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

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, encodingβ-agarases, had been cloned from genomic DNA of Agarivorans albus strain QM38. The open reading frame of agaDOl, 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 98.7~0 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.

Agarase Production by Marine Pseudoalteromonas sp.MHS:Optimization,and Purification

Agar is an essential polysaccharide that has been utilized in numerous fields.Many kinds of literature have been published regarding agarolytic microorganisms’isolation and agarases biochemical studies.In this search,a local marine agarolytic bacterium associated with marine alga Ulva lactuca surface was isolated and identified as Pseudoalteromonas sp.MHS.The agarase production was parallel to the growth of Pseudoalteromonas sp.MHS as cells displayed a lag phase(2 h),subsequently an exponential growth that prolonged till 10 h where maximum growth(OD550nm=3.9)was achieved.The enzyme activity increased rapidly as cells increased exponentially where the maximum activity of 0.22 U/mL was achieved after 8h and remained constant till 12 h during the stationary phase of growth.Agarase production was optimized using Plackett-Burman statistical design by measuring enzyme activity as a response and the design was validated using a verification experiment;the activity of the enzyme increased from 0.22 U/mL to 0.29 U/mL.Pseudoalteromonas sp.MHS agarase was partially purified and its molecular weight(MW)was determined by SDS-PAGE(15-25 kDa).Agarase showed approximately 94% of its activity at 40℃.The enzyme stability decreased as the temperature increased;the enzyme could retain about 98,90,80,75,and 60% of its activity at 20,30,40,50,and 60℃,respectively.Biomass of the red alga Pterocladia capillacea proved to be a suitable substrate for agarase production using Pseudoalteromonas sp.MHS;the enzyme activity recorded after 24 h of incubation was 0.35 U/mL compared to 0.29 U/mL from the optimized medium.

Applying Both Chemical Liquefaction and Enzymatic Catalysis Can Increase Production of Agaro-Oligosaccharides from Agarose

Red algae represents an important marine bioresource.One high-value utilization of red algae is the production of agarooligosaccharides which have many positive biological effects.However,the lack of an efficient production route seriously limits the application of agaro-oligosaccharides.In this study,we established a green route that combines chemical liquefaction and enzymatic catalysis for the efficient production of agaro-oligosaccharides,and we used the production of neoagarotetraose(NA4)as an example.Agarose(150 g L^−1)liquefaction by citric acid was controlled with respect to two targets:a 100%liquefaction rate and a high average degree of polymerization(>4)of the liquesced agaro-oligosaccharides,which were then catalyzed byβ-agarase into an oligosaccharides mixture with a high concentration of NA4(30.8 g L^−1)in a 1-L reaction volume.After purification,we obtained 25.5 g of NA4 with a purity of 92%.This work establishes an easy route for the efficient production of pure agaro-oligosaccharides from agarose.