谷氨酰胺转氨酶在Yarrowia lipolytica中的活性表达
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摘要
谷氨酰胺转氨酶(EC2. 3. 2. 13,Transglutaminase,TGase)是一种重要的食品酶。基于N-端酶原区对折叠的重要影响,TGase通常以无活性的酶原(pro-TGase)形式在异源宿主中表达。以茂源链霉菌(Streptomyces mobaraensis) pro-TGase为基因来源,重组解脂耶氏酵母(Yarrowia lipolytica po1h)为研究对象,通过在pro-TGase插入宿主Kex2蛋白酶识别位点(策略1)和共表达pro-TGase与谷氨酰胺转氨酶激活金属蛋白酶(TAMEP,策略2),使proTGase在Y. lipolytica中表达后被切除酶原区,而直接转化为活性TGase。摇瓶发酵结果显示,策略1和策略2构建得到的重组菌的TGase活力分别为5. 26 U/m L和6. 77 U/m L。酶学性质研究表明,策略1和策略2得到的重组菌的TGase比酶活、Km及kcat/Km均明显优于S. mobaraensis TGase。基于Y. lipolytica食品安全性,研究结果为TGase的工业化生产提供了新型高产菌种。
Transglutaminase( EC2. 3. 2. 13,TGase) is an important food enzyme. Since the N-terminal pro-region of TGase has great effect on its folding,TGase is often expressed as its non-active form( pro-TGase) in heterologous hosts. In this study,Streptomyces mobaraensis pro-TGase was used as the gene source and Yarrowia lipolytica po1 h was selected as host. By inserting the Kex2 protease recognition site in pro-TGase( strategy 1) and co-expressing pro-TGase and transglutaminase activating metalloprotease( TAMEP)( strategy 2),pro-TGase was expressed in Y. lipolytica at first,then the pro-region of pro-TGase was excised and the mature TGase was achieved. The results of shake flask fermentation showed that the TGase activities of recombinant strains constructed by strategy 1 and strategy 2 were 5. 26 U/m L and 6. 77 U/m L,respectively. Moreover,the enzymatic properties were further studied. The results showed that the specific activities,Kmand kcat/Kmof the recombinant TGase obtained by strategy 1 and strategy 2 were significantly higher than those of S. mobaraensis TGase. Based on the food safety of Y. lipolytica,the results provide two new high-yield strains for the industrial production of TGase.
Transglutaminase( EC2. 3. 2. 13,TGase) is an important food enzyme. Since the N-terminal pro-region of TGase has great effect on its folding,TGase is often expressed as its non-active form( pro-TGase) in heterologous hosts. In this study,Streptomyces mobaraensis pro-TGase was used as the gene source and Yarrowia lipolytica po1 h was selected as host. By inserting the Kex2 protease recognition site in pro-TGase( strategy 1) and co-expressing pro-TGase and transglutaminase activating metalloprotease( TAMEP)( strategy 2),pro-TGase was expressed in Y. lipolytica at first,then the pro-region of pro-TGase was excised and the mature TGase was achieved. The results of shake flask fermentation showed that the TGase activities of recombinant strains constructed by strategy 1 and strategy 2 were 5. 26 U/m L and 6. 77 U/m L,respectively. Moreover,the enzymatic properties were further studied. The results showed that the specific activities,Kmand kcat/Kmof the recombinant TGase obtained by strategy 1 and strategy 2 were significantly higher than those of S. mobaraensis TGase. Based on the food safety of Y. lipolytica,the results provide two new high-yield strains for the industrial production of TGase.
引文
[1]UMEZAWA Y,OHTSUKA T,YOKOYAMA K,et al. Comparison of enzymatic properties of microbial transglutaminase from Streptomyces sp.[J]. Food Sci Technol Res,2002,8(2):113-118.
[2]KIELISZEK M,MISIEWICZ A. Microbial transglutaminase and its application in the food industry. A review[J]. Folia Microbiol,2014,59(3):241-50.
[3]SANTHI D,KALAIKANNAN A,MALAIRAJ P,et al. Application of microbial transglutaminase in meat foods:a review[J]. Critical Reviews in Food Science and Nutrition,2017,57(10):2071-2076.
[4]TATSUKAWA H,FURUTANI Y,HITOMI K,et al. Transglutaminase 2 has opposing roles in the regulation of cellular functions as well as cell growth and death[J]. Cell Death Dis,2016,7(6):e2244.
[5]MARINIELLO L,PORTA R,SORRENTINO A,et al. Transglutaminase-mediated macromolecular assembly:production of conjugates for food and pharmaceutical applications[J]. Amino Acids,2014,46(3):767-776.
[6]ECKERT R L,KAARTINEN M T,NURMINSKAYA M,et al.Transglutaminase regulation of cell function[J]. Physiol Rev,2014,94(2):383-417.
[7]KUO T F,TATSUKAWA H,KOJIMA S. New insights into the functions and localization of nuclear transglutaminase 2[J]. Febs J,2011,278(24):4756-4767.
[8]FU J,SU J,WANG P,et al. Enzymatic processing of protein-based fibers[J]. Applied Microbiology and Biotechnology, 2015,99(24):10387-10397.
[9]GRIFFIN M,CASADIO R,BERGAMINI C M. Transglutaminases:nature's biological glues[J]. Biochem J,2002,368(Pt 2):377-396.
[10]ITAYA H,KIKUCHI Y. Secretion of Streptomyces mobaraensis protransglutaminase by coryneform bacteria[J]. Applied Microbiology and Biotechnology,2008,78(4):621-625.
[11]刘松. Streptomyces hygroscopicus谷氨酰胺转胺酶的异源表达研究[D].无锡:江南大学,2011.
[12]PASTERNACK R,DORSCH S,OTTERBACH J T,et al. Bacterial pro-transglutaminase from Streptoverticillium mobaraense—purification,characterisation and sequence of the zymogen[J]. Eur J Biochem,1998,257(3):570-576.
[13]SOMMER C,HERTEL T C,SCHMELZER C E,et al. Investigations on the activation of recombinant microbial pro-transglutaminase:in contrast to proteinase K,dispase removes the histidine-tag[J]. Amino Acids,2012,42(2-3):997-1006.
[14]KIKUCHI Y,DATE M,YOKOYAMA K,et al. Secretion of activeform 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.
[15]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 Methylotrophic yeasts[J]. Bioscience,Biotechnology,and Biochemistry,2004,68(10):2058-2069.
[16]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.
[17]KRZYCZKOWSKA J,FABISZEWSKA A U. Yarrowia lipolyticanon-conventional yeast in biotechnology[J]. Postep Mikrobiol,2015,54(1):33-43.
[18]DULERMO R,BRUNEL F,DULERMO T,et al. Using a vector pool containing variable-strength promoters to optimize protein production in Yarrowia lipolytica[J]. Microb Cell Fact,2017,16(1):31.
[19]ZOTZEL J,PASTERNACK R,PELZER C,et al. Activated transglutaminase from Streptomyces mobaraensis is processed by a tripeptidyl aminopeptidase in the final step[J]. Eur J Biochem,2003,270(20):4149-55.
[20]ROCKWELL N C,FULLER R S. Interplay between S-1 and S-4 subsites in Kex2 protease:Kex2 exhibits dual specificity for the P-4 side chain[J]. Biochemistry,1998,37(10):3386-3391.
[21]LIU S,WAN D,WANG M,et al. Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization[J]. BMC Biotechnology,2015,15:75.
[22]XUAN J W,FOURNIER P,GAILLARDIN C. Cloning of the LYS5gene encoding saccharopine dehydrogenase from the yeast Yarrowia lipolytica by target integration[J]. Current Genetics, 1988,14(1):15-21.
[23]ZHANG D,WANG M,DU G,et al. Surfactant protein of the Streptomyces subtilisin inhibitor family inhibits transglutaminase activation in Streptomyces hygroscopicus[J]. J Agric Food Chem,2008,56(9):3403-3408.
[24]PASTERNACK R,LAURENT H P,RUETH T,et al. A fluorescent substrate of transglutaminase for detection and characterization of glutamine acceptor compounds[J]. Anal Biochem, 1997,249(1):54-60.
[25]TORRES F A,VILACA R,PEPE DE MORAES L M,et al. Expression of a kexin-like gene from the human pathogenic fungus Paracoccidioides brasiliensis in Saccharomyces cerevisiae[J]. Medical Mycology,2008,46(4):385-388.
[26]RICHARD M,QUIJANO R R,BEZZATE S,et al. Tagging morphogenetic genes by insertional mutagenesis in the yeast Yarrowia lipolytica[J]. Journal of Bacteriology,2001,183(10):3098-3107.
[27]SKROPETA D. The effect of individual N-glycans on enzyme activity[J]. Bioorganic and Medicinal Chemistry,2009,17(7):2645-2653.
[28]王小艳.糖基化设计对β-葡萄糖醛酸苷酶结构和性能的影响[D].天津:天津大学,2016.
[29]YANG M T,CHANG C H,WANG J M,et al. Crystal structure and inhibition studies of transglutaminase from Streptomyces mobaraense[J]. The Journal of Biological Chemistry,2011,286(9):7301-7307.
[2]KIELISZEK M,MISIEWICZ A. Microbial transglutaminase and its application in the food industry. A review[J]. Folia Microbiol,2014,59(3):241-50.
[3]SANTHI D,KALAIKANNAN A,MALAIRAJ P,et al. Application of microbial transglutaminase in meat foods:a review[J]. Critical Reviews in Food Science and Nutrition,2017,57(10):2071-2076.
[4]TATSUKAWA H,FURUTANI Y,HITOMI K,et al. Transglutaminase 2 has opposing roles in the regulation of cellular functions as well as cell growth and death[J]. Cell Death Dis,2016,7(6):e2244.
[5]MARINIELLO L,PORTA R,SORRENTINO A,et al. Transglutaminase-mediated macromolecular assembly:production of conjugates for food and pharmaceutical applications[J]. Amino Acids,2014,46(3):767-776.
[6]ECKERT R L,KAARTINEN M T,NURMINSKAYA M,et al.Transglutaminase regulation of cell function[J]. Physiol Rev,2014,94(2):383-417.
[7]KUO T F,TATSUKAWA H,KOJIMA S. New insights into the functions and localization of nuclear transglutaminase 2[J]. Febs J,2011,278(24):4756-4767.
[8]FU J,SU J,WANG P,et al. Enzymatic processing of protein-based fibers[J]. Applied Microbiology and Biotechnology, 2015,99(24):10387-10397.
[9]GRIFFIN M,CASADIO R,BERGAMINI C M. Transglutaminases:nature's biological glues[J]. Biochem J,2002,368(Pt 2):377-396.
[10]ITAYA H,KIKUCHI Y. Secretion of Streptomyces mobaraensis protransglutaminase by coryneform bacteria[J]. Applied Microbiology and Biotechnology,2008,78(4):621-625.
[11]刘松. Streptomyces hygroscopicus谷氨酰胺转胺酶的异源表达研究[D].无锡:江南大学,2011.
[12]PASTERNACK R,DORSCH S,OTTERBACH J T,et al. Bacterial pro-transglutaminase from Streptoverticillium mobaraense—purification,characterisation and sequence of the zymogen[J]. Eur J Biochem,1998,257(3):570-576.
[13]SOMMER C,HERTEL T C,SCHMELZER C E,et al. Investigations on the activation of recombinant microbial pro-transglutaminase:in contrast to proteinase K,dispase removes the histidine-tag[J]. Amino Acids,2012,42(2-3):997-1006.
[14]KIKUCHI Y,DATE M,YOKOYAMA K,et al. Secretion of activeform 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.
[15]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 Methylotrophic yeasts[J]. Bioscience,Biotechnology,and Biochemistry,2004,68(10):2058-2069.
[16]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.
[17]KRZYCZKOWSKA J,FABISZEWSKA A U. Yarrowia lipolyticanon-conventional yeast in biotechnology[J]. Postep Mikrobiol,2015,54(1):33-43.
[18]DULERMO R,BRUNEL F,DULERMO T,et al. Using a vector pool containing variable-strength promoters to optimize protein production in Yarrowia lipolytica[J]. Microb Cell Fact,2017,16(1):31.
[19]ZOTZEL J,PASTERNACK R,PELZER C,et al. Activated transglutaminase from Streptomyces mobaraensis is processed by a tripeptidyl aminopeptidase in the final step[J]. Eur J Biochem,2003,270(20):4149-55.
[20]ROCKWELL N C,FULLER R S. Interplay between S-1 and S-4 subsites in Kex2 protease:Kex2 exhibits dual specificity for the P-4 side chain[J]. Biochemistry,1998,37(10):3386-3391.
[21]LIU S,WAN D,WANG M,et al. Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization[J]. BMC Biotechnology,2015,15:75.
[22]XUAN J W,FOURNIER P,GAILLARDIN C. Cloning of the LYS5gene encoding saccharopine dehydrogenase from the yeast Yarrowia lipolytica by target integration[J]. Current Genetics, 1988,14(1):15-21.
[23]ZHANG D,WANG M,DU G,et al. Surfactant protein of the Streptomyces subtilisin inhibitor family inhibits transglutaminase activation in Streptomyces hygroscopicus[J]. J Agric Food Chem,2008,56(9):3403-3408.
[24]PASTERNACK R,LAURENT H P,RUETH T,et al. A fluorescent substrate of transglutaminase for detection and characterization of glutamine acceptor compounds[J]. Anal Biochem, 1997,249(1):54-60.
[25]TORRES F A,VILACA R,PEPE DE MORAES L M,et al. Expression of a kexin-like gene from the human pathogenic fungus Paracoccidioides brasiliensis in Saccharomyces cerevisiae[J]. Medical Mycology,2008,46(4):385-388.
[26]RICHARD M,QUIJANO R R,BEZZATE S,et al. Tagging morphogenetic genes by insertional mutagenesis in the yeast Yarrowia lipolytica[J]. Journal of Bacteriology,2001,183(10):3098-3107.
[27]SKROPETA D. The effect of individual N-glycans on enzyme activity[J]. Bioorganic and Medicinal Chemistry,2009,17(7):2645-2653.
[28]王小艳.糖基化设计对β-葡萄糖醛酸苷酶结构和性能的影响[D].天津:天津大学,2016.
[29]YANG M T,CHANG C H,WANG J M,et al. Crystal structure and inhibition studies of transglutaminase from Streptomyces mobaraense[J]. The Journal of Biological Chemistry,2011,286(9):7301-7307.