两个苹果酸酶的表达和酶学性质分析
|
摘要
为了探讨深黄被孢霉M6-22中不同苹果酸酶的性质,研究从深黄被孢霉M6-22中克隆得到苹果酸酶MIME1基因并在大肠杆菌BL21中诱导表达,经镍柱纯化和酶活分析表明所纯化蛋白能催化苹果酸脱羧生成丙酮酸和NADPH,具有苹果酸酶的特性.同时与前期获得的MIME2进行酶学性质分析.结果显示,重组表达的MIME1和MIME2都具有苹果酸酶的性质,但MIME1的最适温度和最适pH分别为28℃和7.5,MIME2的最适温度和最适pH分别为30℃和5.8,而且在最适温度和最适pH条件下所纯化的MIME1和MIME2酶活分别为292.84 U/mg和235.14 U/mg.进一步酶学性质分析表明,Mg~(2+)、Co~(2+)、Cu~(2+)、Zn ~(2+)可以提高MIME1的活性,其中Cu~(2+)激活作用最为明显,而EDTA、Ca~(2+)、Mn~(2+)对MIME1的活性则有抑制作用;Mn~(2+)、Mg~(2+)、Co~(2+)和Ca~(2+)对MIME2表现出不同程度的激活作用,其中Mn~(2+)激活作用最为明显,而EDTA、Cu~(2+)和Zn~(2+)则对MIME2的酶活表现出明显的抑制作用.这些分析结果表明,同一种细胞中不同的苹果酸酶具有不同的酶学性质,这可能与它们执行不同的生物学功能有关.通过异源表达和分离纯化,研究初步揭示了2个深黄被孢霉M6-22苹果酸酶的一些酶学性质特点,这为以后深入研究该苹果酸酶结构和功能以及与M6-22菌株油脂积累的关系提供了理论依据和参考.
To investigate the properties of different malic enzymes from M6-22, MIME1 gene was cloned from Mortierella isabellina M6-22 and expressed in E. coli BL21, enzymatic activity was analyzed after purification by nickel affinity chromatography. The recombinant expression of MIME1 protein showed the property of malic enzyme which could catalyze the dehydrogenation of malic acid and produce pyruvate and NADPH. The enzymatic property of MIME1 was compared with MIME2 from the previous study. The results showed that both MIME1 and MIME2 had the property of malic enzyme; The optimum temperature and pH of MIME1 and MIME2 were 28 ℃ and 7.5, 30 ℃ and 5.8, respectively; The activities of purified MIME1 and MIME2 were 292.84 U/mg and 235.14 U/mg; Mg~(2+), Co~(2+), Cu~(2+) and Zn~(2+) increased the activity of MIME1 with Cu~(2+) showing the strongest effect, while EDTA, Ca~(2+) and Mn~(2+) inhibited the activity of MIME1. Mn~(2+), Mg~(2+), Co~(2+)and Ca~(2+) showed different degrees of activation on MIME2 with Mn~(2+) showing the strongest effect, while EDTA,Cu~(2+) and Zn~(2+) significantly inhibited the activity of MIME2; Different malic enzymes in the same cell had different enzymatic properties, which may be related to their different biological functions. By purification,isolation and heterologous expression, some characteristics of enzymatic properties of two Mortierella isabellina M6-22 malic enzymes are showed. A theoretical basis and reference for the structure and function of the malic enzymes and the relationship with the lipid accumulation in M6-22 strain is provided.
To investigate the properties of different malic enzymes from M6-22, MIME1 gene was cloned from Mortierella isabellina M6-22 and expressed in E. coli BL21, enzymatic activity was analyzed after purification by nickel affinity chromatography. The recombinant expression of MIME1 protein showed the property of malic enzyme which could catalyze the dehydrogenation of malic acid and produce pyruvate and NADPH. The enzymatic property of MIME1 was compared with MIME2 from the previous study. The results showed that both MIME1 and MIME2 had the property of malic enzyme; The optimum temperature and pH of MIME1 and MIME2 were 28 ℃ and 7.5, 30 ℃ and 5.8, respectively; The activities of purified MIME1 and MIME2 were 292.84 U/mg and 235.14 U/mg; Mg~(2+), Co~(2+), Cu~(2+) and Zn~(2+) increased the activity of MIME1 with Cu~(2+) showing the strongest effect, while EDTA, Ca~(2+) and Mn~(2+) inhibited the activity of MIME1. Mn~(2+), Mg~(2+), Co~(2+)and Ca~(2+) showed different degrees of activation on MIME2 with Mn~(2+) showing the strongest effect, while EDTA,Cu~(2+) and Zn~(2+) significantly inhibited the activity of MIME2; Different malic enzymes in the same cell had different enzymatic properties, which may be related to their different biological functions. By purification,isolation and heterologous expression, some characteristics of enzymatic properties of two Mortierella isabellina M6-22 malic enzymes are showed. A theoretical basis and reference for the structure and function of the malic enzymes and the relationship with the lipid accumulation in M6-22 strain is provided.
引文
[1]Voegele R T,Mitsch M J,Finan T M.Characterization of two members of a novel malic enzyme class[J].Biochimica et Biophysica Acta(BBA)-Protein Structure and Molecular Enzymology,1999,1432(2):275-285.DOI:10.1016/S0167-4838(99)00112-0.
[2]Vongsangnak W,Zhang Y,Chen W,et al.Annotation and analysis of malic enzyme genes encoding for multiple isoforms in the fungus Mucor circinelloides CBS277.49[J].Biotechnology Letters,2012,34(5):941.DOI:10.1007/s10529-012-0859-x.
[3]Ren J G,Seth P,Everett P,et al.Induction of erythroid differentiation in human erythroleukemia cells by depletion of malic enzyme 2[J].Plos One,2010,5(9):e12 520.DOI:10.1371/journal.pone.0012520.
[4]Shearer H L,Turpin D H,Dennis D T.Characterization of NADP-dependent malic enzyme from developing castor oil seed endosperm[J].Archives of Biochemistry&Biophysics,2004,429(2):134-144.
[5]Dolezalp,Vanacova S,Tachezy J,et al.Malic enzymes of Trichomonas vaginalis:two enzyme families,two distinct origins[J].Gene,2004,329(1):81-92.
[6]Lerondel G,Doan T,Zamboni N,et al.YtsJ has the major physiological role of the four paralogous malic enzyme isoforms in Bacillus subtilis[J].Journal of Bacteriology,2006,188(13):4 727-4 736.DOI:10.1128/JB.00167-06.
[7]程万,林辉,赵宇华,等.苹果酸酶调控微生物油脂积累的研究进展[J].科技通报,2010,26(6):853-857.DOI:10.3969/j.issn.1001-7119.2010.06.011.Cheng W,Lin H,Zhao Y H,et al.Research progress on malic enzyme regulating the accumulation of microbial oils[J].Bulletin of Science and Technology,2010,26(6):853-857.
[8]Thorpe R F,Ratledge C.Fatty acid distribution in triglycerides of yeasts grown on glucose or n-alkanes[J].Microbiology,1972,72(1):151-163.
[9]Zhang H,Zhang L,Chen H,et al.Regulatory properties of malic enzyme in the oleaginous yeast,Yarrowia lipolytica,and its non-involvement in lipid accumulation[J].Biotechnology Letters,2013,35(12):2 091-2 098.DOI:10.1007/s10529-013-1302-7.
[10]Wynn J P,Hamid A A,Li Y,et al.Biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi Mucor circinelloides and Mortierella alpina[J].Microbiology,2001,147(10):2 857-2 864.DOI:10.1099/00221287-147-10-2857.
[11]Ratledge C,Wynn J P.The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms[J].Advances in Applied Microbiology,2002,51:1-51.
[12]张怀渊,赵建新,陈永泉,等.耶氏解脂酵母油脂积累分子机制的初步探索[C]//中国食品科学技术学会第八届年会暨第六届东西方食品业高层论坛论文摘要集,2011:2.Zhang H Y,Zhao J X,Chen Y Q,et al.Molecular mechanism of lipid accumulation in Yarrowia lipolytica[C]//Chinese Food Science and Technology Association Eighth Annual Meeting and the Sixth East-West Food Summit Forum Abstract,2011:2.
[13]崔锦锦,李凌彦,季秀玲,等.深黄被孢霉苹果酸酶基因的克隆和表达[J].应用与环境生物学报,2017,23(1):129-133.Cui J J,Li L Y,Ji X L,et al.Cloning and expression of a malic enzyme gene from Mortierella isabellina[J].Chinese Journal of Applied and Environmental Biology,2017,23(1):129-133.
[14]Kuo C W,Hung H C,Tong L,et al.Metal-induced reversible structural interconversion of human mitochondrial NAD(P)+-dependent malic enzyme[J].Proteinsstructure Function&Bioinformatics,2004,54(3):404-411.
[15]Vorapreeda T,Thammarongtham C,Cheevadhanarak S,et al.Repertoire of malic enzymes in yeast and fungi:insight into their evolutionary functional and structural significance[J].Microbiology,2013,159(12):2 548-2 557.
[16]董庆龙,余贤美,刘丹丹,等.苹果NAD-苹果酸酶基因的克隆及在不同组织和果实发育阶段的表达分析[J].园艺学报,2013,40(4):739-748.Dong Q L,Yu M X,Liu D D,et al.Cloning of NAD-malic enzymes and their expression analysis during tissues and fruit development of apple[J].Acta Horticulturae Sinica,2013,40(4):739-748.
[17]Edwards G E,Andreo C S.NADP-malic enzyme from plants[J].Phytochemistry,1992,31(6):1 845-1 857.DOI:10.1016/0031-9422(92)80322-6.
[18]Holaday A S,Lowder G W.Effect of pH on the kinetic parameters of NADP-malic enzyme from a C4 Flaveria(Asteraceae)species[J].Plant Physiology,1989,90(2):401-405.DOI:10.1104/pp.90.2.401.
[19]Drincovich M F,Casati P,Andreo C S.NADP-malic enzyme from plants:a ubiquitous enzyme involved in different metabolic pathways[J].FEBS Letters,2001,490(1-2):1-6.DOI:10.1016/S0014-5793(00)02331-0.
[20]Ferreyra M L F,Andreo C S,Podesta F E.Purification and physical and kinetic characterization of a photosynthetic NADP-dependent malic enzyme from the CAMplant Aptenia cordifolia[J].Plant Science,2003,164(1):95-102.DOI:10.1016/S0168-9452(02)00340-0.
[21]Yang J,Hu X,Zhang H,et al.Expression,purification,and characterization of NADP+-dependent malic enzyme from the oleaginous fungus Mortierella alpina[J].Applied Biochemistry and Biotechnology,2014,173(7):1 849-1 857.DOI:10.1007/s12010-014-0971-6.
[22]吴四平,陆阳,刘均忠,等.两种不同来源富马酸酶酶学性质比较[J].发酵科技通讯,2016,45(1):12-17.DOI:10.3969/j.issn.1674-2214.2016.01.003.Wu S P,Lu Y,Liu J Z,et al.Comparison of enzymatic properties of fumarase from Corynebacterium glutamicum and Escherichia coli[J].Bulletin of Fermentation Science and Technology,2016,45(1):12-17.
[23]张映曈,陈海琴,宋元达,等.卷枝毛霉中苹果酸酶同工酶V的酶学性质[J].微生物学报,2016,56(2):309-316.Zhang Y T,Chen H Q,Song Y D,et al.Characterization of a malic enzyme isoform V from Mucor circinelloides[J].Acta Microbiologica Sinica,2016,56(2):309-316.
[24]Song Y,Wynn J P,Li Y,et al.A pre-genetic study of the isoforms of malic enzyme associated with lipid accumulation in Mucor circinelloides[J].Microbiology,2001,147(6):1 507-1 515.DOI:10.1099/00221287-147-6-1507.
[25]Li Z,Sun H,Mo X,et al.Overexpression of malic enzyme(ME)of Mucor circinelloides improved lipid accumulation in engineered Rhodotorula glutinis[J].Applied Microbiology and Biotechnology,2013,97(11):4 927-4 936.DOI:10.1007/s00253-012-4571-5.
[26]Zhang Y,Adams I P,Ratledge C.Malic enzyme:the controlling activity for lipid production?Overexpression of malic enzyme in Mucor circinelloides leads to a 2 .5-fold increase in lipid accumulation[J].Microbiology,2007,153(7):2 013-2 025.DOI:10.1099/mic.0.2006/002683-0.
[2]Vongsangnak W,Zhang Y,Chen W,et al.Annotation and analysis of malic enzyme genes encoding for multiple isoforms in the fungus Mucor circinelloides CBS277.49[J].Biotechnology Letters,2012,34(5):941.DOI:10.1007/s10529-012-0859-x.
[3]Ren J G,Seth P,Everett P,et al.Induction of erythroid differentiation in human erythroleukemia cells by depletion of malic enzyme 2[J].Plos One,2010,5(9):e12 520.DOI:10.1371/journal.pone.0012520.
[4]Shearer H L,Turpin D H,Dennis D T.Characterization of NADP-dependent malic enzyme from developing castor oil seed endosperm[J].Archives of Biochemistry&Biophysics,2004,429(2):134-144.
[5]Dolezalp,Vanacova S,Tachezy J,et al.Malic enzymes of Trichomonas vaginalis:two enzyme families,two distinct origins[J].Gene,2004,329(1):81-92.
[6]Lerondel G,Doan T,Zamboni N,et al.YtsJ has the major physiological role of the four paralogous malic enzyme isoforms in Bacillus subtilis[J].Journal of Bacteriology,2006,188(13):4 727-4 736.DOI:10.1128/JB.00167-06.
[7]程万,林辉,赵宇华,等.苹果酸酶调控微生物油脂积累的研究进展[J].科技通报,2010,26(6):853-857.DOI:10.3969/j.issn.1001-7119.2010.06.011.Cheng W,Lin H,Zhao Y H,et al.Research progress on malic enzyme regulating the accumulation of microbial oils[J].Bulletin of Science and Technology,2010,26(6):853-857.
[8]Thorpe R F,Ratledge C.Fatty acid distribution in triglycerides of yeasts grown on glucose or n-alkanes[J].Microbiology,1972,72(1):151-163.
[9]Zhang H,Zhang L,Chen H,et al.Regulatory properties of malic enzyme in the oleaginous yeast,Yarrowia lipolytica,and its non-involvement in lipid accumulation[J].Biotechnology Letters,2013,35(12):2 091-2 098.DOI:10.1007/s10529-013-1302-7.
[10]Wynn J P,Hamid A A,Li Y,et al.Biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi Mucor circinelloides and Mortierella alpina[J].Microbiology,2001,147(10):2 857-2 864.DOI:10.1099/00221287-147-10-2857.
[11]Ratledge C,Wynn J P.The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms[J].Advances in Applied Microbiology,2002,51:1-51.
[12]张怀渊,赵建新,陈永泉,等.耶氏解脂酵母油脂积累分子机制的初步探索[C]//中国食品科学技术学会第八届年会暨第六届东西方食品业高层论坛论文摘要集,2011:2.Zhang H Y,Zhao J X,Chen Y Q,et al.Molecular mechanism of lipid accumulation in Yarrowia lipolytica[C]//Chinese Food Science and Technology Association Eighth Annual Meeting and the Sixth East-West Food Summit Forum Abstract,2011:2.
[13]崔锦锦,李凌彦,季秀玲,等.深黄被孢霉苹果酸酶基因的克隆和表达[J].应用与环境生物学报,2017,23(1):129-133.Cui J J,Li L Y,Ji X L,et al.Cloning and expression of a malic enzyme gene from Mortierella isabellina[J].Chinese Journal of Applied and Environmental Biology,2017,23(1):129-133.
[14]Kuo C W,Hung H C,Tong L,et al.Metal-induced reversible structural interconversion of human mitochondrial NAD(P)+-dependent malic enzyme[J].Proteinsstructure Function&Bioinformatics,2004,54(3):404-411.
[15]Vorapreeda T,Thammarongtham C,Cheevadhanarak S,et al.Repertoire of malic enzymes in yeast and fungi:insight into their evolutionary functional and structural significance[J].Microbiology,2013,159(12):2 548-2 557.
[16]董庆龙,余贤美,刘丹丹,等.苹果NAD-苹果酸酶基因的克隆及在不同组织和果实发育阶段的表达分析[J].园艺学报,2013,40(4):739-748.Dong Q L,Yu M X,Liu D D,et al.Cloning of NAD-malic enzymes and their expression analysis during tissues and fruit development of apple[J].Acta Horticulturae Sinica,2013,40(4):739-748.
[17]Edwards G E,Andreo C S.NADP-malic enzyme from plants[J].Phytochemistry,1992,31(6):1 845-1 857.DOI:10.1016/0031-9422(92)80322-6.
[18]Holaday A S,Lowder G W.Effect of pH on the kinetic parameters of NADP-malic enzyme from a C4 Flaveria(Asteraceae)species[J].Plant Physiology,1989,90(2):401-405.DOI:10.1104/pp.90.2.401.
[19]Drincovich M F,Casati P,Andreo C S.NADP-malic enzyme from plants:a ubiquitous enzyme involved in different metabolic pathways[J].FEBS Letters,2001,490(1-2):1-6.DOI:10.1016/S0014-5793(00)02331-0.
[20]Ferreyra M L F,Andreo C S,Podesta F E.Purification and physical and kinetic characterization of a photosynthetic NADP-dependent malic enzyme from the CAMplant Aptenia cordifolia[J].Plant Science,2003,164(1):95-102.DOI:10.1016/S0168-9452(02)00340-0.
[21]Yang J,Hu X,Zhang H,et al.Expression,purification,and characterization of NADP+-dependent malic enzyme from the oleaginous fungus Mortierella alpina[J].Applied Biochemistry and Biotechnology,2014,173(7):1 849-1 857.DOI:10.1007/s12010-014-0971-6.
[22]吴四平,陆阳,刘均忠,等.两种不同来源富马酸酶酶学性质比较[J].发酵科技通讯,2016,45(1):12-17.DOI:10.3969/j.issn.1674-2214.2016.01.003.Wu S P,Lu Y,Liu J Z,et al.Comparison of enzymatic properties of fumarase from Corynebacterium glutamicum and Escherichia coli[J].Bulletin of Fermentation Science and Technology,2016,45(1):12-17.
[23]张映曈,陈海琴,宋元达,等.卷枝毛霉中苹果酸酶同工酶V的酶学性质[J].微生物学报,2016,56(2):309-316.Zhang Y T,Chen H Q,Song Y D,et al.Characterization of a malic enzyme isoform V from Mucor circinelloides[J].Acta Microbiologica Sinica,2016,56(2):309-316.
[24]Song Y,Wynn J P,Li Y,et al.A pre-genetic study of the isoforms of malic enzyme associated with lipid accumulation in Mucor circinelloides[J].Microbiology,2001,147(6):1 507-1 515.DOI:10.1099/00221287-147-6-1507.
[25]Li Z,Sun H,Mo X,et al.Overexpression of malic enzyme(ME)of Mucor circinelloides improved lipid accumulation in engineered Rhodotorula glutinis[J].Applied Microbiology and Biotechnology,2013,97(11):4 927-4 936.DOI:10.1007/s00253-012-4571-5.
[26]Zhang Y,Adams I P,Ratledge C.Malic enzyme:the controlling activity for lipid production?Overexpression of malic enzyme in Mucor circinelloides leads to a 2 .5-fold increase in lipid accumulation[J].Microbiology,2007,153(7):2 013-2 025.DOI:10.1099/mic.0.2006/002683-0.