拉达克轮丝菌TGase在大肠杆菌中的分子改造
|
摘要
【背景】谷氨酰胺转氨酶是一种能够催化酰基转移反应的酶,催化各种蛋白质分子之间或之内发生交联反应,在食品、化妆品、医药等领域具有重要的潜在价值。【目的】克隆来自拉达克轮丝菌(Streptoverticillium ladakanum) B1的谷氨酰胺转氨酶(TGase)基因并对其进行分子改造,使其在大肠杆菌中获得高效异源表达。【方法】分别克隆来自拉达克轮丝菌谷氨酰胺转氨酶的自身前导肽(pro)和除前导肽以外的成熟谷氨酰胺转氨酶(TGase)基因,以pET-22b为表达载体构建pro、TGase共表达和融合表达两种表达模式,在这两种表达模式的基础上进一步用定点突变的方法对成熟TGaseN端前4个氨基酸进行改造,检测不同表达模式以及突变对酶活的影响。【结果】当采用前导肽与TGase共表达时,可以直接得到活性形式的TGase,比酶活达到37.71 U/mg。在融合表达的基础上,将TGaseN端前3个氨基酸DSD突变为AAA,比酶活达到14.04U/mg,相较于原始表达模式提高了14.05%。【结论】前导肽与TGase共表达可以直接产生活性TGase,对于融合表达模式,合适位点的突变有利于提高TGase酶活。
[Background] Transglutaminase(TGase) is an enzyme that catalyzes the acyl transfer reaction,which can catalyze cross-linking reactions between or within various protein molecules, and has important potential value in the food, cosmetics, medicine and other fields. [Objective] Molecular engineering and site-directed mutagenesis were carried out by cloning glutamyl transferase from Streptoverticillium ladakanum B1 to obtain efficient heterologous expression in E. coli. [Methods] The gene of pro and TG from Streptoverticillium ladakanum were cloned into pET-22 b to generate co-expression and fusion expression, respectively. Based on these two expression patterns, the first four amino acids of the mature TGase N-terminal were modified by site-directed mutagenesis to detect the effect of different expression patterns and mutation on enzyme activity. [Results] When co-expression of the pro-peptide with TGase,the active form of TGase can be obtained directly, and the specific activity reaches 37.71 U/mg. Based on the fusion expression, the first three amino acid DSD of the N-terminal of TGase were mutated to AAA,and the specific enzyme activity reached 14.04 U/mg, which was 14.05% higher than the original expression pattern. [Conclusion] The co-expression of pro-peptide with TGase can directly produce the active form of TGase. For the fusion expression pattern, the mutation of the appropriate site is beneficial to increase the TGase activity.
[Background] Transglutaminase(TGase) is an enzyme that catalyzes the acyl transfer reaction,which can catalyze cross-linking reactions between or within various protein molecules, and has important potential value in the food, cosmetics, medicine and other fields. [Objective] Molecular engineering and site-directed mutagenesis were carried out by cloning glutamyl transferase from Streptoverticillium ladakanum B1 to obtain efficient heterologous expression in E. coli. [Methods] The gene of pro and TG from Streptoverticillium ladakanum were cloned into pET-22 b to generate co-expression and fusion expression, respectively. Based on these two expression patterns, the first four amino acids of the mature TGase N-terminal were modified by site-directed mutagenesis to detect the effect of different expression patterns and mutation on enzyme activity. [Results] When co-expression of the pro-peptide with TGase,the active form of TGase can be obtained directly, and the specific activity reaches 37.71 U/mg. Based on the fusion expression, the first three amino acid DSD of the N-terminal of TGase were mutated to AAA,and the specific enzyme activity reached 14.04 U/mg, which was 14.05% higher than the original expression pattern. [Conclusion] The co-expression of pro-peptide with TGase can directly produce the active form of TGase. For the fusion expression pattern, the mutation of the appropriate site is beneficial to increase the TGase activity.
引文
[1]Folk JE.Transglutaminases[J].Annual Review of Biochemistry,1980,49:517-531
[2]Li QZ,Gui P,Huang Z,et al.Effect of transglutaminase on quality and gel properties of pork and fish mince mixtures[J].Journal of Texture Studies,2018,49(1):56-64
[3]Sárdy M,Kárpáti S,Merkl B,et al.Epidermal transglutaminase(TGase 3)is the autoantigen of dermatitis herpetiformis[J].Journal of Experimental Medicine,2002,195(6):747-757
[4]Han X,Yu YY,Wang Q,et al.Facilitation of pretreatment for TGase catalyzingε-PLL grafted wool fabrics[J].New Chemical Materials,2015,43(3):92-95(in Chinese)韩雪,余圆圆,王强,等.预处理对TGase催化羊毛接枝ε-PLL的促进作用[J].化工新型材料,2015,43(3):92-95
[5]Zhao HM,Wang MX,Mou ZC,et al.Study on transglutiminase in food industry[J].Food Science and Technology,2008,33(9):137-141(in Chinese)赵华梅,王曼霞,牟志春,等.食品工业中的谷氨酰胺转氨酶及其酶制剂[J].食品科技,2008,33(9):137-141
[6]Yokoyama K,Nio N,Kikuchi Y.Properties and applications of microbial transglutaminase[J].Applied Microbiology and Biotechnology,2004,64(4):447-454
[7]Kobayashi K,Hashiguchi K,Yokozeki K,et al.Molecular cloning of the transglutaminase gene from Bacillus subtilis and its expression in Escherichia coli[J].Bioscience,Biotechnology,and Biochemistry,1998,62(6):1109-1114
[8]Lin YS,Chao ML,Liu CH,et al.Cloning and expression of the transglutaminase gene from Streptoverticillium ladakanum,in Streptomyces lividans[J].Process Biochemistry,2004,39(5):591-598
[9]Ando H,Adachi M,Umeda K,et al.Purification and characteristics of a novel transglutaminase derived from microorganisms[J].Agricultural and Biological Chemistry,2006,53(10):2613-2617
[10]Kawai M,Takehana S,Takagi H.High-level expression of the chemically synthesized gene for microbial transglutaminase from Streptoverticillium in Escherichia coli[J].Bioscience,Biotechnology,and Biochemistry,2014,61(5):830-835
[11]Yu YJ,Wu SC,Chan HH,et al.Overproduction of soluble recombinant transglutaminase from Streptomyces netropsis in Escherichia coli[J].Applied Microbiology and Biotechnology,2008,81(3):523-532
[12]Liu S,Zhang DX,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]Shimba N,Shinohara M,Yokoyama KI,et al.Enhancement of transglutaminase activity by NMR identification of its flexible residues affecting the active site[J].FEBS Letters,2002,517(1/3):175-179
[14]Chen KK,Liu S,Ma JL,et al.Deletion combined with saturation mutagenesis of N-terminal residues in transglutaminase from Streptomyces hygroscopicus results in enhanced activity and thermostability[J].Process Biochemistry,2012,47(12):2329-2334
[15]Buettner K,Hertel TC,Pietzsch M.Increased thermostability of microbial transglutaminase by combination of several hot spots evolved by random and saturation mutagenesis[J].Amino Acids,2012,42(2/3):987-996
[16]Chen KK.The study on the molecular modificatuion of Streptomyces hygroscopicus transglutaminase for enhanced catalytic properties[D].Wuxi:Doctoral Dissertation of Jiangnan University,2013(in Chinese)陈康康.分子改造强化Streptomyces hygroscopicus谷氨酰胺转胺酶催化性能研究[D].无锡:江南大学博士学位论文,2013
[17]Bradford MM.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(1/2):248-254
[18]Grossowicz N,Wainfan E,Borek E,et al.The enzymatic formation of hydroxamic acids from glutamine and asparagine[J].Journal of Biological Chemistry,1950,187(1):111-125
[2]Li QZ,Gui P,Huang Z,et al.Effect of transglutaminase on quality and gel properties of pork and fish mince mixtures[J].Journal of Texture Studies,2018,49(1):56-64
[3]Sárdy M,Kárpáti S,Merkl B,et al.Epidermal transglutaminase(TGase 3)is the autoantigen of dermatitis herpetiformis[J].Journal of Experimental Medicine,2002,195(6):747-757
[4]Han X,Yu YY,Wang Q,et al.Facilitation of pretreatment for TGase catalyzingε-PLL grafted wool fabrics[J].New Chemical Materials,2015,43(3):92-95(in Chinese)韩雪,余圆圆,王强,等.预处理对TGase催化羊毛接枝ε-PLL的促进作用[J].化工新型材料,2015,43(3):92-95
[5]Zhao HM,Wang MX,Mou ZC,et al.Study on transglutiminase in food industry[J].Food Science and Technology,2008,33(9):137-141(in Chinese)赵华梅,王曼霞,牟志春,等.食品工业中的谷氨酰胺转氨酶及其酶制剂[J].食品科技,2008,33(9):137-141
[6]Yokoyama K,Nio N,Kikuchi Y.Properties and applications of microbial transglutaminase[J].Applied Microbiology and Biotechnology,2004,64(4):447-454
[7]Kobayashi K,Hashiguchi K,Yokozeki K,et al.Molecular cloning of the transglutaminase gene from Bacillus subtilis and its expression in Escherichia coli[J].Bioscience,Biotechnology,and Biochemistry,1998,62(6):1109-1114
[8]Lin YS,Chao ML,Liu CH,et al.Cloning and expression of the transglutaminase gene from Streptoverticillium ladakanum,in Streptomyces lividans[J].Process Biochemistry,2004,39(5):591-598
[9]Ando H,Adachi M,Umeda K,et al.Purification and characteristics of a novel transglutaminase derived from microorganisms[J].Agricultural and Biological Chemistry,2006,53(10):2613-2617
[10]Kawai M,Takehana S,Takagi H.High-level expression of the chemically synthesized gene for microbial transglutaminase from Streptoverticillium in Escherichia coli[J].Bioscience,Biotechnology,and Biochemistry,2014,61(5):830-835
[11]Yu YJ,Wu SC,Chan HH,et al.Overproduction of soluble recombinant transglutaminase from Streptomyces netropsis in Escherichia coli[J].Applied Microbiology and Biotechnology,2008,81(3):523-532
[12]Liu S,Zhang DX,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]Shimba N,Shinohara M,Yokoyama KI,et al.Enhancement of transglutaminase activity by NMR identification of its flexible residues affecting the active site[J].FEBS Letters,2002,517(1/3):175-179
[14]Chen KK,Liu S,Ma JL,et al.Deletion combined with saturation mutagenesis of N-terminal residues in transglutaminase from Streptomyces hygroscopicus results in enhanced activity and thermostability[J].Process Biochemistry,2012,47(12):2329-2334
[15]Buettner K,Hertel TC,Pietzsch M.Increased thermostability of microbial transglutaminase by combination of several hot spots evolved by random and saturation mutagenesis[J].Amino Acids,2012,42(2/3):987-996
[16]Chen KK.The study on the molecular modificatuion of Streptomyces hygroscopicus transglutaminase for enhanced catalytic properties[D].Wuxi:Doctoral Dissertation of Jiangnan University,2013(in Chinese)陈康康.分子改造强化Streptomyces hygroscopicus谷氨酰胺转胺酶催化性能研究[D].无锡:江南大学博士学位论文,2013
[17]Bradford MM.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(1/2):248-254
[18]Grossowicz N,Wainfan E,Borek E,et al.The enzymatic formation of hydroxamic acids from glutamine and asparagine[J].Journal of Biological Chemistry,1950,187(1):111-125