摘要
本文以Pseudoalteromonas carrageenovora ASY5菌株中的car基因序列为研究对象,通过PCR克隆卡拉胶酶基因,将它克隆至表达载体pET-28a,以期获得稳定性较高的酶,并实现大量制备卡拉胶酶的目的。利用生物信息学方法分析该基因及其推测出的编码蛋白理化性质、蛋白的亲水性或疏水性、预测信号肽、跨膜结构域、蛋白二级结构、三级结构等特征。结果表明,ASY5菌株的的cai基因开放阅读框全长1194 bp,卡拉胶蛋白包含397个氨基酸,分子量为35.8 kD、理论等电点为6.17,结构域分析表明该κ-卡拉酶符合GH16家族的特点,是一种含有信号肽的可溶性亲水蛋白质。二级结构以α-螺旋、β-折叠、β-转角和不规则卷曲为其基本结构单元。利用SWISS-MODEL对PseudoalTeromonas carrageenovoraκ-卡拉胶酶序列进行三维模建,Pymol软件观察Pseudosalteromonas carrageenovoraκ-卡拉胶酶与模版的重叠情况较好。
Nucleotide and deduced amino acid sequence of car gene were studied,which cloned from K-carrageenase producing bacteria strain Pseudoalteromonas carrageenovora ASY5.The gene(car) was amplified by PCR and cloned into the pET-28 a vector,expressed in Escherichia coli BL21(DE3) and characterized to increase stability of K-carrageenase for large scale preparation.K-Carrageenase gene were assessed by using bioinformatic methods,which including analysis of physical and chemical characteristics,hydrophobicity or hydrophilicity,prediction of signial peptide,transmembrane spaning domain,secondary structure and tertiary structure according to protein sequence.The results showed that K-carrageenase was comprised of 1194 bp.It encoded 397 amino acid polypeptide with a molecular weight of 35.8 kD and the isoelectric point of 6.17.Based on domain structure analysis,the K-carrageenase belongs to glycohydrolase family.It was a hydrophyilic soluble protein and had signal peptide.The main structural elements of the secondary structure are α-helix,β-sheet and random coil.Tertiary structure was also obtained using SWISS-MODEL and Pymol analysis.
引文