Investigation of the Enzymatic Kinetic Model of Immobilized Penicillin G Acylase Reaction and the Partition/Reaction in Aqueous Two-Phase System
| Autor: | Jian-Liang Pan, 潘建亮 |
|---|---|
| Rok vydání: | 2005 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 93 In this study, cephalosporin G (Ceph-G) as the substrate was hydrolyzed by penicillin G acylase. Its hydrolyzed products are 7-aminodesacetylcephalosporanic acid (7-ADCA) and phenylacetic acid (PAA). The research work can be concluded into three parts: the kinetic study of cephalosporin hydrolysis by penicillin G acylase; the effect of adding polyethylene glycol (PEG) on the hydrolysis reaction of PGA; and the effect of aqueous two-phase system composed of different polyethylene glycols and different salts, respectively, on the partition coefficient of 7-ADCA, PAA and Ceph-G.. The factors affecting the kinetics of PGA are temperature, pH, 7-ADCA and PAA. Variations of initial rate (Vi), conversion of 7-ADCA (XA), specific velocity (V/Vi), rate constant of specific velocity (ksv), deactivation rate constant (kd), activation energy (Ea), activation energy of thermal denature ((Ea)TD), energy of specific velocity ((Ea)sv), thermal deactivation energy (Ed), thermodynamic properties (ΔH, ΔS and ΔG) and kinetics constant (Km and Vm) obtained from different temperatures and pH were all investigated. The inhibition mechanism was estimated from a Lineweaver-Burk plot by adding different amounts of 7-ADCA and PAA. The PGA velocity model was determined taken into consideration of the inhibition mechanism of both 7-ADCA and PAA. The inhibition constants (KiA, KiP and KiS) were also calculated. Different concentrations of polyethylene glycols were applied to the PGA catalyzed reaction of cephalosporin G and the influence on the parameters such as activation energy (Ea), deactivation rate constant (kd), thermal deactivation energy (Ed), thermodynamic properties (ΔH, ΔS and ΔG) and kinetics constant (Km and Vm) were all discussed. The inhibition mechanism was estimated from a Lineweaver-Burk plot by adding different concentrations of polyethylene glycol. Similarly, the PGA velocity model was established from considering the inhibition mechanism of polyethylene glycol. The inhibition constants (KiPEG) were also calculated. The partition coefficients (K7-ADCA, KPAA and KCeph-G.) were obtained from the aqueous two-phase systems composed of different polyethylene glycols and different salts. The experimental results obtained from different concentrations of polyethylene glycols and salts, volume ratio (Vt/Vb), pH or variations of the partition coefficient by adding an electrolyte, yield in the top phase (Yt), yield in the bottom phase (Yb), 7-ADCA separation coefficient (gA) and Ceph-G separation coefficient (gC) were helpful to search for an aqueous two-phase system of optimum composition. Thus, the purification of Ceph-G hydrolyzed by PGA in an aqueous two-phase system could be successfully performed. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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