The role of phenylacetic acid in biosynthesis of penicillin amidase in E. coli
TL;DR: For improved biosynthesis of penicillin amidase by E. coli, NCIM 2400, the participation of different carboxylic acids and polyols has been studied in association with the effect of phenylacetic acid in a modified defined medium.
Abstract: For improved biosynthesis of penicillin amidase by E coli, NCIM 2400, the participation of different carboxylic acids and polyols has been studied in association with the effect of phenylacetic acid A modified defined medium has been devised for this purpose
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TL;DR: The whole cell immobilization technique has been optimized for different process parameters and the granular catalyst has good mechanical strength, low protein leachability, and high retention of penicillin amidase activity.
24 citations
TL;DR: The secretion of the biologically active enzyme into the medium indicated that the postranslational processing of the PAC molecule, including the excision of a 54 amino acid spacer peptide between the α and β subunit, is not carried out in the periplasmic space, but occurs at the cytoplasmic membrane or autocatalytically.
Abstract: L-form strains of Proteus mirabilis and Escherichia coli lacking the cell wall represent an alternative prokaryotic cell system for the production of recombinant proteins (KLESSEN et al. 1988, LAPLACE et al. 1988a, 1989b). We could demonstrate that they are also able to synthesize the enzyme penicillin G acylase (PAC)1). PAC was processed and secreted into the medium by recombinant L-form strains. The synthesis of PAC was growth-associated and stably regulated. Expression, secretion, and processing were not temperature-dependent and occurred at 26 degrees C, 32 degrees C and even 37 degrees C. The expression vector pHC1 carried the pac gene under the control of the lac UV promotor and a kanamycin resistance gene. It could be maintained in L-form cells, showing low structural as well as segregational instability. The secretion of the biologically active enzyme into the medium indicated that the postranslational processing of the PAC molecule, including the excision of a 54 amino acid spacer peptide between the alpha and beta subunit, is not carried out in the periplasmic space, but occurs at the cytoplasmic membrane or autocatalytically.
19 citations
01 Jan 2008
TL;DR: In this paper, the authors define peptides as heteropolymers composed by amino acid residues linked by peptidic bonds between the carboxyl group of one amino acid residue and the α-amino group of the next one.
Abstract: Peptides are heteropolymers composed by amino acid residues linked by peptidic bonds between the carboxyl group of one amino acid residue and the α-amino group of the next one. The definition is rather vague in terms of chain length, peptides ranging from two residues to a few dozens residues. Its upper limit of molecular mass has been set rather arbitrarily in 6,000 Da. The size of the molecule determines the technology most suitable for its production. Recombinant DNA technology is particularly suitable for the synthesis of large peptides and proteins, as illustrated by the case of insulin and other hormones (Walsh 2005). Chemical synthesis is a viable technology for the production of small and medium size peptides ranging from about 5 to 80 residues (Kimmerlin and Seebach 2005). Enzymatic synthesis is more restricted and has been hardly applied for the synthesis of peptides exceeding 10 residues. Its potential relies on the synthesis of very small peptides and, in fact, most of the cases reported correspond to dipeptides and tripeptides (Kumar and Bhalla 2005). In this sense, the technologies for peptide production are not competitive with each other in most of the cases. © Springer Science+Business Media B.V. 2008.
11 citations
TL;DR: In this investigation cell-free culture filtrate was used instead of process water and an extracellular protein factor was found to influence the synthesis of penicillin amidase.
6 citations
TL;DR: Experimental findings show that the periplasmic penicillin amidase does not show any variation by the chloroform treatment, and this analysis was also extended to the E. coli cells grown at various concentrations of phenylacetic acid plus glucose and lactic acid.
Abstract: 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.
4 citations
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TL;DR: The enzyme was shown to be inhibited by excess substrate, benzylpenicillin, and by both of the products of hydrolysis, which was found to be competitive and by 6-aminopenicillanic acid to be non-competitive.
282 citations
TL;DR: In this paper, the adsorption of n-alkyltrimethylammonium corrosion inhibitors at a dropping mercury electrode in N-potassium chloride solution was taken as far as -1.9 volts S.C.E.
Abstract: In further experiments on the adsorption of n-alkyltrimethylammonium corrosion inhibitors at a dropping mercury electrode in N-potassium chloride solution, the potential applied to the dropping mercury electrode was taken as far as -1.9 volts S.C.E. The electrocapillary and differential capacitance curves for n-dodecyl-and n-hexadecyltrimethylammonium bromides in N-potassium chloride were generally similar to those in N-sulphuric acid, and the calculated surface concentrations of the adsorbed quaternary ammonium ions were almost the same.
The wider range of potential in the present experiments enabled the desorption peaks in the differential capacitance curves for the more dilute solutions to be demonstrated. The stronger solutions gave subsidiary peaks, or steps, which may be associated with the formation of double or multiple layers.
The results of these and previous experiments are explained on the basis that the adsorbed layer of n-alkyltrimethylammonium ions does not completely exclude solvated ions, but hinders their approach to the metal surface.
34 citations
TL;DR: The differential rate of synthesis of penicillinamidohydrolase was studied in Escherichia coli growing in some chemically defined media and in a complex medium to find the highest rate of the enzyme synthesis is reached in a medium containing phenylacetic acid as the only source of carbon and energy.
Abstract: Synthesis of penicillinamidohydrolase (penicillin acylase, EC 3.5.1.11) inEscherichia coli is subjected to the absolute catabolite repression by glucose and partial repression by acetate. Both types of catabolite repression of synthesis of the enzyme inEscherichia coli are substantially influenced by cyclic 3,′5′-adenosinemonophosphate (cAMP). Growth diauxie in a mixed medium containing glucose and phenylaoetic acid serving as carbon and energy sources is overcome by cAMP. cAMP does not influence the basal rate of the enzyme synthesis (without the inducer). Derepression of synthesis of penicillinamidohydrolasa by cAMP in a medium with glucose and inducer (phenylacetic acid) is associated with utilization of the inducer, due probably to derepression of other enzymes responsible for degradation of phenylacetic acid. Lactate can serve as a “catabolically neutral” source of carbon suitable for the maximum production of penicillinamidohydrolase. The gratuitous induction of the enzyme synthesis in a medium with lactate as the carbon and energy source and with phenylacetic acid is not influenced by cAMP; however, cAMP overcomes completely the absolute catabolite repression of the enzyme synthesis by glucose.
28 citations
01 Jan 1980
TL;DR: In the process of developing a most efficient immobilized cell system for the production of 6 APA, two routes have been followed: increase of the specific activity of the cell system itself and selection of the appropriate immobilization technique.
Abstract: In the process of developing a most efficient immobilized cell system for the production of 6 APA, two routes have been followed: (1) increase of the specific activity of the cell system itself and (2) selection of the appropriate immobilization technique.
22 citations
TL;DR: The case is argued for an increased use of stirred deep cultures because a greater rate of output, at a higher cell concentration and with improved control of cultural conditions can be achieved.
Abstract: Present-day chemical and physical studies of the nucleic acid system of bacteria frequently call for quantities of cells which are beyond the capacity of traditional culture methods such as that of the shake flask. The case is therefore argued for an increased use of stirred deep cultures because a greater rate of output, at a higher cell concentration and with improved control of cultural conditions can be achieved. For the isolation of deoxyribonucleic acid and transfer ribonucleic acid, low growth-rate cells should be the most prolific source: whereas for ribosomes, ribosomal ribonucleic acid and messenger ribonucleic acid, high growth cells should give bigger yields. It is concluded that the information is somewhat obscure on what are the best growth conditions for the production of the related-enzymes.
A batch method for producing low growth-rate cells of Escherichia coli, in lots of 1 kg dry wt., is then described, followed by a continuous method for producing high growth-rate cells, with an output of 3 kg of dry cells per day. Both cultures have been designed to give maximum yield of cells by promoting maximum assimilation of the carbon substrate. From the point of view of designing a culture, the section dealing with the continuous process discusses the rationale of medium formulation, the calculation of aeration requirements and briefly reviews earlier work on the kinetics of continuous culture.
18 citations