不动杆菌JH250-8邻苯二酚双加氧酶基因的克隆及分析

目的研究不动杆菌JH250-8的石油降解机制,克隆并分析其邻苯二酚双加氧酶基因.方法应用数据库的同源序列设计引物,对不动杆菌JH250-8邻苯二酚1,2-双加氧酶(C12O)和邻苯二酚2,3-双加氧酶(C23O)基因进行扩增,并用生物信息学软件及在线生物分析工具等分析编码蛋白质.结果不动杆菌JH250-8中同时含有C12O和C23O两种邻苯二酚双加氧酶;扩增的DNA序列长度分别为1 299和1 118 bp,蛋白质编码区长度为1047和930 bp,分别编码358和309个氨基酸;两个邻苯二酚双加氧酶等电点(p I)分别为5.03和4.79,均为酸性蛋白质;酶分子中都含有较多极性和可电离氨基酸,在水中有较好的溶解性,但由于N端都有一段疏水性肽段,可能会影响其水溶液的稳定性;在蛋白质二级结构上,两个酶分子中都有较丰富的二级结构单元,既有较好的稳定性,又有较好的底物适应性.C12O的三级结构由同型二聚体构成,每个亚基上都结合有Fe3+;C23O三级结构的核心由两组β-折叠构成,另有4个α-螺旋结构分布于外侧.结论不动杆菌JH250-8有两种邻苯二酚双加氧酶,两种酶均为酸性蛋白质,有较好的水溶性及结构稳定性.分析结果对邻苯二酚双加氧酶的后续研究有重要的参考价值及指导意义.

Clone and Analysis of Catechol Dioxygenase Gene from Strain JH250-8 of Acinetobacter

Objective To investigate the oil degradation mechanism, the genes of catechol dioxygenases from strain JH250-8 of Acinetobacter were cloned and analyzed. Method Homologous sequences from data base online were used to design PCR primers. Then the genes of catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase ( C23 O ) from JH250-8 were amplified and analyzed by bioinformatics software and online bioanalysis tools. Results Both catechol dioxygenases (C12O and C23O) were detected in the strain JH250-8. The length of DNA sequences were 1299 bp and 1118 bp and coding regions were 1047 bp and 930 bp separately. The length of amino acid sequences were 358 and 309. Both catechol dioxygenases are acidic protein with pI value 5. 03 and 4. 79. For the presence of polar amino acids and charged amino acids,the two proteins were more stable in aqueous solution,while the hydrophobic peptides exist at the N-terminus should affect their stabilities in water. Prediction of protein secondary structure results showed that,for the rich of constitutional units both C12O and C23O were stable in structure and adaptable for substrates. The results of tertiary structure prediction showed that there is Fe3+binding site in C12O,and the nature structure C12O might be a dimer formed by two enzyme molecules. While there was a hydrophobic core formed with two groups ofβ-folding structures,and four α-helical structures distribute outside of protein. Conclusion There are two kinds of catechol dioxygenases in strain JH250-8. Both of them are belong to acidic protein, and with better water solubility and structural stability. Those analytical results were important to investigate catechol dioxygenases of JH250-8.