Phase Transfer Bioconversion of Penicillin G into 6-APA by Immobilized Penicillin Acylase in Recycling Aqueous Two-phase Systems With Light-pH Sensitive Copolymers / 中国生物工程杂志

Immobilized penicillin acylase was used for bioconversion of penicillin G into 6-APA in aqueous two-phase systems consisted of a light-sensitive polymer PNBC and a pH-sensitive polymer PADB.Partition coefficients of 6-APA was found to be:about 5.78,in the presence of 1% NaCl.Enzyme kinetic showed that reaction reached equilibrium at 7h or so.The 6-APA mole yields were 85.3%(pH 7.8,and 20 ℃) and this value was about 20%higher than control in reaction of single aqueous phase buffer.Partition coefficient of penicillin G(Na) washardly changeable,while partition coefficient of product,6-APA and phenylacetate acid was significantly changeable.Reason is due to Donnan effect of phase systems andhydrophobicity of products.The change of partition coefficients of products also affects bioconversion yield of products.In the aqueous two-phase systems,substrate,penicillin G,products 6-APA and phenylacetate acid are biased in top phase,while immobilized penicillin acylase is completely partitioned in bottom.Substrate,penicillin G enters into bottom phase,and it is catalyzed into 6-APA and phenylacetate acid,then the products enter into top phase.Finally,inhibition of substrate and products is removed to result in improvement of products yield.Moreover,immobilized enzymehashigher efficiency than immobilized cells and occupy smaller volume.Comparing with free enzyme,immobilized enzymehashigher stability,longer use life,completely partitioned in bottom phase and recycle.Bioconversion in two-phase systems using immobilized penicillin acylase showed outstanding advantage.The light-sensitive copolymer forming aqueous two-phase systems could be recovered by laser radiation at 488 nm or filtrated 450 nm light,while pH-sensitive polymer PADB could be recovered by isoelectric point(pH 4.1).The recovery of the two copolymers was 95%~99%.

IMMOBILIZATION STUDIES OF PENICILLIN ACYLASE ON THE POROUS BEAD WITH OXIRANE GROUPS / 微生物学通报

Penicillin Acylase from B. megaterium was immobilized on the porous bead carriers based on methacrylate, N,N-methelene-bis-methacrymide, glycidyl methacrylate, Allyl ether copolymers (Eupergit-c) either directly or after chemical modification with 1.6-deaminohexane and L-Lysine. Directly binding with oxirane groups, the most efficient immobilization results were achieved. The immobilization yield was markedly influenced by the ratio of amount of free enzyme to the weight of the carrier. The specific activities of 89 up to 475IU/g (wet) and binding protein of 6.9 to 112 mg/g (dry) were obtained when the free enzyme added to the immobilization solution was from 323IU/g (dry) up to 6250IU/g (dry). The residual activity of immobilized PGA in a recycling system at the 20th was about 92.5% of the initial value.

Immobilization of Kluyvera citrophila penicillins acylase on controlledpore ceramics.

Penicillin acylase was purified from Kluyvera citrophila and immobilized on glutaraldehyde derivatives of silanized controlled-pore ceramics. The behaviour of the enzyme attached to TiO2, Al2O3 and SiO2 in the hydrolytic reaction are compared with that of the native enzyme as well as of the enzyme bound to CNBr-activated Sepharose 4B. The enzyme immobilized on TiO2 shows an efficiency of about 95% on the basis of protein bound. The penicillin acylase attached to SiO2, unlike the enzyme immobilized on TiO2, Al2O3 and Sepharose looses activity markedly in every cycle of use.