Site directed mutagenesis studies of horseradish peroxidase
Site directed mutagenesis studies of horseradish peroxidase
复制标题
DOI：
--
复制
发表时间：
2006
期刊：
影响因子：
--
通讯作者：
B. M. Ryan
中科院分区：
文献类型：
--
作者：
B. M. Ryan
过氧化物酶是在动物界和植物界都发现的酶的普遍存在的子集。在所有的过氧化物酶中，大多数研究集中在III类辣根过氧化物酶（E.C. 1.11.1.7基本形式HRP-C是最常见的，并在本研究中使用。重组HRP-C在十五年前首次表达;然而，其生产一直受到包涵体形成和低产量的困扰。在本研究中，HRP基因和PelB前导序列定向克隆形成融合蛋白，表达到细菌周质包膜。通过操纵随后的表达条件，成功地产生了全功能HRP，其中包含允许单步纯化的多组氨酸标签。利用定点诱变来探测重组酶的稳定性。采用两种方法来选择用于突变的残基，最初是理性方法，基于先前的知识提出了16个突变。第二种方法基于过氧化物酶序列比对，利用新的生物信息学软件，提出了6个突变。这些突变体通过优化的条件产生、表达和纯化。所有突变体的特征在于基于热，溶剂和H2O2的稳定性，以及动力学分析。注意到谷氨酸238、谷氨酰胺106和HRP蛋白内的螺旋二级结构的重要稳定作用。过氧化物酶结构的进化可以通过一个新的原型过氧化物酶序列产生。增加赖氨酸232/赖氨酸241轴中氢键的突变提高了稳定性。还采用基因工程来产生重组HRP-C，其允许简单的定向固定化（一个三重突变体和一个五重突变体），允许最大程度地接近酶的活性位点。然而，以游离和固定化稳定性为代价实现了改进的固定化能力。研究了野生型重组HRP-C在丝网印刷和蚀刻铂电极生物传感器中的应用。对于两种配置，重组HRP的直接电子转移均优于植物HRP。
The peroxidases are a ubiquitous subset of enzymes found in both the animal and plant kingdoms. Of all the peroxidases, the majority of research has focussed on the Class III Horseradish Peroxidase (E.C. 1.11.1.7). The basic form, HRP-C, is the most common and is utilised in this study. Recombinant HRP-C was first expressed over fifteen years ago; however, its production has been plagued by the formation of inclusion bodies and low yields. In this present study, the HRP gene and a PelB leader sequence were directionally cloned to form a fusion protein, expressed to the bacterial periplasmic envelope. By manipulating subsequent expression conditions, fully functional HRP was successfully produced, with the inclusion of a poly-Histidine tag permitting single step purification. Site directed mutagenesis was utilised to probe the stability of the recombinant enzyme. Two methodologies were employed to select residues for mutation, initially a rational approach, based on previous knowledge proposed 16 mutations. The second method based on a peroxidase sequence alignment utilising novel bioinformatic software, proposed 6 mutations. These mutants were generated, expressed and purified via optimised conditions. All mutants were characterised based on thermal, solvent and H2O2 stabilities, as well as kinetic analysis. Important stabilising roles for Glutamic Acid 238, Glutamine 106 and the helical secondary structure within the HRP protein were noted. Peroxidase structure evolution could be followed via a novel archetype peroxidase sequence generated. Mutations, which increased hydrogen bonding in the Lysine 232/Lysine 241 axis, improved stability. Genetic engineering was also employed to generate a recombinant HRP-C that permitted simple directed immobilisation (one triple mutant and one pentuple mutant), allowing maximal accessibility to the enzymes’ active site. Improved immobilisation capacity was achieved, however, at the cost of free and immobilised stability. Application o f wildtype recombinant HRP-C to screen-printed and etched platinum electrode biosensors was investigated. Improved direct electron transfer was noted for recombinant HRP over plant HRP for both configurations.