The Design And Synthesis Of Carrier For Immobilized PGA

Penicillin G acylase(PGA)is a kind of important industrial enzyme,catalyzes hydrolysis penicillin G into 6-amino penicillium alkanes acid(6-APA)which is an important intermediate of semi-synthetic penicillin drug.PGA has the advantage of strong specificity,high yield,mild reaction conditions,sa ving cost,green environmental protection and so on.However,PGA exists the disadvantage of difficult recycle,poor environmental tolerance and stability,etc,which restricts its application and development in catalytic conversion industry.Immobiliz ation is a major method to overcome the above problems.The related researches have made significant progresses,simultaneously,some immobilized PGAs have already been applied in the catalytic conversion industr y.However,it was so difficult to characterize the functional group and its distribution in the solid phase environment that the influence of carrier microenvironment on catalytic performance of PGA was hard to be studied.The relevant theoretical researches about carrier microenvironment influencing on the catalytic performance of PGA were laged behind.The design and synthesis of immobilized carrier was lack of theoretical guidance.Consequently,the loss of catalytic activity immobilized PGA that the present studied on was high.In this paper,on the basis of being familiar with research methods in the traditional immobilized PGA,innovating research method,starting from the study of PGA immobilized carrier microenvironment,design ing and synthesizing carrier that could maintain the catalytic performance of PGA in a large extent,to be used for PGA immobilization and catalytic conversion research,which has obtained some certain results.The main research contents were summarized as follows:1)Penicillin G acylase was immobilized through a novel immob ilization method,that is,nucleophilic substitution,using macroporous adsorption resin CLX1180 as carrier.The structure of CLX1180 and its immobilization product were characterized by FTIR.Then,the influence of reaction conditions,such as immobilizat ion time,temperature,p H,and the amount of PGA,were investigated systematically.Based on the condition investigation,the optimum immobilization conditions were obtained as follows: the optimum immobilization time was 10 h,temperature was 36 oC,p H value of the solution was 6.0,and the amount of PGA was 35.00 m L(The original free PGA activity was 7360 U/g),the activity of immobilized PGA was 2200 U/g,but the activity recovery was only 29.89 %.Finally,the reusability of immobilized PGA study showed that there was no obvious decrease in the activity of immobilized PGA after using 50 cycles.Besides,the storage stability study of immobilized PA also presented an excellent result.2)The catalytic performance of PGA treated with organic solvents containing a variety of functions at room temperature for different interval,such as alkanes,alcohols,acids,halogenated hydrocarbon and ethers,was investigated.The result indicated:(1)With the extension of time,as for hydrophobic organic solvent,activ ity retention rate(Cr)decreased and achieved equilibrium gradually,the smaller log P,the smaller Cr;as for hydrophilic organic solvent,Cr increased first and then decreased to a equilibrium value,the smaller log P,the bigger Cr.(2)With the increasing of the solvent molecular size l,equilibrium Cr achieved showed different changing trends: when l < 4.4 ?,equilibrium Cr achieved was fast and not affected by l;when 4.4 ? < l < 6.9 ?,it was slow and increased with the increasing of l,when l > 6.9 ?,it was fast and decreased with the increasing of l.(3)PGA treated with polyol,its Cr was maximum and maintained stability.3)A series segmented copolymers of poly(N,N-diethylacrylamide)-b-poly(glycidyl methacrylate)-b-poly(hydroxyethyl methylacrylate)(DGH)and poly(N,N-diethylacrylamide)-b-poly(hydroxyethyl methylacrylate)-b-poly(glycidyl methacrylate)(DHG)were synthesized by Reversible Addition-Fragmentation Chain Transfer Polymerization(RAFT)using N,N-diethylacrylamide(DEA),glycidyl methacrylate(GMA)and hydroxyethyl methylacrylate(HEMA)as temperature-sensitivity,therapeutic target and building microenvironment monomers,azobisisobutyronitrile(AIBN)as initiator,and dimethyl formamide(DMF)as solvent.The relationship between the monomer proportion in carrier and the catalytic performance of immobilized PGA was investigated,and obtained the following results:(1)The relative molecular weight of copolymer increased linearly with time and its molecular weight distribution was the range of 1.10-1.35 through 1H NMR and GPC,it demonstrated that the synthesis reaction of copolymer was living polymerization.Besides,the temperature-sensitivity of prepared segmented copolymers by acenaphthylene(ACE)tagged were studied with fluorescence photometry instrument.With the increase of HEMA in segmented copolymer,the Lowe Critical Solution Temperature(LCST)increased,but the phase transition temperature range became wider,that is,temperature-sensitivity was weakened;with the increase of GMA,LCST decreased,the phase transition temperature range became wider,that is,temperature-sensitivity was weakened.(2)With the increasing of monomer HEMA or GMA,PGA capacity(Ac)increased gradually and then got shallower,PGA activity(ag)and acticity recovery(Ar)increased to maximum first and then decreased slightly to a shallower value.The optimal PGA immobilization carrier was DHG4724,its LCST was 36 oC,and ag of immobilized PGA was 780 U/g,Ac was 910 U(The original free PGA activity was 886 U/g),Ar was 88 %,Cr was 83 % after using 10 cycles.4)The influence of immobilized conditions on the catalytic performance of immobilized PGA was investigated using DHG4724,and obtained the optimizing immobilized conditions: immobilized time was 10 h,temperature was 36 oC,p H value was 8.0,and PGA concentration was 10 %(the volume is 5 m L),and ag of immobilized PGA was 830 U/g,Ac was 1100 U(The original free PGA activity was 886 U/g),Ar was 94 %.