Cloning and characterisation of four catA genes located on the chromosome and large plasmid of Pseudomonas putida ND6
Cloning and characterisation of four catA genes located on the chromosome and large plasmid of Pseudomonas putida ND6
复制标题
恶臭假单胞菌ND6染色体上4个catA基因及大质粒的克隆及鉴定
DOI：
10.1016/j.ejbt.2018.06.001
复制
发表时间：
2018-07-01
影响因子：
2.7
通讯作者：
Zhao, Huabing
中科院分区：
文献类型：
--
作者：
Li, Shanshan;Qin, Kun;Zhao, Huabing
背景资料：虽然在几种微生物中已经报道了儿茶酚1，2-双加氧酶（C120）基因的功能冗余，但是有限的酶被表征，更不用说C120基因的多个拷贝共存的优点了。在本研究中，在萘降解菌Pseudomonasputida ND 6中发现了4个新的C120基因，命名为catA、catA（I）、catA（II）和catA（III），被克隆并鉴定。氨基酸序列的系统发育分析表明，这4种C120同工酶与其他生物的同源物一起形成独立的子树。所有四种酶在pH 7.4时表现出最大活性，并且在碱性条件下比在酸性条件下具有更高的活性。此外，CatA、CatA（I）和CatA（III）在45 ℃的温度下具有最大活性，而CatA（II）在50 ℃的温度下观察到更高的最适温度。CatA（I）的温度稳定性上级其它3种C120同工酶，动力学分析表明CatA、CatA（I）和CatA（III）的酶活性相似，CatA（III）的酶活性较低。值得注意的是，在金属离子的测试，只有铜2+基本上抑制这些C120同工酶的活性，从而表明它们有可能促进在金属的存在下与芳烃污染的环境中的生物修复。结论：catA基因的冗余增加了恶臭假单胞菌ND 6中C120的可利用量，有利于其在逆境中的生存。(C)2018年瓦尔帕莱索天主教大学。Elsevier B. V.制作和主持。保留所有权利。
Background: Although the functional redundancy of catechol 1,2-dioxygenase (C120) genes has been reported in several microorganisms, limited enzymes were characterised, let alone the advantage of the coexistence of the multiple copies of C120 genes.Results: In this study, four novel C120 genes, designated catA, catA(I), catA(II) and catA(III), in the naphthalene-degrading strain Pseudomonas putida ND6, were cloned and characterised. Phylogenetic analysis of their deduced amino acid sequences revealed that the four C120 isozymes each formed independent subtrees, together with homologues from other organisms. All four enzymes exhibited maximum activity at pH 7.4 and higher activity in alkaline than in acidic conditions. Furthermore, CatA, CatA(I) and CatA(III) were maximally active at a temperature of 45 degrees C, whereas a higher optimum temperature was observed for CatA(II) at a temperature of 50 degrees C. CatA(I) exhibited superior temperature stability compared with the other three C120 isozymes, and kinetic analysis indicated similar enzyme activities for CatA, CatA(I) and CatA(III), whereas that of CatA(III) was lower. Significantly, among metal ions tested, only Cu2+ substantially inhibited the activity of these C120 isozymes, thus indicating that they have potential to facilitate bioremediation in environments polluted with aromatics in the presence of metals. Moreover, gene expression analysis at the mRNA level and determination of enzyme activity dearly indicated that the redundancy of the catA genes has increased the levels of C120.Conclusion: The results clearly imply that the redundancy of catA genes increases the available amount of C120 in P. putida ND6, which would be beneficial for survival in challenging environments. (C) 2018 Pontificia Universidad Catolica de Valparaiso. Production and hosting by Elsevier B.V. All rights reserved.