Physiology of Amidase Production by Methylophilus methylotrophus: Isolation of Hyperactive Strains Using Continuous Culture

Abstract

~~~~ ~~The obligately methylotrophic bacterium Methylophilus methylotrophus hydrolyses aliphatic amides to ammonia and aliphatic acid using a cytoplasmic amidase. Physiological regulation of amidase activity was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. The results showed that synthesis of the enzyme was induced by various amides (acrylamide > acetamide) and repressed by ammonia. Growth of the wildtype organism in acetamide-limited continuous culture at very low dilution rate (D 0.025 h-l) led to the selection of a hyperactive strain (MM6), the subsequent growth of which under acrylamide limitation led to the selection of another strain (MM8) which showed even higher activity. The amidase activities of strains MM6 and MM8 were respectively approximately four and twelve times higher than that of the wild-type organism following growth under similar conditions, whereas the concentrations of the enzyme as determined by SDS-PAGE and scanning densitometry were approximately four times higher than the wild-type organism in both strains. The amidase in strain MM8 exhibited a K , for acrylamide that was approximately one-third lower than that of the wild-type organism or of strain MM6. It is concluded that the hyperactivity of strain MM6 was due predominantly to the production of more wild-type enzyme, whilst the hyperactivity of strain MM8 was due to the production of approximately the same amount of enzyme as strain MM6 (up to 25% of the total cell protein depending on the nature of the limiting amide) but with a substantially enhanced catalytic activity (Kcat). These changes were apparently the result of spontaneous mutations that occurred in response to growth at extremely low amide concentrations, giving the novel strains a strong selective advantage under these conditions (possibly by enhancing the rate of diffusion of amide into the cell).

Crystallization and preliminary X-ray analysis of a γ-lactamase

“…smegmatis acetamidase. This is in agreement with the observed amidase activity of the puri®ed protein, which was found to be 0.07 mM NH 3 per minute per microgram of enzyme (Silman et al, 1989). The (+)-lactamase was inhibited by PMSF and BAM, which suggests the presence of a serine residue in Figure 1 Triclinic crystals of (+)-lactamase.…”

“…Amidase activity was determined colorimetrically by measuring the rate of ammonia formation at pH 6.0 with 50 mM acetamide as the substrate at 310 K (Silman et al, 1989). The absorbance was measured at 630 nm and the amount of ammonia released was determined from a standard curve with ammonium chloride.…”

Identification and characterization of an aliphatic amidase in Helicobacter pylori

“…A very similar enzymatic assay has been described previously for amidase activity determination (Silman et al, 1989). Amidase activity was measured as the release of ammonia after cleavage from its amide substrate.…”

“…This might reflect derepression of amiE expression or AmiE activity in the absence of urease, possibly because of a low intracellular ammonia concentration. Synthesis of the M. methylotrophus aliphatic amidase has been shown to be repressed by ammonia (Silman et al, 1989). Alternatively, the amidase may also contribute to the carbon source supply to H. pylori.…”

Purification and properties of extracellular lipase from Pseudomonas aeruginosa EF2

“…Discontinuous SDS-PAGE and determination of protein using the Bradford method was carried out as described previously (Silman et al, 1989;Gilbert et al, 1991). The gels were stained for protein with Kenacid blue R, then destained and, where appropriate, scanned at 633 nm using an LKB laser densitometer linked to a recording integrator.…”