Extraction And Characteristics Of Cathepsin D And Protein Isolates From Sea Cucumber (Stichopus Japonicus) Guts

Sea cucumber is popular with consumers for its high nutritional value.In recent years,the aquaculture of sea cucumber has rapidly developed in china.During the processing of sea cucumber,a large amount of sea cucumber guts and eggs were generated as by-products.The by-products are usually discarded as industrial wastes,which result in resources waste and environmental pollution.It has been reported that sea cucumber guts were rich in proteases and proteins.Therefore,the gut could be a good resource for the extraction of proteases and proteins.The objectives of this study were to:?1?to optimize the extraction conditions of cathepsin D of sea cucumber?Stichopus japonicus?guts and provide basic information about its enzymatic properties;?2?to optimize the extraction conditions of protein isolates of sea cucumber?S.japonicus?guts and characterise the functional and physiochemical properties of the protein isolates.The cathepsin D of sea cucumber?S.japonicus?guts was extracted by using 6 volumes buffer?50 mmol/L Glycine-HCl,pH 3.0?at 4°C for 1 h.The activity of the cathepsin D was investigated by using acid denatured hemoglobin as a substrate.The results of this study showed that the cathepsin D exhibited optimum activity at pH 3.0 and 50°C.The cathepsin D showed high pH stability within the range of pH 3.0⁷.0 and thermostability up to 40°C.The cathepsin D activity was markedly activated by Mg²⁺,Ca²⁺,Ni²⁺,Zn²⁺,Cd²⁺and K⁺,whereas strongly inactivated by Fe³⁺and Fe²⁺.Pepstatin A,E-64 and IAA also had significant inhibitory effects on the cathepsin D.The chemical composition showed that protein content of defatted powder of sea cucumber?S.japonicus?guts was 75.78%.Protein isolates were extracted from the defatted powder using an acid-alkaline process.The extraction conditions were as follows:the extraction temperature was 25°C,the pH of alkali solution was pH 11.5,the ratio of material to liquid was 1:20?w/v?,the extraction time was 2 h,and the pH of acid precipitation was pH 3.6.In this case,the extraction rate of the protein isolates reached 32.23%and the protein purity was 80.54%.The amino acid composition showed that the protein isolates contained all essential amino acids.The content of essential amino acids in the protein isolates was 39.56%.The results of SDS-PAGE showed that the molecular weight of the protein isolates was less than 44 kDa.The hydrophobicity was 1211.7 for the protein isolates.The disulfide bond content of the protein isolates was 37.16?mol/g.The secondary structure composition of the protein isaolates was as follows:19.53%?-helix,6.90%?-sheet,39.30%?-turn and 33.67%random coil.The protein isolates had better solubility at alkaline condition.The water and oil holding capacities of the protein isolates were 5.38⁶.4 g/?g·protein?and 6.25⁷.53 g/?g·protein?,respectively.As comparison with soy protein isolates,the protein isolates had better emulsifying properties and oil holding capacities.This study showed that sea cucumber?S.japonicus?guts contained cathepsin D,and the protein isolates of the guts had good functional properties,which indicate that sea cucumber?S.japonicus?guts can be used as a good source for the preparation of proteases and proteins.