Research On Isolation And Characterization Of Agarase-producing Strain And Enzymology Properties

The thesis was aimed to obtain the high yield of agarase-producing strains from the seawater sample.Then the fermentation conditions were optimized. In addition, agarase-fst, a novel agarase, was firstly purified to homogeneity from a newly isolated agarolytic bacterium, Thalassospira profundimaris fst-13007. In the end, the degraded product by agarase-fst agar oligosaccharide’s components were identified and analyzed by thin-layer chromatography (TLC) and liquid chromatograph-mass spectrometer(LC-MS). The antioxidant ability of agar oligosaccharides was also studied in this research.The main research and results are summarized as follows:(1) Eight agarase-producing strains were successfully separated from the seawater sample collected from the offshore sea of Xiamen, China. Fst-13007, showed the highest agarase activity was selected to cultivate for the following investigation. Its 16S rDNA gene sequence was deposited in GenBank with an Accession Number KU578316. Finally, strain fst-13007 was grouped into the clade composed of annotated Thalassospira profundimaris species, and named Thalassospira sp.fst-13007.(2) The single factor test and orthogonal design were applied to optimizethe fermentation conditions for Thalassospira sp. fst-13007. The optimal medium components were agar 0.8%(w/v), salinity 3%(w/v), yeast 0.10%(w/v) and tryptone 0.50%(w/v). The best fermentation conditions were initial pH 8.0,8% ofinoculation quantity,35℃ of culture temperature,200 rpm of shaking speed,70 ml/250 ml by loaded liquid and 12 h for fermentation time. Under the optimal conditions, the enzyme activity of the agarase in the crude enzyme liquid was 2.76 U/ml, which was enhanced by 340% compared to the without fermentation optimization (0.81 U/ml).(3) Agarase-fst was successfully purified and appeared on the gel as a single protein band in SDS-PAGE after DEAE-Sepharose Fast flow column and Sephacryl S-100. It was purified 18.97-fold with a yield of 13.87%. The specific activity of purified agarase was 1418 Umg-1 and the molecular weight was approximately 66.20 kDa.(4) The activity of Agarase-fst was optimal at 45 ℃ and pH 8 and was stable at pH 5-9 or 30-50 ℃. Agarase-fst required Mn2+ and DTT for agarase activity and inhibition by Cu2+ Fe3+、Ca2+、Na+、Mg2+、K+、SDS and EDTA.(5) Tests of hydrolysis pattern and substrate specificity, TLC analysis and mass spectrometry of the hydrolysis products revealed that it is an endo-type (3-agarase hydrolyzing agarose into neoagarobiose, neoagarotetraose and neoagarohexaose. And results of MALDI-TOF-TOF/MS indicated that it lack of homology to previously identified proteins and present conserved domain of β-agarase belonging to glycoside hydrolases (GH)-16 family.(6) To determine the molecular mass distribution and identified of hydrolysis products, the hydrolysis products were analysed by LC-MS/MS and TLC. According to the relative peak intensities, hydrolysis products contained 40% NA2,53% NA4 and 7% NA6. And then the antioxidant test was applied to hydrolysis products for the ability of removing DPPH. The result showed that the hydrolysis products possessed certain antioxidant ability.