巨大芽孢杆菌青霉素G酰化酶的定点突变及其动力学性质研究

Site-directed mutagenesis of B.megaterium penicillin G acylase and its kinetics

为了提高青霉素G酰化酶 (PGA)在酸性及有机溶剂中的稳定性 ,以大肠杆菌的晶体结构为模板 ,用软件PMODELING同源模建巨大芽孢杆菌青霉素G酰化酶的三维结构并且选择PGA分子表面的合适碱性氨基酸突变为丙氨酸。通过三种不同的快速PCR介导定点突变的方法 ,将位于PGA的α亚基 2 1位、12 8位和β亚基 4 92位、5 12位的赖氨酸残基分别突变为丙氨酸 ,获得四个突变酶Kα0 2 1A、Kα12 8A、Kβ4 92A和Kβ5 12A。其中Kα12 8A和Kβ5 12A保持与野生型相近的酶活力 ,其动力学性质如最适温度、最适pH、Km及Kcat没有明显变化 ;突变酶Kα0 2 1A和Kβ4 92A则丧失了酶活力。上述结果表明 ,PGA分子表面非活性中心的赖氨酸→丙氨酸点突变使突变子的性状发生了分化 ,突变效应呈现出丰富的多样性。该有理设计不但可以提高酶的稳定性 。

Penicillin G acylase (PGA) with higher stability in anhydrous media and acidic media is an attractive candidate in β-lactam antibiotics synthetic conversions. Hence, manipulation of rational design was performed to improve stability of B.megaterium PGA. Based on the crystal structure of Escherichia coli PGA, a three-dimensional model of B.megaterium PGA was constructed by using PMODELING program. Four basic amino acids of lysine, which located on the molecular surface, were selected as mutant candidate and mutated to alanine. Different methods based on PCR technique were developed in the site- directed mutagenesis protocol and four PGA mutants, Kα021A、Kα128A、Kβ492A and Kβ512A were obtained. Among these PGA, mutants of Kα128A、Kβ512A showed similar properties as wild type PGA. However, mutants of Kα021A and Kβ492A showed no specific activity. This study not only successfully improved stability of PGA, but also supplied us with a surprising diversity, which probably became a model of investigating the relationship between structure and function of this enzyme.