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S W Queener

Publications and source records attributed to S W Queener.

27 records · Page 2Linked to original sources
PubMed1975

Glutamate dehydrogenase specific activity and cephalosporin C synthesis in the M8650 series of Cephalosporium acremonium mutants.

Conditions for the accurate measure of glutamic dehydrogenase (GDH) from Cephalosporium acremonium were determined. K(m) values for alpha-ketoglutarate and ammonium ion were 7 and 15 mM, respectively. The half-saturation for reduced nicotinamide adenine dinucleotide phosphate was 5 muM. Reduced nicotinamide adenine dinucleotide did not serve as a cofactor for the enzyme. The specific activity of GDH was measured in six mutants of C. acremonium which varied in their ability to synthesize cephalosporin C. The mutants represented two separately derived lines, A and B. The four mutants in line B were characterized by a derepression of the GDH upon entry into stationary phase. The two mutants in line A were characterized by repressed levels of GDH during the same period. Both lines exhibited high GDH activity early in their fermentations, but activity decreased during the period of active cell growth. Cytochrome c concentrations followed the same pattern as total soluble intracellular protein. Line A mutants were low in cephalosporin C productivity and line B encompassed low, intermediate, and high productivity mutants. The relative frequency of yield improvements in line A and B indicate that the altered regulation pattern for GDH in line B may have removed a nitrogen limitation for cephalosporin C synthesis.

Acremonium↗
PubMed1975

Differentition of mutants of Cephalosporium acremonium in complex medium: the formation of unicellular arthrospores and their germination.

Differentiation of swollen hyphal fragments to unicellular arthrospores accompanied the synthesis of cephalosporin C by a series of Cephalosporium acremonium mutants during propagation in a complex medium. The complex medium supported significantly higher synthesis than the defined medium used in previous studies of differentiation in C. acremonium. The mutants differed in their ability to form unicellular arthrospores and to synthesize cephalosporin C, but a one-to-one correspondence between the two properties was not observed. An inverse relation was observed between the growth rates of the mutants and their ability to synthesize cephalosporin C: each mutant produced more antibiotic but grew more slowly than its parent strain. Germination of the unicellular arthrospores occurred in complex medium but differed significantly from the germination of conidia in seed medium. The unicellular arthrospores were examined by electron microscopy and compared with swollen hyphal fragments and slender hyphal filaments. The unicellular arthrospores had a thicker cell wall, rougher cell surface, and had one or more small identations in their surface. The internal structure of the unicellular arthrospore resembled those of the swollen hyphal fragment and slender hyphal filament. Filaments had lower concentrations of lipid-containing vacuoles which were prevalent in both the swollen hyphal fragments and the unicellular arthrospores.

Acremonium↗
PubMed1974

Synthesis of deactoxycephalosporin C by a mutant of Cephalosporium acremonium.

A number of Cephalosporium acremonium mutants blocked in the synthesis of cephalosporin C were investigated for accumulation of other beta-lactam compounds. The non-cephalosporin C producers were isolated after exposing the superior cephalosporin C-producing strain M8650-4 to ultraviolet light (268 nm). One of the blocked mutants, MH63, accumulated deacetylcephalosporin C (0.4 mg/ml), deacetoxycephalosporin C (1.5 mg/ml), and penicillin N (2.7 mg/ml). In contrast, the parent of MH63 produced high levels of cephalosporin C as well as deacetylcephalosporin C (2.2 mg/ml) and penicillin N (1.0 mg/ml), but only traces of deacetoxycephalosporin C (about 0.1 mg/ml). Deacetoxycephalosporin C was isolated from the mutant strain and identified by ultraviolet light, nuclear magnetic resonance, bioactivity spectrum, and co-migration with authentic standard in three chromatography systems.

Acremonium↗
PubMedDate not supplied

Isolation, sequence determination and expression in Escherichia coli of the isopenicillin N synthetase gene from Cephalosporium acremonium.

The enzyme isopenicillin N synthetase (IPS) catalyses the oxidative condensation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (LLD-ACV) to isopenicillin N, which is a central reaction in the pathway to clinically important penicillins and cephalosporins. Here we report the cloning, characterization and expression in Escherichia coli of the gene encoding the IPS protein in Cephalosporium acremonium. The IPS gene was identified by purifying IPS protein, determining the first 23 amino-terminal amino acids, preparing a set of synthetic oligonucleotides encoding a portion of the determined amino-acid sequence, and probing a cosmid genome library with the mixed oligonucleotides. A cosmid hybridizing with the probe was isolated and the IPS gene was localized and sequenced. The IPS gene encodes a polypeptide of relative molecular mass (Mr) 38,416. When this open reading frame was cloned into an E. coli expression vector and inserted into E. coli, the recombinant E. coli produced a new protein co-migrating with authentic IPS as the major protein of the cell (approximately 20% of cell protein). Crude cell extracts condensed LLD-ACV to a penicillinase-sensitive molecule whose antibacterial activity indicated that it was isopenicillin N.

Acremonium↗
PubMedDate not supplied

Enzymatic synthesis of diastereospecific carbacephem intermediates using serine hydroxymethyltransferase.

The serine hydroxymethyltransferase (SHMT) gene glyA was over-expressed in Escherichia coli and the enzyme was purified to near homogeneity. Reaction conditions for E. coli and rabbit liver SHMTs were optimized using succinic semialdehyde methyl ester (SSAME) and glycine. The catalytic efficiency (kcat/K(m)) of E. coli SHMT for SSAME was 2.8-fold higher than that of rabbit liver enzyme. E. coli SHMT displayed a pH-dependent product distribution different from that of rabbit liver enzyme. For the pyridoxal-5'-phosphate (PLP)-dependent reaction, E. coli and rabbit liver SHMTs showed a high product diastereospecificity. The stoichiometric ratio of PLP to the dimeric E. coli SHMT was 0.5-0.7, indicating a requirement for external PLP for maximal activity. Using SSAME or its analog at a high temperature, E. coli SHMT mediated efficient condensation via a lactone pathway. In contrast, at a low temperature, the enzyme catalyzed efficient conversion of 4-penten-1-al via a non-lactone mechanism. Efficient conversion of either aldehyde type to a desirable diastereospecific product was observed at a pilot scale. E. coli SHMT exhibited a broad specificity toward aldehyde substrates; thus it can be broadly useful in chemo-enzymatic synthesis of a chiral intermediate in the manufacture of an important carbacephem antibiotic.

Animals↗