Extraction, Purification And Characterization Ofβ-1,3-glucanases From The Gut Of The Sea Cucumber

Sea cucumber is an important kind of economically aquatic animal because of its abundant nutrition and bioactive substance. The abilities of marine species to utilize ingested nutrients depend on the activities of digestive enzymes existing in their digestive tract. As an important cultured aquatic species, sea cucumber Stichopus japonicus has strong digestive capabilities. There are lots of ingestive enzymes in digestive tract of sea cucumber, and the isolation and characterization of which in combination with molecular studies would provide valuable information on the digestive physiology of sea cucumber.In this article, an β-1,3-glucanases was extracted and purified from digestive tract of sea cucumber Stichopus japonicus and its character was studied. The optimal extracted conditions were confirmed. The enzyme was purified to homogeneity by ammonium sulfate precipitation, ion exchange chromatography with DEAE-Sepharose CL-6B, hydrophobic interaction chromatography with Butyl-Sepharose and preparative electrophoresis with PAGE. The molecular weight of β-1,3-glucanases was measured through SDS-PAGE and the character of the purified protease was studied.The results indicated that the best extracted conditions of the β-1,3-glucanases were50mmol/L phosphate buffer (pH6.0) containing0.05mol/L NaCl and precipitation with80%of sulfate ammonium.The purity of purified enzyme was23.24-fold increase in the specific activity of the enzyme and3.58%recovery rate over the crude extract. The molecular weight of the purified enzyme was estimated to be37.5kDa by SDS-PAGE. The enzyme exhibited optimum activity at pH5.5and40℃. The enzyme showed high pH stability within the range of pH5.0～8.0and thermostability up to40℃. The enzyme activity was markedly activated by Mn2+, whereas strongly inactivated by Cu2+and Ag+. The Km and Fmax of the β-1,3-glucanase were]9.8μg/ml and2000μg/minmg, respectively.