| The remarkable transition in the manufacture of β-lactam antibiotics and their derivants is undergoing. To this effect, traditional chemical procedures are gradually being replaced by biotransformations. Penicillin acylase (penicillin G acylase or penicillin G amidase, E.C. 3.5.1.11, PGA or PAC) is a kind of critical enzyme for the industrial production of semi-synthetic β-lactam antibiotics, mainly in use for hydro-lyzing penicillin and cephalosporin to producing nuclei 6-aminopenicillanic acid (6-APA) and 7-aminodesacctoxyccphalosporanic acid (7-ADCA), respectively. It can also catalyze the renction of producing novel antibiotics using 6-APA or 7-ADCA with side-chains, which is kinctically controlled. The main obstacle for kinetically controlled syntheses is the hydrolysis accompanied. Therefore, synthesis/hydrolysis ratio is often regarded as an effective index for the synthetic process. Improving S/H is an effective means to increase antibiotic production and decrease hydrolysis. DNA family shuffling was used to increase PGA S/H in a certain synthetic reaction. The main results of this study were as follow:A general method for the expression and purification of PGAs was developed by fusing HisTag into a PGA using PCR and expressing it within a pET-24a (+) plasmid vector. Crude enzymes after fermentation were purified using Co~2+-IDA agar in one step. Activity recovery ratios of four kinds of recombinant wild-type PGAs (AfPGA, EcPGA, KcPGA, PrPGA) were 27.6%, 36.3%, 33.6% and 19.7%, respectively. The purification folds of enzymes were 40, 183, 107 and 17, respectively. The S/H ratios of four purified recombinant PGAs in the synthesis of cephalexin were respectively 2.5,13,17 and 3.3.Achimeric pool of PGA a subunits was obtained using DNA family shuffling by digesting five PGA gen(?) (?)alis,B. megaterium,E. coli,K. citrophila,P. rettgeri PGAs) with rare restrietion enzymes. A framework vector for receiving the chimera library was constructed by introduction a KpnI site at 1054 bp of KcPGA gene through silent site-mutation. Both library and framework vector were digested withNdeVKpnl, digestion product were then ligated and transformed. 5000 chimera vector transformants in E. coli JM109 (DE3) were screened through halos (25 mmol/L 7-ADCA and 50 mmol'L D-PGA) and 400 candidate clones were got. Selected mutants were fermented in lest tubes and collected cells were suspended by two kinds of substrate solutions and incubated at 30°C. After 24h, reaction components were determined by HPT.C ond mi!f:nt Ftrnins exhibiting an at least 2-fold increased cephalexin/phenyh1':?■'?''■!■.' p^;.': Kiiios compared to KcPGA in different time courses were picked for fur'Vr * ';n- rid their sequences were analyzed. Although after sequencing no signifirnni H : ik i were found, they have mutations and are different to former subunitreco'iH'1 ion.A general mf^rv! < felon;"{, expression and purification various recombinant PGA was establishe ■'. " ' ;ch m: 'e it a reality to compare the properties of various PGA including wn^'i u??■-' o!" ^i-d from site-mutation and direct evolution (subunit recombination, PXTA I1 !v slnlTline, genome shuffling, etc.). A whole route of high-throughout sr-^r;ri". S/II property of PGA variants was established and well used to screen a civ■-?--a library constructed by DNA family shuffling. This study lay the basis for further -■?<-'■:i em';:"vringon PGAs. |
PDF Full Text Request