通过发酵策略及诱变选育来提高谷氨酰胺转胺酶酶活
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摘要
微生物谷氨酰胺转胺酶(Microbial Transglutaminase,简称MTG,EC 2.3.2.13)能催化多种蛋白质分子间、分子内的酰基转移反应,改善各种蛋白质的功能性质,在食品、化妆品、制药等工业领域具有广阔的应用前景。
本研究以提高吸水链霉菌(Streptomyces hygroscopicus)WSH03-13发酵产谷氨酰胺转胺酶的产量为目标,分别研究了吸水链霉菌的高密度培养和溶氧浓度对发酵过程的影响并通过不同的诱变手段来筛选高酶活的菌株。主要内容如下:
1.对吸水链霉菌的高密度培养进行了研究。通过考察不同初始葡萄糖浓度下的分批发酵,确定了最佳初始葡萄糖浓度为10 g/L。通过比较了不同的流加速率,确定在8-16 h采用较高的的比生长速率(0.15 h-1),后期降低比生长速率(0.10 h-1)这种较优的葡萄糖流加速率组合可以获得较高的菌体量。
2.考察了添加氮源对Streptomyces hygroscopicus发酵产酶的影响。在菌体生长到最大值后添加氮源,促进产酶。实验结果表明,添加50 g/L的豆饼粉酶活最高可达到5.79 U/mL。
3.在3 L罐中考察了不同的溶氧浓度对谷氨酰胺转胺酶分批发酵的影响,确定了在0-20 h保持30%的溶氧浓度,通气量1.25vvm;20 h后保持10%的溶氧浓度,通气量1.25vvm的条件来进行发酵,最终酶活可达到5.88 U/mL,实现了谷氨酰胺转胺酶的高产量、高生产强度的统一。
4.采用N+注入、紫外照射等常规诱变手段,并筛选抗2-脱氧-D-葡萄糖(2-deoxy-D-glucose,2-DG)突变株。由此分离纯化获得的高产菌株DG-1具有抗葡萄糖代谢阻遏的能力。最高酶活可达到8.19 U/mL,比出发菌株提高了96%。
Microbial transglutaminase (MTG; protein-glutamineγ-glutamyltransferase, EC 2.3.2.13) is a recently developed enzyme and is capable of catalyzing acyl transfer reactions, which introduces covalent cross-links between proteins, peptides and various primary amines. So it has potential application in food processing, cosmetic, pharmacy and other industries.
This study is mainly aimed at enhancing the production of microbial transglutaminase (MTG) by Streptomyces hygroscopicus. The relatively high cell density culture was invesgated. The effect of DO concentration and the effect of mutagenesis of Streptomyces hygroscopicus were researched, respectively. The following researches have been done:
1. The relatively high cell density culture of Streptomyces hygroscopicus was investigated, the optimized initial concentration of glucose was 10 g/L.The effects of exponential feeding of glucose and the addition of nitrogen source on MTG production were investigated. The results showed that keeping a high specific growth rate (0.15 h-1) in 8-16 h and a relative low specific growth rate (0.10 h-1) during later time could obtain high-cell density.
2. Effect of nitrogen sources addition on MTG fermentation by Streptomyces hygroscopicus was investigated. Nitrogen sources were added until the end of cells growing. The results showed that MTG activity reached 5.79 U/mL by adding 50 g/L soybean.
3. The effect of dissolved oxygen (DO) concentration on the batch production of MTG in a 3 L stirred fermentor by Streptomyces hygroscopicus was studied. It was found that keep the DO at 30% during 0-20 h, and 10% during later time, the air flow rate was controlled at 1.25 vvm all the time in the fermentation process, the final MTG activity reached 5.88 U/mL achieving the unification of high MTG yield and high productivity.
4. Streptomyces hygroscopicu CCTCC M203062 was mutagenized by UV and N~+-Implantation, and screened by 2-deoxy-D-glucose. DG-1, which is resistant to catabolite repression, was screened. The highest activity reached 8.19 U/mL, increased by 96% over the parent strain.
本研究以提高吸水链霉菌(Streptomyces hygroscopicus)WSH03-13发酵产谷氨酰胺转胺酶的产量为目标,分别研究了吸水链霉菌的高密度培养和溶氧浓度对发酵过程的影响并通过不同的诱变手段来筛选高酶活的菌株。主要内容如下:
1.对吸水链霉菌的高密度培养进行了研究。通过考察不同初始葡萄糖浓度下的分批发酵,确定了最佳初始葡萄糖浓度为10 g/L。通过比较了不同的流加速率,确定在8-16 h采用较高的的比生长速率(0.15 h-1),后期降低比生长速率(0.10 h-1)这种较优的葡萄糖流加速率组合可以获得较高的菌体量。
2.考察了添加氮源对Streptomyces hygroscopicus发酵产酶的影响。在菌体生长到最大值后添加氮源,促进产酶。实验结果表明,添加50 g/L的豆饼粉酶活最高可达到5.79 U/mL。
3.在3 L罐中考察了不同的溶氧浓度对谷氨酰胺转胺酶分批发酵的影响,确定了在0-20 h保持30%的溶氧浓度,通气量1.25vvm;20 h后保持10%的溶氧浓度,通气量1.25vvm的条件来进行发酵,最终酶活可达到5.88 U/mL,实现了谷氨酰胺转胺酶的高产量、高生产强度的统一。
4.采用N+注入、紫外照射等常规诱变手段,并筛选抗2-脱氧-D-葡萄糖(2-deoxy-D-glucose,2-DG)突变株。由此分离纯化获得的高产菌株DG-1具有抗葡萄糖代谢阻遏的能力。最高酶活可达到8.19 U/mL,比出发菌株提高了96%。
Microbial transglutaminase (MTG; protein-glutamineγ-glutamyltransferase, EC 2.3.2.13) is a recently developed enzyme and is capable of catalyzing acyl transfer reactions, which introduces covalent cross-links between proteins, peptides and various primary amines. So it has potential application in food processing, cosmetic, pharmacy and other industries.
This study is mainly aimed at enhancing the production of microbial transglutaminase (MTG) by Streptomyces hygroscopicus. The relatively high cell density culture was invesgated. The effect of DO concentration and the effect of mutagenesis of Streptomyces hygroscopicus were researched, respectively. The following researches have been done:
1. The relatively high cell density culture of Streptomyces hygroscopicus was investigated, the optimized initial concentration of glucose was 10 g/L.The effects of exponential feeding of glucose and the addition of nitrogen source on MTG production were investigated. The results showed that keeping a high specific growth rate (0.15 h-1) in 8-16 h and a relative low specific growth rate (0.10 h-1) during later time could obtain high-cell density.
2. Effect of nitrogen sources addition on MTG fermentation by Streptomyces hygroscopicus was investigated. Nitrogen sources were added until the end of cells growing. The results showed that MTG activity reached 5.79 U/mL by adding 50 g/L soybean.
3. The effect of dissolved oxygen (DO) concentration on the batch production of MTG in a 3 L stirred fermentor by Streptomyces hygroscopicus was studied. It was found that keep the DO at 30% during 0-20 h, and 10% during later time, the air flow rate was controlled at 1.25 vvm all the time in the fermentation process, the final MTG activity reached 5.88 U/mL achieving the unification of high MTG yield and high productivity.
4. Streptomyces hygroscopicu CCTCC M203062 was mutagenized by UV and N~+-Implantation, and screened by 2-deoxy-D-glucose. DG-1, which is resistant to catabolite repression, was screened. The highest activity reached 8.19 U/mL, increased by 96% over the parent strain.
引文
1. Okurmura K, Ikura K, Youshikawa M, Sasaki R and Chiba H. Incorporation of lysyldipeptides into food proteins by transglutaminase. Agric. Biol. Chem. 1984, 48: 2435.
2. Nonaka M, Tanaka H, Okiyama A, Motoki M, Ando H, Umeda K, et al. Polymerization of several proteins by Ca2+-independent transglutaminase derived from microorganism. Agric Biol Chem. 1989, 53: 2619–23.
3. Folk, J.E, Cole, P.W. Structural requirements of specific substrates for guinea pig liver transglutaminase. J. Biol. Chem. 1965, 240: 2951-9.
4. Steinknaus KH. Nutritional significance of fermented foods. Food Res Int 1994, 27: 259–67.
5. Motoki M, Kiyama A, Nonaka M, Tanaka H, Uchio R, Matsura A, et al. Novel transglutaminase manufacture for preparation of protein gelling compounds. Jpn Kokai Kokkyo Koho. 1989, JP 0217471.
6. Zhu Y, Rinzema A, Tramper J, Bol J. Microbial. Transglutaminase-a review on its production and application in food processing. Appl Microbiol Biotechnol 1995, 44: 277–82.
7. Folk J.E., Cole P.W. Mechanism of action of guinea pig liver transglutaminase. I. Purification and properties of the enzyme: identification of a functional cysteine essential for activity, J Biol Chem. 1966, 241: 5518–5525.
8. Folk J.E. Transglutaminases, Annu. Rev. Biochem. 1980, 49: 517–531.
9. Nonaka M, Sakamoto H, Toiguchi S, Kawajiri H, Soeda T,& Motoki M.. Sodiumcaseinate and skim milk gels formed by incubation with microbial transglutaminase. Journal of Food Science. 1992, 57: 1214-1218.
10. Laslo L. Transglutaminase. Molecular and Celluar Biochemistry. 1984, 58: 9-35.
11. Ikura K, Okumura K, Yoshikawa M, Sasaki R, Chiba H, Incorporation of lysyldipeptides into food proteins by transglutaminase. Agric. Biol. Chem. 1985, 49: 1877-85.
12. Sakamoto H, Yamazaki K, Kaga C, Yamamoto Y, Ito R, Kurosawa Y. Strength enhancement by addition of microbial transglutaminase during Chinese noodle processing. Nippon Shokuhin Kagaku Kaishi. 1996, 43: 598–602.
13. Kuraishi C, Sakamoto J, Soeda T. The usefulness of transglutaminase for food processing, biotechnology for improved foods and flavors. In: Takeoka GR, Teranishi R, Williams PJ, Kobayashi A. Biotechnology for improved foods and flavors. ACS symposium series. 1996, 637: 29–38.
14. Kumazawa Y, Sakamoto H, Kawajiri H, Seguro K, Motoki M. Determination ofε-(γ-glutamyl) lysine in several fish eggs and muscle proteins. Fish Sci.1996, 62: 331–332.
15.卢雨正,高卫东等.利用MTG酶提高精纺羊毛织物易护理性能.毛纺科技. 2004, 7: 5-7.
16. Ando H, Adachi M, Umeda K, Matsuura A, Nonaka M, Uchio R, et al. Purification and characterization of a novel transglutminase derived from microorganism. Agric Biol Chem. 1989, 53: 2613–7.
17. Motoki M, Nio N, & Takinami K. Functional properties of food proteins polymerized by transglutaminase. Agricultural and Biological Chemistry. 1984, 48: 1257–1261.
18. Motoki M, Seguro K, Transglutaminase and its use for. Food processing. Trends Food Sci. Technol. 1998, 9: 204-211.
19. Zheng M. Y, Du G. C, Chen J, et al. A temperature-shift strategy in batch microbial transglutaminase fermentation. Process Biochem. 2001, 36: 525-530.
20. Zheng M. Y, Du G. C, Chen J, et al. Modeling of temperature effects on batch microbial transglutaminase fermentation with Streptoverticillium mobaraense. World J. Microb. Biotechnol. 2002, 18: 767–771.
21. Zheng M. Y, Du G. C, Chen J, et al. pH control strategy of batch microbial transglutaminase production with Streptoverticillium mobaraense. Enzyme Microb. Technol. 2002, 31(4):477-481.
22.柏映国,燕国梁,堵国成,陈坚.Streptomyces hygroscopicus产谷氨酰胺转胺酶的摇瓶发酵条件优化.食品与发酵工业,2004年,30(2): 27-32.
23.张程杰.吸水链霉菌WSH03-13产谷氨酰胺转胺酶发酵条件优化及中试生产研究。江南大学硕士学位论文,2005.
24.程力,以氮源优化及调控酶原激活过程的手段促进Streptomyces hygroscopicus产谷氨酰胺转胺酶[D].江南大学硕士学位论文, 2007.
25. Washizu K, Ando K, Koikeda S, Hirose S, Matsnura A, Takagi H and Motoki M Biosci. Biotechnol. Biochem, 1994, 58(1): 82-87.
26. S. Takehana, K. Washizu, K. Ando, S. Koikeda, K. Takeuchi, H. Matui, M. Motoki, H. Takagi, Chemical synthesis of the gene for microbial transglutaminase from Streptoverticillium and its expression in Escherichia coli, Biosci. Biotechnol. Biochem, 1994, 58: 88–92.
27. Masayo Date, Kikuchi Yokoyama, Yukiko Umezawa, Hiroshi Matsui, Yoshimi Kikuchi High level expression of Streptomyces mobaraensis transglutaminase inCorynebacterium glutamicum using a chimeric pro-region from Streptomyces cinnamoneus transglutaminase. Journal of Biotechnology, 2004, 110: 219–226.
28.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.江南大学博士学位论文, 2008.
29.俞俊堂,唐孝宣.生物工艺学[M].上海:华东理工大学出版社, 1997, 75-107.
30. Grossowicz N, Wainfan E, Borek E et al. The enzymatic formation of hydroxamic acids from glutamine and asparagine. J Biol Chem., 1950, 187: 111-125.
31. Yin G L, He J M, Ren S X, et al. Production of vitamin C precursor 2-keto-L-gulonic acid from L-sorbose bu a novel bacterial component system of SCB329-SCB933 I. The biological characteristics of a novel bacterial component system of SCB329-SCB933 [J]. Industrial Microorganism, 1997, 24(1): 1 - 7.
32.顾其丰.生物化工原理[M].上海:上海科学技术出版社, 1997, 73-89.
33. Pirt S J. The dynamics of microbial processes: a personal view in microbial growth dynamics[M]. Tokyo: IRL Press, 1990, 28: 1-16.
34.陈坚,堵国成,李寅,等.发酵工程实验技术[M].北京:化学工业出版社, 2003, 199-239.
35. Zotzel J, Keller P, Fuchsbauer HL. Transglutaminase from Streptomyces mobaraensis is activated by an endogenous metalloprotease. Eur J Biochem, 2003, 270:3214–3222.
36. Zhu Y. Rinzema J. Tramper E. Fed-batch fermentation dealing with nitrogen limitation in microbial transglutaminase production by Stretoverticillium mobaraense. Appl. Microbiol Biotechnol. 1998, 49: 251-257.
37.任增亮, Streptomyces hygroscopicus谷氨酰胺转胺酶酶原基因的鉴定、表达及序列分析.江南大学硕士论文, 2007.
38.梅乐和,姚善泾,林东强.生化生产工艺学[M].北京:科学出版社, 1999, 186-201.
39.李寅,陈坚.发酵过程优化原理与实践[M].北京:化学工业出版社, 2002, 24-28.
40.蒋世春,吴建平,白骅.应用等离子体辐射技术选育柔红霉素产生菌—天兰淡红链霉菌[J].辐射研究与辐射工艺学报(JIANG Shi-chun, WU Jian-ping, BAI Hua. Selection of daunorubicin-producing strains S.coeruleorubidus by plasma radiation technology [J]. Journals Radiat Res Radiat Process), 2001, 19(2): 133–137.
2. Nonaka M, Tanaka H, Okiyama A, Motoki M, Ando H, Umeda K, et al. Polymerization of several proteins by Ca2+-independent transglutaminase derived from microorganism. Agric Biol Chem. 1989, 53: 2619–23.
3. Folk, J.E, Cole, P.W. Structural requirements of specific substrates for guinea pig liver transglutaminase. J. Biol. Chem. 1965, 240: 2951-9.
4. Steinknaus KH. Nutritional significance of fermented foods. Food Res Int 1994, 27: 259–67.
5. Motoki M, Kiyama A, Nonaka M, Tanaka H, Uchio R, Matsura A, et al. Novel transglutaminase manufacture for preparation of protein gelling compounds. Jpn Kokai Kokkyo Koho. 1989, JP 0217471.
6. Zhu Y, Rinzema A, Tramper J, Bol J. Microbial. Transglutaminase-a review on its production and application in food processing. Appl Microbiol Biotechnol 1995, 44: 277–82.
7. Folk J.E., Cole P.W. Mechanism of action of guinea pig liver transglutaminase. I. Purification and properties of the enzyme: identification of a functional cysteine essential for activity, J Biol Chem. 1966, 241: 5518–5525.
8. Folk J.E. Transglutaminases, Annu. Rev. Biochem. 1980, 49: 517–531.
9. Nonaka M, Sakamoto H, Toiguchi S, Kawajiri H, Soeda T,& Motoki M.. Sodiumcaseinate and skim milk gels formed by incubation with microbial transglutaminase. Journal of Food Science. 1992, 57: 1214-1218.
10. Laslo L. Transglutaminase. Molecular and Celluar Biochemistry. 1984, 58: 9-35.
11. Ikura K, Okumura K, Yoshikawa M, Sasaki R, Chiba H, Incorporation of lysyldipeptides into food proteins by transglutaminase. Agric. Biol. Chem. 1985, 49: 1877-85.
12. Sakamoto H, Yamazaki K, Kaga C, Yamamoto Y, Ito R, Kurosawa Y. Strength enhancement by addition of microbial transglutaminase during Chinese noodle processing. Nippon Shokuhin Kagaku Kaishi. 1996, 43: 598–602.
13. Kuraishi C, Sakamoto J, Soeda T. The usefulness of transglutaminase for food processing, biotechnology for improved foods and flavors. In: Takeoka GR, Teranishi R, Williams PJ, Kobayashi A. Biotechnology for improved foods and flavors. ACS symposium series. 1996, 637: 29–38.
14. Kumazawa Y, Sakamoto H, Kawajiri H, Seguro K, Motoki M. Determination ofε-(γ-glutamyl) lysine in several fish eggs and muscle proteins. Fish Sci.1996, 62: 331–332.
15.卢雨正,高卫东等.利用MTG酶提高精纺羊毛织物易护理性能.毛纺科技. 2004, 7: 5-7.
16. Ando H, Adachi M, Umeda K, Matsuura A, Nonaka M, Uchio R, et al. Purification and characterization of a novel transglutminase derived from microorganism. Agric Biol Chem. 1989, 53: 2613–7.
17. Motoki M, Nio N, & Takinami K. Functional properties of food proteins polymerized by transglutaminase. Agricultural and Biological Chemistry. 1984, 48: 1257–1261.
18. Motoki M, Seguro K, Transglutaminase and its use for. Food processing. Trends Food Sci. Technol. 1998, 9: 204-211.
19. Zheng M. Y, Du G. C, Chen J, et al. A temperature-shift strategy in batch microbial transglutaminase fermentation. Process Biochem. 2001, 36: 525-530.
20. Zheng M. Y, Du G. C, Chen J, et al. Modeling of temperature effects on batch microbial transglutaminase fermentation with Streptoverticillium mobaraense. World J. Microb. Biotechnol. 2002, 18: 767–771.
21. Zheng M. Y, Du G. C, Chen J, et al. pH control strategy of batch microbial transglutaminase production with Streptoverticillium mobaraense. Enzyme Microb. Technol. 2002, 31(4):477-481.
22.柏映国,燕国梁,堵国成,陈坚.Streptomyces hygroscopicus产谷氨酰胺转胺酶的摇瓶发酵条件优化.食品与发酵工业,2004年,30(2): 27-32.
23.张程杰.吸水链霉菌WSH03-13产谷氨酰胺转胺酶发酵条件优化及中试生产研究。江南大学硕士学位论文,2005.
24.程力,以氮源优化及调控酶原激活过程的手段促进Streptomyces hygroscopicus产谷氨酰胺转胺酶[D].江南大学硕士学位论文, 2007.
25. Washizu K, Ando K, Koikeda S, Hirose S, Matsnura A, Takagi H and Motoki M Biosci. Biotechnol. Biochem, 1994, 58(1): 82-87.
26. S. Takehana, K. Washizu, K. Ando, S. Koikeda, K. Takeuchi, H. Matui, M. Motoki, H. Takagi, Chemical synthesis of the gene for microbial transglutaminase from Streptoverticillium and its expression in Escherichia coli, Biosci. Biotechnol. Biochem, 1994, 58: 88–92.
27. Masayo Date, Kikuchi Yokoyama, Yukiko Umezawa, Hiroshi Matsui, Yoshimi Kikuchi High level expression of Streptomyces mobaraensis transglutaminase inCorynebacterium glutamicum using a chimeric pro-region from Streptomyces cinnamoneus transglutaminase. Journal of Biotechnology, 2004, 110: 219–226.
28.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.江南大学博士学位论文, 2008.
29.俞俊堂,唐孝宣.生物工艺学[M].上海:华东理工大学出版社, 1997, 75-107.
30. Grossowicz N, Wainfan E, Borek E et al. The enzymatic formation of hydroxamic acids from glutamine and asparagine. J Biol Chem., 1950, 187: 111-125.
31. Yin G L, He J M, Ren S X, et al. Production of vitamin C precursor 2-keto-L-gulonic acid from L-sorbose bu a novel bacterial component system of SCB329-SCB933 I. The biological characteristics of a novel bacterial component system of SCB329-SCB933 [J]. Industrial Microorganism, 1997, 24(1): 1 - 7.
32.顾其丰.生物化工原理[M].上海:上海科学技术出版社, 1997, 73-89.
33. Pirt S J. The dynamics of microbial processes: a personal view in microbial growth dynamics[M]. Tokyo: IRL Press, 1990, 28: 1-16.
34.陈坚,堵国成,李寅,等.发酵工程实验技术[M].北京:化学工业出版社, 2003, 199-239.
35. Zotzel J, Keller P, Fuchsbauer HL. Transglutaminase from Streptomyces mobaraensis is activated by an endogenous metalloprotease. Eur J Biochem, 2003, 270:3214–3222.
36. Zhu Y. Rinzema J. Tramper E. Fed-batch fermentation dealing with nitrogen limitation in microbial transglutaminase production by Stretoverticillium mobaraense. Appl. Microbiol Biotechnol. 1998, 49: 251-257.
37.任增亮, Streptomyces hygroscopicus谷氨酰胺转胺酶酶原基因的鉴定、表达及序列分析.江南大学硕士论文, 2007.
38.梅乐和,姚善泾,林东强.生化生产工艺学[M].北京:科学出版社, 1999, 186-201.
39.李寅,陈坚.发酵过程优化原理与实践[M].北京:化学工业出版社, 2002, 24-28.
40.蒋世春,吴建平,白骅.应用等离子体辐射技术选育柔红霉素产生菌—天兰淡红链霉菌[J].辐射研究与辐射工艺学报(JIANG Shi-chun, WU Jian-ping, BAI Hua. Selection of daunorubicin-producing strains S.coeruleorubidus by plasma radiation technology [J]. Journals Radiat Res Radiat Process), 2001, 19(2): 133–137.