Study On The Preparation Of Novel Metal Chelating Carriers And Their Application In Immobilizing Papain

Papain is a natural plant protease,which not only has strong proteolysis and synthesis ability,but also has curd,lipolytica and lysozyme activity.Therefore,it is widely used in food,medicine,feed,cosmetics,leather and other industrial fields.But the free enzyme was easy to be inactivated and decreased catalytic efficiency,which limit its application in industrial production.Immobilization is an effective method to solve the problem of free enzyme,which not only can keep the catalytic properties of free enzyme,but also has the advantages of good stability and reuse,easy separation and recycle,recovery and continuous operational.However,traditional methods of immobilization have many disadvantages,such as the high cost of immobilized reagents and carriers,the complicated operation of immobilization process,which limit the industrial application of the immobilized enzyme.Therefore,it's important and meaningful to develop immobilized enzyme carriers and methods.In this work,two kinds of immobilized carriers with excellent properties were prepared.The principle of metal chelating chromatography was used for the immobilization of the papin enzyme,and it was used to hydrolyzed soybean protein.Metal chelating carriers are simple and convenient,and also have little influence on protein conformation and enzyme activity in the immobilization process.Moreover,the carriers also can be reused.The main contents are as follows:(1)The chitosan powder was used as raw material,nano Fe3O4 as magnetic material,Si02 as the pore forming agent,to prepare magnetic multi-porous chitosan microspheres carrier PCMM.At the same time,The ECH has been used as the activating agent,and the optimum conditions such as activation temperature,activation time,dimethyl sulfoxide concentration,sodium hydroxide concentration,epichlorohydrin volume fraction have been investigated according to the change of epoxy matrix density.To prepare two kinds of porous magnetic metal chelating carriers(PCMM-IDA-Cu2+ and PCMM-CM-ASP-Ni2+)were prepared by using iminodiacetic acid(IDA)and carboxymethylated aspartate(CM-ASP)as chelating ligands to chelate the transition state of metal ions(Cu2+ and Ni2+),respectively.The PCMM-IDA-Cu2+ and PCMM-CM-ASP-Ni2+ were characterized by optical microscopy,scanning electron microscopy(SEM),vibrating sample magnetometer(V SM),infrared spectroscopy(FT-IR)and thermal gravimetric analysis(TGA).(2)Two kinds of magnetic metal chelating carriers(PCMM-IDACu2+ and PCMM-CM-ASP-Ni2+)were used to study the immobilization condition of papain.Through the single factor test,the factors which affect enzyme immobilization such as the immobilized time,the amount of enzyme,the immobilized pH and the immobilized temperature was been investigated.On the basis of single factor experiments,the response surface analysis(RSM)method has been used to optimize the conditions of enzyme immobilization,and the optimum conditions for enzyme immobilization were obtained:the obtained optimum conditions for PCMM-IDA-Cu2+carrier was:immobilized pH 6.73,the amount of enzyme 1.56 mg per 15.0 mg carrier,immobilization temperature 30.9?,the optimum immobilized enzyme activity was 7.976 U/mg;the optimum conditions for PCMM-CM-ASP-Ni2+ carrier was:immobilized pH 7.29,the amount of enzyme 1.90 mg per 15.0mg carrier,immobilization temperature 30.7?,the optimum immobilized enzyme activity was 7.397 U/mg.Under optimal conditions of immobilization,the amount of immobilized enzyme was 94.18 mg/g(PCMM-IDA-Cu2+)and 89.16 mg/g(PCMM-CM-ASP-Ni2+),recovery rate of enzyme activity reached 87.21%(PCMM-IDA-Cu2+)and 81.63%(PCMM-CM-ASP-Ni2+).(3)The enzymatic activity of immobilized papain was investigated by researching the enzymatic properties of free enzyme and immobilized enzyme,for which casein was chosen as reaction substrate,which to provide some theoretical basis for practical applications.The results showed that the optimum reaction temperature was 70? for immobilized papain,and the immobilized enzyme maintained a higher enzyme activity from 60? to 90?.The optimum reaction pH for free enzyme and immobilized enzyme were 7.0 and 8.0,respectively,the immobilized enzyme still maintained a higher enzyme activity in the range of 6.0-7.0 and 9.0?10.0,the enzyme activity of the immobilized enzyme was still higher than that of the free enzyme.The thermal stability of immobilized enzyme were higher than the free enzyme.The Km value of the free enzyme and immobilized papain were 1.59mg/mL,0.99 mg/mL(PCMM-IDA-Cu2+)and 0.25mg/mL(PCMM-CM-ASP-Ni2+),indicating that the immobilized enzyme exhibited a higher affinity to the substrates.After 9 times of repeated use of the immobilized enzyme,the residual enzyme activity of PCMM-IDA-Cu2+and PCMM-CM-ASP-Ni2+immobilized enzyme were still retained more than 60%.After six times of reuses,the amount of immobilized enzyme was 68.81 mg/g(PCMM-IDA-Cu2+)and 65.21 mg/g(PCMM-CM-ASP-Ni2+).(4)Two kinds of immobilized enzyme(PCMM-IDA-Cu2+and PCMM-CM-ASP-Ni2+)was preliminary applicated in food,which was used in hydrolyzing soybean protein.Set hydrolysis degree as index,the factors affecting such as hydrolysis temperature,hydrolysis time,substrate concentration,the amount of enzyme,pH have been investigated according to the change of the degree of hydrolysis of the soy protein.The optimum immobilized conditions for two kinds of immobilized the amount of immobilized enzyme 6000 U/g,reaction time 2 h,substrate concentration 4%,temperature 70?,pH 8.0,which provides a theoretical basis for the actual production and application.