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食品与发酵工业  2020, Vol. 46 Issue (15): 1-7    DOI: 10.13995/j.cnki.11-1802/ts.023938
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
谷氨酰胺转氨酶活化蛋白酶在大肠杆菌中的表达及性质研究
高慧1,2, 刘松1,2,*
1(粮食发酵工艺与技术国家工程实验室(江南大学),江苏 无锡,214122)
2(江南大学 生物工程学院,江苏 无锡,214122;
Expression and characterization of transglutaminase-activating metalloprotease in Escherichia coli
GAO Hui1, 2, LIU Song1, 2, *
1(National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China)
2(School of Biotechnology,Jiangnan University,Wuxi 214122,China)
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摘要 茂源链霉菌(Streptomyces mobaraensis)谷氨酰胺转氨酶(transglutaminase,TGase)活化蛋白酶(activating metalloprotease,TAMEP)能够专一性切割TGase的酶原区,其高效制备对于重组TGase的生产有重要意义。将S. mobaraensis 来源的TAMEP成功表达于大肠杆菌。通过对信号肽类型、信号肽N端密码子及诱导表达条件的优化,使胞外TAMEP酶活力达到186.3 U/mL。酶学性质分析显示,重组TAMEP的最适反应温度和最适反应pH分别为55 ℃和7.0;TAMEP在30~50 ℃孵育60 min酶活力保持在50%以上,且在pH 6.2~8.9较稳定;0.133 μmol/L重组 TAMEP能够在30 min内使6.91 μmol/L TGase基本活化。研究结果为TAMEP规模化制备提供了生产菌株和基础数据。
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高慧
刘松
关键词:  谷氨酰胺转氨酶  活化蛋白酶  大肠杆菌  表达优化  酶学性质    
Abstract: Streptomyces mobaraensis transglutaminase (TGase)-activating metalloprotease (TAMEP) can specifically cleave the zymogen region of TGase to form the active form. In this study,TAMEP from S. mobaraensis was successfully expressed in Escherichia coli. Through the optimizations of signal peptide type,N-terminal codon of the signal peptide,and induction conditions,the extracellular activity of TAMEP reached 186.3 U/mL. As indicated by enzymatic analysis,that the optimal reaction temperature and pH of the recombinant TAMEP were 55 ℃ and pH 7.0,respectively. 50% activity of TAMEP was retained after incubating at 30-50 ℃ for 60 min,and the enzyme was also stable at pH 6.2-8.9. 0.133 μmol/L recombinant TAMEP can basically activated 6.91 μmol/L TGase in 30 minutes. The research results provided strains and basic data for the large-scale preparation of TAMEP.
Key words:  transglutaminase    activating protease    Escherichia coli    optimization of expression    enzymatic properties
收稿日期:  2020-03-12      修回日期:  2020-03-31           出版日期:  2020-08-15      发布日期:  2020-08-15      期的出版日期:  2020-08-15
作者简介:  硕士研究生(刘松副教授为通讯作者,E-mail: liusong@jiangnan.edu.cn)
引用本文:    
高慧,刘松. 谷氨酰胺转氨酶活化蛋白酶在大肠杆菌中的表达及性质研究[J]. 食品与发酵工业, 2020, 46(15): 1-7.
GAO Hui,LIU Song. Expression and characterization of transglutaminase-activating metalloprotease in Escherichia coli[J]. Food and Fermentation Industries, 2020, 46(15): 1-7.
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http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.023938  或          http://sf1970.cnif.cn/CN/Y2020/V46/I15/1
[1] YOKOYAMA K,NIO N,KIKUCHI Y.Properties and applications of microbial transglutaminase[J].Applied Microbiology and Biotechnology,2004,64(4):447-454.
[2] TAGHI G S M,SHIMA Y,CHRONAKIS I S.Microbial transglutaminase in noodle and pasta processing[J].Critical Reviews in Food Science and Nutrition,2019,59(2):313-327.
[3] SOO K C,THEAM S L.Bioengineering of microbial transglutaminase for biomedical applications[J].Applied Microbiology and Biotechnology,2019,103(7):2 973-2 984.
[4] DUARTE L,MATTE C R,BIZARRO C V,et al.Review transglutaminases: part II-industrial applications in food,biotechnology,textiles and leather products[J].World Journal of Microbiology & Biotechnology,2019,36(1):11.
[5] STROP P.Versatility of microbial transglutaminase[J].Bioconjugate Chemistry,2014,25(5):855-862.
[6] ANDO H,ADACHI M,UMEDA K,et al.Purification and characteristics of a novel transglutaminase derived from microorganisms[J].Agricultural and Biological Chemistry,1989,53(10):2 613-2 617.
[7] LIU S,ZHANG D,WANG M,et al.The pro-region of Streptomyces hygroscopicus transglutaminase affects its secretion by Escherichia coli[J].FEMS Microbiology Letters,2011,324(2):98-105.
[8] YURIMOTO H,YAMANE M,KIKUCHI Y,et al.The pro-peptide of Streptomyces mobaraensis transglutaminase functions in cis and in trans to mediate efficient secretion of active enzyme from methylotropic yeasts[J].Bioscience Biotechnology and Biochemistry,2004,68(10):2 058-2 069.
[9] LIU X,YANG X,XIE F,et al.On-column refolding and purification of transglutaminase from Streptomyces fradiae expressed as inclusion bodies in Escherichia coli[J].Protein Expression and Purification,2007,51(2):179-186.
[10] KIKUCHI Y,DATE M,YOKOYAMA K,et al.Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-Like protease from Streptomyces albogriseolus[J].Applied and Environmental Microbiology 2003,69(1):358-366.
[11] ZHAO X,SHAW A C,WANG J,et al.A novel high-throughput screening method for microbial transglutaminases with high specificity toward Gln141 of human growth hormone[J].Journal of Biomolecular Screening,2010,15(2):206-212.
[12] LIU S,ZHANG D,WANG M,et al.The order of expression is a key factor in the production of active transglutaminase in Escherichia coli by co-expression with its pro-peptide[J].Microbial Cell Factories,2011,10(1):112.
[13] 李鹏飞,孙红兵,游丽金,等.利用毕赤酵母系统直接分泌表达具有活性的谷氨酰胺转胺酶[J].生物工程学报,2013,29(2):180-188.
[14] ITAYA H,KIKUCHI Y.Secretion of Streptomyces mobaraensis pro-transglutaminase by coryneform bacteria[J].Applied Microbiology and Biotechnology,2008,78(4):621-625.
[15] DATE M,YOKOYAMA K I,UMEZAWA Y,et al.Highlevel expression of Streptomyces mobaraensis transglutaminase in Corynebacterium glutamicum using a chimeric pro-region from Streptomyces cinnamoneus transglutaminase[J].Journal of Biotechnology,2004,110(3):219-226.
[16] ZOTZEL J,KELLER P,Fuchsbauer H L.Transglutaminase from Streptomyces mobaraensis is activated by an endogenous metalloprotease[J].European Journal of Biochemistry,2003,270(15):3 214-3 222.
[17] ZOTZEL J,PASTERNACK R,PELZER C,et al.Activated transglutaminase from Streptomyces mobaraensis is processed by a tripeptidyl aminopeptidase in the final step[J].European Journal of Biochemistry,2003,270(20):4 149-4 155.
[18] TAGUCHI S,ARAKAWA K,YOKOYAMA K,et al.Overexpression and purification of microbial pro-transglutaminase from Streptomyces cinnamoneum and in vitro processing by Streptomyces albogriseolus proteases[J].Journal of Bioscience and Bioengineering,2002,94(5):478-481.
[19] MARX C K,HERTEL T C,PIETZSCH M.Purification and activation of a recombinant histidine-tagged pro-transglutaminase after soluble expression in Escherichia coli and partial characterization of the active enzyme[J].Enzyme and Microbial Technology,2008,42(7):568-575.
[20] JUETTNER N E,CLASSEN M,COLIN F,et al.Features of the transglutaminase-activating metalloprotease from Streptomyces mobaraensis DSM 40847 produced in Escherichia coli[J].Journal of Biotechnology,2018,281:115-122.
[21] ZHANG L,HAN X,ZHANG L.Determination of key protease for TGase yield from Streptomyces mobaraensis[J].Food and Fermentation Industries,2014,40(2):6-9.
[22] 任蕊蕊,刘松,李江华,等.分子改造提高谷氨酰胺转氨酶的催化活性[J].食品与发酵工业,2018,44(9):9-14.
[23] ZHAO W,LIU L,DU G,et al.A multifunctional tag with the ability to benefit the expression,purification,thermostability and activity of recombinant proteins[J].Journal of Biotechnology,2018,283:1-10.
[24] CHEN W,NIE Y,XU Y.Signal peptide-independent secretory expression and characterization of pullulanase from a newly isolated Klebsiella variicola SHN-1 in Escherichia coli[J].Applied Biochemistry and Biotechnology,2013,169(1):41-54.
[25] YANG H,LIU L,SHIN H,et al.Comparative analysis of heterologous expression,biochemical characterization optimal production of an alkalineα-amylase from alkaliphilic Alkalimonas amylolytica in Escherichia coli and Pichia pastoris[J].Biotechnology Progress,2013,29(1):39-47.
[26] CHEONG D E,KO K C,HAN Y,et al.Enhancing functional expression of heterologous proteins through random substitution of genetic codes in the 5′ coding region[J].Biotechnology and Bioengineering,2015,112(4):822-826.
[27] BOSTROM M,MARKLAND K,SANDEN A M,et al.Effect of substrate feed rate on recombinant protein secretion,degradation and inclusion body formation in Escherichia coli[J].Applied Microbiology and Biotechnology,2005,68(1):82-90.
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