Purification And Characterization Of β-1,3-glucanase From The Viscera Of Abalone Haliotis Discus Hannai

β-1,3 glucanases (EC 3.2.1.39) is a hydrolase,which can specifically hydrolyze β-1,3 glycosidic bond. It is widely distributed in microorganisms, plants and animals. β-1,3-glucanase exist in marine invertebrates, and it plays an important role in the biological process of carbohydrate metabolism as one of the important digestive enzymes.β-1,3-glucanases was extracted and purified from abalone viscera and the character was studied. The enzyme was extracted and separated by ammonium sulfate precipitation. The ion exchange chromatography of DEAE-52 Sephacryl S-200 HR gel filtration and preparative electrophoresis was used to purify the enzyme. The molecular weight of β-1,3-glucanases was measured through SDS-PAGE and the characters of the purified protease was studied. Under certain conditions, phenylmethylsulfonyl fluoride (PMSF), N-bromosuccinimide (NBS), dimercapto Jisu sugar alcohols (DTT), succinic anhydride (SUAN) and chloramine-T were used to modified the moleculars of abalone viscera β-1,3-glucanases. Finally, the enzyme hydrolysis products were analysed by the method of thin layer chromatography (TLC).The results indicated that the abalone viscera β-1,3-glucanases were extracted from abalone viscera by 0.1 mol/L disodium hydrogen phosphate-citrate buffer (pH 6.0) and precipitated with 60% of sulfate ammonium. After the purification by ion chromatography DEAE-52, Sephacryl S-200 HR gel chromatography, The purified enzyme activity was 5.11 fold increased reaching to the elative enzyme activity of 21.02 U/g. The molecular weight of the purified enzyme was estimated to be 28.5 kDa detected by SDS-PAGE. The enzyme exhibited optimum activity at pH 5.0. The enzyme showed high pH stability within the range of pH 3.0-pH 6.0 and thermostability up to 40℃. The enzyme activity was markedly inhibitied by Ni+, Cu2+, Mg2+, Hg+, and Fe3+ had no influence on the activity. The Km of the β-1,3-glucanase was 0.4034 mmol/L.Distinct enzyme activity inhibition was not only observed in the presence of glycosidic bond inhibitors Nojirimycin, but also existed in the presence of Amino acids modifiers of N-bromosuccini-mide (NBS) and Chloramine-T. The effect degree of thiol-activating agent DTT, Succinic anhydride and Phenyl methyl sulfonyl fluoride (PMSF) had the relationships with different concentrations of these agents. Therefore, tryptophan and state-of-art 1-methionine might be the necessary functional groups of abalone visceral β-1,3-glucanases.