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Penicillin amidase

About: Penicillin amidase is a research topic. Over the lifetime, 576 publications have been published within this topic receiving 15563 citations. The topic is also known as: penicillin amidohydrolase & ampicillin acylase.


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Journal ArticleDOI
TL;DR: In this paper, the effects of reaction variables such as enzyme concentration, pH, temperature, and molar ratio of the substrates on PVM synthesis were investigated using an immobilized Escherichia coli penicillin acylase.
Abstract: Enzymatic synthesis of pivampicillin (PVM) from D-α-phenylglycine methyl ester (PGM) and pivaloyloxymethyl 6-aminopenicillanic acid (POM-6-APA) was investigated using an immobilized Escherichia coli penicillin acylase. The effects of reaction variables such as enzyme concentration, pH, temperature, and molar ratio of the substrates on PVM synthesis were investigated. The time-course profiles of the PVM synthesis reaction followed a typical pattern of the kinetically-controlled synthesis of β-lactam antibiotics: the concentration of PVM reached a maximum and then decreased gradually. By lowering the reaction temperature, the maximum yield was enhanced significantly. This was mainly attributed to the suppressed hydrolysis of PVM and PGM at low temperatures. A higher yield of PVM was also attained with increasing the molar ratio of PGM to POM-6-APA. When the molar ratio of PGM to POM-6-APA was 10, the maximum yield of 61.8% was obtained at 4°C. The addition of organic cosolvents, on the other hand, showed no improvement in the PVM synthesis yields due to inhibitory effect of the solvent; both synthetic and hydrolytic activities of penicillin acylase were reduced by organic solvents. The degree of inhibition was found to be more profound in the presence of less polar solvents.

30 citations

Journal ArticleDOI
TL;DR: Data suggest that transport and translation of the penicillin acylase precursor are coupled and that the short Shine-Dalgarno-AUG distance interferes with a competent interaction between the translation initiation complex and the export system at high temperature.
Abstract: The enzyme penicillin acylase is synthesized by Escherichia coli only at growth temperatures below 30 degrees C. The biochemical basis of this strict temperature-dependent formation of an enzyme was investigated. When the gene (pac) was under the control of the lacUV5 promoter it showed the same temperature-dependent expression as the chromosomally encoded gene transcribed from its own promoter. This indicates that translation of the pac mRNA rather than transcription of the gene is temperature-dependent. This conclusion could be further confirmed by Northern hybridisation and by analysis of pac-lacZ transcriptional fusions. TnphoA insertion mutagenesis and experiments in which the promoter and 5' sequence encoding the signal peptide of the pac gene was exchanged with those of the cyclodextrin glycosyltransferase gene from Klebsiella oxytoca localised the region of pac mRNA responsible for the temperature-sensitive translation to the 5'-untranslated region and/or the signal peptide. Extension of the 5 nucleotide long spacer separating the Shine-Dalgarno motif from the AUG initiation codon by one or three nucleotides lead to partial or full synthesis of penicillin acylase precursor at 40 degrees C, respectively. The precursor of penicillin acylase formed at 40 degrees C by the mutant variants or when placed under the control of a heterologous upstream region was associated with the membrane but could not be translocated. Taken together these data suggest that transport and translation of the penicillin acylase precursor are coupled and that the short Shine-Dalgarno-AUG distance interferes with a competent interaction between the translation initiation complex and the export system at high temperature. Moreover, evidence was also provided which indicates a direct effect of temperature on the conformation of the precursor and it is proposed that the lack of translation at high temperatures has been selected to prevent the accumulation of transport-incompetent protein locked in the membrane.

30 citations

Journal ArticleDOI
TL;DR: It is shown that the pro-sequence in cis functions as a folding catalyst and accelerates the folding rate by seven orders of magnitude and that Ca2+, found in the crystal structure, is not directly involved in the folding process.

30 citations

Journal ArticleDOI
TL;DR: Compared to the penicillin acylase of E. coli, PAS2 showed superior potential for the synthesis of 6-aminopenicillanic acid (6-APA)-derived antibiotics, allowing the accumulation of up to 2.3-fold more target product at significantly higher conversion rates.

30 citations

Journal ArticleDOI
TL;DR: If the reaction is carried out at the thermodynamically optimum pH of synthesis (low pH), penicillin can be obtained in high yield and the possibility of using activated acid derivatives in synthesis and the advantages of using computer calculations for process optimization are discussed.

30 citations

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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
20234
20222
20183
20175
20165
20153