N端缺失的锰过氧化氢酶生理生化特性

Physiological and biochemical characteristics of a manganese catalase lacking n-terminal

摘要:

摘要:【目的】在红球菌(Rhodococcus sp.)R04中发现了一种高表达，N端缺失的锰过氧化氢酶(Mn-CAT)，为了明确其在活性氧(Reactive oxygen species，ROS)清除与多氯联苯(Polychlorinated biphenyls，PCBs)代谢中所起的作用，本文对其生理生化特性进行了研究。【方法】利用DNAMAN 对Rhodococcus sp. R04与Rhodococcus sp. R1101Mn-CAT的核酸和蛋白序列进行比对。化学合成和PCR搭桥法获取Mn-CAT全长基因。分别构建了原核表达载体pETm3c-Mn-CAT，pETm3c-MnCAT-C，转入大肠杆菌(Escherichia coli) BL21，得到重组菌pETm3c-Mn-CAT/BL21，pETm3c-MnCAT-C/BL21。工程菌诱导表达后，粗酶液经Q-sepharose和硫铵沉淀进行纯化。构建了锰过氧化氢酶C端(MnCAT-C)基因的敲除载体pK18mobsacB-ΔMnCAT-C，电击法转入Rhodococcus sp. R04。荧光极化测定ROS的含量，HPLC分析多氯联苯的降解率。【结果】与Rhodococcus sp. R1101Mn-CAT基因序列相比，Rhodococcus sp. R04Mn-CAT缺少N端(R1101的Mn-CAT序列长度为915bp，R04的MnCAT-C序列长度为468bp)。获得了纯度较高的MnCAT-C，SDS-PAGE分析表明分子量约为23 kDa。以H2O2为底物时，MnCAT-CKm比Mn-CATKm大，约为0.02357mol/L。通过基因同源重组的方式，得到菌株R04的MnCAT-C敲除菌株，与野生菌株相比，敲除菌株体内ROS浓度显著增高，生长速率和多氯联苯降解速率明显下降。【结论】发现了一种N端缺失的锰过氧化氢酶，该酶具有原酶的大部分 活性，且可以清除体内的ROS。MnCAT-C基因的缺失影响了菌株的生长速率和多氯联苯的降解速率。

Abstract:

Abstract:［Objective］ We characterizeda manganese catalase lacking n-terminal (MnCAT-C)，to revealits roles in bacterial growth，reactive oxygen species (ROS) removal and degradation of polychlorinated biphenyls (PCBs) in Rhodococcus sp. R04．［Methods］Manganese catalase (Mn-CAT) sequence of the strain R04 was aligned with that of Rhodococcus sp. R1101．Mn-CAT and MnCAT-C were expressed in E．coli BL21 (DE3)，and the target protein was purified with Q-sepharose and ammonium sulphate precipitation．Knockout strain was obtained by homologous recombination. ROS was measured by fluorescence polarization，and the degradation rate of PCBs was measured by HPLC．［Results］ MnCAT-C protein was purified，and SDS-PAGE analysis showed that its molecular weight was 23 kDa．Compared with wild strains，the ROS concentration increased，and the growth rate was inhibited in knockout strains．Moreover，the degradationrate of PCBs decreased．［Conclusion］MnCAT-C retained the majority of the active properties of the original enzyme，including ROS clearance．The lack of MnCAT-C gene affected the growth rate and the PCBsdegradationrate instrain R04.