Directed evolution of biphenyl dioxygenase: Emergence of enhanced degradation capacity for benzene, toluene, and alkylbenzenes
Directed evolution of biphenyl dioxygenase: Emergence of enhanced degradation capacity for benzene, toluene, and alkylbenzenes
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DOI：
10.1128/jb.183.18.5441-5444.2001
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发表时间：
2001-09-01
影响因子：
3.2
通讯作者：
Furukawa, K
中科院分区：
文献类型：
--
作者：
Suenaga, H;Mitsuoka, M;Furukawa, K
联苯双加氧酶(BPH Dox)催化联苯及相关化合物的初始氧化。BPH Dox是一种多组分的酶，其中一个大亚基(由bphA1基因编码)与底物特异性密切相关。利用假单胞菌KF707和洋葱伯克霍尔德氏菌LB400的bphA1 DNA改组过程，产生了一系列进化的BPH Dox酶。其中，表达BPH Dox嵌合体的大肠杆菌克隆对苯、甲苯和烷基苯的降解率显著提高。在这个进化的BphA1中，四个氨基酸(H255Q、V258I、G268A和F277Y)从KF707酶改变为LB400酶。随后的定点突变使我们能够确定导致单环芳香烃降解的氨基酸。
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.