Preparations Of Immobilized Metal Ion Affinity Membranes And Their Applications In Separating And Purifying Papain

With the instant development of biological engineering and life science,the affinity membrane chromatography has a fast development in separation and purification of proteins and enzymes.The affinity membrane chromatography possesses a number of advantages of high convective flow,high flow-rate operation for process,low pressure drop,high production capacity, high selective,easy to scale production.Papain(EC 3.4.22.2) exists primarily from the latex and unripe fruit of Carica papaya (tropical melon or papaw),widely used in food,leather,textile,cosmetic and pharmaceutical industries.Purification of papain from papaya latex has traditionally been achieved by complicated precipitation methods.However,the low purity can not meet the needs of modern industry,difficult to scale production.In this research based on nylon membrane,four different metal ion as ligands were used to prepare a novel material to separate papain.Their physical properties were studied by scanning electron microscopy(SEM),infrared spectroscopy(IR) and inductively coupled plasma atomic emission spectroscopy(ICP-AES).The optical condition which chelated metal ions metal including absorption time,solution pH value and adsorption dosages were determined.The adsorption-desorption properties of papain on immobilized metal ion affinity membranes were studied.The effects of factors,such as contact time,pH value,ionic strength,flow rate of adsorption and desorption,elution style,pH value and ionic strength of elution were determined. The papain activity eluted was calculated and it also characterized by SDS-PAGE.The interactions between different metal ion and papain were investigated by adsorption isotherms and kinetics experiments.The results show that the best conditions of copper ion chelating on membranes are 30 minutes,pH 4.0.SEM photographs show that the pore size of immobilized metal ion affinity membranes is decreased,but it is sufficient to allow large protein molecules pass.Metal amount chelated on the membrane are 4402.1,658.1,339.6,1258.3μg/g by ICP-AES(immobilized copper,nickel,zinc,cobalt metal ion affinity membrane),respectively.The results also show that the optical constant time of papain adsorption by immobilized Cu,Ni,Zn,Co metal ion affinity membranes are respectively 60,50,50,30 min;the optical pH are respectively 8.0,10.0,9.0,9.0; optical ion strength(concentration of NaCl) are 0 mol/L;all optical elutions are NaSCN;all optical flow rates of adsorption are 2.0 ml/min;all optical flow rates of desorption are 1.0 ml/min; all optical pH and ion strength(concentration of NaCl) of elutions are 5.0 and 1.0 mol/L.The papain activity desorped by immobilized Zn²⁺ affinity membrane is highest,and its purification fold is 27.06.The adsorption isotherm for papain adsorption by four immobilized metal ion affinity membranes fits with Freundlich equation.The pseudo-second-order kinetic model provides the better correlation to the experimental results.This study will provide important theoretical basis for application of separation and purification of papain.