联苯双加氧酶的定向进化：对苯、甲苯和烷基苯的降解能力增强的出现
Directed evolution of biphenyl dioxygenase: emergence of enhanced degradation capacity for benzene, toluene, and alkylbenzenes.
作者信息
Suenaga H, Mitsuoka M, Ura Y, Watanabe T, Furukawa K
机构信息
Department of Biosciences and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan.
出版信息
J Bacteriol. 2001 Sep;183(18):5441-4. doi: 10.1128/JB.183.18.5441-5444.2001.
Abstract
Biphenyl dioxygenase (Bph Dox) catalyzes the initial oxygenation of biphenyl and related compounds. Bph Dox is a multicomponent enzyme in which a large subunit (encoded by the bphA1 gene) is significantly responsible for substrate specificity. By using the process of DNA shuffling of bphA1 of Pseudomonas pseudoalcaligenes KF707 and Burkholderia cepacia LB400, a number of evolved Bph Dox enzymes were created. Among them, an Escherichia coli clone expressing chimeric Bph Dox exhibited extremely enhanced benzene-, toluene-, and alkylbenzene-degrading abilities. In this evolved BphA1, four amino acids (H255Q, V258I, G268A, and F277Y) were changed from the KF707 enzyme to those of the LB400 enzyme. Subsequent site-directed mutagenesis allowed us to determine the amino acids responsible for the degradation of monocyclic aromatic hydrocarbons.
摘要
联苯双加氧酶（Bph Dox）催化联苯及相关化合物的初始氧化反应。Bph Dox是一种多组分酶，其中一个大亚基（由bphA1基因编码）对底物特异性起重要作用。通过对假产碱假单胞菌KF707和洋葱伯克霍尔德菌LB400的bphA1进行DNA改组过程，产生了许多进化的Bph Dox酶。其中，一个表达嵌合Bph Dox的大肠杆菌克隆表现出极其增强的苯、甲苯和烷基苯降解能力。在这种进化的BphA1中，四个氨基酸（H255Q、V258I、G268A和F277Y）从KF707酶变为LB400酶的氨基酸。随后的定点诱变使我们能够确定负责单环芳烃降解的氨基酸。
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