伯克霍尔德氏菌(Burkholderia sp.)2-萘酸单加氧酶基因(nmo)的克隆及表达

通过酶切连接将Burkholderia sp．JTl500的一段DNA片段(4．8kb)亚克隆到表达载体pUC18上，得到重组子pEKl23。测序后的pEKl23重组子4．8kb插入片段的序列已经登陆欧洲EMBL基因库，序列接受号为AJ566333。对这一DNA片段的序列分析显示，此DNA片段含有3个阅读框，且在这3个阅读框5’端发现一启动子特异序列。再用酶切连接方法得到仅含一个阅读框的重组子pXK3，其阅读框长度为1158bp，编码386个氨基酸，与已报道的Ralstonia eutropha HF39羟化酶(单加氧酶，bec)氨基酸序列有64％的同源性。pEKl23对2-萘酸代谢途径中4个关键底物的转化实验结果显示，其基因产物仅对2-萘酸发生加氧转化反应，而且2-萘酸浓度有明显的降低，证实此基因是2-萘酸单加氧酶基因(nmo)。同时发现其基因产物也可以转化苯甲酸钠。该酶对苯甲酸的加氧转化途径正在研究中。SDS-PAGE结果表明，pXK3、pEKl23两重组子中2-萘酸单加氧酶表达量并没明显区别，但加氧酶酶活却存在显著的差别。推测在启动子后，单加氧酶阅读框前的两个阅读框的基因产物，对单加氧酶活有促进作用。

Cloning and Expression of a 2-Naphthoate Monooxygenase Gene (nmo) in Burkholderia sp. JT1500

A 4.8kb DNA fragment from one blue colony of the pLARF1 gene library of Burkholderia sp. JT1500 was subcloned to pUC18, designated as pEK123. The sequence of the inserted 4.8kb DNA' of pEK123 was analyzed and submitted to EMBL nucleotide database, the accession # is AJ566333. The transformants of pEK123 could also become blue in LB agar and sequence analysis showed that three open reading frames and a putative promoter sequence were located in this inserted fragment. Then the 4.4kb insert fragment of pEK123 was double digested with Xba I / Kpn I and EcoR I / Xba I respectively to construct plamsids pXK3 and pEX12. The pXK3 contained only one 1158bp open reading frame (ORF) and pEX12 with other two ORFs. Unlike pEK123, the colonies of pEX12 did not show any blue color even incubated for 72h in LB agar, but the transformants of pXK3 did oxidize indole into indigo. The deduced 43kD protein of 1158bp ORF showed 64% homology of amino acid composition to Ralstonia eutropha HF39 hydroxylase (bec). Results of substrate transformation analysis showed that the transformants of pEK123 was able to catalyze the oxidation of 2-naphthoate but not other key intermediates in 2-naphthoate metabolic pathway. These results confirmed that the product of 1158bp ORF is 2-naphthoate monooxygenase. Though the oxygenase activity of pEK123 is much higher than that of pXK3, SDS-PAGE analysis found no difference between the amount of the band of monooxygenase produced by pXK3 or pEK123, but one more band was found produced by pEK123. According to the difference of substrate analysis between pXK3 and pEK123, it is supposed that the products of two open reading frames up stream of nmo gene had strong influence on the activity of the monooxygenase. Benzoate was oxidized by free-cell extracts of the transformants of pEK123 in the transformation experiment with different aromatic substrates. As the DNA sequence and amino acid sequence of 2-naphthoate monoxygenase (nmo) did no show any homology with the DNA sequence and amino acid sequence of benzoate oxygenases reported, the pathway of benzoate oxidation conducted by nmo is on the investigation.