Showing papers on "Penicillin amidase published in 1986"
TL;DR: A simple and versatile procedure to clone penicillin acylase genes has been developed that involves the construction of a plasmid library in a host presenting an amino acid auxotrophy and the production of those clones carrying the E. coli acyl enzyme was more sensitive to the growth temperature than that of the clones containing the K. citrophila gene.
58 citations
TL;DR: The Bacillus sphaericus gene encoding penicillin V amidase, which catalyzes the hydrolysis of penicillus V, has been characterized and high expression of the gene was obtained in Escherichia coli using an inducible promoter, showing that the gene product is stable in this heterologous host.
33 citations
TL;DR: The nt sequence of the regulatory region of this gene, the identification of a functional promoter, the transcriptional start point, and the description of possible regulatory regions are reported.
32 citations
Patent•
25 Mar 1986
TL;DR: In this article, the ribosome binding site of the penicillin amidase gene and its peptide signal peptide are fused with the structural gene for human serum albumin.
Abstract: Human serum albumin is produced by culturing a bacterium (e.g. E. coli) capable of maintaining a plasmid containing an inducible promoter (e.g. P trp ) upstream of the penicillin amidase promoter, the ribosome binding site of the penicillin amidase gene and the penicillin amidase signal peptide, fused with the structural gene for human serum albumin.
28 citations
TL;DR: The present method is suitable not only for screening penicillin G acylase-production by a variety of bacteria but also for detection from a large number of transformant colonies of clones containing a gene encoding for the enzyme.
24 citations
TL;DR: The results of the computer simulations indicate that a high conversion of Pen‐G may be achieved (80–90%) at bulk pH values of about 7.5–8.5, and the effectiveness factor and the conversion in a CSTR at different enzyme loadings are computed.
Abstract: A mathematical model has been developed for the internal pH control in immobilized enzyme particles. This model describes the kinetics of a coupled system of two enzymes, immobilized in particles of either planar, cylindrical, or spherical shape. The enzyme kinetics are assumed to be of a mixed type, including Michaelis-Menten kinetics, uncompetitive substrate inhibition, and competitive and noncompetitive product inhibition. In a case study we have considered the enzyme combination urease and penicillin acylase, whose kinetics are coupled through the pH dependence of the kinetic parameters. The hydrolysis of urea by urease yields ammonia and carbon dioxide, whereas benzylpenicillin (Pen-G) is converted to 6-animo penicillanic acid and phenyl acetic acid by penicillin acylase. The production of acids by the latter enzyme will cause a decrease in pH. Because of the presence of the ammonia-carbon dioxide system, however, the pH may be kept under control. In order to obtain information about the optimum performance of this enzymatic pH controller, we have computed the effectiveness factor and the conversion in a CSTR at different enzyme loadings. The results of the computer simulations indicate that a high conversion of Pen-G may be achieved (80-90%) at bulk pH values of about 7.5 - 8.more » 27 references.« less
10 citations
TL;DR: Two fluorogenic substrates are described for the detection of Penicillin-G-acylase activity in coloured cell-containing media and inside single cells and it is possible to label enzyme-containing cells and to distinguish them from enzyme-free cells by fluorescence microscopy or laser-flow cytometry.
9 citations
Patent•
24 Mar 1986
TL;DR: In this article, the culturing of a bacterium (E.coli) capable of providing for the stable maintenance of a plasmid containing an inducible promoter (Ptrp) upstream of the penicillin amidase gene, was described.
Abstract: The method comprises the culturing of a bacterium (E.coli) capable of providing for the stable maintenance of a plasmid containing an inducible promoter (Ptrp) upstream of the penicillin amidase promoter, the ribosome binding site of the penicillin amidase gene, and the penicillin amidase signal peptide fused with the human serum albumin structural gene.
7 citations
Journal Article•
6 citations
TL;DR: This paper deals with the modelling of the hydrolysis of benzylpenicillin to 6-aminopenicillanic acid and phenyl acetic acid in a small pilot plant batch recirculated reactor by an immobilised penicillin amidase preparation.
Abstract: This paper deals with the modelling of the hydrolysis of benzylpenicillin to 6-aminopenicillanic acid (6-APA) and phenyl acetic acid (PAA) in a small pilot plant batch recirculated reactor by an immobilised penicillin amidase preparation. By using the following linearised form for an integrated Michaelis-Menten equation Et/V0X=α+β| In (1-X)/X| where α and β are reaction kinetic parameters, good correlations are obtained of α and β with linear velocity across the reactor, substrate concentration and temperature of operation. A process to determine α and β from initial velocity measurements is outlined. The applicability of the above equation to published data is also analysed.
5 citations
TL;DR: A rapid and specific procedure was developed for the simultaneous detection of bacterial acylases and beta-lactamases, using ampicillin and cephalexin as substrates and has been successfully applied to screening for acylase activity in a variety of bacteria.
Abstract: A rapid and specific procedure was developed for the simultaneous detection of bacterial acylases and beta-lactamases, using ampicillin and cephalexin as substrates. Bacterial suspensions from agar plates were incubated separately with each beta-lactam substrate for 1 h at 37 degrees C. The supernatant of the reaction mixture was dansylated, and the dansyl derivatives were separated by two-dimensional thin-layer chromatography on polyamide sheets. The end products resulting from acylase hydrolysis, including the intact beta-lactam nucleus, 6-aminopenicillanic acid or 7-aminodeacetoxycephalosporanic acid, and the acyl side chain acid, D-(-)-alpha-aminophenylacetic acid, and the end product resulting from beta-lactamase hydrolysis (D-phenylglycylpenicilloic acid or D-phenylglycyldeacetoxycephalosporoic acid) were separated from each unhydrolyzed substrate and amino acids by this procedure. The presence of the intact beta-lactam nucleus in the reaction mixture is the indication of acylase activity. This method is sensitive and reproducible and has been successfully applied to screening for acylase activity in a variety of bacteria. It may be pharmaceutically useful for identifying organisms capable of removing the acyl side chain from naturally occurring beta-lactam antibiotics such as penicillin G, penicillin V, and cephalosporin C for production of the beta-lactam nuclei which serve as the starting materials for semisynthetic beta-lactam antibiotics.