Study On Screening And Selecting An Effectively Agarase-producing Strain, Its Optimal Cultivation And Characteristics Of Agarase

With the purposes of screening and selecting an effectively agarase-producing strain, studying the biological effect and characteristics of agarase and using agarase to produce agarooligosaccharides, a marine agarolytic bacterium F-6, an effectively producing agarase producer, was isolated and selected from marine environment. According to the criteria of Bergey's Manual of Systematic Bacteriology and the analysis of sequence of 16S rDNA, strain F-6 was identified as Vibrio sp.In one-factor-at-a-time methodology and orthogonal experiment, the optimal conditions of strain F-6 to produce agarase were studied. The optimal fermentation medium of strain F-6 contained agar 0.7%; yeast powder 0.3%; peptone 0.5%; NaCl 2.5%; K₂HPO₄ 0.1%; MgSO₄.7H₂O 0.05%; FeSO₄.7H₂O 0.002%; CaCl₂ 0.02%; initial pH 7.5. The optimal temperature and time of fermentation conditions for F-6 to produce agarase were 26℃and 36h, respectively.The biological effect of agarase on cells of Porphyra yezoensis was also studied. The main steps were as follows: 1) fermentation broth was centrifuged at 6000g for 30min to get culture supernatant; 2) the culture supernatant was ultrafiltrated and then mixed with 1mol/L permeabilization agents; 3) the mixture was filtrated to remove bacteria by 0.22μm separate film; 4) Porphyra yezoensis tissue block was decomposed to produce protoplasts; 5)protoplasts were cultivated in 20℃in the light scheme of 12h/day and at light intensity of 1500-2000lux and salt concentration was gradually declined to normal sea water. Results showed that the agarase could easily decompose Porphyra yezoensis tissue block to get protoplasts; Production rate was about 3×106 protoplasts per gram of fresh tissue block. The optimal time of enzymatic hydrolysis to Porphyra yezoensis was 3.5h. The permeabilization agent of glucose (1mol/L) could obviously improve the production rate and survival rate of protoplasts. Protoplasts could develop into callus and survival rate was approximately 60%.Agarase produced from strain F-6 was an inducible and secreting extracellar enzyme. Two types of agarases (Agarase-a and Agarase-b) from the culture supernatant of...