嗜热芽孢杆菌丙酮酸激酶和乳酸脱氢酶的基因克隆、蛋白表达纯化及活性分析
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摘要
偶联法测定酶的活性是常用酶活测定方法之一。核苷二磷酸(NDP)是核苷三磷酸的β,γ高能磷酸键水解产物之一,丙酮酸激酶(PK)和乳酸脱氢酶(LDH)是用来测定NDP生成的常用偶联酶。为了分析嗜热菌苍白空气芽孢杆菌(Aeribacillus pallidus JH-7)(最适生长温度为50℃)的PK和LDH是否可以作为50℃条件下的偶联酶,研究从Aeribacillus pallidus JH-7基因组中克隆pk和ldh,连接到表达载体pET24a。结果表明,经诱导表达、纯化得到纯度达95%以上的Ap PK和Ap LDH。并利用分光光度法,分别对Ap LDH和Ap PK的动力学常数及催化效率进行了分析。结果表明,Ap PK对NDP的最适底物为ADP,催化效率为5×104 mol/(L·s),最适p H值为8.0,最适温度为45℃;NADH为Ap LDH的最适底物,催化效率为3.7×105 mol/(L·s),最适pH值为5.8,最适温度为35℃。虽然50℃不是Ap LDH和Ap PK的最适温度,但它们在此温度下的kcat分别为16.4,64.5 s-1,暗示它们可以作为此温度下的偶联酶。
The enzyme coupling to measuring enzyme activity is one of the commonly method for enzyme activity assays. Nucleoside diphosphate(NDP)is one of the high energy phosphate bond hydrolysates of nucleoside triphosphate. Pyruvate kinase(PK)and lactate dehydrogenase(LDH) are commonly coupling enzymes used to assay NDP production. To analyze whether PK and LDH of the thermophilic bacteria Aeribacillus pallidus JH-7(optimal growth temperature was 50 ℃)could be used as a coupling enzyme at 50 ℃,we cloned pk and ldh from Aeribacillus pallidus JH-7 genome, and they were constructed into the expression vector pET24 a. By induction and purify, we got Ap PK and Ap LDH with a purity of over 95%. The paper respectively analysed kinetic constants and catalytic efficiency of Ap LDH and Ap PK by spectrophotometry. The results showed that the optimum substrate for Ap PK was ADP, and the catalytic efficiency was 5×104 mol/(L·s), the optimum p H was 8.0, and the optimum temperature was 45 ℃. NADH was the optimum substrate for Ap LDH, the catalytic efficiency was 3.7 × 105 mol/(L·s), the optimum pH was 5.8, and the optimum temperature was 35 ℃.Although 50 ℃ is not the optimum temperature for Ap LDH an Ap PK, but at this temperature, Kcatwas 16.4, 64.5 s-1, respectively,indicating that they can be used as coupling enzyme at this temperature.
The enzyme coupling to measuring enzyme activity is one of the commonly method for enzyme activity assays. Nucleoside diphosphate(NDP)is one of the high energy phosphate bond hydrolysates of nucleoside triphosphate. Pyruvate kinase(PK)and lactate dehydrogenase(LDH) are commonly coupling enzymes used to assay NDP production. To analyze whether PK and LDH of the thermophilic bacteria Aeribacillus pallidus JH-7(optimal growth temperature was 50 ℃)could be used as a coupling enzyme at 50 ℃,we cloned pk and ldh from Aeribacillus pallidus JH-7 genome, and they were constructed into the expression vector pET24 a. By induction and purify, we got Ap PK and Ap LDH with a purity of over 95%. The paper respectively analysed kinetic constants and catalytic efficiency of Ap LDH and Ap PK by spectrophotometry. The results showed that the optimum substrate for Ap PK was ADP, and the catalytic efficiency was 5×104 mol/(L·s), the optimum p H was 8.0, and the optimum temperature was 45 ℃. NADH was the optimum substrate for Ap LDH, the catalytic efficiency was 3.7 × 105 mol/(L·s), the optimum pH was 5.8, and the optimum temperature was 35 ℃.Although 50 ℃ is not the optimum temperature for Ap LDH an Ap PK, but at this temperature, Kcatwas 16.4, 64.5 s-1, respectively,indicating that they can be used as coupling enzyme at this temperature.
引文
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[7]李鸿艳,周思甜,马建辉,等.大肠杆菌II型丙酮酸激酶的纯化分析及其作为偶联酶的应用[J].中国生物工程杂志,2013,33:68-74.
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[12]MALCOVATI M,VALENTINI G. AMP-and fructose 1,6-bisphosphate-activated pyruvate kinases from Escherichia coli[J]. Methods in Enzymology,1982,90(6):170-179.
[13]EICHNER R D. L(+)-lactate dehydrogenase from Homarus americanus[J]. Methods in Enzymology,1982,89:359-262.
[14] SHOEMARK D K,CLIFF M J,SESSIONS R B,et al. Enzymatic properties of the lactate dehydrogenase enzyme from Plasmodium falciparum[J]. Febs Journal,2007,274(11):2738-2748.
[15] KOUASSI G K,ANANTHESWARAN R C,KNABEL S J,et al.Effect of high-pressure processing on activity and structure of alkaline phosphatase and lactate dehydrogenase in buffer and milk[J]. J Agric Food Chem,2007,55:9520-9529.
[16] STEINB?CHEL A,SCHLEGEL H G. NAD-linked L(+)-lactate dehydrogenase from the strict aerobe Alcaligenes eutrophus. 2.Kinetic properties and inhibition by oxaloacetate.[J]. Febs Journal,1983,130(2):329-334.
[17]DENTON R M,HALESTR A P. Regulation of pyruvate metabolism in mammalian tissues[J]. Essays Biochem,1979,15:37-77.
[18]贡莎莎,孟庆玲,乔军,等.少孢节丛孢菌XJ-A1几丁质酶基因AO-190的克隆、表达及其酶活性的测定[J].华北农学报,2018,33(4):71-78.
[19]贺飞燕,闫建俊,白云凤,等.籽粒苋C4关键酶丙酮酸磷酸双激酶基因的原核表达及酶活性测定[J].华北农学报,2017,32(2):61-65.
[20] BRADFORD M M. A rapid method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem,1976,72(1/2):248-254.
[21]WAYGOOD E B,SANWAL B D. The control of pyruvate kinases of Escherichia coli. I. Physicochemical and regulatory properties of the enzyme activated by fructose 1,6-diphosphate[J]. J Biol Chem,1974,249(1):265-274.
[22]GARVIE E I. Bacterial lactate dehydrogenases[J]. Microbiol Rev,1980,44:106-139.
[23]张君,张静,张晓,等.芽孢杆菌TH07代谢产物抑菌成分的研究[D].金华:浙江师范大学,2017.
[2] MUNOZ M E,PONCE E. Pyruvate kinase:current status of regulatory and functional properties[J]. Comp Biochem Physiol B Biochem Mol Biol,2003,135:197-218.
[3]GUNTHER T. Concentration,compartmentation and metabolic function of intracellular free Mg2+[J]. Magnes Res,2006,19:225-236.
[4] LUTHI D,GUNZEL D,MCGUIGAN J A. Mg-ATP binding:its modification by spermine,the relevance to cytosolic Mg2+buffering,changes in the intracellular ionized Mg2+concentration and the estimation of Mg2+by 31P-NMR[J]. Exp Physiol,1999,84:231-252.
[5] RUBIN H. Magnesium:The missing element in molecular views of cell proliferation control[J]. Bioessays,2005,27:311-320.
[6] IRELAND R J,DE LUCA V,DENNIS D T. Characterization and kinetics of isoenzymes of pyruvate kinase from developing castor bean endosperm[J]. Plant Physiol,1980,65:1188-1193.
[7]李鸿艳,周思甜,马建辉,等.大肠杆菌II型丙酮酸激酶的纯化分析及其作为偶联酶的应用[J].中国生物工程杂志,2013,33:68-74.
[8] IMAMURA K,TANAKA T. Pyruvate kinase isozymes from rat[J].Methods in Enzymology,1982,90(6):150-165.
[9] ORIA-HERNANDEZ J,CABRERA N,PEREZ-MONTFORT R,et al. Pyruvate kinase revisited:the activating effect of K+[J]. J Biol Chem,2005,280:37924-37929.
[10] ANDRE C,BENNING C. Arabidopsis seedlings deficient in a plastidic pyruvate kinase are unable to utilize seed storage compounds for germination and establishment[J]. Plant Physiol,2007,145:1670-1680.
[11] SAITO T,NISHI M,LIM M I,et al. A novel GDP-dependent pyruvate kinase isozyme from Toxoplasma gondii localizes to both the apicoplast and the mitochondrion[J]. Journal of Biological Chemistry,2008,283(20):14041-14052.
[12]MALCOVATI M,VALENTINI G. AMP-and fructose 1,6-bisphosphate-activated pyruvate kinases from Escherichia coli[J]. Methods in Enzymology,1982,90(6):170-179.
[13]EICHNER R D. L(+)-lactate dehydrogenase from Homarus americanus[J]. Methods in Enzymology,1982,89:359-262.
[14] SHOEMARK D K,CLIFF M J,SESSIONS R B,et al. Enzymatic properties of the lactate dehydrogenase enzyme from Plasmodium falciparum[J]. Febs Journal,2007,274(11):2738-2748.
[15] KOUASSI G K,ANANTHESWARAN R C,KNABEL S J,et al.Effect of high-pressure processing on activity and structure of alkaline phosphatase and lactate dehydrogenase in buffer and milk[J]. J Agric Food Chem,2007,55:9520-9529.
[16] STEINB?CHEL A,SCHLEGEL H G. NAD-linked L(+)-lactate dehydrogenase from the strict aerobe Alcaligenes eutrophus. 2.Kinetic properties and inhibition by oxaloacetate.[J]. Febs Journal,1983,130(2):329-334.
[17]DENTON R M,HALESTR A P. Regulation of pyruvate metabolism in mammalian tissues[J]. Essays Biochem,1979,15:37-77.
[18]贡莎莎,孟庆玲,乔军,等.少孢节丛孢菌XJ-A1几丁质酶基因AO-190的克隆、表达及其酶活性的测定[J].华北农学报,2018,33(4):71-78.
[19]贺飞燕,闫建俊,白云凤,等.籽粒苋C4关键酶丙酮酸磷酸双激酶基因的原核表达及酶活性测定[J].华北农学报,2017,32(2):61-65.
[20] BRADFORD M M. A rapid method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem,1976,72(1/2):248-254.
[21]WAYGOOD E B,SANWAL B D. The control of pyruvate kinases of Escherichia coli. I. Physicochemical and regulatory properties of the enzyme activated by fructose 1,6-diphosphate[J]. J Biol Chem,1974,249(1):265-274.
[22]GARVIE E I. Bacterial lactate dehydrogenases[J]. Microbiol Rev,1980,44:106-139.
[23]张君,张静,张晓,等.芽孢杆菌TH07代谢产物抑菌成分的研究[D].金华:浙江师范大学,2017.