| Penicillins are the most important antibiotics in terms of annual production and prescription volume, despite the availability of an ever-growing number of alternative antimicrobials. The excessive use of these antibiotics has led to the development of resistance in pathogens and newer semisynthetic antibiotics are produced to overcome the resistance.Penicillin G and penicillin V are the natural penicillins produced in bulk by fermentation. However, only a small amount of these penicillins is used directly as therapeutic agents while the majority is used for the production of 6-amino penicillanic acid (6-APA). Penicillin G acylases (PGAs) are the enzymes used to catalyze the hydrolysis of natural penicillins to yield 6-APA, the β-lactam nucleus from which a wide range of semisynthetic penicillins are made. The semisynthetic penicillin derivatives not only exhibit better properties such as increased stability, easier absorption and fewer side effects than penicillin G or penicillin V, but also address the problem of microbial resistance to antibiotics. The industrial production of β-lactam antibiotics and their intermediates is undergoing a remarkable transformation.Traditional chemical conversions based on stoichiometry are being replaced by enzyme-catalyzed processes. Still, the starting materials for these transformations are fermentatively obtained chiral structures such as cephalosporin C, penicillin G, Benzylpenicillin,Amoxicilli,Formidacillin and penicillin V. The ever-increasing insight into biochemical pathways and the resulting optimization of fermentation processes will make this application of the chiral pool even more profitable in the years to come.Type I enzymes preferentiallyhydrolyze phenoxymethylpenicillin (penicillin V); type II enzymes primarily act on benzyl penicillin (penicillin G),although they exhibit a broader range of specificities; and type III enzymes hydrolyze ampicillin (D-_-aminobenzylpenicillin).The PGA's S/H ratio influence that PGA was used in the industry ofβ-lactam antibiotics synthesis.In this thesis,we studied content that A General Method of Cloning,Expression and Purification of Recombinant Penicillin G Acylases; and Set Up a System to Determine the PGA's S/H Ratio.With the advent of PGA, The sequence determined penicillin G acylases ,are from Alcaligenes faecalis(Afa),Esherichia coli(Ecoli), Kluyvera citrophila(Kc), Providencia rettgeri(Pr), Bacillus megaterium (Bm) and Arthrobacter viscosus(Av)..The former four kinds of the PGA's gene had been cloned and expressed in E.coli .To research the quality of this PGAs further more, the four kinds of PGAs have been purified under various methods.Because of the different quality every enzyme with others,the way to purify PGA was various from each other and two columns were needed generally.To compared equally with the PGAs which were gained by the way of directed-mutation , DNA family shuffling and so on.We found a general method of cloning ,expression and purification of recombinant penicillin G acylases.In this thesis ,we evaluate a method to purify PGAs by immobilized metal affinity chromatography(IMAC).The PGA sequences were cloned into pET28b+,expressed them in E.coli.JM109(DE3) and we purified them with Co2+–IDA agarose.12% SDS-PAGE indicated that we got the four kinds of recombinant PGAs and two subunits were found in the map ,the large subunit (β subunit ) is at 63KD;the small subunit(α subunit ) is at 23KD.The results indicated that purification of PGAs (EcPGA,KcPGA,PrPGA,AfaPGA) from a crude extract and elution by one –step with 400mM imidazole:the purified fold were 183,107,17,40;the purified yield were 36.3%,33.6%,19.7%,27.6%. The penicillin G acylase is the key enzyme used in industrial production of β-lactam antibiotics,High S/H ratio was important for this PGA to used in ndustry.In this thesis ,we set up a system to determine the PGA's S/H ratio .Recombinant PGAs were determined under this system ,and we filterd the PGA which had a high S/H ratio,now this PGA...
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