Preparation And Study Of Magnetic Nanocomposite Carrier For Penicillin Acylase Immobilized

As an important industrial biocatalyst in the field of bio-pharmaceuticals,immobilized penicillin G acylase（PGA）is mainly used to catalyze the industrial synthesis of 6-aminopenicillanic acid（6-APA）from penicillin G potassium（PG）,and as a key enzyme to compound the different types ofβ-lactam antibiotics（BAs）from6-APA.Due to the overuse of BAs,and the discharge ofsewage during the preparation of BAs resulted in antibiotic-resistant bacteria,which seriously weakened the clinical efficacy of BAs.At present,performing various derivatives of 6-APA to prepare BAs with high efficiency,broad spectrum,and different antibacterial spectrum are the main methods to overcame above problem.Immobilized PGA technology has aroused wide enterprises’attention due to the performance of enzyme activity recovery rate,operational stability and reusability of immobilized PGA has a very close relationship with their economic costs and benefits.Accordingly,how to prepare immobilized PGA with high enzyme activity and high operational stability has become a key issue.This paper based on the research results of our group and research progress at home and abroad,a series of excellent performance of immobilized carriers have been prepared.Besides,PGA immobilization and catalytic performance of immobilized PGA were studied in this work and the following results were obtained:1)Firstly,Fe₃O₄ NPs were synthesized by a reverse microemulsion method.Then,PTA was coated on the surface of Fe₃O₄ NPs,and PGA was immobilized in Fe₃O₄@PTA NPs via physical adsorption.Meanwhile,the structure of the different stage products was characterized and the process conditions were further optimized.The result shown that the enzyme activity（EA）,and enzyme recovery（EAR）and maximum load（ELC）of the immobilized PGA under the optimal conditions were27479 U/g,82.1%,156 mg/g,respectively;what’s more,the immobilized PGA shown the more excellent stability and reusability than free PGA.2)Based on acetal reaction,Fe₃O₄@PTA NPs were modified by glutaraldehyde（GA）.The grafting rate of GA(Gr-_GA)was used as an index to optimize the process conditions,and the optimal G_r-GA was 30.0%.Afterwards,PGA was covalently bound with Fe₃O₄@PTA-GA nanocarriers by the Schiff base reaction between the aldehyde group and the amino group under mild conditions.And further optimized immobilized process conditions by characterized the structure of the products at each stage.Under the optimal conditions,the results shown that the enzyme activity（EA）,enzyme recovery（EAR）and the maximum load（ELC）were 26843 U/g,80.2%,and 125 mg/g respectively.Moreover,the Fe₃O₄@PTA-GA-PGA NPs showed higher storage stability and reusability than Fe₃O₄@PTA-PGA NPs,and the affinity of Fe₃O₄@PTA-GA-PGA NPs with the substrate has also been further improved.3)Based on the experimental fact that Mn²⁺had a certain activating effect on the catalytic activity of PGA.In order to introduce Mn²⁺into the composite material,Mn Cl₂ was added during the process of TA coating on the surface of Fe₃O₄ NPs.Then,the C=C bond was introduced via the ring-opening reaction of the phenolic hydroxyl group of TA and the epoxy group of glycidyl methacrylate（GMA）.Afterward,using GMA as the target monomer,methyl methacrylate（MMA）as the monomer for the controltargetspacing,themagneticnanocompositecarrier Mn²⁺-Fe₃O₄@PTA-GMA-b-P（MMA-co-GMA）NPs with the molal ratio of MMA/GMA is 5/2 was prepared via RAFT polymerization.And it was applied for immobilized PGA by covalent.Subsequently,the immobilized process conditions were further optimized by characterized the structure of the different stage product.Under the optimal conditions,the enzyme activity（EA）,enzyme recovery（EAR）,and the maximum load（ELC）of the immobilized PGA were 31361 U/g,93.7%,120 mg/g,respectively.Compared with the above immobilized PGA,this immobilized PGA shown better stability,reusability and substrate affinity.