Rashamuse K J, Burton S G, Cowan D A
CSIR Biosciences, Modderfontein, Johannesburg, South Africa.
J Appl Microbiol. 2007 Nov;103(5):1610-20. doi: 10.1111/j.1365-2672.2007.03394.x.
Isolation and identification of bacterial isolates with specific ferulic acid (FA) esterase activity and cloning of a gene encoding activity.
A micro-organism with ethyl ferulate hydrolysing (EFH) activity was isolated by culture enrichment techniques. Detailed molecular identification based on species-specific primers and two conserved genes (16S rRNA and recA) led to the identification of the isolate as Burkholderia multivorans UWC10. A gene (designated estEFH5) encoding an EFH enzyme was cloned and its nucleotide sequence determined. Translational analysis revealed that estEFH5 encoded a polypeptide of 326 amino acids with an estimated molecular weight of 34.83 kDa. The EstEFH5 primary structure showed a typical serine hydrolase motif (G-H-S-L-G). The estEFH5 gene was over-expressed in Escherichia coli in an insoluble form. Following urea denaturation and in vitro refolding, the enzyme was purified using one-step His Select Nickel chromatographic column.
Purified EstEFH5 showed a preference for short-chain rho-nitrophenyl esters (C2 and C3) a typical feature for carboxylesterase. Furthermore, the recombinant enzyme also retained the activity against ethyl ferulate (EF).
A biocatalytic process for the production of FA from EF as a model substrate was demonstrated. This is the first report that describes the cloning and expression of a gene encoding FA esterase activity from the genus Burkholderia.
分离和鉴定具有特定阿魏酸（FA）酯酶活性的细菌菌株，并克隆编码该活性的基因。
通过培养富集技术分离出具有阿魏酸乙酯水解（EFH）活性的微生物。基于种特异性引物和两个保守基因（16S rRNA和recA）进行详细的分子鉴定，结果表明该分离株为多食伯克霍尔德菌UWC10。克隆了一个编码EFH酶的基因（命名为estEFH5）并测定了其核苷酸序列。翻译分析表明，estEFH5编码一个由326个氨基酸组成的多肽，估计分子量为34.83 kDa。EstEFH5的一级结构显示出典型的丝氨酸水解酶基序（G-H-S-L-G）。estEFH5基因在大肠杆菌中以不溶性形式过表达。经过尿素变性和体外重折叠后，使用一步His Select镍色谱柱对该酶进行纯化。
纯化后的EstEFH5对短链对硝基苯酯（C2和C3）表现出偏好，这是羧酸酯酶的典型特征。此外，重组酶对阿魏酸乙酯（EF）也保留了活性。
证明了以EF作为模型底物生产FA的生物催化过程。这是首次报道从伯克霍尔德菌属克隆和表达编码FA酯酶活性的基因。
