Novel Encapsulation Of Papain And Study Of The Encapsulated Papain

Three novel encapsulated enzymes are described by this dissertation.The modified-encapsulated enzyme combined the modification and entrapment, modified-encapsulated enzyme was multipoint connected to the carrier by the unsaturated double bonds modified on the enzyme; the final enzyme conformation of UV irradiation initiated encapsulated enzyme is rigid;due to the interconnected macropores network structure cryogel encapsulated enzyme exhibited lower resistance of mass transfer. And their catalytic properties in aqueous solution were investigated. Then the influences of temparature and pH on the specific activities of the three encapsulated enzymes were also disucussed. At last, their specific activities and stabilities were compared in this work.Comparing the activity yield of three encapsulated PA, the result was as follows: CryogelPA-0.6(90.3%)>A1-PA(83.6%)>Ca-PA (65.2%); At60℃and70℃, after lOh of incubation, comparing the thermostability of three encapsulated PA, the result was as follows:Ca-PA>Al-PA>CryogelPA-0.6; At pH2.4and pH10.83, after180min of incubation, comparing the pH stability of three encapsulated PA, the result was as follows:CryogelPA-0.6>Al-PA> Ca-PA.Km (app) value of them are14.9、21.7、45.3mg/mL.From the SEM image we found the cyyogel has a super-macroporous structure with interconnected pores(14-50μm)surrounded by thin walls.The preparation of modified-encapsulated papain(PA):Amino groups of PA were modified by N-acryloxysuccinimide(NAS), when the mole ratio of papain and NAS was about1:28and the modification time was45min, the optimum result was showed.; then four additives cysteine(Al), bovine serum albumin(A2), xylose(A3), CAB35(A4) were added into a copolymer system which contained PEG400-diacrylate ester as monomer and papain.; the polymerization was initiated by free radicals, then four modified-encapsulated PA could be obtained. When2mmolAl>1.5μmolA2、0.03molA3.7.5mLA4was added to lmLPA(5mg/mL) respectively the best results were attained.Under the activity yield of four modified-encapsulated PA (Al-PA, A2-PA, A3-PA, A4-PA)was83.6%、78.5%、80.1%、82.2%respectively. The immobilization process brought significant enhancement of stability to temperature and extreme pH values with respect to free papain. In the condition of pH=2.4, pH=10.83or100℃, the residual activity of Al-PA was34.2%、62%、34%.The optimum temparature of Al-PA shifted from40to60℃. When Al-PA was reused by ten times, the relative activity of Al-PA was retained56.5%of its initial activities. And the activity of Al-PA remained75%of its initial activities after storage for60days at4℃. The preparation of encapsulated PA:PA was added into a W/O system, which contained four different kinds of PEG200(400)-di(meth)acrylate ester and trimethylolpropane trimethacrylate.; the polymerization was initiated by ultraviolet irradiation. The irradiation time was lmin. The best activity yield (65.2%) was attained by Ca-PA. In the condition of pH=2.4, pH=10.83or100℃, the residual activity of Ca-PA was35.6%,57.9%,34.7%.The optimum temparature of Ca-PA shifted from40to50℃. When Ca-PA was reused by ten times, the relative activity of Ca-PA was retained83.1%of its initial activities. And the activity of Ca-PA remained85%of its initial activities after storage for60days at4℃.The preparation of cryogel encapsulate papain:papain,PEG200-diacrylate ester,acrylamide, initiator(H2O2) were added under stirring to obtain a homogeneous aqueous solution. The solution was ftozen for2h; then the frozen crystal was irradiated by UV-vis light to initiate the polymerization. The optimum chilling temparature was minus15℃. when the mass ratio of crosslinking agent and monomer was about0.6, The best activity yield (90.3%) was attained, In the condition of pH=2.4, pH=10.83or100℃, the residual activity of CryogelPA-0.6was46.7%,67.8%,43.5%. The optimum temparature of CryogelPA-0.6shifted from40to50℃. When CryogelPA-0.6was reused by ten times, the relative activity of CryogelPA-0.6was retained67.7%of its initial activities. And the activity of CryogelPA-0.6remained77.1%of its initial activities after storage for60days at4℃.