假单胞菌ND6菌株儿茶酚2，3-双加氧酶的性质和蛋白质工程研究-生物化学与分子生物学专业论文.docxVIP

南开大学硕士研究生(学位)毕业论文摘 南开大学硕士研究生(学位)毕业论文 摘 要 在大多数芳香化合物的生物好氧代谢过程中，各种化合物通过不同的降解 途径降解，汇合到儿茶酚(邻苯二酚)，再由儿茶酚．2，3双加氧酶(C230)或 儿茶酚1，2一双加氧酶(大多数是儿茶酚2,3双加氧酶)分解成开环产物2一羟基 粘康酸半醛，再由共同的途径降解成可进入三羧酸循环的小分子物质，因此C230 基因的结构和功能多样性及进化关系的研究受到广泛重视。 本实验室分离到的假单胞菌ND6是一个萘降解菌株，其萘降解基因位于一 个102kb的质粒pND6．1上。其中的儿茶酚2,3．双加氧酶基因(nahH)己被克 隆到大肠杆菌中。本研究对此基因表达产物C230进行纯化，测定其酶活及耐 热性，并与国外报道的萘降解质粒NAH7中nahH基因编码的酶进行对比。结 果表明，ND6菌株中的C230酶活力更高，耐热性更好。 用PCR方法从环境污染物中分离C230基因的中间片段和pND6．1质粒的 C230基因两端片段，再通过第二轮PCR将三个片段相连，得到杂合的ncIhH 基因。希望通过这种盒式PCR(cassette-PCR)的方法得到性能更加优越的儿茶 酚2，3．双加氧酶。本研究将NAH7的nahH基因中间片段与pND6一l质粒的C230 基因两端片段相连得到了一个杂合的nahH基因，并克隆到大肠杆菌中进行表 达，纯化表达产物，杂合的C230具有较高的酶活。 关键词：儿茶酚2,3．双加氧酶，酶的纯化和性质，盒式PCR，杂合基因 南开大学硕士研究生(学位)毕业论文Abstract 南开大学硕士研究生(学位)毕业论文 Abstract In the broad outline aerobic aromatic catabolism，a variety of pathways will converge on some common intermediates，catechols，which are then converted by the catechol 2，3-dioxygenase(c230)Or catechol 1，2-dioxygenase(c 1 20)．In most cases，the catechol is catalyzed by the C230 into the 2-hydroxymuconic semialdehyde， which is further assimilated by a small number of common pathways into small aliphatic compounds that Call enter central metabolism directly． Pseudomonas SP ND6 is a naphthalene-degrading strain isolated by our lab．Its naphthalene degrading genes are located on a 102kb plasmid，pND6-1 The nahH gene，which encodes C230，has been cloned and expressed in E coli This gene product was purified and its enzymatic activity and thermostability were compared with the C230 from NAH7 strain．The results revealed that the C230 from ND6 strain has higher activity and thermostability． A method was developed to isolate central segments of C230 genes from environmental samples and to insert these C230 gene segments into nahH(the structural gene for C230 encoded by catabolic plasmid pND6—1)by replacing the corresponding nahH sequence with the isolated segments．The hybrid gene WaS expressed in the E coli and the expressed product has the activity of C230 Key words：Catechol 2,3一dioxygena