Whole-cell biocatalysis in organic media

The release of Penicillin acylase from Escherichia coli cells through mechanical cell disruption using high-pressure homogenization and sonication was studied. From these cell disruption processes, the enzyme activity was totally released although with low specific activities, 0.1–0.3 IU(mg prot)−1. Intracellular total soluble protein release was quantified and modelled by a first order kinetic model. The effect of the driving force for each mechanical method, namely acoustic power input and homogenization pressure, on the respective kinetic disruption constants was also analysed. The release of Penicillin acylase by cell permeabilization using osmotic shock was also evaluated. The effects of cell concentration, penicillin acylase activity in E coli cells, type of buffer, pH, hypertonic solution composition, temperature and time used for osmotic shock were evaluated. Using cold osmotic shock, highly selective penicillin acylase release was attained with specific enzyme activities of about 4 IU(mg prot)−1 and enzyme activity release yields higher than 90%. The high purity of the penicillin acylase was a consequence of the optimized differential enzyme release method which was validated by SDS gel electrophoresis.



© 2002 Society of Chemical Industry

Penicillin acylase release from Escherichia coli cells by mechanical cell disruption and permeabilization

Background: L-glutamic acid is one of the major amino acids that is present in a wide variety of foods. It is mainly used as a food additive and flavor enhancer in the form of sodium salt. Corynebacterium glutamicum (C. glutamicum) is one of the major organisms widely used for glutamic acid production. Methods: The study was dealing with immobilization of C. glutamicum and mixed culture of C. glutamicum and Pseudomonas reptilivora (P. reptilivora) for L-glutamic acid production using submerged fermentation. 2, 3 and 5% sodium alginate concentrations were used for production and reusability of immobilized cells for 5 more trials. Results: The results revealed that 2% sodium alginate concentration produced the highest yield (13.026±0.247 g/l by C. glutamicum and 16.026±0.475 g/l by mixed immobilized culture). Moreover, reusability of immobilized cells was evaluated in 2% concentration with 5 more trials. However, when the number of cycles increased, the production of L-glutamic acid decreased. Conclusion: Production of glutamic acid using optimized medium minimizes the time needed for designing the medium composition. It also minimizes external contamination. Glutamic acid production gradually decreased due to multiple uses of beads and consequently it reduces the shelf life.

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Production of L-glutamic Acid with Corynebacterium glutamicum (NCIM 2168) and Pseudomonas reptilivora (NCIM 2598): A Study on Immobilization and Reusability

Secretion of levansucrase from Zymomonas mobilis in Escherichiacoli by glycine supplement was investigated. A significant amount of levansucrase (about 25% of total activity) was found in intact whole-cells. Cell fractionation experiments showed that levansucrase was found both in the periplasmic space and in the cytoplasmic fraction of E. coli. None or only trace amounts of levansucrase was detected in the extracellular culture broth at 24 h of cultivation and it accrued with the increasing concentration of glycine in the culture medium and duration of the culture period. Optimal glycine concentration for the maximum secretion of levansucrase was in the range of 0.8–1%, in which approximately 20–50% of levansucrase was released into the extracellular fraction at 24 h of cultivation, although glycine retarded the bacterial growth.

Extracellular secretion of levansucrase from Zymomonas mobilis in Escherichia coli

Production of glutamic acid using brevibacterium roseum with free and immobilized cells

Penicillin amidase is a periplasmic enzyme in Escherichia coli. Conventionally, the periplasmic enzymes are released into the medium by osmotic shock which is tedious involving a number of centrifugation steps. The present communication deals with a simple technique for the release of penicillin amidase by chloroform shock. Experimental findings show that the periplasmic penicillin amidase does not show any variation by the chloroform treatment. This analysis was also extended to the E. coli cells grown at various concentrations of phenylacetic acid, optimal concentration of phenylacetic acid plus glucose and lactic acid.

Release of periplasmic penicillin amidase from Escherichia coli by chloroform shock

A novel method of coimmobilized whole cells of Pseudomonas reptilivora and Micrococcus glutamicus, entrapped in calcium alginate beads have been used for the production of L-glutamic acid in a single stage fermentation process, using selected production medium enriched with glucose as substrate. The results obtained were compared with the L-glutamic acid production by free cells of Micrococcus glutamicus and by mixed culture of Pseudomonas reptilivora and Micrococcus glutamicus. The yield of glutamic acid obtained with mixed culture is relatively more than that the yield obtained with Micrococcus glutamicus alone. The properties of coimmobilized whole cells of Pseudomonas reptilivora and Micrococcus glutamicus in calcium alginate gel matrix have been investigated thoroughly and compared with those of free cells under most suitable conditions of fermentation.

Coimmobilized whole cells of Pseudomonas reptilivora and Micrococcus glutamicus in calcium alginate gel for the production of L-glutamic acid

Different operational mode of bioreactors influence the biosynthesis of the enzyme and related products as well as the growth of industrial microorganisms. This communication deals with the effect of mode of operation of various bioreactors with different geometric configurations, viz., batch (includes commercially available batch stirred tank, and custom-designed cylindrical and tapered reactors), batch-fed, continuous flow stirred tank reactors on the biosynthesis of penicillin amidase in Escherichia coli. Experimental findings show that the biosynthesis of penicillin amidase in E. coli show a little variation among batch reactor modes and significant variation on the continuous mode of operation. Further analysis show that the different reactor modes also influence periplasmic localization of the enzyme in the cell.

K. Jagannadha Rao

Papers

Release of periplasmic penicillin amidase from Escherichia coli by chloroform shock

Coimmobilized whole cells of Pseudomonas reptilivora and Micrococcus glutamicus in calcium alginate gel for the production of L-glutamic acid

Effect of mode of operation of bioreactors on the biosynthesis of penicillin amidase in Escherichia coli

Coimmobilized whole cells of