Cloning, sequencing, and overexpression in Escherichia coli of the Enterobacter sp Px6-4 gene for ferulic acid decarboxylase
Cloning, sequencing, and overexpression in Escherichia coli of the Enterobacter sp Px6-4 gene for ferulic acid decarboxylase
复制标题
阿魏酸脱羧酶肠杆菌 Px6-4 基因在大肠杆菌中的克隆、测序和过表达
DOI：
10.1007/s00253-010-2978-4
复制
发表时间：
2011-03-01
影响因子：
5
通讯作者：
Yang, Jinkui
中科院分区：
文献类型：
--
作者：
Gu, Wen;Li, Xuemei;Yang, Jinkui
阿魏酸脱羧酶（Ferulic acid decarboxylase，FADase）是一种能催化阿魏酸脱羧生成4-乙烯基愈创木酚的酶。本研究利用简并引物和基因组步移技术，首次从肠杆菌属Px 6 -4中分离到编码FAD酶的基因。FAD酶的推定编码基因（fad）由507 bp核苷酸组成，编码168个氨基酸残基的多肽。此外，一个假定的基因编码的转录调控被确定从上游的fad基因。肠杆菌属Px 6 -4的FAD酶与其它细菌的脱羧酶的同源性为51.2-53.3%。在大肠杆菌BL 21中成功表达了fad基因，并纯化了重组FAD酶。23 kDa，在pH 4.0和28 ℃下具有最佳活性。纯化的FAD酶能有效地将阿魏酸转化为4-乙烯基愈创木酚，Cu 2+和Hg 2+对酶的抑制率分别为99%和99.5%。来自细菌的FADase蛋白的系统发育分析揭示了几个不同的进化枝。本研究结果为进一步研究阿魏酸转化途径和提高香草醛的产量奠定了基础。
Ferulic acid decarboxylase (FADase) can catalyze the transformation of ferulic acid into 4-vinyl guaiacol via decarboxylation in microorganisms. In this study, a gene encoding FADase was first isolated from the bacterium Enterobacter sp. Px6-4 using degenerate primers and a genome walking technique. The putative encoding gene (fad) of FADase consists of 507-bp nucleotides, coding a polypeptide of 168 amino acid residues. In addition, a putative gene encoding the transcriptional regulator was identified from the upstream of the fad gene. The deduced peptide sequence of the FADase from Enterobacter sp. Px6-4 showed a 51.2-53.3% sequence identity to decarboxylases from other bacteria. The gene fad was successfully expressed in Escherichia coli BL21, and the recombinant FADase was purified as a protein of ca. 23 kDa with an optimal activity at pH 4.0 and 28 degrees C. The purified FADase could convert ferulic acid to 4-vinyl guaiacol effectively, and its hydrolytic activity could be inhibited by Cu2+ (99%) and Hg2+ (99.5%). A phylogenetic analysis of the FADase protein from bacteria revealed several different clades. Our result provided a basis for further studies of the ferulic acid transformation pathway and for enhanced production of vanillin in the future.