Purification and preliminary characterization of alcohol dehydrogenase from Aspergillus nidulans

Abstract

Aspergillus alcohol dehydrogenase is produced in response to growth in the presence of a wide variety of inducers, of which the most effective are short-chain alcohols and ketones, e.g. butan-2-one and propan-2-ol. The enzyme can be readily extracted from fresh or freeze-dried cells and purified to homogeneity on Blue Sepharose in a single step by using specific elution with NAD+ and pyrazole. The pure enzyme has Mr 290 000 by electrophoresis or gel filtration; it is a homopolymer with subunit Mr 37 500 by electrophoresis in sodium dodecyl sulphate; its amino acid composition corresponds to Mr 37 900, and the native enzyme contains one zinc atom per subunit. The enzyme is NAD-specific and has a wide substrate activity in the forward and reverse reactions; its activity profile is not identical with those of other alcohol dehydrogenases.

Regulation of the Aldehyde Dehydrogenase Gene (aldA) and Its Role in the Control of the Coinducer Level Necessary for Induction of the Ethanol Utilization Pathway in Aspergillus nidulans

“…One possibility would be by reduction to ethanol, catalyzed by the alcA-encoded ADHI that is expressed pseudo-constitutively in aldA mutants. Acetaldehyde has been shown to be an excellent substrate for ADHI in vitro; reduction by ADHI is considerably faster than oxidation by ALDH (50). One has to note however that other mechanisms of detoxification may be operational, such as the presence of another ALDH activity that would not be sufficient by itself to support growth on ethanol.…”

Developmental repression of growth and gene expression in Aspergillus.

“…1). alcR-strains are unable to induce either alcA or aldA and cannot grow on ethanol (10,11). The mechanism of L-threonine use has not been elucidated for A. nidulans but probably proceeds by threonine aldolasemediated cleavage to yield glycine and the alcR coinducer acetaldehyde (10).…”

“…DH1 (11) and AldDH activity or the other pathway. Instead, metabolic alterations occurletermined at the indicated hour and are reported as change ring as a consequence of developmental induction must stop nce per min per mg of protein.…”

“…Alcohol dehydrogenase 1 (ADH1) and aldehyde dehydrogenase (AldDH) activities were determined as described (10)(11)(12), except that cell-free protein extracts were made by using lyophilized cells. Protein concentrations were determined by the Bradford procedure (13).…”

Either alpha-tubulin isogene product is sufficient for microtubule function during all stages of growth and differentiation in Aspergillus nidulans.

“…To upregulate expression of tubB, we used the previously characterized A. nidulans alcA(p), which is repressed in hyphae grown in the presence of glucose but is readily induced in the presence of various alcohols (13,53). The level of expression from alcA(p) can be regulated by the type of alcohol used as inducing agent (7,41). If the tubB a-tubulin isotype is capable of functioning in place of the tubA isotype, strains carrying the tubA4 allele and the tubB coding region under VOL.…”