| In the previous work of our research group, a strain, Flammeovirga pacifica WPAGA1, which could directly degrade Gracilaria lemaneiformis to produce oligosaccharides was isolated from deep sea sediment of the west Pacific Ocean. In this study, the genome of the strain was sequenced by shotgun method, and nine agarase genes were found from the genome. Sequence analysis revealed that all of the nine agarase genes encoded β-agarases. Three of them could be classified into glucoside hydrolase family 16(GH16) and the other six belonged to GH86 family.The agarase genes were cloned and expressed in E.coli BL21. Only three protein products which had agarase activities were induced by IPTG successfully, which were designated as Aga P2049, Aga P4255 and Aga P4383, respectively. The agarases were purified through Ni-chelating affinity chromatography, and protein products at electrophoresis level were obtained.The study of enzymatic properties of the three agarases indicated that the optimum temperature and p H of Aga P2049 were 45 ℃ and 7.0, respectively. As for Aga P4255,the number were 50 ℃ and 7.0, and it were 50 ℃ and 9.0 for Aga P4383. The three agarases performed good thermal stability and p H stability, which given it the potential application in industry. The enzyme activities were enhanced by 1mmol/L Co2+ and Mn2+, while were strongly inhibited by Ag+ and Cu2+, and were slightly inhibited by EDTA. Denaturants,such as SDS, urea, guanidine hydrochloride, DTT and β-mercaptoethanol had little impact on the agarase activity under the test concentrations. The three agarases have substrate specificity, which only hydrolyze the polysaccharide chain of agar. The enzymatic products of the agarases were agarotriose, neoagarotetraose and neoagarohexaose. The optimium concentration of agar substrate for the three agarases were 0.4 %.According to the results of enzymatic properties, Aga P4383 performed the best among the three agarases. So we particularly studied the kinetic parameters of Aga P4383. Results showed that the Km value of Aga P4383 towards agar was 8.33mg/m L, while it was 19.81 mg/m L towards Gracilaria lemaneiformis. It turned outthat the agarase Aga P4383 has the ability to hydrolyze Gracilaria lemaneiformis and produce oligosaccharides directly. In order to express the agarase Aga P4383 on a large scale, the induction conditions were optimized. The result of the orthogonal optimization test showed that the optimum induction conditions for Aga P4383 were:induced at 20 ℃for 12 h with a final concentration of IPTG under 200 μg/m L.The agarase Aga P4383 was successfully applied to prepare oligosaccharides by using Gracilaria lemaneiformis as the substrate, and a productivity of 10 % was reached. The prepared oligosaccharides were used to study its bioactivities. Results indicated that oligosaccharides performed well on antioxidation in vitro. On the one hand, the oligosaccharides could scavenge DPPH free radicals efficiently; One the other hand, the oligosaccharides could protect cell from oxidative damage which caused by H2O2. Furthermore, with the help of fluorescence labeling, the oligosaccharides were observed to gather on cytomembrane in three minutes and were absorbed by cell immediately. It was found that oligosaccharides also has the ability on fruit preservation. Oligosaccharides with a concentrations of 160 mg/kg could form a thin film on the surface of cherry tomatoes, which kept the qualities of the fruit and prolong its shelf life efficiently. |
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