Identification of a marine agarolytic bacterium Agarivorans albus QM38 and cloning and sequencing its beta-agarase genes

摘要

A total of 117 agar-decomposing cultures were isolated from coastal seawater around Qingdao,China.The phenotypic and agarolytic features of an agarolytic isolate,QM38,were investigated.The strain was gram negative,strictly aerobic,curved rod and polar flagellum.On the basis of several phenotypic characters,biochemical and morphological characters and phylogenetic analysis of the gene coding for the 16S rRNA,the strain was identified as Agarivorans albus strain QM38.This strain can liquefy the agar on the solid agar plate.An excellular agarase activity was determined in liquid culture.The enzyme exhibited maximal activity at 40℃,pH 7.6.Its activity was greatly affected by different concentrations of agarose.The highest activity 32 U/ml was achieved in the culture supernatant.The hydrolytic product was analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE).After complete hydrolysis of agarose,a series of agaro-oligosaccharides were produced.The main products of the enzymes were oligosaccharides in the degree of polymerization (DP) of 2,4,6 and 8.Three genes agaD01,agaD02 and agaD03,encoding β-agarases,had been cloned from genomic DNA of Agarivorans albus strain QM38.The open reading frame of agaD01,consisted of 2 988 bp,and shared 95.5%-98.9% identity to the β-agarase genes of some strains of Vibrio and Agarivorans.Gene agaD02 comprised 2 868 bp and encoded a 955-amino-acid protein.It showed 97.4% and 98.7% identity to the β-agarase genes of strain Vibrio sp.PO-303 and strain Vibrio sp.JT0107,respectively.Only partial sequence of agaD03 gene has been cloned.It showed 96.5% identity to β-agarase gene (agaB) of Pseudoalteromonas sp.CY24,and shared 96.8% identity to β-agarase-c gene of Vibrio sp.PO-303.