多聚赖氨酸对双亚基肌酸酶表面的修饰

Surface Modification on Homodimeric Creatinase by Poly-Lysine Binding

DOI：10.3969/j.issn.1673-1689.2019.07.019

中文关键词: 肌酸酶 Pseudomonas putida 多聚赖氨酸 表面修饰 稳定性

英文关键词: creatinase,Pseudomonas putida,poly-lysine,surface modification,stability

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中文摘要:

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L9（34）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-NH2、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K/im和kcat分别为4.75×10^-2mol/L和2.50×10²S^-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。

英文摘要:

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 L9（34）orthogonal experiment， the optimal condition were set as molar ratios of CRE-COOH to poly-lysine-NH2and CRE-COOH to EDC were 1∶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 （Km andkcat） of modified enzyme were determined as 4.75×10^-5mol/L and 2.50×10²S^-1， respectively. Moreover， compared to the native enzyme， the thermal and pH stabilities of the modified creatinase were improved， asTm of modified enzyme was raised by 2.07 ℃， and more than 50% of initial activity of modified enzyme was maintained at pH 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.

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