酮古龙酸菌醇醛脱氢酶基因的克隆及表达

目的：从酮古龙酸菌SCB329株中分离山梨糖生物氧化相关酶的基因并进行表达验证。方法：根据酮古龙酸菌SCB329株基因组序列设计引物,通过PCR从SCB329株基因组中扩增醇醛脱氢酶基因aadh;构建载体pBMP3-aadh并在大肠杆菌中表达,经活性染色、体外转化反应等方法考察表达产物的活性。结果：目的产物能够催化山梨糖、葡萄糖、果糖、木糖等多种含羟基及羰基化合物脱氢,并能将L-山梨糖直接转化为2-酮基-L-古龙酸。结论：从酮古龙酸菌SCB329株中分离到一种醇醛脱氢酶基因,可为该菌株糖酸转化机制的研究提供帮助。

Cloning and Expression of an Alcohol/Aldehyde Dehydrogenase Gene of Ketogulonigenium vulgare

Objective： To isolate a sorbose bio-oxidation related gene from Ketogulonigenium vulgare SCB329 and to verify it by expressing it in E.coli. Methods： The primers were designed according to the genome sequence of K.vulgare SCB329 and the gene fragment aadh encoding alcohol/aldehyde dehydrogenase was amplified by PCR using SCB329 genome as template. A vector for aadh expression, pBMP3-aadh, was constructed and expressed in E.coli DH5α. The bioactivity of the enzyme was tested by native PAGE and in vitro bioconversion. Results： The protein of interest was able to catalyze dehydrogenation of multiple compounds with hydroxyl or carbonyl and convert L-sorbose directly to 2-keto-Lgulonic acid. Conclusion： A gene coding alcoholic aldehyde dehydrogenase was isolated from SCB329, which would be useful for further research on mechanism of bio-oxidation of sorbose in Ketogulonigenium.