Isolation, Purification And Application Of Agarase From A Marine Bacteria

Viable single cells and protoplasts of algae can be used in physiological, biochemical and genetic studies,biotechnology and mariculture.In addition,they can be employed to obtain economically valuable compounds.Single cells and protoplasts can be isolated by enzyme treatment of seaweed thalli.Complex enzyme systems including cellulases,pectinases,xylanases and agarases are necessary for obtaining protoplasts.Red algae are a very heterogeneous group with respect to the chemical composition of the cell walls and intercellular matrix,which can change depending on the life-cycle stage,physiological status and cultural conditions.Therefore,finding new sources of enzymes degrading red algal polysaccharides and selecting enzyme complexes for thalli maceration are beneficial to the development of red seaweed cell technologies.Agarases are specific enzymes catalyzing the hydrolysis of agar,which is the major component of the cell wall matrix in red algae.The Agarase-Producing bacteria was isolated from red algae collected in Yantai. The strain is Gram-negative,couldn't hydrolyze the cellulose and starch,and couldn't fluidify glutin.16S rDNA sequence analysis of the strain revealed high similarity with the Agarivorans sp.:therefor it was named Agarivorans sp.SM0527.At the same time,the liquid state fermentation media for agarase production by SM0527 was optimized.The optimized medium contained agarose 0.5%;yeast extract 0.5%;each 500 ml gular flask containing 100 ml of medium,inoculum size 5%and a harvest time of 60h.Under these optimized conditions,agarase activity of 3300 IU/ml in the crude culture filtrate was obtained.By centrifugation,ammonium sulfate fractionation and anion- exchange,a homogenousβ-agarase was purified from the crude culture filtrate of SM0527 with a gain rate of 10.2%and a purification of 8.2 folds.The molecular weight of the purified agarase was approximately 90 kDa.The optimal pH for the agarase activity was at pH 8.5,and agarase was optimally active at 45℃.Furthermore,the optimal reaction time of the enzyme assay was l0 min.The agarase from SM0527 and the cellulases excreted by Trichoderma pseudokoningii K9301(a strain preserved in our lab)were employed to isolate protoplasts from some species of red algae.Experiments revealed that this complex enzyme system which contained the agarase of 10%to 15%and the cellulases of 1% to 2%had good effects on red algae including Porphyra yezoensis,Gracilaria verrucosa,Ceramium koudoi and Plocamium telfariae.With glucose of 2 M,an effective and economical penetrate in preparing protoplasts,yields of these protoplasts reached about 9.4×10~5 To 2.3×10~6 cells per gram of fresh weight algae.