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

Abstract

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.

Improvement of Catalytic Properties of Escherichia coli Penicillin G Acylase Immobilized on Glyoxyl Agarose by Addition of a Six-Amino-Acid Tag

“…The periplasmic extract (3) was purified on a Q-Sepharose column (Pharmacia), with a 0-to-75 mM NaCl gradient in 50 mM Tris HCl (pH 8.5) (average yield was 18 mg of purified protein per 30 g of wet cells). pET28-3G3KPAC was expressed in BL21 Star(DE3) cells as described previously, except that the temperature for the induction of expression was lowered to 20°C, due to the increased sensitivity and lower yield of processing and maturation (2,4,8,13,18,19) of the modified enzyme (data not shown). The periplasmic extract was precipitated with 75% (NH 4 ) 2 SO 4 and the pellet, resuspended in 1 M (NH 4 ) 2 SO 4 and 50 mM potassium phosphate (pH 7), was purified on a phenyl Sepharose column with a gradient of 0 to 30% ethylene glycol in 50 mM potassium phosphate (pH 7).…”

Significance of location of enzymes on their release during microbial cell disruption

“…The reason for location factor value of 0.59 for penicillin acylase, from E.coli cells of 10-h culture, indicating it as active cytoplasmic enzyme is not yet known. It is reported that incubation under aerobic conditions with high dissolved oxygen concentration have resulted in higher penicillin acylase activity (De Leon et al, 1996). Sonication is found to be useful for oxidation of potassium iodide (Gogate et al, 2001;Senthil Kumar et.…”

“…Penicillin acylase is reported to undergo posttranslation mechanism of translocation. The polypeptide precursor of penicillin acylase is translocated to the periplasmic space where the signal peptide is removed to give the active penicillin acylase enzyme (De Leon et al, 1996). The reason for location factor value of 0.59 for penicillin acylase, from E.coli cells of 10-h culture, indicating it as active cytoplasmic enzyme is not yet known.…”

Design and characterization of a one‐compartment scale‐down system for simulating dissolved oxygen tension gradients

“…For instance, DOT oscillations can be exploited to produce polymer families of particular molecular weight characteristics during production of alginate by Azotobacter vinelandii, 13 or to increase recombinant penicillin acylase activity produced by E. coli. 15 Monte Carlo-type oscillatory algorithms simulate better the frequency distribution of circulation times in large-scale bioreactors than algorithms using constant oscillation periods. 27 However, www.interscience.wiley.com/jctb Reonse rate (%DOT s …”

“…Cortés et al 14 reported that DOT oscillations lead to increased volumetric and specific lactase activity, without affecting maximum yeast concentration. De León et al 15 reported that variations in the DOT oscillation axis affected the specific growth rate, biomass yield, and production of penicillin acylase by a recombinant E. coli compared with cultures without oxygen limitation.…”