产谷氨酰胺转胺酶茂源链轮丝菌诱变育种及发酵条件研究
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摘要
谷氨酰胺转胺酶(Transglutaminase,EC 2.3.2.13,简称TGase)可以催化多种蛋白质及氨基酸或谷氨酸残基之间的共价交联或酰基转移反应。由于该酶在食品、固定化酶、生物医药及纺织皮革加工等工业领域的应用,引起了人们极大的关注。
本研究以提高茂源链轮丝菌(Streptoverticillium mobaraense)03-10发酵生产TGase的产量为目标,对S.mobaraense 03-10进行诱变,筛选高产突变株,并进行发酵条件优化研究。主要内容如下：
(1)以S.mobaraense 03-10为出发菌株,采用大气压辉光放电的冷等离子体技术对链霉菌孢子进行诱变。根据双层平板法菌落显色及诱变处理后菌落形态差异快速筛选TGase高产突变株。突变率、正突变率分别达到42.8%和20.6%。最后复筛选育出具有较好遗传稳定性和形态稳定性的高产突变株G2-1,酶活达到2.73 U/mL,比出发菌株提高了82%。
(2)对突变株S.mobaraense 03-10 G2-1种子培养基、种子培养条件及发酵培养基进行了优化。优化后的结果为：种子培养最佳碳源为25 g/L可溶性淀粉,最佳的孢子接种量106个/mL,最佳的种龄及最佳的种子接种量分别为20 h和8%。发酵培养基中最佳钙离子为2g/L无水氯化钙,最佳单一碳源为30g/L甘油,优化后的摇瓶条件下酶活达到3.71 U/mL,与优化前相比(2.71 U/mL)提高了40%。
(3)考察了发酵培养基中混合碳源对发酵的影响。得出最佳的混合碳源为甘油与玉米淀粉；并比较了不同搅拌转速下不同混合碳源配比时3 L罐中发酵的情况,得出当搅拌转速为500 r/min,甘油与玉米淀粉比例为10：20时,酶活最高为3.54 U/mL。
(4)在3 L发酵罐上研究了不同pH控制策略和甘油流加策略对突变菌株发酵生产TGase的影响。确定发酵过程中pH控制策略为：初始pH7.4,发酵过程中用NH3·H20控制pH≥6.5；考察了不同初始甘油浓度及不同甘油恒速流加速率下对发酵过程的影响,确定了10-20 h控制甘油流加速率为1.67g/L/h,20-30 h流加速率为0.83g/L/h,30-40 h流加速率为0.41g/L/h的分阶段甘油流加策略,与优化前结果相比较(酶活2.07 U/mL,生产强度42.71 U/(L·h)),酶活和生产强度获得了很大的提高,分别达到3.69 U/mL,76.88u/(L·h)。
Transglutaminases are a family of enzymes (EC 2.3.2.13) that catalyse the formation of covalent cross-links between a free amine group and the y-carboxamide group of protein-bound or peptide-bound glutamine. It has gained interest due to its attractive potential applications in food sector, immobilization of enzymes, biomedical engineering, textiles and leather processing.
This study is mainly aimed at enhancing the production of TGase by Streptoverticillium mobaraense 03-10. Firstly we mutagenized and selected the TGase producing strain and then optimized fermentation conditions. The main results are listed as following:
(1) The atmospheric pressure glow discharge (APGD) plasma jet driven by a radio frequency (RF) power was used to treat the spores of S.mobaraense 03-10 for the selection of TGase producer. The mutant with high TGase production was quickly screened according to the formation of color on the double-layered plate and the different appearances of colonies. The total mutation rate was over 42.8% and the positive mutant rate was 20.6%. The obtained mutant G2-1 has good genetic and morphology stability and TGase activity reached 2.73 U/mL, which was 82% higher than that of original strain.
(2) The effects of environmental conditions on fermentation by the mutant strain S. mobaraense 03-10 G2-1 were optimized in flask culture. The proper culture conditions in seed medium were as the following:the carbon source was 25 g/L soluble starch, and inoculation spores was 106per mL, and the seed age was 20 h, and the inoculum size was 8%. The optimal fermentation conditions were 2 g/L CaCl2 and 30 g/L glycerol as single carbon source. The TGase activity in flask reached 3.71 U/mL, which was improved by 40%.
(3) The effects of different mixture carbon sources on fermentation were investigated. The optimal mixture carbon source were glycerol and corn starch in flask culture. Then compared the fermentation situations in different agitator speeds and different ratios of carbon sources in a 3 L stirred fermentor, the best results were that the agitator speed was 500 r/min, and the ratio was 10:20. The maximum activity was 3.54 U/mL.
(4) Influence of different pH and feeding strategies on fermentation in a 3 L stirred fermentor by the screened mutant were investigated. The optimal pH control strategy in fementation processing was as the following:the initial pH7.4 and using NH3·H2O to control pH≥6.5. Based on the optimal pH control strategy, the effect of different initial glycerol concentrations and glycerol feeding rates were studied. A three-stage feeding-shift strategy was proposed:feeding rate 1.67 g/L/h during 10～20 h,0.83 g/L/h during 20～30 h,0.41 g/L/h during 30～40 h. By applying this feeding-shift strategy in fermentation, the maximal activity and productivity were significantly improved and reached 3.69 U/mL and 76.88 U/(L-h) respectively, compared with the results of before optimization(2.07 U/mL and 42.71 U/(L-h)).
本研究以提高茂源链轮丝菌(Streptoverticillium mobaraense)03-10发酵生产TGase的产量为目标,对S.mobaraense 03-10进行诱变,筛选高产突变株,并进行发酵条件优化研究。主要内容如下：
(1)以S.mobaraense 03-10为出发菌株,采用大气压辉光放电的冷等离子体技术对链霉菌孢子进行诱变。根据双层平板法菌落显色及诱变处理后菌落形态差异快速筛选TGase高产突变株。突变率、正突变率分别达到42.8%和20.6%。最后复筛选育出具有较好遗传稳定性和形态稳定性的高产突变株G2-1,酶活达到2.73 U/mL,比出发菌株提高了82%。
(2)对突变株S.mobaraense 03-10 G2-1种子培养基、种子培养条件及发酵培养基进行了优化。优化后的结果为：种子培养最佳碳源为25 g/L可溶性淀粉,最佳的孢子接种量106个/mL,最佳的种龄及最佳的种子接种量分别为20 h和8%。发酵培养基中最佳钙离子为2g/L无水氯化钙,最佳单一碳源为30g/L甘油,优化后的摇瓶条件下酶活达到3.71 U/mL,与优化前相比(2.71 U/mL)提高了40%。
(3)考察了发酵培养基中混合碳源对发酵的影响。得出最佳的混合碳源为甘油与玉米淀粉；并比较了不同搅拌转速下不同混合碳源配比时3 L罐中发酵的情况,得出当搅拌转速为500 r/min,甘油与玉米淀粉比例为10：20时,酶活最高为3.54 U/mL。
(4)在3 L发酵罐上研究了不同pH控制策略和甘油流加策略对突变菌株发酵生产TGase的影响。确定发酵过程中pH控制策略为：初始pH7.4,发酵过程中用NH3·H20控制pH≥6.5；考察了不同初始甘油浓度及不同甘油恒速流加速率下对发酵过程的影响,确定了10-20 h控制甘油流加速率为1.67g/L/h,20-30 h流加速率为0.83g/L/h,30-40 h流加速率为0.41g/L/h的分阶段甘油流加策略,与优化前结果相比较(酶活2.07 U/mL,生产强度42.71 U/(L·h)),酶活和生产强度获得了很大的提高,分别达到3.69 U/mL,76.88u/(L·h)。
Transglutaminases are a family of enzymes (EC 2.3.2.13) that catalyse the formation of covalent cross-links between a free amine group and the y-carboxamide group of protein-bound or peptide-bound glutamine. It has gained interest due to its attractive potential applications in food sector, immobilization of enzymes, biomedical engineering, textiles and leather processing.
This study is mainly aimed at enhancing the production of TGase by Streptoverticillium mobaraense 03-10. Firstly we mutagenized and selected the TGase producing strain and then optimized fermentation conditions. The main results are listed as following:
(1) The atmospheric pressure glow discharge (APGD) plasma jet driven by a radio frequency (RF) power was used to treat the spores of S.mobaraense 03-10 for the selection of TGase producer. The mutant with high TGase production was quickly screened according to the formation of color on the double-layered plate and the different appearances of colonies. The total mutation rate was over 42.8% and the positive mutant rate was 20.6%. The obtained mutant G2-1 has good genetic and morphology stability and TGase activity reached 2.73 U/mL, which was 82% higher than that of original strain.
(2) The effects of environmental conditions on fermentation by the mutant strain S. mobaraense 03-10 G2-1 were optimized in flask culture. The proper culture conditions in seed medium were as the following:the carbon source was 25 g/L soluble starch, and inoculation spores was 106per mL, and the seed age was 20 h, and the inoculum size was 8%. The optimal fermentation conditions were 2 g/L CaCl2 and 30 g/L glycerol as single carbon source. The TGase activity in flask reached 3.71 U/mL, which was improved by 40%.
(3) The effects of different mixture carbon sources on fermentation were investigated. The optimal mixture carbon source were glycerol and corn starch in flask culture. Then compared the fermentation situations in different agitator speeds and different ratios of carbon sources in a 3 L stirred fermentor, the best results were that the agitator speed was 500 r/min, and the ratio was 10:20. The maximum activity was 3.54 U/mL.
(4) Influence of different pH and feeding strategies on fermentation in a 3 L stirred fermentor by the screened mutant were investigated. The optimal pH control strategy in fementation processing was as the following:the initial pH7.4 and using NH3·H2O to control pH≥6.5. Based on the optimal pH control strategy, the effect of different initial glycerol concentrations and glycerol feeding rates were studied. A three-stage feeding-shift strategy was proposed:feeding rate 1.67 g/L/h during 10～20 h,0.83 g/L/h during 20～30 h,0.41 g/L/h during 30～40 h. By applying this feeding-shift strategy in fermentation, the maximal activity and productivity were significantly improved and reached 3.69 U/mL and 76.88 U/(L-h) respectively, compared with the results of before optimization(2.07 U/mL and 42.71 U/(L-h)).
引文
1. Nonaka M, Tanaka H, Okiyama A,et al.Polymerization of several proteins by Ca2+ independent transglutaminase derived from microorganisms. Agricultural and Biological Chemistry,1989,53(10):2619-2623.
2. Lorand L, Conrad S M. Transglutaminases.Molecular and cellular biochemistry,1984, 58(1):9-35.
3. Nemes Z, Marekov L N, Fesus L, et al. A novel function for transglutaminase 1: Attachment of long-chain ω-hydroxyceramides to involucrin by ester bond formation. Proceedings of the National Academy of Sciences,1999,96(15):8402-8407.
4. Grossowicz N, Wainfan E, Borek E, et al. The enzymatic formation of hydroxamic acids from glutamine and asparagine. J Biol Chem,1950,187(1):111-125.
5. Bergamini C M, Signorini M, Barbato R, et al. Transglutaminase-catalyzed polymerization of troponin in vitro. Biochemical and biophysical research communications,1995,206(1): 201-206.
6. Pasternack R, Laurent H P, Kaiser A, et al. A Fluorescent Substrate of Transglutaminase for Detection and Characterization of Glutamine Acceptor Compounds. Analytical biochemistry, 1997,249(1):54-60.
7. Folk J E, Cole P W. Structural requirements of specific substrates for guinea pig liver trans-glutaminase. Journal of Biological Chemistry,1965,240(7):29-51.
8. Folk J E, Gross M. Mechanism of action of guinea pig liver transglutaminase. Journal of Biological Chemistry,1971,246(21):66-83.
9. Clarke D D, Neidle A, Sarkar N K, et al. Metabolic activity of protein amide groups. Arch Biochem Biophys,1957,71(1):277-279.
10. Folk J E. Transglutaminases. Annu Rev Biochem,1980,49:517-531.
11. Aeschlimann D, Paulsson M. Transglutaminases:protein cross-linking enzymes in tissues and body fluids. Thromb Haemost,1994,71(4):402-415.
12. Folk J E, Cole P W. Mechanism of action of guinea pig liver transglutaminase I. J Biol Chem,1966,241(23):5518-5525.
13. Zhu Y, Rinzema A, Tramper J, et al. Microbial transglutaminase-a review of its production and application in food processing. Applied Microbiology and Biotechnology,1995,44(3): 277-282.
14. Icekson I, Apelbaum A. Evidence for transglutaminase activity in plant tissue. Plant physiology,1987,84(4):972-974.
15. Serafini-Fracassini D, Del Duca S, Beninati S. Plant transglutaminases. Phytochemistry, 1995,40(2):355-365.
16. Ando H, Adachi H, Umeda K, et al. Purification and characteristics of a novel transglutaminase derived from microorganisms. Agricultural and Biological Chemistry,1989, 53(10):2613-2617.
17. Junqua M, Duran R, Gancet C, et al. Optimization of microbial transglutaminase production using experimental designs. Applied Microbiology and Biotechnology,1997, 48(6):730-734.
18. Suzanne Schleehauf Negus B.Sc, M.Scst. A Novel Microbial Transglutaminase Derived From Streptoverticillium baldccii[D]:[A thesis of degree of doctor]. Australia:Griffith University, Nathan Campus,2001.
19. Barros Soares L De, Assmann F. Purification and properties of a transglutaminase produced by a Bacillus circulans strain isolated from the Amazon environment. Biotechnology and applied biochemistry,2003,37:295-299.
20.燕国梁,堵国成,等.谷氨酰胺转胺酶发酵条件的优化研究[J].工业微生物,2003,33(1)：4-8.
21. Simon J.Tellez-Luis, Juan J. GonzaLez-cabriales, et al. Production of transglutaminase by Streptoverticillium ladakanum NRRL-3191 using glycerol as carbon source. Food Technology and Biotechnology,2004,42(2):75-81.
22. Lin Y S, Chao M L, et al. Cloning of the gene coding for transglutaminase from Streptomyces platensis and its expression in Streptomyces lividans. Process Biochemistry, 2006,41(3):519-524.
23. Liu X, Yang X, et al. Cloning of transglutaminase gene from Streptomyces fradiae and its enhanced expression in the original strain. Biotechnology Letters,2006,28(17):1319-1325.
24. Yu Y J, Wu S C, et al. Overproduction of soluble recombinant transglutaminase from Streptomyces netropsis in Escherichia coli. Applied Microbiology and Biotechnology,2008, 81 (3):523-532.
25. Du G C, Cui L, et al. Improvement of shrink-resistance and tensile strength of wool fabric treated with a novel microbial transglutaminasc from Streptomyces hygroscopicus. Enzyme and Microbial Technology,2007,40(7):1753-1757.
26. Clare D A, Gharst G, et al. Transglutaminase polymerization of peanut proteins. J. Agric. Food Chem,2007,55(2):432-438.
27. Mariniello L, Giosafatto C VI, et al. Synthesis and resistance to in vitro proteolysis of transglutaminase cross-linked phaseolin, the major storage protein from Phaseolus vulgaris. J. Agric. Food Chem,2007,55(12):4717-4721.
28. Mizuno A, Mitsuiki M, et al. Effect of transglutaminase treatment on the glass transition of soy protein. J. Agric. Food Chem,2000,48(8):3286-3291.
29. Autio K, Kruus K, et al. Kinetics of transglutaminase-induced cross-linking of wheat proteins in dough. J. Agric. Food Chem,2005,53(4):1039-1045.
30. Clare D A, Catignani G L, et al. Cross-linking and rheological changes of whey proteins treated with microbial transglutaminase. J. Agric. Food Chem,2004,52(5):1170-1176.
31. Nieuwenhuizen W F, Dekker H L, et al. Modification of Glutamine and Lysine Residues in Holo and Apo a-Lactalbumin with Microbial Transglutaminase. J. Agric. Food Chem,2003, 51 (24):7132-7139.
32. Date M, Yokoyama K, et al. Production of native-type Streptoverticillium mobaraense transglutaminase in Corynebacterium glutamicum. Applied and Environmental Microbiology, 2003,69(5):30-11.
33. Garcia Y, Wilkins B, et al. Towards development of a dermal rudiment for enhanced wound healing response.Biomaterials,2008,29(7):857-868.
34. Villalonga R. Thermal stabilization of trypsin by enzymatic modification with β-cyclodextrin derivatives. Biotechnol. Appl Biochem,2003,38:53-59.
35. Beninati S, Bergamini C M, et al. An overview of the first 50 years of transglutaminase research. Amino Acids,2009,36(4):591-598.
36. Cui L, Wang Q, et al. Transglutaminase-Mediated Crosslinking of Gelatin onto Wool Surfaces to Improve the Fabric Properties. Journal of Applied Polymer Science,2009,113(4): 2598-2604.
37. Portilla-Rivera O M, Tellez-Luis S J, et al. Production of Microbial Transglutaminase on Media Made from Sugar Cane Molasses and Glycerol. Food Technology and Biotechnology, 2009,47(1):19-26.
38.王灼维,王璋.土壤分离转谷氨酰胺酶生产菌株[J].食品与发酵工业,2003,29(4)：5-10.
39.宋敏,曹娟,等.不同菌落形态的链霉菌对产谷氨酰胺转胺酶的影响[J].广西农业生物科学,2008,27(4)：435-444.
40.祖海珍,陆兆新,等.转谷氨酰胺酶产生菌的筛选和鉴定[J].淮海工学院学报,2001,10(2)：44-46.
41. Date M, Yokoyama K I, Umezawa Y, et al. High level expression of Streptomyces mobaraensis transglutaminase in Corynebacterium glutamicum using a chimeric pro-region from Streptomyces cinnamoneus transglutaminase. Journal of Biotechnology,2004,110(3): 219-226.
42. Washizu K, Ando K, Koikeda S, et al. Molecular cloning of the gene for microbial transglutaminase from Streptoverticillium and its expression in Streptomyces lividans. Bioscience, biotechnology, and biochemistry,1994,58(1):82-87.
43. Yokoyama K, Nakamura N, Seguro K, et al. Overproduction of microbial transglutaminase in Escherichia coli, in vitro refolding, and characterization of the refolded form. Bioscience, biotechnology, and biochemistry,2000,64(6):1263-1270.
44. Taguchi S, Arakawa K, Yokoyama K, et al. Overexpression and purification of microbial protransglutaminase from Streptomyces cinnamoneum and in vitro processing by Streptomyces albogriseolus proteases. Journal of bioscience and bioengineering,2002,94(5): 478-481.
45. Zotzel J, Pasternack R, Pelzer C, et al. Activated transglutaminase from Streptomyces mobaraensis is processed by a tripeptidyl aminopeptidase in the final step. European Journal of Biochemistry,2003,270(20):4149-4155.
46. Zotzel J, Keller P, Fuchsbauer H L. Transglutaminase from Streptomyces mobaraensis is activated by an endogenous metalloprotease. European Journal of Biochemistry,2003, 270(15):3214-3222.
47. Marx C K, Hertel T C, Pietzsch M. Soluble expression of a pro-transglutaminase from Streptomyces mobaraensis in Escherichia coli. Enzyme and Microbial Technology,2007, 40(6):1543-1550.
48.崔艳华,张兰威.谷氨酰胺转氨酶研究进展[J].生物技术通报,2009,1：31-36.
49. Wu J W, Tsai C J, Jiang S T. Screening the microorganism and some factors for the production of transglutaminase. JOURNAL-CHINESE AGRICULTURAL CHEMICAL SOCIETY,1996,34:228-240.
50. Motoki M, Kumazawa Y. Recent research trends in transglutaminase technology for food processing. Food Science and Technology Research,2000,6(3):151-160.
51. Zhu Y, Tramper J. Novel applications for microbial transglutaminase beyond food process-ing. Trends in Biotechnology,2008,26(10):559-565.
52. Zhang D, Zhu Y, Chen J. Microbial Transglutaminase Production:Understanding the Mechanism. Biotechnology & genetic engineering reviews,2010,26:205-221.
53.王灼维,王璋,刘新征,等.产谷氨酰胺转胺酶菌株筛选方法初步探讨.第十届全国生物化工学术会议论文集,2002：425-428.
54.王璋,王灼维,莫湘筠.微生物转谷氨酰胺酶的生产菌种选育和发酵生产分析[J].生物加工过程,2003,1(1)：52-59.
55.崔国燕,胡青平.蛋白质交联-絮凝沉淀法筛选产转谷氨酰胺酶放线菌株的效果分析[J].沈阳农业大学学报,2008,39(5)：632-634.
56.王璋,王灼维,莫湘筠.微生物谷氨酰胺转胺酶生产菌株的育种研究[J].中国生物 工程杂志,2003,23(6)：1-5.
57. Li G, Li H P, Wang L Y, et al. Genetic effects of radio-frequency, atmospheric-pressure glow discharges with helium. Applied Physics Letters,2008,92:221-504.
58. Wang L Y, Huang Z L, Li G, et al. Novel mutation breeding method for Streptomyces avermitilis using an atmospheric pressure glow discharge plasma. Journal of Applied Microbiology,2010,108(3):851-858.
59.清华大学.大气压低温等离子体育种装置[P].中国专利,ZL 200820079382.1.2009.
60. Kieser T, Bibb M J, Buttner M J, et al. Practical streptomyces genetics[M].2 edition. England:The John Innes Foundation Norwich,2000:417-417.
61.孙伟伟,曹维强,王静.DNS法测定玉米秸秆中总糖[J].食品研究与开发,2006,27(6)：120-123.
62.张永生,高辉,王艳萍.克拉维酸发酵液中碳源—甘油含量的比色法测定[J].天津科技大学学报,2006,21(1)：15-17.
63.许晓娟.通过发酵控制策略及诱变选育来提高谷氨酰胺转胺酶酶活[D]：[硕士学位论文].无锡：江南大学生物工程学院,2009.
64. Li H P, Sun W T, Wang H B, et al. Electrical features of radio-frequency, atmospheric-pressure, bare-metallic-electrode glow discharges. Plasma Chemistry and Plasma Processing, 2007,27(5):529-545.
65.周德庆.微生物学教程[M].第二版.北京：高等教育出版社,2002.213-216.
66. Hopwood D A. Towards an Understanding of Gene Switching in Streptomyces, the Basis of Sporulation and Antibiotic Production. Proceedings of the Royal Society of London. Series B, Biological Sciences,1988,235(1279):121-138.
67.张东旭.吸水链霉菌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：江南大学生物工程学院,2008.
68.熊宗贵.发酵工艺原理[M].北京：中国医药科技出版社,2000.201-202.
69.常忠义,江波,王璋.培养基组成对轮枝链霉菌合成谷氨酰胺转胺酶的影响.无锡轻工大学学报,2001,20(1)：51-54.
70. Yan G L, Du G C, Li Y, et al. Enhancement of microbial transglutaminase production by Streptoverticillium mobaraense:application of a two-stage agitation speed control strategy. Process Biochemistry,2005,40(2):963-968.
71.蔡谨,孙章辉.补料发酵工艺的应用及其研究进展[J].工业微生物,2005,35(1)：42-48.
2. Lorand L, Conrad S M. Transglutaminases.Molecular and cellular biochemistry,1984, 58(1):9-35.
3. Nemes Z, Marekov L N, Fesus L, et al. A novel function for transglutaminase 1: Attachment of long-chain ω-hydroxyceramides to involucrin by ester bond formation. Proceedings of the National Academy of Sciences,1999,96(15):8402-8407.
4. Grossowicz N, Wainfan E, Borek E, et al. The enzymatic formation of hydroxamic acids from glutamine and asparagine. J Biol Chem,1950,187(1):111-125.
5. Bergamini C M, Signorini M, Barbato R, et al. Transglutaminase-catalyzed polymerization of troponin in vitro. Biochemical and biophysical research communications,1995,206(1): 201-206.
6. Pasternack R, Laurent H P, Kaiser A, et al. A Fluorescent Substrate of Transglutaminase for Detection and Characterization of Glutamine Acceptor Compounds. Analytical biochemistry, 1997,249(1):54-60.
7. Folk J E, Cole P W. Structural requirements of specific substrates for guinea pig liver trans-glutaminase. Journal of Biological Chemistry,1965,240(7):29-51.
8. Folk J E, Gross M. Mechanism of action of guinea pig liver transglutaminase. Journal of Biological Chemistry,1971,246(21):66-83.
9. Clarke D D, Neidle A, Sarkar N K, et al. Metabolic activity of protein amide groups. Arch Biochem Biophys,1957,71(1):277-279.
10. Folk J E. Transglutaminases. Annu Rev Biochem,1980,49:517-531.
11. Aeschlimann D, Paulsson M. Transglutaminases:protein cross-linking enzymes in tissues and body fluids. Thromb Haemost,1994,71(4):402-415.
12. Folk J E, Cole P W. Mechanism of action of guinea pig liver transglutaminase I. J Biol Chem,1966,241(23):5518-5525.
13. Zhu Y, Rinzema A, Tramper J, et al. Microbial transglutaminase-a review of its production and application in food processing. Applied Microbiology and Biotechnology,1995,44(3): 277-282.
14. Icekson I, Apelbaum A. Evidence for transglutaminase activity in plant tissue. Plant physiology,1987,84(4):972-974.
15. Serafini-Fracassini D, Del Duca S, Beninati S. Plant transglutaminases. Phytochemistry, 1995,40(2):355-365.
16. Ando H, Adachi H, Umeda K, et al. Purification and characteristics of a novel transglutaminase derived from microorganisms. Agricultural and Biological Chemistry,1989, 53(10):2613-2617.
17. Junqua M, Duran R, Gancet C, et al. Optimization of microbial transglutaminase production using experimental designs. Applied Microbiology and Biotechnology,1997, 48(6):730-734.
18. Suzanne Schleehauf Negus B.Sc, M.Scst. A Novel Microbial Transglutaminase Derived From Streptoverticillium baldccii[D]:[A thesis of degree of doctor]. Australia:Griffith University, Nathan Campus,2001.
19. Barros Soares L De, Assmann F. Purification and properties of a transglutaminase produced by a Bacillus circulans strain isolated from the Amazon environment. Biotechnology and applied biochemistry,2003,37:295-299.
20.燕国梁,堵国成,等.谷氨酰胺转胺酶发酵条件的优化研究[J].工业微生物,2003,33(1)：4-8.
21. Simon J.Tellez-Luis, Juan J. GonzaLez-cabriales, et al. Production of transglutaminase by Streptoverticillium ladakanum NRRL-3191 using glycerol as carbon source. Food Technology and Biotechnology,2004,42(2):75-81.
22. Lin Y S, Chao M L, et al. Cloning of the gene coding for transglutaminase from Streptomyces platensis and its expression in Streptomyces lividans. Process Biochemistry, 2006,41(3):519-524.
23. Liu X, Yang X, et al. Cloning of transglutaminase gene from Streptomyces fradiae and its enhanced expression in the original strain. Biotechnology Letters,2006,28(17):1319-1325.
24. Yu Y J, Wu S C, et al. Overproduction of soluble recombinant transglutaminase from Streptomyces netropsis in Escherichia coli. Applied Microbiology and Biotechnology,2008, 81 (3):523-532.
25. Du G C, Cui L, et al. Improvement of shrink-resistance and tensile strength of wool fabric treated with a novel microbial transglutaminasc from Streptomyces hygroscopicus. Enzyme and Microbial Technology,2007,40(7):1753-1757.
26. Clare D A, Gharst G, et al. Transglutaminase polymerization of peanut proteins. J. Agric. Food Chem,2007,55(2):432-438.
27. Mariniello L, Giosafatto C VI, et al. Synthesis and resistance to in vitro proteolysis of transglutaminase cross-linked phaseolin, the major storage protein from Phaseolus vulgaris. J. Agric. Food Chem,2007,55(12):4717-4721.
28. Mizuno A, Mitsuiki M, et al. Effect of transglutaminase treatment on the glass transition of soy protein. J. Agric. Food Chem,2000,48(8):3286-3291.
29. Autio K, Kruus K, et al. Kinetics of transglutaminase-induced cross-linking of wheat proteins in dough. J. Agric. Food Chem,2005,53(4):1039-1045.
30. Clare D A, Catignani G L, et al. Cross-linking and rheological changes of whey proteins treated with microbial transglutaminase. J. Agric. Food Chem,2004,52(5):1170-1176.
31. Nieuwenhuizen W F, Dekker H L, et al. Modification of Glutamine and Lysine Residues in Holo and Apo a-Lactalbumin with Microbial Transglutaminase. J. Agric. Food Chem,2003, 51 (24):7132-7139.
32. Date M, Yokoyama K, et al. Production of native-type Streptoverticillium mobaraense transglutaminase in Corynebacterium glutamicum. Applied and Environmental Microbiology, 2003,69(5):30-11.
33. Garcia Y, Wilkins B, et al. Towards development of a dermal rudiment for enhanced wound healing response.Biomaterials,2008,29(7):857-868.
34. Villalonga R. Thermal stabilization of trypsin by enzymatic modification with β-cyclodextrin derivatives. Biotechnol. Appl Biochem,2003,38:53-59.
35. Beninati S, Bergamini C M, et al. An overview of the first 50 years of transglutaminase research. Amino Acids,2009,36(4):591-598.
36. Cui L, Wang Q, et al. Transglutaminase-Mediated Crosslinking of Gelatin onto Wool Surfaces to Improve the Fabric Properties. Journal of Applied Polymer Science,2009,113(4): 2598-2604.
37. Portilla-Rivera O M, Tellez-Luis S J, et al. Production of Microbial Transglutaminase on Media Made from Sugar Cane Molasses and Glycerol. Food Technology and Biotechnology, 2009,47(1):19-26.
38.王灼维,王璋.土壤分离转谷氨酰胺酶生产菌株[J].食品与发酵工业,2003,29(4)：5-10.
39.宋敏,曹娟,等.不同菌落形态的链霉菌对产谷氨酰胺转胺酶的影响[J].广西农业生物科学,2008,27(4)：435-444.
40.祖海珍,陆兆新,等.转谷氨酰胺酶产生菌的筛选和鉴定[J].淮海工学院学报,2001,10(2)：44-46.
41. Date M, Yokoyama K I, Umezawa Y, et al. High level expression of Streptomyces mobaraensis transglutaminase in Corynebacterium glutamicum using a chimeric pro-region from Streptomyces cinnamoneus transglutaminase. Journal of Biotechnology,2004,110(3): 219-226.
42. Washizu K, Ando K, Koikeda S, et al. Molecular cloning of the gene for microbial transglutaminase from Streptoverticillium and its expression in Streptomyces lividans. Bioscience, biotechnology, and biochemistry,1994,58(1):82-87.
43. Yokoyama K, Nakamura N, Seguro K, et al. Overproduction of microbial transglutaminase in Escherichia coli, in vitro refolding, and characterization of the refolded form. Bioscience, biotechnology, and biochemistry,2000,64(6):1263-1270.
44. Taguchi S, Arakawa K, Yokoyama K, et al. Overexpression and purification of microbial protransglutaminase from Streptomyces cinnamoneum and in vitro processing by Streptomyces albogriseolus proteases. Journal of bioscience and bioengineering,2002,94(5): 478-481.
45. Zotzel J, Pasternack R, Pelzer C, et al. Activated transglutaminase from Streptomyces mobaraensis is processed by a tripeptidyl aminopeptidase in the final step. European Journal of Biochemistry,2003,270(20):4149-4155.
46. Zotzel J, Keller P, Fuchsbauer H L. Transglutaminase from Streptomyces mobaraensis is activated by an endogenous metalloprotease. European Journal of Biochemistry,2003, 270(15):3214-3222.
47. Marx C K, Hertel T C, Pietzsch M. Soluble expression of a pro-transglutaminase from Streptomyces mobaraensis in Escherichia coli. Enzyme and Microbial Technology,2007, 40(6):1543-1550.
48.崔艳华,张兰威.谷氨酰胺转氨酶研究进展[J].生物技术通报,2009,1：31-36.
49. Wu J W, Tsai C J, Jiang S T. Screening the microorganism and some factors for the production of transglutaminase. JOURNAL-CHINESE AGRICULTURAL CHEMICAL SOCIETY,1996,34:228-240.
50. Motoki M, Kumazawa Y. Recent research trends in transglutaminase technology for food processing. Food Science and Technology Research,2000,6(3):151-160.
51. Zhu Y, Tramper J. Novel applications for microbial transglutaminase beyond food process-ing. Trends in Biotechnology,2008,26(10):559-565.
52. Zhang D, Zhu Y, Chen J. Microbial Transglutaminase Production:Understanding the Mechanism. Biotechnology & genetic engineering reviews,2010,26:205-221.
53.王灼维,王璋,刘新征,等.产谷氨酰胺转胺酶菌株筛选方法初步探讨.第十届全国生物化工学术会议论文集,2002：425-428.
54.王璋,王灼维,莫湘筠.微生物转谷氨酰胺酶的生产菌种选育和发酵生产分析[J].生物加工过程,2003,1(1)：52-59.
55.崔国燕,胡青平.蛋白质交联-絮凝沉淀法筛选产转谷氨酰胺酶放线菌株的效果分析[J].沈阳农业大学学报,2008,39(5)：632-634.
56.王璋,王灼维,莫湘筠.微生物谷氨酰胺转胺酶生产菌株的育种研究[J].中国生物 工程杂志,2003,23(6)：1-5.
57. Li G, Li H P, Wang L Y, et al. Genetic effects of radio-frequency, atmospheric-pressure glow discharges with helium. Applied Physics Letters,2008,92:221-504.
58. Wang L Y, Huang Z L, Li G, et al. Novel mutation breeding method for Streptomyces avermitilis using an atmospheric pressure glow discharge plasma. Journal of Applied Microbiology,2010,108(3):851-858.
59.清华大学.大气压低温等离子体育种装置[P].中国专利,ZL 200820079382.1.2009.
60. Kieser T, Bibb M J, Buttner M J, et al. Practical streptomyces genetics[M].2 edition. England:The John Innes Foundation Norwich,2000:417-417.
61.孙伟伟,曹维强,王静.DNS法测定玉米秸秆中总糖[J].食品研究与开发,2006,27(6)：120-123.
62.张永生,高辉,王艳萍.克拉维酸发酵液中碳源—甘油含量的比色法测定[J].天津科技大学学报,2006,21(1)：15-17.
63.许晓娟.通过发酵控制策略及诱变选育来提高谷氨酰胺转胺酶酶活[D]：[硕士学位论文].无锡：江南大学生物工程学院,2009.
64. Li H P, Sun W T, Wang H B, et al. Electrical features of radio-frequency, atmospheric-pressure, bare-metallic-electrode glow discharges. Plasma Chemistry and Plasma Processing, 2007,27(5):529-545.
65.周德庆.微生物学教程[M].第二版.北京：高等教育出版社,2002.213-216.
66. Hopwood D A. Towards an Understanding of Gene Switching in Streptomyces, the Basis of Sporulation and Antibiotic Production. Proceedings of the Royal Society of London. Series B, Biological Sciences,1988,235(1279):121-138.
67.张东旭.吸水链霉菌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：江南大学生物工程学院,2008.
68.熊宗贵.发酵工艺原理[M].北京：中国医药科技出版社,2000.201-202.
69.常忠义,江波,王璋.培养基组成对轮枝链霉菌合成谷氨酰胺转胺酶的影响.无锡轻工大学学报,2001,20(1)：51-54.
70. Yan G L, Du G C, Li Y, et al. Enhancement of microbial transglutaminase production by Streptoverticillium mobaraense:application of a two-stage agitation speed control strategy. Process Biochemistry,2005,40(2):963-968.
71.蔡谨,孙章辉.补料发酵工艺的应用及其研究进展[J].工业微生物,2005,35(1)：42-48.