Synthesis And Application Of Disordered Block Thermo-sensitive Copolymer Carrier

Immobilized penicillin G acylase?PGA?is an important industrial catalyst that can catalyze the reaction of penicillin G potassium to form 6-aminopenicillanic acid?6-APA?,6-APA is an important intermediate of penicillin drugs,occupying a huge market space in the field of antibacterial and anti-inflammatory.Therefore,the performance of immobilized PGA,such as enzyme recovery,response rate,reusability,etc.,directly affects the economic benefits of the enterprise.The performance of immobilized PGA is closely related to the interaction between PGA and carrier.Therefore,designing and synthesizing the appropriate carrier type is of great significance to improve the performance of immobilized PGA.At present,the types of immobilized PGA carriers can be mainly divided into inorganic carriers,organic carriers and composite carriers.Although the above carriers have made different degrees of progress in terms of response rate and reusability of immobilized PGA,the activity recovery rate of immobilized PGA has progressed slowly.One of the main reasons for the low activity recovery rate is that the carrier microenvironment tends to cause conformational deformation of PGA active center during the immobilization.Considering the compatibility of activity recovery rate,response rate and reusability of immobilized PGA,we designed and synthesized two disordered block thermo-sensitive copolymer carrier for PGA.It was proposed to use the temperature sensitive polymer sol/gel phase transition process to achieve the fast response rate and good reusability of immobilized PGA.Designing the polyhydroxy microenvironment of the carrier to improve the hydrophilicity of the carrier,thereby maintaining the high catalytically active conformation of PGA.Adjusting the target spacing further consolidates the high catalytic activity conformation of PGA,thereby improving the recovery rate of enzyme activity,and the research has achieved good some results.The main research contents and results were summarized as follows:1)N,N-diethyl acrylamide?DEA?and?-hydroxyethyl methacrylate?HEMA?was as functional monomer.The thermo-sensitive copolymer PDEA-b-PHEMA with different HEMA molar ratio was synthesized via the reversible addition-fragmentation chain transfer?RAFT?polymerization method.The effect of HEMA molar ratio in PDEA-b-PHEMA on the activity of PGA was investigated.Then,the reactivity ratios of?-hydroxyethyl methacrylate?HEMA?and glycidyl methacrylate?GMA?in the random copolymer P?HEMA-co-GMA?were explored under specific conditions.On the basis of the above work,the disordered block polymer carriers,PDEA-b-PHEMA-b-P?HEMA-co-GMA?with different target spacings were synthesized.Their structure and molecular weight of the copolymer was investigated by nuclear magnetic resonance spectroscopy?¹HNMR?,results showed that the error range between the theoretical values and the experimental value of HEMA and GMA in the copolymer was 1.76-3.47%.The molecular weight distribution was explored by gel permeation chromatography?GPC?,which concluded the reaction was active/controlled radical polymerization.The temperature sensitivity study results showed that the LCST of copolymer decreased with the increase of HEMA in the random polymer;the response rate of immobilized PGA was as fast as free PGA;The activity recovery of immobilized PGA increased with increase of target spacing.When the molar ratio of HEMA to GMA in the copolymer was 8.15:1(the copolymer was PDEA₁₀₀-b-PHEMA₁₀-b-P(HEMA₆₅-co-GMA₈)),the activity recovery of immobilized PGA could reach 51.20%,which was 16.90%higher than that of pure GMA.2)HEMA in the random copolymer was changed into?methyl methacrylate?MMA,andtheblockdisorderedpolymercarrier,PDEA-b-PHEMA-b-P?MMA-co-GMA?,was prepared by the similar method.The structure and molecular weight of the copolymer were investigated by ¹HNMR,results showed that the error range between the theoretical values and the experimental value of MMA and GMA in the copolymer was 1.32-3.71%.The molecular weight distribution was explored by GPC,which concluded that reaction was active/controlled radical polymerization.The temperature sensitivity study results showed that LCST of copolymer decreased with the increase of MMA in the random polymer;the response rate of immobilized PGA was as fast as free PGA;The activity recovery rate of immobilized PGA increased with increase of target spacing,when the molar ratio of MMA to GMA in the copolymer was about 8.75:1(It was PDEA₁₀₀-b-PHEMA₁₀-b-P(MMA₈₇-co-GMA₁₀)),the recovery rate of immobilization PGA could be as high as 63.51%,which was 12.31%higher than that of PDEA₁₀₀-b-PHEMA₁₀-b-P(HEMA₆₅-co-GMA₈)and was 21.70%higher than that of pure GMA.Most important of all,when HEMA and MMA were used as comonomers,the activity recovery rate of immobilized PGA increased with the target spacing.It could be concluded that when the density of immobilized target was large,the immobilized PGA molecular deformation due to mutual extrusion caused a serious deformation of the active center configuration,which was the main reason for the low activity recovery rate.At the same time,multi-point fixation also affected the activity recovery rate of immobilized PGA.3)By comparing the effects of the above two series of disordered block copolymercarriersontherecoveryrateofimmobilizedPGA,(PDEA₁₀₀-b-PHEMA₁₀-b-P(MMA₈₇-co-GMA₁₀))was as the best carrier,it mainly studied the influence of immobilization conditions,such as concentration of enzyme solution,time,pH and temperature,on the activity recovery rate of immobilized PGA.Results showed that when the concentration,time,pH and temperature were 1.0%,16hours,8.0 and 37^oC,respectively,the recovery rate was the highest,and the value was68.50%.In addition,the reusability and storage stability of the immobilized carrier were also investigated.The results showed that when the carrier was repeatedly used10 times,the carrier recovery rate was 99%,and the relative activity of the immobilized PGA was 94%;After 250 days,the relative activity of the immobilized PGA was 92%,which displayed that the immobilized PGA has excellent reusability and storage stability.