茂源链霉菌MTG基因pMA5载体的构建及鉴定
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摘要
[目的]提高MTG产量并实现简便有效的纯化过程,以枯草芽孢杆菌为宿主构建pMA5_(mtg)重组质粒。[方法]通过提取茂源链霉菌的基因组DNA,扩增mtg基因片段,再将异源信号肽与mtg基因片段进行融合,融合片段和pMA5质粒双酶切后连接,转化到大肠杆菌感受态细胞中,菌落PCR和测序获得阳性单克隆。然后将pMA5_(mtg)重组质粒转入枯草芽孢杆菌感受态中,通过抗性筛选和双酶切鉴定获取重组菌株。[结果]成功扩增出mtg基因片段,构建出重组质粒pMA5_(mtg)。[结论]获得pMA5_(mtg)重组表达菌株,为MTG的纯化及其未来生物工程的改造提供了有效的方法。
[Objective]In order to increase the MTG yield and achieve a simple and effective purification process,the recombinant vector pMA5_(mtg) was constructed and expressed in Bacillus subtilis.[Method]The mtg gene fragment was amplified from the genomic DNA of Streptoverticillium mobaraensis,and the heterologous signal peptide was fused in front of the mtg gene fragment.The fusion fragment and the pMA5 vector were digested and ligated,and then transformed into E.coli competent cells.The positive strains were identified by PCR and extracted to check by DNA sequencing.The recombinant vector pMA5_(mtg) was then transformed into the competent cells of B.subtilis,then the recombinant strain was obtained by resistance screening and double restriction enzyme digestion.[Result]The mtg gene fragment was successfully amplified,and the recombinant vector pMA5_(mtg) was constructed.[Conclusion]The recombinant expression strain of pMA5_(mtg) was obtained,which provided an effective method for the purification of MTG and its future bioengineering transformation.
[Objective]In order to increase the MTG yield and achieve a simple and effective purification process,the recombinant vector pMA5_(mtg) was constructed and expressed in Bacillus subtilis.[Method]The mtg gene fragment was amplified from the genomic DNA of Streptoverticillium mobaraensis,and the heterologous signal peptide was fused in front of the mtg gene fragment.The fusion fragment and the pMA5 vector were digested and ligated,and then transformed into E.coli competent cells.The positive strains were identified by PCR and extracted to check by DNA sequencing.The recombinant vector pMA5_(mtg) was then transformed into the competent cells of B.subtilis,then the recombinant strain was obtained by resistance screening and double restriction enzyme digestion.[Result]The mtg gene fragment was successfully amplified,and the recombinant vector pMA5_(mtg) was constructed.[Conclusion]The recombinant expression strain of pMA5_(mtg) was obtained,which provided an effective method for the purification of MTG and its future bioengineering transformation.
引文
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[2] GASPAR A L C,DE GóES-FAVONI S P.Action of microbial transglutaminase (MTGase) in the modification of food proteins:A review[J].Food Chem,2015,171:315-322.
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[6] LIU L,LIU Y F,SHIN H D,et al.Developing Bacillus spp.as a cell factory for production of microbial enzymes and industrially important biochemicals in the context of systems and synthetic biology[J].Appl Microbiol Biotechnol,2013,97(14):6113-6127.
[7] GRIFFIN M,CASADIO R,BERGAMINI C M.Transglutaminases:Nature’s biological glues[J].Biochem J,2002,368(Pt 2):377-396.
[8] HONG G P,XIONG Y L.Microbial transglutaminase-induced structural and rheological changes of cationic and anionic myofibrillar proteins[J].Meat Sci,2012,91(1):36-42.
[9] MACEDO J A,SETTE L D,SATO H H.Purification and characterization of a new transglutaminase from Streptomyces sp.isolated in Brazilian soil[J].J Food Biochem,2011,35(4):1361-1372.
[10] MARX C K,HERTEL T C,PIETZSCH M.Soluble expression of a pro-transglutaminase from Streptomyces mobaraensis in Escherichia coli[J].Enzyme Microb Technol,2007,40(6):1543-1550.
[11] ITAYA H,KIKUCHI Y.Secretion of Streptomyces mobaraensis pro-transglutaminase by coryneform bacteria[J].Appl Microbiol Biotechnol,2008,78(4):621-625.
[2] GASPAR A L C,DE GóES-FAVONI S P.Action of microbial transglutaminase (MTGase) in the modification of food proteins:A review[J].Food Chem,2015,171:315-322.
[3] JAROS D,PARTSCHEFELD C,HENLE T,et al.Transglutaminase in dairy products:Chemistry,physics,applications[J].J Texture Stud,2006,37(2):113-155.
[4] YANG M T,CHANG C H,WANG J M,et al.Crystal structure and inhibition studies of transglutaminase from Streptomyces mobaraense[J].J Biol Chem,2011,286(9):7301-7307.
[5] GUERRA-RODRíGUEZ E,VáZQUEZ M.Evaluation of a novel low-cost culture medium containing exclusively milk,potato and glycerol for microbial transglutaminase production by Streptomyces mobaraensis[J].Chem Eng Res Des,2014,92(4):784-791.
[6] LIU L,LIU Y F,SHIN H D,et al.Developing Bacillus spp.as a cell factory for production of microbial enzymes and industrially important biochemicals in the context of systems and synthetic biology[J].Appl Microbiol Biotechnol,2013,97(14):6113-6127.
[7] GRIFFIN M,CASADIO R,BERGAMINI C M.Transglutaminases:Nature’s biological glues[J].Biochem J,2002,368(Pt 2):377-396.
[8] HONG G P,XIONG Y L.Microbial transglutaminase-induced structural and rheological changes of cationic and anionic myofibrillar proteins[J].Meat Sci,2012,91(1):36-42.
[9] MACEDO J A,SETTE L D,SATO H H.Purification and characterization of a new transglutaminase from Streptomyces sp.isolated in Brazilian soil[J].J Food Biochem,2011,35(4):1361-1372.
[10] MARX C K,HERTEL T C,PIETZSCH M.Soluble expression of a pro-transglutaminase from Streptomyces mobaraensis in Escherichia coli[J].Enzyme Microb Technol,2007,40(6):1543-1550.
[11] ITAYA H,KIKUCHI Y.Secretion of Streptomyces mobaraensis pro-transglutaminase by coryneform bacteria[J].Appl Microbiol Biotechnol,2008,78(4):621-625.