Recombinant whole cell penicillin acylase biocatalyst: Production, characterization and use in the synthesis and hydrolysis of antibiotics

Ospina, S.; Merino, Enrique; Ramı́rez, Octavio T.; López-Munguı́a, Agustı́n

doi:10.1007/bf00129388

Cited by 9 publications

(3 citation statements)

References 9 publications

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“…In contrast to intact cells, the enzyme penicillin G acylase in immobilized cells was much stable at either 30 or 50°C. Ospina et al (1995) also reported that the whole-cell catalyst in the immobilized form had a considerably higher stability than the free enzyme. In comparison with 50°C, the decay in enzymatic activity was much slower when the membrane reactor was operated at 30°C.…”

Section: Conversion Of Penicillin To 6-apa In the Membrane Enzyme Reamentioning

confidence: 97%

“…As indicated in a recent review article (Parmar et al, 2000), the production of 6-APA using whole-cell penicillin acylase became more attractive because of the improvement of activity by using a recombinant strain. Ospina et al (1995) used a recombinant E. coli JM101 (pPA102) strain for whole-cell immobilization by entrapment in agar. A speci®c activity of 2 U/mg was obtained at the end of a fed-batch culture and the immobilized preparation had a higher activity (134 U/ g preparation) of penicillin hydrolysis.…”

Section: Introductionmentioning

confidence: 99%

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A novel enzyme reactor using gluten membrane entrapping cell‐associated enzyme

Lee

Guo²

2001

Biotech & Bioengineering

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A membrane enzyme reactor consisting of variable pieces of replaceable cell-immobilized membranes was proposed for the continuous production of bioproducts. To demonstrate the characteristics of the reactor, cell-immobilized membranes were prepared by the entrapment of permeabilized recombinant Escherichia coli cells containing penicillin G acylase within the gluten matrices. A stainless-steel net that was created with a mesh frame was used to support each gluten membrane so that the membranes could be filled into the rectangular-shaped reactor. The reactor equipped with either six or 12 pieces of cell-immobilized gluten membranes containing a biomass concentration of 5%, w/w was effective in catalyzing the production of 6-aminopenicillanic acid from penicillin G. In comparison with intact cells, the cell-immobilized preparation was more stable and the half-life time of the immobilized cell-associate enzyme in gluten membrane was estimated to be 36 days by a long-term operation. As the substrate solution was forced to flow through the reactor equipped with six membranes and in the direction perpendicular to the membranes, the pressure drop was determined to be less than 50 cm H(2)O with a flow-rate up to 50 mL/min. This low pressure due to the porous structure of gluten membrane would lead to a lower operational cost. Increasing either the number of membranes or the area of each cell-immobilized membrane can easily do scaling-up of this membrane reactor.

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Section: Conversion Of Penicillin To 6-apa In the Membrane Enzyme Reamentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

A novel enzyme reactor using gluten membrane entrapping cell‐associated enzyme

Lee

Guo²

2001

Biotech & Bioengineering

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“…Nonetheless, from a commercial standpoint, higher specific PA activities, than most of the presently reported, are still required to produce economically attractive biocatalysts (Rarrdrez et al, 1994b;Ospina et al, 1995). Further improvements in specific enzyme activity could result from manipulation of the environmental variables that affect PA processing.…”

Section: Introductionmentioning

confidence: 99%

A postfermentative stage improves penicillin acylase production by a recombinant E. coli

1996

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Active penicillin acylase is formed only after an in vivo post-translational processing of the polypeptide precursor. Such a maturation process is rare in procaryotes. In this work, incubation under aerated conditions, of whole recombinant E. coli cells after glucose depletion and growth cessation, i.e., during the postfermentative stage, consistently resulted in 2-to 4-fold increases in penicillin acylase activity. Such results suggest that penicillin acylase maturation occurs to a high extent even during the postfermentative stage. Accordingly, the effect of different incubation conditions, during the postfermentative stage, on penicillin acylase was determined. Incubation under anaerobic conditions resulted only in a 1.27-fold increase of enzyme activity, with respect to the end of the batch culture, whereas a 3-and 4-fold increase occurred during incubation under dissolved oxygen concentrations of 100 and 43% (with respect to air sat.), respectively. Only a small negative effect, on the maturation process, was observed during incubation with acetate concentrations above 0.6 g/L. No effect of pH, in the range of 6.0 to 8.0, was observed.

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