Study On Extraction,Isolation,Purification And Characterization Of Lectin From Loach Skin Mucus

Loach(Misgurnus anguillicaudatus)skin mucus was usually wasted during transportation and process of loach.Making the best of loach skin mucus could increase the economic benefit for companies.In this work,a kind of lectin from loach skin mucus was successfully isolated and purified,and its characterization was explored for further use in food and medicine industry.The basic chemical composition of the freeze-dring mucus was analyzed,indicating total protein content being 73.96%,soluble protein content being 36.95%,total fat content being 5.12%,and total carbohydrate content being 4.46%.Amino acid compositions analysis indicated total amino acid content was 560.16 mg/g,and therein,the contents of histidine,lysine and leucine were 118.80 mg/g,82.68 mg/g and 64.41 mg/g,respectively.The gas composition of loach skin mucus was analyzed by GC-MS,indicating there were six kinds of hydrocarbons(17.08%),two kinds of ketones(2.97%),six kinds of alcohols(33.98%),three kinds of aldehydes(9.27%),one kind of amines(35.43%)and one kind of phenolic substance(1.27%).The effects of traditional extraction and ultrasound-assisted extraction,respectively,on the yield of the lectin were compared by experiments of single factor and response surface.The optimal parameter of extracting lectin by traditional method was liquid-solid ratio of 5.4 mg/mL,pH of 6.7,and extraction time of 2.7 h.Under this condition,the extraction yield of lectin was 36.63%.The optimal parameter of extracting lectin by ultrasound-assisted method was liquid-solid ratio of 3.6 mg/mL,pH of 8.8,ultrasound power of 264 W,ultrasonic time of 36.8 min.Under this condition,the extraction yield of lectin was 71.02%.The extraction solution was concentrated,dialyzed,and lyophilized to obtain the crude lectin.A novel Ca2+-dependent lectin,specific binding D-iactose,from loach skin mucus was successfully isolated and purified by DEAE-52 and Sephadex-G200.During the process of purification,the protein recovery percent was 9.80%,while the sample purity increased to 17.37 folds.SDS-PAGE and high performance gel permeation chromatography was applied to determine the purity and molecular weight,indicating the molecular weight of the lectin was 245 KDa.Moreover,the structure change of the lectin indifferent pH and temperature was studied by fluorescence spectrum.The result of fluorescence spectrum suggested the unfolding of protein structure was accompanied with heat treatment.There are a certain functional relationship among hemagglutination activity,heat treatment temperature and time.The analysis of kinetic data suggested that the heat inactivation of the lectin followed first-order reaction kinetics.The purified lectin from loach skin mucus was stable under extensive alkaline conditions,and optimal pH was 8.5.The experiment ofβ-elimination reaction suggested the lectin contained the structure of O-glycosylation.Based on the photographs observed by scanning electron microscope,the structure of the lectin exhibited random sheet with many round holes.Fourier transform infrared spectroscope suggested the secondary structure of the lectin contained a-helix of 4.97%,β-sheet of 27.55%,turns sturcture of 49.78%,unordered structure of 17.70%.