金属离子对氯过氧化物酶在25℃到55℃激活作用以及机理的研究

基于氯过氧化物酶（CPO）的卤化活性分析，发现某些碱土金属（Ca2+, Mg2+）和过渡金属（Co2+, Ni2+）对CPO具有明显的激活及稳定化作用。例如25 ºC时与CPO在纯缓冲溶液中相比，在75 μmol·L-1 Ca2+，90 μmol·L-1 Mg2+，90 μmol·L-1 Ni2+及105 μmol·L-1 Co2+存在时CPO可分别获得1.33，1.37，1.34 及1.27倍的最大相对活性。而在55 ºC，没有金属离子存在时，CPO 30分钟后仅能保留40%的活性，但在Ca2+，Mg2+离子的介质中，CPO的活性可分别保留81% 和 75%。推测这是由于金属离子结合在CPO活性中心周围的酸-碱催化位点Glu183, His105 and Asp106上，通过底物浓集和诱导有利构象来激活CPO. 同时动力学研究表明金属离子对CPO的激活归因于催化效率（kcat）的提高，以及CPO对底物亲和性及选择性的改善。

Activation Function of Chloroperoxidase in the Presence of Metal Ions at Elevated Temperature from 25 to 55°C

The investigation and comparison of chlorination activity of chloroperoxidase (CPO) from Caldariomyces fumago in metal ion solutions to those in pure buffer indicated that CPO could be effectively activated by some alkaline‐earth metals and transition metals. The obtained maximum relative activity of CPO was 1.33 time at 75 µmol·L^?1 Ca²⁺, 1.37 time at 90 µmol·L^?1 Mg²⁺, 1.34 time at 90 µmol·L^?1 Ni²⁺, and 1.27 time at 105 µmol·L^?1 Co²⁺ at 25°C. Moreover, the CPO stability against temperature was improved in the presence of the above metal ions. At 55°C, CPO could retain only about 40% of activity whereas 75% and 81% of activity were maintained in Mg²⁺ and Ca²⁺ media, respectively. It was suggested that the metal ions bind to the acid‐base catalytic groups Glu183, His105 and Asp106 around the active site of CPO, and activate CPO by both an enrichment of substrate concentration and the conformational change of CPO, which are favorable to the substrate access. The analysis of kinetic parameters indicated that the activation was mainly due to an increase in k_cat values. The affinity and specificity of CPO to substrates were also improved in these metal ion media. The results in this work are promising in view of industrial applications of this versatile biological catalyst.