarninase for food processing Ⅰ: effects of microbial transglutaminase on the texture of gel prepared from skuetouderasurimi. [J].J of Japanese Society of Food Science and Technology, 1997, 43 (11):345-350
25. SOEDAT, LSHIIT, YAMAZUKIK, etal.The functionalities of MTGase for food application. Ⅰ: effect of transglutimanase on texture of tofu [J].J of Japanese Society of Food Science and Technology, 1997, 43 (2):453-458
26. ELFADIL, BABIKER. Polymerization of soy protein digests by MTGase for improvement of the functional properties [J] Food Research international, 1996, 29(7): 627-634.
27. MOTOKIM, NION, TAKINAMIK. Functional properties of food protein polymerized by transglutaminase [J] Food Research international, 1984, 48: 1257-1261
28. TANIMOTOSY, KINSELLAJE.Enzymatic modification of proteins: effects of transglutimanase cross linking of some physical properties of 13-1acto globulin. [J].J of Agricultural and Food Chemistry, 1988, 28: 281-285
29. Zhu Y, Rinzema A and Tramper J. Applied Microbiology and Biotchnology, 1995, 44: 277～282
30．特许公报．JP03168059，1991(日)
31．特许公报．JP043 60641，1991(日)特许公报．JP02286031，1990(日)
32. AtanabeM, SuzukiT, IkezawaZ, etal. Controlled Enzymatic Treatment of Wheat Proteins for Production of Hyper allergenic Flour[J]Biosci Biotech Biochem, 1994, 58(2): 388～390.
33. Neilson PM. Reaction sand Potential Industrial Applications of Transglutaminase.
[J]Food Biotech, 1995, 9(3): 199～156
34. ZhuY, RinzemaA, Tramper J, etal. Microbial transglutaminase a review of its production and application in food processing [J]. Appl.Microbiol Biotechnol, 1995, 44: 277～282
35．特许公报．JP02171160，1990(日)
36．常忠义，江波，王璋．碳源对轮枝链霉菌SK-2产谷氨酰胺转胺酶的影响生物技术，2000, 10(5)：20～22
37．吴介文；蔡国珍；江善宗．谷氨酰胺转胺酶生产菌株之筛选及影响产量之因子之探讨．中国农业化学会志(台湾)．1996，53，2613-2617