| In recent years, with the continuous development of preparation technology of oligosaccharide and the research on glycobiology, the enormous application potential of agaro-oligosaccharide had obtained a great many concerns, which has become the hotspots of research and development on high-valued polysaccharides, Pharmaceuticals, new health food and new chemical product. The preparation of oligosaccharide is still a world difficulty, and chemical method has many defects, such as the problems of no single product, limitation of operation conditions and product recovery difficulty. The agarase secreted by bacteria can degrade agar and obtain agaro-oligosaccharide. Enzymolysis approach is not only easy in the controlling of reaction conditions, but also low in energy consumption and high in catalytic efficiency. Moreover, enzyme catalysis has specificity, and can selectively cut glycosidic bond, thus the existing problem of acid process can be overcome.This study used agar as the carbon source, and obtained several bacterial strains that can degrade agar by separation from tropical marine environment in South China Sea. It also purified and screened out three high-activity bacterial strains on improved culture medium, which were respectively numbered as FG1 bacteria, FG2 bacteria and FG3 bacteria. Moreover, this paper had taken bacterial genomic DNA as the template, designed and synthesized the universal primer 1492R and 968F of bacteria 16S rDNA, and carried out PCR amplification on the target fragment of 16S rDNA. After undergoing AGE (agarose gel electrophoresis), recovery, connection and transformation by the obtained target fragment, positive clones were screened out by blue and white screening method. T carrier specificity primer M13R (-48) and M13F (-47) was used to carry out PCR amplification to inocula, so as to further identify positive clones.BLAST comparative analysis was carried out between sequencing results and 16S rDNAs sequence recorded in GenBank database, and MEGA5 software was used to construct phylogenetic tree, so as to determine taxonomic status of bacteria.3,5-dinitrosalicylic acid (DNS) colorimetric method was used to determine the output of reducing oligosaccharide; orthogonal experiment was used to study the influence of agar concentration, medium salinity, and cultivated bacterial strain temperature to enzyme production,optimize the conditions of enzyme production;℃Moreover, this paper studied the enzymatic property of three bacterial strains were carried out from enzyme reaction pH and reaction temperature. Ammonium sulfate precipitation was done to the fermented supernatant of agar-decomposing strain, so as to obtain the crude enzyme. Then SDS-PAGE technology and coomassie brilliant blue were applied to detect the relative molecular massofagarase.This study purified and screened out three high activity bacterial strains, which were respectively numbered as FG1, FG2 and FG3. Respectively inoculate the fresh bacteria into solid plate culture medium taking agar as the carbon source (without glucose), after cultivating for 2-3 days, the solid medium surrounding the bacterial colony would occur obvious liquidation or generate the hollow left after the decomposing of agar, and the solid medium would be completely liquefied within one week. Through long-term experimental observation and sub-culturing, it was found that such three bacterial strains always kept efficient agar decomposing activity. FG1, FG2 and FG3 were separated and identified, which were all gram-negative bacterium. And the recording number of 16S rDNAs in GenBank was respectively JX073661, JX073662 and JX073663. The 16S rDNA sequence homology between FG1 bacteria and Labrenzia agregata was as high as 99%, thus it can be preliminarily identified as Labrenzia sp. bacterial genus. The 16S rDNA sequence homology between FG2 bacteria and Vibrio harveyi was 99%, as the sequence homology was vary high, it can also be identified as Vibrio sp.. The 16S rDNA sequence homology between FG3 bacteria and Sagittula stellata was 97%, thus it can be preliminarily identified as Sagittula sp..The results of study on the optimization of conditions for enzyme production showed that, the activity of FG1 and FG3 was the highest in culture medium with agar concentration of 0.9-1.5% for enzyme production, the optimal agar concentration for FG1 was 1.2% while that for FG3 was 1.5%, the concentration of enzyme activity were 2793U/mL and 3266.6U/mL respectively; FG2 had high activity in culture medium with agar concentration of 0.5-0.9%, and the optimal agar concentration was 0.7%, the concentration of enzyme activity was 2066.6U/mL. The activity of FG1 was high for enzyme production when salinity in the medium was 31.320-32.625, while it was the highest when salinity was 31.97, the concentration of enzyme activity was 2786.6U/mL. The activity of FG2 and FG3 was higher for enzyme production when salinity in the medium was 30.67-31.32 and the optimal salinity for FG2 and FG3 was 30.67, the concentration of enzyme activity were 1914.6U/mL and 3135.4U/mL respectively. The activity of FG1 and FG2 was higher for enzyme production when the culture temperature was 27-31℃, the optimal temperature for FG1 was 31℃ while that for FG2 was 27℃, the concentration of enzyme activity were 2715.4U/mL and 1653.4U/mL respectively. FG3 was adaptive in wide-range temperature and it kept active for enzyme production at 20～31℃ while 27℃ was the optimal temperature, the concentration of enzyme activity was 2906.6U/mL.The optimal culture time of the three bacteria for enzyme production was 96h, the concentration of enzyme activity were 2789.6U/mL,2139.9U/mL and 3333.4U/mL respectively.The results of above orthogonal test for factors showed that, for FG1, the optimal conditions for enzyme production was the agar concentration 1.2%, salinity 31.97, temperature 31℃ and pH 7.2, the concentration of enzyme activity was 2815.4U/mL; for FG2, the optimal conditions for enzyme production was the agar concentration 0.5%, salinity 30.67,27℃, pH7.2, the concentration of enzyme activity was 2213.8U/mL; for FG3, the optimal conditions for enzyme production was agar concentration 1.5%, salinity 31.97, temperature 27℃, pH7.2, the concentration of enzyme activity was 3415.8U/mL. Study on the conditions of enzyme reaction activity showed that:when pH of enzyme reaction solution was at 7.4-8.4, the activity of enzyme FG1 to degrade substrate was high, When pH 8, enzyme activity reached the highest, the specific activity was 3480U/mL; when pH of enzyme reaction solution was at 8.0-9.0, the activity of enzyme FG2 to degrade substrate was high, When pH 8, enzyme activity reached the highest, the specific activity was 2106.6U/mL; when pH of enzyme reaction solution was at 7.4-9.0 the activity of enzyme FG3 to degrade substrate was high, When pH 8, enzyme activity reached the highest, the specific activity was 3746.6U/mL. When the reaction temperature was kept at 30～40℃, the activity of FG1 and FG2 kept high, when it was 35℃, the activity of FG1 reached the highest while when it was at 30℃, that of FG2 reached the highest, the specific activity were 3654U/mL and 2016U/mL respectively; When the reaction temperature is at 30～50℃, the activity of FG3 was high, and when it was 40℃, the activity reaches the highest, the specific activity was 4041U/mL.The study also found, the gene expression regulation of the three marine bacteria agarase was the induced expression regulation type which meant in the case of agar as the only carbon source, the agarase gene in the cells was able to express. The regulation of agar-degrading enzyme gene expression remains to be researched and analysed further. |
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