香茅醇假单胞菌SJTE-3的短链脱氢酶SDR-X1的克隆及酶性质测定

Gene Cloning and Enzymatic Properties of the Short Chain Dehydrogenase SDR-X1 from Pseudomonas citronellolis SJTE-3

香茅醇假单胞菌SJTE-3能够以17β-雌二醇为唯一碳源并将其高效降解,但其催化雌二醇转化的关键酶仍不明确。本文鉴定了该菌株中降解雌二醇的短链脱氢酶SDR-X1(WP_043267487.1),并对其功能进行了研究。首先利用荧光定量PCR,检测了不同碳源条件下基因sdr-x1的转录水平;克隆基因sdr-x1,在大肠杆菌BL21(DE3)菌株中诱导表达,利用亲和层析纯化获得了重组蛋白SDR-X1;体外检测了重组蛋白SDR-X1的催化活性与酶学性质,并利用高效液相色谱鉴定了其转化17β-雌二醇的产物。蛋白SDR-X1可被17β-雌二醇诱导表达;蛋白序列比对显示蛋白SDR-X1含有短链脱氢酶的保守基序与氨基酸。该酶以NAD+为辅因子,将17β-雌二醇氧化为雌酮;其K_(m)值为(0.03986±0.004061)mmol/L,V_(max)值为(3.168±0.135)mmol/L/min/mg,可在15 min内转化61.75%以上的雌二醇。该酶对温度具有一定耐受性,最佳反应温度为50℃,偏碱性pH可促进其酶促反应。不同二价金属离子对该酶活性具有不同的影响,Mg^(2+)、Mn^(2+)和Ca^(2+)可增强其酶活性。香茅醇假单胞菌SJTE-3中的SDR-X1可高效催化17β-雌二醇转化,参与该菌株的雌激素降解过程,其功能研究将推进细菌的雌激素代谢机制解析。

Pseudomonas citronellolis SJTE-3 can efficiently degrade 17β-estradiol as the sole carbon source,but the key enzyme catalyzing the transformation of estradiol remains unclear.The short chain dehydrogenase SDR-X1(WP_043267487.1)in strain SJTE-3 degrading estradiol was identified and its function was studied.First fluorescence quantitative PCR was used to detect the transcription levels of gene sdr-x1 under different carbon sources.Gene sdr-x1 was cloned and over-expressed in Escherichia coli BL21(DE3)strain,and the recombinant protein SDR-X1 was purified by affinity chromatography.The catalytic properties of protein SDR-X1 to estrogen were characterized and the conversion products of 17β-estradiol were identified by high performance liquid chromatography.The transcription of gene sdr-x1 was induced by 17β-estradiol.The results of multiple sequence alignment showed that protein SDR-X1 contained the conserved motifs and amino acid residues of short chain dehydrogenase.Using NAD+as its co-factor,17β-estradiol was oxidized into estrone.Its K_(m) value was(0.03986±0.004061)mmol/L and V_(max) value was(3.168±0.135)mmol/L/min/mg,and 61.75%of 17β-estradiol was converted within 15 min.The enzyme SDR-X1 had certain tolerance to temperature and its optimal reaction temperature was 50℃.The alkaline pH promoted the enzymatic reaction of SDR-X1.Different divalent metal ions had different effects on the enzymatic activity,and Mg^(2+)and Mn^(2+)enhanced the activity of the enzyme SDR-X1.The enzyme SDR-X1 in P.citronellolis SJTE-3 effectively catalyzed the transformation of 17β-estradiol and was involved in the estrogen-degrading process.Its characterization can promote the mechanism study of estrogen metabolism in bacteria.