Isolation, Identification And Enzymatic Hydrolysate Analysis Of Extra-agarase From Marinobacter Adhaerens G4

The agaro-oligosaccharides have many important biological activities of antioxidant,antineoplastic, promoting the growth of intestinal probiotics and preventing diabetes, etc.The traditional agaro-oligosaccharide preparation of acid hydrolysis method is limited at industry application because of the degradation product diversity and environmental pollution. Whereas, the agaro-oligosaccharide preparation of agarase enzymatic method have the advantages of high efficiency, non-pollution and homogenous product, which have attracted more and more attention. Most agarases was obtained from marine bacteria.Screening the efficient bacterial strain of agarase production and to analyze its agarase property has important theoretical significance for the enzymatic method application.Therefore, the efficient agarase-producing bacterial strain Marinobacter adhaerens G4 was as research object to analyze the property of extracellular agarase.The obvious enzymolysis circle and liquification circle of strain G4 were found with the diameter of 2.1 cm and 1.2 cm in the solid plate for culture 48 h by Lugol's iodine staining. The results of growth curve and agarase production indicated that the growth exponential phase of 6-33 h and the growth stationary phase of 33-72 h. In growth exponential phase the extracellular agarase activity increased constantly as the extension of time. The maximum extracellular agarase relative activity was 163.88 U/mL at 15 h.Compared to the exponential phase, the extracellular agarase activity was high and held steady at 120 U/mL in stationary phase.The crude enzyme of M. adhaerens G4 was prepared by the methods of ammonium sulfate precipitation and ultrafiltration centrifugation. The LC-MS analysis results of crude enzyme indicated that 29 of 34 identificated proteins could be associated with the process of agar degradation. According to protein function, these proteins was classfied into six group: agarase (1), ABC transporter, molecular chaperone (11), dehydrogenase (4),antioxidant proteins (3) and other proteins (8). The similar agarase (NCBI access number of 737500643) was from the bacteria of Aliagarivorans marinus, which the molecular weight of was 127.36 kD and the amino acid sequence similarity was 57%-62% with other agarase .Zymogram analysis was performed by the methods of SDS-PAGE and Lugol's iodine staining The results showed that two protein bands had the agarase activity and the key band with the molecular weight of 48 kDa. The MS analysis result indicated that the key protein was P-agarase and the retrieved similar protein also was composed by 955 aa and the molecular weight of 127.36 kDa. Comprehensive analysis presumed that the post-translational modifications occurred in the process of agarase exocytosis.The enzymatic products of agarase was was analyzed by the method of TLC. The results showed the enzymatic products was only composed of NA4 which indicated M.adhaerens G4 had a good application prospect.The optimal conditions for enzyme production were determined by the methods of singer factor analysis and response surface analysis. The optimal conditions of culture medium was yeast extract of 0.60 g/L, tryptone of 4.93 g/L, ferric citrate pentahydrate of 0.01 g/L, agar powder of 0.1% (w/v). The optimal culture temperature and pH value were 30.64 ? and initial pH value of 7.54. The measured value of agarase activity at the optimal conditions was 216.78 U/mL and had minor difference to the predicted value of 215.83 U/mL, which indicated the model was reliable.