多聚赖氨酸对双亚基肌酸酶表面的修饰
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摘要
利用多聚赖氨酸(poly-lysine)对同源二聚体肌酸酶进行化学修饰,研究该法对酶稳定性的影响。选用L_9(3~4)正交试验方案,研究不同修饰条件对修饰酶的催化效率和热稳定性的影响,确定多聚赖氨酸修饰肌酸酶(CRE)的最佳条件为CRE-COOH与poly-lysine-NH_2、EDC的摩尔比分别为1∶100和1∶10,pH为7.0。通过SDS-PAGE鉴定,探测多聚赖氨酸可在酶分子表面产生共价结合。修饰酶的催化动力学参数Km和kcat分别为4.75×10~(-2)mol/L和2.50×10~2S~(-1)。与游离酶相比,修饰酶的热稳定性和pH稳定性均明显提高。修饰酶比游离酶的Tm值高了2.07℃,当pH值为4.0和10.0时,修饰酶的酶活力仍保留在50%以上,而游离酶活性几乎丧失。进一步优化研究表明,多聚赖氨酸表面修饰法有可能成为有效提高肌酸酶稳定性的一种方法。
The surface modification of homodimeric creatinase by poly-lysine was carried out to study the effect of enhancing enzyme stability. The influences of different modification condition on enzyme catalytic efficiency and stability were studied by L_9(3~4)orthogonal experiment, the optimal condition were set as molar ratios of CRE-COOH to poly-lysine-NH_2 and CRE-COOH to EDC were1∶100 and 1∶10, in pH 7.0. The covalently binding of poly-lysine onto enzyme surface was tested by SDS-PAGE. The catalytic kinetic parameters(K_m and k_(cat)) of modified enzyme were determined as 4.75 ×10~(-5) mol/L and 2.50 ×10~2 S~(-1), respectively. Moreover, compared to the native enzyme, the thermal and p H stabilities of the modified creatinase were improved, as Tm of modified enzyme was raised by 2.07 ℃, and more than 50% of initial activity of modified enzyme was maintained at p H 4.0 and 10.0, but the control sample was almost no activity. After further exploitation,poly-lysine modification would be one of the effective methods for improving stability of homodimeric creatinase.
The surface modification of homodimeric creatinase by poly-lysine was carried out to study the effect of enhancing enzyme stability. The influences of different modification condition on enzyme catalytic efficiency and stability were studied by L_9(3~4)orthogonal experiment, the optimal condition were set as molar ratios of CRE-COOH to poly-lysine-NH_2 and CRE-COOH to EDC were1∶100 and 1∶10, in pH 7.0. The covalently binding of poly-lysine onto enzyme surface was tested by SDS-PAGE. The catalytic kinetic parameters(K_m and k_(cat)) of modified enzyme were determined as 4.75 ×10~(-5) mol/L and 2.50 ×10~2 S~(-1), respectively. Moreover, compared to the native enzyme, the thermal and p H stabilities of the modified creatinase were improved, as Tm of modified enzyme was raised by 2.07 ℃, and more than 50% of initial activity of modified enzyme was maintained at p H 4.0 and 10.0, but the control sample was almost no activity. After further exploitation,poly-lysine modification would be one of the effective methods for improving stability of homodimeric creatinase.
引文
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[21]KANG K,KAN C,YEUNG A,et al.The immobilization of trypsin on soap-free P(MMA-EA-AA)latex particles[J].Materials Science and Engineering:C,2006,26(4):664-669.
[2]WANG Y Y,MA X H,ZHAO W F,et al.Study on the creatinase from Paracoccus sp strain WB1[J].Process Biochemistry,2006,41(9):2072-2077.
[3]YOSHIMOTO T,OKA I,TSURU D.Creatine amidinohydrolase of Pseudomonas putida:crystallization and some properties[J].Archives of Biochemistry and Biophysics,1976,177(2):508-515.
[4]SUZUKI K,SAGAI H,SUGIYAMA M,et al.Molecular cloning and high expression of the Bacillus creatinase gene in Escherichia coli[J].Journal of Fermentation and Bioengineering,1993,76(2):77-81.
[5]PADMANABHAN B,PAEHLER A,HORIKOSHI M.Structure of creatine amidinohydrolase from Actinobacillus[J].Acta Crystallographica Section D-Biological Crystallography,2002,58:1322-1328.
[6]ZHI Q,KONG P Y,ZANG J T,et al.Biochemical and molecular characterization of a novel high activity creatine amidinohydrolase from Arthrobacter nicotianae strain 02181[J].Process Biochemistry,2009,44(4):460-465.
[7]YOSHIMOTO T,IMAO O,TSURU D.Purification,crystallization,and some properties of creatine amidinohydrolase from Pseudomonus putida[J].Journal of Biochemistry,1976,79(6):1381-1383.
[8]HOEFFKEN H W,KNOF S H,BARTLETT P A,et al.Crystal structure determination,refinement and molecular model of creatine amidinohydrolase from Pseudomonas putida[J].Journal of Molecular Biology,1988,204(2):417-433.
[9]COLL M,KNOF S H,OHGA Y,et al.Enzymatic mechanism of creatine amidinohydrolase as deduced from crystal structures[J].Journal of Molecular Biology,1990,214(2):597-610.
[10]SCHUMANN J,MOLLERING H,JAENICKE R.Intrinsic stability and extrinsic stabilization of creatinase from Pseudomonas putida[J].Biological Chemistry Hoppe-Seyler,1993,374(7):427-434.
[11]SCHUMANN J,BOHM G,SCHUMACHER G,et al.Stabilization of creatinase from Pseudomonas putida by random mutagenesis[J].Protein Science,1993,2(10):1612-1620.
[12]BERBERICH J A,YANG L W,BAHAR I,et al.A stable three enzyme creatinine biosensor.2.Analysis of the impact of silver ions on creatine amidinohydrolase[J].Acta Biomaterialia,2005,1(2):183-191.
[13]LAD U,KHOKHAR S,KALE G M.Electrochemical creatinine biosensors[J].Analytical Chemistry,2008,80(21):7910-7917.
[14]FERNANDEZ-LAFUENTE R.Stabilization of multimeric enzymes:Strategies to prevent subunit dissociation[J].Enzyme and Microbial Technology,2009,45(6-7):405-418.
[15]DAI Jun,KANG Zhen,ZHANG Linpei,et al.Expression and application analysis of creatinase from Arthrobacter nicotianae[J]Journal of Food Science and Biotechnology,2016(5):465-470.(in Chinese）
[16]DAI J,ZHANG L,KANG Z,et al.High-level production of creatine amidinohydrolase from Arthrobacter nicotianae 23710 in Escherichia coli[J].Applied Biochemisty Biotechnology,2015,175(5):2564-2573.
[17]TONG Y,XIN Y,YANG H,et al.Efficient improvement on stability of sarcosine oxidase via poly-lysine modification on enzyme surface[J].International Journal of Biological Macromolecules,2014,67:140-146.
[18]LOPEZ-ALONSO J P,DIEZ-GARCIIA F,FONT J,et al.Carbodiimide EDC induces cross-links that stabilize RNase A C-dimer against dissociation:EDC adducts can affect protein net charge,conformation,and activity[J].Bioconjugate Chemistry,2009,20(8):1459-1473.
[19]SWARNALATHA V,ALURI ESTHER R,DHAMODHARAN R.Immobilization ofα-amylase on gum acacia stabilized magnetite nanoparticles,an easily recoverable and reusable support[J].Journal of Molecular Catalysis B:Enzymatic,2013,96:6-13.
[20]仝艳军.肌氨酸氧化酶高效表达、亲和纯化及酶稳定性研究[D].无锡：江南大学，2015.
[21]KANG K,KAN C,YEUNG A,et al.The immobilization of trypsin on soap-free P(MMA-EA-AA)latex particles[J].Materials Science and Engineering:C,2006,26(4):664-669.