吸水链霉菌谷氨酰胺转氨酶的活化机理研究
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摘要
谷氨酰胺转氨酶(Transglutaminase, EC 2.3.2.13,简称TGase)能催化蛋白质分子内、分子间发生酰基转移反应,从而改变蛋白质结构和功能特性,在食品、生物医药、组织工程、纺织等领域具有广泛的应用前景。链霉菌TGase是酶原(Pro-TGase)经活化而成的,但是关于链霉菌TGase的活化机理还不清楚。为了深入地研究吸水链霉菌(Streptomyces hygroscopicus)产TGase的活化机理,本论文对S. hygroscopicus发酵产生的Pro-TGase和TGase的高效分离纯化及TGase的活化过程进行了研究。主要研究结果如下：
1.在培养基中分别添加不同浓度的Cu2+, Mn2+, Zn2+, Fe2+, Co2+五种金属离子,摇瓶发酵培养发现,Cu2+, Mn2+, Zn2+对S. hygroscopicus产TGase有明显的促进作用,并且确定0.05 mmol/L Cu2+是最适合添加的离子。确定了S. hygroscopicus利用优化培养基液体培养过程中,培养60 h时Pro-TGase可以完全转化成TGase。
2.利用硫酸铵沉淀和阴离子交换层析以及凝胶过滤层析对S. hygroscopicus发酵上清液中的Pro-TGase和TGase进行了高效分离纯化,并得到两种活性较高而电荷量不同的TGase (TGase A和TGase B)。本研究是首次利用阴离子交换层析法对吸水链霉菌Pro-TGase和TGase分离纯化。
3.通过Hiprep 16 10 phenyl FF柱和LC-MS以及CD谱对两种TGase的疏水性、天然相对分子质量以及二级结构进行分析,发现TGase A和TGase B的疏水性、相对分子质量和二级结构均存在差异。研究了两种TGase的动力学,发现两种TGase的Vmax、Km和Kcat差异也很大。
4.测定了S. hygroscopicus来源的Pro-TGase的N端氨基酸序列为ASGGDG, TGase A和TGase B的N端前五个氨基酸序列均为DAADE。S.hygroscopicus来源Pro-TGase的N端氨基酸序列与已报道的S. cinnamoneus、S.fradiae、S. netropsis来源的相似性很高,但TGase的N端序列与这三种链霉菌来源的相似性却不高。本研究是首次对S.hygroscopicus来源的TGase的N端氨基酸序列进行报道。
5.对纯化后的Pro-TGase与TGase A和TGase B的相互作用进行研究发现,Pro-TGase可以被自身的TGase B活化但不能被TGase A活化。热处理后的Pro-TGase对TGase A和TGase B的活性均有抑制作用,而且对TGase B的抑制作用更显著。本研究是首次对S. hygroscopicus来源的Pro-TGase可以被自身的TGase活化进行报道。
6.分别对不同培养时间点的发酵液进行分离纯化,发现了TGase的活化机理：S.hygroscopicus经过24 h的培养后Pro-TGase被分泌到胞外,一部分Pro-TGase经胞外的活化蛋白酶切割成为成熟的TGase B; TGase B也具有活化自身的Pro-TGase的功能,被切去酶原区的部分空间结构发生变化,生成性质不同的TGase A。
Transglutaminase (TGase; EC 2.3.2.13) is a functional enzyme, which could introduce covalent cross-links between proteins as well as amines and peptides by catalyzing acyl transfer reactions. It has great wide applications in the fields of food processing, biomedical engineering, material science, textiles. It has been reported that Streptomyces TGase is secreted as zymogen (Pro-TGase) and activated by protease, but the mechanism of TGase activation was still unclear. To further explore this mechanism, the purification and activation process of TGase from S. hygroscopicus were studied in this paper.
1. S. hygroscopicus was cultured in fermentation liquid added Cu2+, Mn2+, Zn2+, Fe2+ and Co2+ of different concentration respectively. Through the analysis of TGase activity, the activity of TGase could be promote by Cu2+, Mn2+, Zn2+, and the optimal was Cu2+in the concentration of 0.05 mmol/L. After cultured 60 h, Pro-TGase could entirely transformed into mature TGase in optimized culture.
2. Two active TGases (TGase A and TGase B) and Pro-TGase were effectively purified from Streptomyces hygroscopicus fermentation liquid by ammonium sulfate precipitation, Hitrap Q HP and Superdex 7510/300GL columns. This is the first report for the purification of TGase and Pro-TGase using anion chromatography.
3. The two TGases exhibit different hydrophobicities, molecular weights, and second structures by analysis using Hiprep 1610 phenyl FF、LC-MS and CD spectra. They also have different Vmax, Km, and kcat values.
4. The N-terminal amino acid sequence of purified two TGase were determined as DAADER and N-terminal amino acid sequence of Pro-TGase was ASGGDG. According to the amino acid sequence alignment, the N-terminal sequences of Pro-TGase from S. hygroscopicus exhibited significant sequence similarity to those from S. cinnamoneus, S. fradiae and S. netropsis, but the N-terminal sequence of TGase from Streptomyces hygroscopicus exhibited low sequence similarity to them. This is the first report of the N-terminal amino acid sequence of TGase from Streptomyces hygroscopicus.
5. The results of interaction between Pro-TGase and TGase indicated that Pro-TGase could be activated by one type of mature TGase (TGase B), while the activity of both TGases was inhibited by the heat-treated Pro-TGase.
6. The activation process was studied using the fermentation liquid in different time. The results indicate that Pro-TGase could be activated by some enzyme to TGase B, then TGase B activate the Pro-TGase to TGase A.
1.在培养基中分别添加不同浓度的Cu2+, Mn2+, Zn2+, Fe2+, Co2+五种金属离子,摇瓶发酵培养发现,Cu2+, Mn2+, Zn2+对S. hygroscopicus产TGase有明显的促进作用,并且确定0.05 mmol/L Cu2+是最适合添加的离子。确定了S. hygroscopicus利用优化培养基液体培养过程中,培养60 h时Pro-TGase可以完全转化成TGase。
2.利用硫酸铵沉淀和阴离子交换层析以及凝胶过滤层析对S. hygroscopicus发酵上清液中的Pro-TGase和TGase进行了高效分离纯化,并得到两种活性较高而电荷量不同的TGase (TGase A和TGase B)。本研究是首次利用阴离子交换层析法对吸水链霉菌Pro-TGase和TGase分离纯化。
3.通过Hiprep 16 10 phenyl FF柱和LC-MS以及CD谱对两种TGase的疏水性、天然相对分子质量以及二级结构进行分析,发现TGase A和TGase B的疏水性、相对分子质量和二级结构均存在差异。研究了两种TGase的动力学,发现两种TGase的Vmax、Km和Kcat差异也很大。
4.测定了S. hygroscopicus来源的Pro-TGase的N端氨基酸序列为ASGGDG, TGase A和TGase B的N端前五个氨基酸序列均为DAADE。S.hygroscopicus来源Pro-TGase的N端氨基酸序列与已报道的S. cinnamoneus、S.fradiae、S. netropsis来源的相似性很高,但TGase的N端序列与这三种链霉菌来源的相似性却不高。本研究是首次对S.hygroscopicus来源的TGase的N端氨基酸序列进行报道。
5.对纯化后的Pro-TGase与TGase A和TGase B的相互作用进行研究发现,Pro-TGase可以被自身的TGase B活化但不能被TGase A活化。热处理后的Pro-TGase对TGase A和TGase B的活性均有抑制作用,而且对TGase B的抑制作用更显著。本研究是首次对S. hygroscopicus来源的Pro-TGase可以被自身的TGase活化进行报道。
6.分别对不同培养时间点的发酵液进行分离纯化,发现了TGase的活化机理：S.hygroscopicus经过24 h的培养后Pro-TGase被分泌到胞外,一部分Pro-TGase经胞外的活化蛋白酶切割成为成熟的TGase B; TGase B也具有活化自身的Pro-TGase的功能,被切去酶原区的部分空间结构发生变化,生成性质不同的TGase A。
Transglutaminase (TGase; EC 2.3.2.13) is a functional enzyme, which could introduce covalent cross-links between proteins as well as amines and peptides by catalyzing acyl transfer reactions. It has great wide applications in the fields of food processing, biomedical engineering, material science, textiles. It has been reported that Streptomyces TGase is secreted as zymogen (Pro-TGase) and activated by protease, but the mechanism of TGase activation was still unclear. To further explore this mechanism, the purification and activation process of TGase from S. hygroscopicus were studied in this paper.
1. S. hygroscopicus was cultured in fermentation liquid added Cu2+, Mn2+, Zn2+, Fe2+ and Co2+ of different concentration respectively. Through the analysis of TGase activity, the activity of TGase could be promote by Cu2+, Mn2+, Zn2+, and the optimal was Cu2+in the concentration of 0.05 mmol/L. After cultured 60 h, Pro-TGase could entirely transformed into mature TGase in optimized culture.
2. Two active TGases (TGase A and TGase B) and Pro-TGase were effectively purified from Streptomyces hygroscopicus fermentation liquid by ammonium sulfate precipitation, Hitrap Q HP and Superdex 7510/300GL columns. This is the first report for the purification of TGase and Pro-TGase using anion chromatography.
3. The two TGases exhibit different hydrophobicities, molecular weights, and second structures by analysis using Hiprep 1610 phenyl FF、LC-MS and CD spectra. They also have different Vmax, Km, and kcat values.
4. The N-terminal amino acid sequence of purified two TGase were determined as DAADER and N-terminal amino acid sequence of Pro-TGase was ASGGDG. According to the amino acid sequence alignment, the N-terminal sequences of Pro-TGase from S. hygroscopicus exhibited significant sequence similarity to those from S. cinnamoneus, S. fradiae and S. netropsis, but the N-terminal sequence of TGase from Streptomyces hygroscopicus exhibited low sequence similarity to them. This is the first report of the N-terminal amino acid sequence of TGase from Streptomyces hygroscopicus.
5. The results of interaction between Pro-TGase and TGase indicated that Pro-TGase could be activated by one type of mature TGase (TGase B), while the activity of both TGases was inhibited by the heat-treated Pro-TGase.
6. The activation process was studied using the fermentation liquid in different time. The results indicate that Pro-TGase could be activated by some enzyme to TGase B, then TGase B activate the Pro-TGase to TGase A.
引文
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2. Nicholas B, Smethurst P, Verderio E, et al. Cross-linking of cellular proteins by tissue transglutaminase during necrotic cell death:a mechanism for maintaining tissue integrity. Biochem J,2003,371:413-422
3. Ohtake Y, Nagashima T, Suyama-Satoh S, et al. Transglutaminase catalyzed dissociation and association of protein-polyamine complex. Life Sci,2007, 81(7):577-584
4. Ikura K, Otomo C, Natsuka S, et al. Inhibition of transglutaminase by synthetic tyrosine melanin. Biosci Biotechnol Biochem,2002,66(6):1412-1414
5. Griffin M, Casadio R, and Bergamini C M. Transglutaminases:nature's biological glues. Biochem J,2002,368:377-396
6. Ando H, Adachi M, Umeda K, et al. Purification and characteristics of a novel transglutaminase derived from microorganisms. Agric Biol Chem,1989, 53:2613-2617
7. Cui L, Du G, Zhang D, et al. Purification and characterization of transglutaminase from a newly isolated Streptomyces hygroscopicus. Food Chem.,2007, 105(2):612-618
8. Duran R, Junqua M, Schmitter J M, et al. Purification, characterisation, and gene cloning of transglutaminase from Streptoverticillium cinnamoneum CBS 683.68. Biochimie,1998,80(4):313-319
9. Hiroyasu A, Akira M, Susumu H. Process for producing a transglutaminase derived from streptomyces [P]. United States Patent,1993,5:252-469.
10. Masao M., Atsushi O, Masahiko N, et al. Transglutaminase [P]. United States Patent, 1992,5:156-469.
11. Suzuki S, Izawa Y, and Kobayashi K. Purification and characterization of novel transglutaminase from Bacillus subtilis spores. Biosci Biotechnol Biochem,2000, 64:2344-2351
12. SLH Barros, Assmann F, and Zachia A M A. Purification and Properties of a transglutaminase produced by a Bacillus circulans strain isolated from the Amazon environment. Biotechnol Appl Biochem,2003,37:295-299
13. Paul P, Brookhart P L, and McMahon M T. Purification of guinea pig liver transglutaminase using a phenylalanine-Sepharose 4B affinity column. Analytical Biochemistry,1983,128(1):202-205
14. Worratao A, and Yongsawatdigul J. Purification and characterization of transglutaminase from Tropical tilapia (Oreochromis niloticus). Food Chem,2005, 93:651-658
15. Villalobos E, Santos M, Talavera D, et al. Molecular cloning and characterization of a maize transglutaminase complementary DNA. Gene,2004,336(1):93-104
16. Kang H, and Cho Y D. Purification and properties of transglutaminase from soybean (Glycine max) leaves. Biochem Biophys Res Commun,1996,223(2):288-292
17. Bishop P D, Teller D C, Smith R A, et al. Expression, purification, and characterization of human factor ⅩⅢ in Saccharomyces cerevisiae. Biochemistry (Mosc).1990,29(7):1861-1869
18. Yee V C, Pedersen L C, Le Trong I, et al. Three-dimensional structure of a transglutaminase:human blood coagulation factor ⅩⅢ. Proc Natl Acad Sci USA, 1994,91(15):7296-7300
19. Ortner E, Schroeder V, Walser R, et al. Sensitive and selective detection of free FⅩⅢ activation peptide:a potential marker of acute thrombotic events. Blood,2010, 115(24):5089-5096
20. Hornyak T J, and Shafer J A. Role of calcium ion in the generation of factor ⅩⅢ activity. Biochemistry.1991,30(25):6175-6182
21. Ahvazi B, Boeshans K M, and Steinert P M. Crystal structure of transglutaminase 3 in complex with GMP:structural basis for nucleotide specificity. J Biol Chem,2004, 279(25):26716-26725
22. Noguchi K, Ishikawa K, Yokoyama K, et al. Crystal structure of red sea bream transglutaminase. J Biol Chem,2001,276(15):12055-12059
23. Kashiwagi T, Yokoyama K, Ishikawa K, et al. Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense. J Biol Chem,2002, 277(46):44252-44260
24. Casadio R, Polverini E, Mariani P, et al. The structural basis for the regulation of tissue transglutaminase by calcium ions. Eur J Biochem,1999,262(3):672-679
25. De Jong G A H, and Koppelman S J. Transglutaminase catalyzed reactions:Impact on food applications. J Food Sci,2002,67(8):2798-2806
26. Yokoyama K, Nio N, and Kikuchi Y. Properties and applications of microbial transglutaminase. Appl Microbiol Biotechnol,2004,64(4):447-454
27. Kuraishi C, Yamazaki K, and Susa Y. Transglutaminase:Its utilization in the food industry. Food Rev Int,2001,17(2):221-246
28. Jaros D, Partschefeld C, Henle T, et al. Transglutaminase in dairy products:Chemistry, physics, applications. Journal of Texture Studies,2006,37(2):113-155
29. Zhu Y, and Tramper J. Novel applications for microbial transglutaminase beyond food processing. Trends Biotechnol,2008,26(10):559-565
30. Zhu Y, Rinzema A, Tramper J, et al. Microbial transglutaminase-a review of its production and application in food processing. Appl Microbiol Biotechnol,1995, 44(5):277-282
31. Ho ML, Leu SZ, and Hsieh JF. Technical Approach to Simplify the Purification Method and Characterization of Microbial Transglutaminase Produced from Streptoverticilliun ladakanu. J Food Science,2000,65:76-80
32. Wu J, and Tsai J. Screening the microorganisms and some factors for the production of transglutaminase. Chinese Agric Chem Society,1996,34(2):228-240
33. Suzuki S, Izawa Y, Kobayashi K, et al. Purification and characterization of novel transglutaminase from Bacillus subtilis spores. Biosci Biotechnol Biochem,2000, 64(11):2344-2351
34. Kumazawa Y. Development and application of transglutaminase for seafood products. Nippon Suisan Gakkaishi,2002,68(5):633-636
35. Dube M, Schafer C, Neidhart S, et al. Texturisation and modification of vegetable proteins for food applications using microbial transglutaminase. Eur Food Res Technol,2007,225(2):287-299
36. Zhu Y, Rinzema A, Tramper J, et al. Microbial transglutaminase—a review of its production and application in food processing. Appl Microbiol Biotechnol,1995, 44(3):277-282
37. Liu X Q, Yang X Q, Xie F H, et al. Cloning of transglutaminase gene from Streptomyces fradiae and its enhanced expression in the original strain. Biotechnol Lett,2006,28(17):1319-1325
38. Lin Y S, Chao M L, Liu C H, et al. Cloning of the gene coding for transglutaminase from Streptomyces platensis and its expression in Streptomyces lividans. Process Biochem,2006,41(3):519-524
39. Gerber U, Jucknischke U, Putzien S, et al. A rapid and simple method for the purification of transglutaminase from S. mobaraense. Biochem J,1994,299:825-829
40. 张东旭.吸水链霉菌产谷氨酰胺转胺酶的活化机制和生理功能[D]:[博士学位论文].无锡：江南大学,2008
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48. Pfleiderer C, Mainusch M, Weber J, et al. Inhibition of bacterial transglutaminase by its heat-treated pro-enzyme. Microbiol Res,2005,160(3):265-267
2. Nicholas B, Smethurst P, Verderio E, et al. Cross-linking of cellular proteins by tissue transglutaminase during necrotic cell death:a mechanism for maintaining tissue integrity. Biochem J,2003,371:413-422
3. Ohtake Y, Nagashima T, Suyama-Satoh S, et al. Transglutaminase catalyzed dissociation and association of protein-polyamine complex. Life Sci,2007, 81(7):577-584
4. Ikura K, Otomo C, Natsuka S, et al. Inhibition of transglutaminase by synthetic tyrosine melanin. Biosci Biotechnol Biochem,2002,66(6):1412-1414
5. Griffin M, Casadio R, and Bergamini C M. Transglutaminases:nature's biological glues. Biochem J,2002,368:377-396
6. Ando H, Adachi M, Umeda K, et al. Purification and characteristics of a novel transglutaminase derived from microorganisms. Agric Biol Chem,1989, 53:2613-2617
7. Cui L, Du G, Zhang D, et al. Purification and characterization of transglutaminase from a newly isolated Streptomyces hygroscopicus. Food Chem.,2007, 105(2):612-618
8. Duran R, Junqua M, Schmitter J M, et al. Purification, characterisation, and gene cloning of transglutaminase from Streptoverticillium cinnamoneum CBS 683.68. Biochimie,1998,80(4):313-319
9. Hiroyasu A, Akira M, Susumu H. Process for producing a transglutaminase derived from streptomyces [P]. United States Patent,1993,5:252-469.
10. Masao M., Atsushi O, Masahiko N, et al. Transglutaminase [P]. United States Patent, 1992,5:156-469.
11. Suzuki S, Izawa Y, and Kobayashi K. Purification and characterization of novel transglutaminase from Bacillus subtilis spores. Biosci Biotechnol Biochem,2000, 64:2344-2351
12. SLH Barros, Assmann F, and Zachia A M A. Purification and Properties of a transglutaminase produced by a Bacillus circulans strain isolated from the Amazon environment. Biotechnol Appl Biochem,2003,37:295-299
13. Paul P, Brookhart P L, and McMahon M T. Purification of guinea pig liver transglutaminase using a phenylalanine-Sepharose 4B affinity column. Analytical Biochemistry,1983,128(1):202-205
14. Worratao A, and Yongsawatdigul J. Purification and characterization of transglutaminase from Tropical tilapia (Oreochromis niloticus). Food Chem,2005, 93:651-658
15. Villalobos E, Santos M, Talavera D, et al. Molecular cloning and characterization of a maize transglutaminase complementary DNA. Gene,2004,336(1):93-104
16. Kang H, and Cho Y D. Purification and properties of transglutaminase from soybean (Glycine max) leaves. Biochem Biophys Res Commun,1996,223(2):288-292
17. Bishop P D, Teller D C, Smith R A, et al. Expression, purification, and characterization of human factor ⅩⅢ in Saccharomyces cerevisiae. Biochemistry (Mosc).1990,29(7):1861-1869
18. Yee V C, Pedersen L C, Le Trong I, et al. Three-dimensional structure of a transglutaminase:human blood coagulation factor ⅩⅢ. Proc Natl Acad Sci USA, 1994,91(15):7296-7300
19. Ortner E, Schroeder V, Walser R, et al. Sensitive and selective detection of free FⅩⅢ activation peptide:a potential marker of acute thrombotic events. Blood,2010, 115(24):5089-5096
20. Hornyak T J, and Shafer J A. Role of calcium ion in the generation of factor ⅩⅢ activity. Biochemistry.1991,30(25):6175-6182
21. Ahvazi B, Boeshans K M, and Steinert P M. Crystal structure of transglutaminase 3 in complex with GMP:structural basis for nucleotide specificity. J Biol Chem,2004, 279(25):26716-26725
22. Noguchi K, Ishikawa K, Yokoyama K, et al. Crystal structure of red sea bream transglutaminase. J Biol Chem,2001,276(15):12055-12059
23. Kashiwagi T, Yokoyama K, Ishikawa K, et al. Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense. J Biol Chem,2002, 277(46):44252-44260
24. Casadio R, Polverini E, Mariani P, et al. The structural basis for the regulation of tissue transglutaminase by calcium ions. Eur J Biochem,1999,262(3):672-679
25. De Jong G A H, and Koppelman S J. Transglutaminase catalyzed reactions:Impact on food applications. J Food Sci,2002,67(8):2798-2806
26. Yokoyama K, Nio N, and Kikuchi Y. Properties and applications of microbial transglutaminase. Appl Microbiol Biotechnol,2004,64(4):447-454
27. Kuraishi C, Yamazaki K, and Susa Y. Transglutaminase:Its utilization in the food industry. Food Rev Int,2001,17(2):221-246
28. Jaros D, Partschefeld C, Henle T, et al. Transglutaminase in dairy products:Chemistry, physics, applications. Journal of Texture Studies,2006,37(2):113-155
29. Zhu Y, and Tramper J. Novel applications for microbial transglutaminase beyond food processing. Trends Biotechnol,2008,26(10):559-565
30. Zhu Y, Rinzema A, Tramper J, et al. Microbial transglutaminase-a review of its production and application in food processing. Appl Microbiol Biotechnol,1995, 44(5):277-282
31. Ho ML, Leu SZ, and Hsieh JF. Technical Approach to Simplify the Purification Method and Characterization of Microbial Transglutaminase Produced from Streptoverticilliun ladakanu. J Food Science,2000,65:76-80
32. Wu J, and Tsai J. Screening the microorganisms and some factors for the production of transglutaminase. Chinese Agric Chem Society,1996,34(2):228-240
33. Suzuki S, Izawa Y, Kobayashi K, et al. Purification and characterization of novel transglutaminase from Bacillus subtilis spores. Biosci Biotechnol Biochem,2000, 64(11):2344-2351
34. Kumazawa Y. Development and application of transglutaminase for seafood products. Nippon Suisan Gakkaishi,2002,68(5):633-636
35. Dube M, Schafer C, Neidhart S, et al. Texturisation and modification of vegetable proteins for food applications using microbial transglutaminase. Eur Food Res Technol,2007,225(2):287-299
36. Zhu Y, Rinzema A, Tramper J, et al. Microbial transglutaminase—a review of its production and application in food processing. Appl Microbiol Biotechnol,1995, 44(3):277-282
37. Liu X Q, Yang X Q, Xie F H, et al. Cloning of transglutaminase gene from Streptomyces fradiae and its enhanced expression in the original strain. Biotechnol Lett,2006,28(17):1319-1325
38. Lin Y S, Chao M L, Liu C H, et al. Cloning of the gene coding for transglutaminase from Streptomyces platensis and its expression in Streptomyces lividans. Process Biochem,2006,41(3):519-524
39. Gerber U, Jucknischke U, Putzien S, et al. A rapid and simple method for the purification of transglutaminase from S. mobaraense. Biochem J,1994,299:825-829
40. 张东旭.吸水链霉菌产谷氨酰胺转胺酶的活化机制和生理功能[D]:[博士学位论文].无锡：江南大学,2008
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