Screening Of Marine Microorganisms For Agarase Production And Optimization Of Fermentation Conditions

An effective agarase-producing bacterium was isolated from seawater samples,laver samples and soil samples in the coastal waters of Zhoushan and Taizhou（Zhejiang province）where laver grew,and also from the enriched seawater and laver for several times.When growing in the agar plate,the bacterium could degrade agar into agaro-oligosaccharides resulting that deep hollows were shaped surrounding central colonies, where transparent circles appeared by iodine solution staining.The preliminary identification results indicted that the strain named HS519-211 was a gram-negative rod strain and it was determined as a member of the Actinobacillus genus by bioMerieux VITEK 32.The Actinobacillus genus HS519-211 was a new member of the agarase-producing bacterial family.Mutation breeding was applied to enhance the ability of producing agarase of the strain HS519-211 by ultraviolet mutation andγ-irradiation and the activity of agarase was increased by 13.2%.The experiments of single factors and orthogonal design were carried out to optimize the culture medium and cultivation conditions.As a result,the optimal medium composition was as follows（%）:agar 0.25,NaNO₃ 0.4,NaH₂PO₄ 0.06, NaCl1.0,MgSO₄·7H₂O 0.5,KCl 0,1,FeSO₄·7H₂O 0.002,CaCl₂ 0.02,MnCl₂·4H₂O 0.015,pH was adjusted to 7.5.The fermentation for agarase production was carried out in 250-mL Erlenmeyer flasks containing 30 mL of medium with inoculation of 9%（v/v）seeds,incubated at a agitation speed of 200 rpm and 30℃for about 45 h.The agarase activity reached 215.83 U/mL under the optimum conditions and it was 135%higher than that before the optimization,which could be used to the further study and applied to the preparation of agarase for bioactive agaro-oligosaccharides production in industry.The agarase was an inducible extracellular enzyme.The enzymatic conditions of degrading agar into oligosaccharides by the agarase were also studied.The results showed that the optimal time,temperature and agitation rate for enzymatic reaction were 30 min,45℃and 200 rpm respectively.The optimal substrate was 0.3%（w/v）agar melted in phosphate buffer（pH7.0）.The agarase could keep higher enzyme activity in the range of pH 7.0～8.5 and the relative agarase activities were all over 93%.In addition,it exhibited substrate specificity to agar.Besides, addition of Mg²⁺would greatly promote the enzymatic reaction,while K⁺ could cause inhibition,and the effect of Na⁺ on the reaction was totally different from that of other agarases reported before.Na⁺ at lower concentration had little effect on the reaction whereas higher Na⁺ inhibited the reaction to some extent.Therefore,it was essential to do some further researches on enzymological properties of the agarase.Subsequently,the preliminary study was carried out on the separation and purification of the agarase and its enzymological properties.Agarase was precipitated by 40%saturation with solid sulfate ammonium by the step-by-step ammonium sulfate precipitation（10-80%saturation）test.The specific activity for the precipitate reached 1364.32 U/mg,1.3 times higher than that before precipitation.After the agarase solution was purified by Sephacryl S-100 gel filtration,ultrafiltration was used to condense the agarase again.The purity of the enzymes was tested by SDS-PAGE and their molecular weights were estimated to be more than 85 kDa.Finally,the test on thermostability of agarase was conducted.The results showed that the enzyme kept a higher agarase activity at 4℃and 15℃（room temperature）and almost kept stable after 6 h.Meanwhile,it was totally feasible to store the enzyme in refrigerators of 4℃for short-term preservation.