中文摘要：

【目的】分别对恶臭假单胞菌（Pseudomonas putida）ND6基因组中基因高度同源的儿茶酚1,2-双加氧酶基因catA1、catA2进行克隆和原核表达,并进行生物信息学分析和酶学性质比较。【方法】通过生物信息学软件对catA1和catA2进行序列分析和进化树构建,利用经典的酶切连接方法和核酸外切酶III介导的无缝连接两种方法分别对catA1和catA2进行了表达载体的构建,对表达产物的酶学性质（最适反应温度,最适反应p H,耐热性,底物特异性等）进行了研究。【结果】catA1和catA2在基因序列上相似性为73.57%,两个酶的最适反应p H均为7.4,最适反应温度分别为45℃、50℃,Km值分别为1.28、1.66μmol/L,酶活力单位分别为9.00、8.35 U/mg,两个酶对儿茶酚和4-甲基儿茶酚具有较好的反应活性,在C12O分类中属于同一类型。【结论】本研究通过两种方式成功构建了高度同源性两个基因的表达载体,对于分离高同源性基因有一定的参考意义,同时通过对ND6菌株中同工酶的酶学性质进行研究和比较,加深了对ND6萘降解机制的理解,进一步探讨了冗余降解基因存在的意义。

英文摘要：

【Objective】To clone two highly homologous catechol 1, 2-dioxygenase genes（named catA, and catA2） in Pseudomonas putida ND6 genome sequence which were then expressed in E.coli BL21（DE3）, and analyze the gene sequence and enzymatic properties.【Methods】The gene sequence of cat A1 and catA2 was analyzed and phylogenetic tree was constructed by bioinformatics tools respectively. The expression vectors of catA1 and catA2 were constructed by traditional polymerase chain reaction（PCR） combined with restriction enzyme digestion（exonuclease III）. The enzymatic properties（optimal temperature, optimal p H, thermostability and substrate-specificity etc.） were detected.【Results】Sequence analysis showed that the catA1 and catA2 exhibited a homology of 73.57% in nucleotide acid levels. Their optimal p H was 7.4. The optimal temperatures of two enzymes were 45℃ and 50℃. The Km values of two enzymes were 1.28 μmol/L and 1.66 μmol/L. The levels of Vmaxwere 9.00 U/mg and 8.35 U/mg. They shared analogical enzymatic properties towards catechol and 4-methylcatechol, and they both belonged to the same class in the catechol 1,2-dioxygenase（C12O） category.【Conclusion】The expression vectors of two highly homologous genes（catA1 and catA2） are successfully constructed in two different ways. It provides a reference for homologous gene isolation. The study on enzymatic properties in Pseudomonas putida ND6 is helpful for interpreting the degradation mechanism of naphthalene in ND6 and the existence meaning of redundancy degradation genes.