深海细菌Pseudomonas sp.IOFA1醇脱氢酶酶学性质研究

Characterization of alcohol dehydrogenase from deep-sea bacterium Pseudomonas sp. IOFA1

[目的]对来自印度洋深海的一株假单胞菌醇脱氢酶(Adh)进行序列分析和酶活性质分析。[方法]首先以同源比对和进化树聚类为手段分析该酶序列信息。其次,在E.coli宿主中进行重组表达和镍柱亲和纯化后,对重组醇脱氢酶的酶活性质进行进一步地研究。[结果]结果显示Adh蛋白与其它物种已知醇脱氢酶的氨基酸序列最高相似性为81%,分属于第三类醇脱氢酶蛋白。酶学性质分析表明,重组酶Adh的最适作用温度为42℃,表现出良好的中低温适应性;最适p H值为5.0,在p H 4~6时具有较高的活性,表明Adh为酸性醇脱氢酶。Zn^(^(2+))、Na+在终浓度为0.5 mmol/L时对Adh有明显的激活作用,尤其是Zn^(2+)可使Adh的酶活显著提高11%。[结论]实现了adh基因在大肠杆菌的高效表达,为Adh的应用提供了理论依据。

[ Objective] The aim of this study was to characterize the DNA/protein sequence and the enzymatic activity of an alcohol dehydrogenase gene （designated as adh） from Pseudomonas sp. IOFA1 ,which was isolated from deep- sea sediments of Indian Ocean. [ Methods ] Using homologous alignment and phylogenetic analysis, the primary sequence of Adh was first studied. After heterologously expressed in Escherichia coli, recombinant Adh was purified with a Ni affinity column and the enzy- matic activity was further characterized. [ Results ] It showed that Adh shared 81% maximum amino acid sequence identity to known alcohol dehydrogenase from other species and belonged to class 3 alcohol dehydrogenase. The recombinant enzyme was most active at 42℃ and retained more than 50% of its maximal activity at temperature range of 0 -52℃ , suggesting it can adapt to medium and low temperature environments. The optimal pH for Adh was determined to be 5 and Adh was found to be a acidic alcohol dehydrogenase as it was highly active at pH ranging from 4 - 6. Some ions （ Zn^2＋, Na^＋） , especially Zn^2＋ could largely increased the activity of Adh by up to 11%. [ Conclusion ] Collectively, Adh was successfully expressed in Escherichia coli,which will provide foundations for application of Adh in industry in the future.