[目的]研究Sphingomonas sp.YL-JM2C菌株的生长特性,确定以三氯卡班作为碳源的生长情况.挖掘菌株YL-JM2C潜在的邻苯二酚1,2-双加氧酶及邻苯二酚2,3-双加氧酶基因,在大肠杆菌(Escherichia coli)中异源表达邻苯二酚双加氧酶基因并研究其酶学性质.[方法]优化S.sp.YL-JM2C菌株以三氯卡班作为碳源时的培养条件,并利用全自动生长曲线测定仪测定菌株生长情况,绘制生长曲线.通过生物信息学方法挖掘潜在的邻苯二酚双加氧酶基因,并分别在Escherichia coli BL21 (DE3)中进行异源表达,通过AKTA快速纯化系统纯化蛋白,分别以邻苯二酚、3-和4-氯邻苯二酚为底物检测重组蛋白的酶学特性.[结果]菌株在pH为7.0-7.5时生长最优.在以浓度为4-8 mg/L的三氯卡班做为底物时,菌株适宜生长.当R2A培养基仅含有0.01％酵母提取物和无机盐时,加入终浓度为4 mg/L的三氯卡班可促进菌株生长.挖掘-6个潜在的邻苯二酚双加氧酶基因stcA1、stcA2、stcA3、stcE1、stcE2和stcE3,表达并通过粗酶液分析证明其中5个基因stcA1、stcA2、stcA3、stcE1和stcE2编码的酶均具有邻苯二酚双加氧酶和氯邻苯二酚双加氧酶的活性;纯化酶的底物范围研究揭示了StcA1、StcA2和StcA3均属于Ⅱ型邻苯二酚1,2-双加氧酶,StcE1和StcE2为两个新型邻苯二酚2,3-双加氧酶;它们酶动力学分析研究证明了5个酶对邻苯二酚的亲和力和催化效率最高,4-氯邻苯二酚次之.[结论]在同一菌株中发现了5个具有功能的邻苯二酚双加氧酶基因,stcA1、stcA2和stcA3编码的酶均属于Ⅱ型邻苯二酚1,2-双加氧酶,stc E1和stcE2为两个新型邻苯二酚2,3-双加氧酶编码基因.5个酶均具有催化邻苯二酚和氯邻苯二酚开环反应的功能,这为更好地理解微生物基因组内代谢邻苯二酚及其衍生物氯代邻苯二酚基因的多样性奠定了基础.%[Objective] We are aiming to study the growth characteristics of Sphingomonas sp.YL-JM2C when triclocarban acts as carbon source;to mine putative genes encoding catechol 1,2-dioxygenase and catechol 2,3-dioxygenase from strain YL-JM2C;and to express,purify and functionally identify the putative catechol dioxygenase,then revealing the characters of catechol dioxygenases in this strain.[Methods] The growth of S.sp.YL-JM2C was measured at different pH values with different triclocarban concentrations on the optimized R2A medium containing extremely low amount of single carbon source.The putative catechol dioxygenase genes were cloned and heterologous expressed in E.coli BL21 (DE3),and purified through AKTA purifier system.The purified proteins were functionally identified based on their abilities to catalyze the ring cleavage of catechol as well as its derivatives 3-chlorocatechol and 4-chlorocatechol.Their enzymatic characters were also determined through enzyme kinetics parameters.[Results] The optimal pH value was 7.0-7.5 for the growth of strain YL-JM2C,and this strain was able to utilize triclocarban as the carbon source and the optimum concentration for its growth was in the range of 4-8 mg/L.With adding 4 mg/L triclocarban,the biomass of strain YL-JM2C increased over time when this strain grew in the optimized R2A medium containing 0.01％ of yeast extract and mineral salts.Six putative catechol dioxygenase genes (stcA1,stcA2,stcA3,stcE1,stcE2 and stcE3) were found through bioinformatics analysis.All exhibited catechol dioxygenase activities through assay using crude enzymes expressed in E.coli,except StcE3.Further,after purification,the substrate range analyses revealed that StcA1,StcA2 and StcA3 belonged to type Ⅱ catechol 1,2-dioxygenase while StcE1 and StcE2 were identified as the novel type catechol 2,3-dioxygenase,which were all able to catalyze the ring cleavage of catechol and chlorocatechol.The kinetics parameters obtained from purified enzymes revealed that the enzymes exhibited highest affinity and catalytic efficiency to catechol,followed by 4-chlorocatechol.[Conclusion] In this research,five active catechol dioxygenase genes are functional identified from a single strain,which all have the ability to catalyze the ring cleavage of catechol and chlorocatechol.Among them,StcA1,StcA2,and StcA3 belong to type Ⅱ catechol 1,2-dioxygenase,and StcE1 and StcE2 are new type catechol 2,3-diocygenase.This study will be greatly helpful to explore the diverse capability for the microbial aromatic degradation of microbes using catechol and chlorocatechol as ring-cleavage substrates.
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