一种新型琼胶酶AgaB的基因克隆及生物信息学分析

Molecular Cloning and Bioinformatic Analysis of a Novel Agarase Enzyme Gene,AgaB

利用从青岛近海发现的一株高产琼胶酶的假别单胞菌CY24(Pseudoaltero-monassp.CY24)建立基因组DNA文库后,用水解圈的方法从中筛选到一个具有琼胶酶活性的阳性克隆pBA1。pBA1的序列分析结果表明,插入DNA片段的长度为6.7 kb,含有4个推测读码框架,并利用亚克隆法确定了琼胶酶的编码基因agaB。agaB基因的开放阅读框架为1 437 bp,编码478个氨基酸,理论分子量为50.9 kDa,在N末端有一个包含38个氨基酸的信号肽。推测的成熟蛋白质的分子量和等电点分别为47.2 kDa和4.83。AgaB的氨基酸序列与已知蛋白质包括所有的糖苷水解酶序列完全没有相似性;HCA的蛋白质二级结构分析结果表明,AgaB不同于其他已知琼胶酶。因此,推测AgaB为一新型的琼胶酶,代表一个新的糖苷水解酶家族。

Based on sole carbon source culture and clearing-zone screening, a strain named as Pseudoalterornonas sp. CY24 with high agarase activity was isolated from the coast of Qingdao. Using the genomie library and clearing-zone screening method, a subclone pBA1 with the agarase activity was obtained. Subelone pBA1 has four open read- ing frames. To determine the location of agarase, double enzymatic cleavage to generate the smaller subelone method was carried on and at last an agarase gene, named agaB was identified with a single open reading frame of 1 437 bp （GenBank accession number AY293310）. The deduced protein of agaB gene is 478 amino acids with a theoretical molecular mass of 50.9 kDa. Sequence analysis showed AgaB did not display obvious overall similarities to any of the previously defined glycoside hydrolase families, and it appeared to be divided into a new family.