| People are attaching more and more importance to the biotechnologies which could help us to find a new source for medicine and sanitarian food from the ocean.As the research on carbohydrate biology goes deeper in recent years,people discovered that agaro-oligosaccharides,derived from agarose, have plenty of important biological activities, such as anti-inflammation, anti-tumor and antioxidant, etc. During the study on agaro-oligosaccharides, researchers believe that the most optimal and less contaminative way to derive them from agar is using agarases. However,as the source for agarases is quite rare and most of their enzymatic activities are low, it is far too expensive for them to be used in indurstry to produce agaro-oligosccharides. Therefore, it has significant practical meanings to screen strains that produce agarases effectively and study on the production of agarase from them.In this study, an effectively agarase-producing strain, Agarivorans albus QM38, which was isolated from sea water is experimented on.The cultivation conditions are optimized and the ability of this strain to produce agarase is strengthened. Two types of agarase are purified from the culture supernatant and the characterization and analysis of hydrolyzed products of these two agarase are also studied.Using one-factor-at-a-time methodology and orthogonal experiment, the optimal conditions for QM38 to produce agarase are researched. The optimal culture medium has been settled as: agar 0.5 %, NaCl 3.0 %,carbamide 0.1 %, yeast powder 0.1 %, MgSO4 0.05 %, K2HPO4 0.1 %, FeSO4 0.002 %, Fe2(SO4)3 0.001 %; the culture conditions are settled as: 30℃,150 rpm/min, inoculated as 1.0 %. After 36h's incubation, the highest enzyme activity is got as 56.58 U/mL.Plenty of fermentation broth is obtained after a massive cultivation of QM38 under the optimal cultivation condition. After the treatment with centrifugation and ammonium sulfate fraction precipitation, crude enzyme in the culture supernatant is obtained. From the result of Native-PAGE and enzymatic activity stain after that, it is dicovered that there were at least three type of agarase in the culture supernatant.Through all the treatments with centrifugation, ammonium sulfate fraction precipitation, successive chromatography on DEAE-Sepharose Fast Flow anion-exchange and Sephacryl S-100 gel-filtration columns, two types of purified agarases from Agarivorans albus QM38 are obtained, identified as agarase A and agarase B. After all the processes mentioned, agarase A was purified 17.6 folds while B was purified 15.4 folds. Their relative molecular weight are estimated by SDS-PAGE to be 127.80kDa and 92.83kDa. The molecular weight of agarase A is higher than that of all the agarases reported so far. The specific phenotypic features of these two agarases, A and B are studied. The mass spectrum is also used to analyze the hydrolyzed products of agarase A.The results of characterization shows that these two agarases are both strongly inhibited by most of the divalent metal ions. The enzyme A was extremely inhibited by Pb2+,Zn2+,Fe3+, and the B was strongly inhibited by Cu2+,Fe3+,Ca2+. The optimum reacting temperature for A is 35℃, while for B is 55℃. It seems that the agarase B has a better thermal stability. Both A and B have their highest enzymatic activity under a alkalescent reaction system, which is similar to most of the other agarases reported, while for A it is pH 7.6 and for B it is pH 8.6. According to the result of optimal concentration of agar for these agarases, it seems that B has a more strong ability to hydrolyze jellied agar. Both of these enzymes purified from the culture supernatant of QM38 have a specialized hydrolase activity on agar and has no effect on the hydrolyzing of other polysaccharides which have a similar structure to agar.Agarooligosaccharides are obtained by agarase A hydrolyzing 0.9 % agar. The components of hydrolytic product analyzed by mass spectrum showed that agarase A mainly produced tetrose and hexose, as long as a small amount of disaccharides.
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