Physiology and biochemistry of amidase production by Methylophilus methylotrophus.

The physiological regulation of amidase production by Methylophilus methylotrophus was studied following growth of the organism under various conditions of nutrient sufficiency and limitation in batch, fed-batch and continuous cultures. Amidase was severely repressed by ammonia, and was induced by short chain aliphatic amides. Growth of the wild-type organism in acetamide-1imited continuous culture led to the selection of a hyperactive strain , the subsequent growth of which under acrylamide limitation led to the production of another strain (MM8) which exhibited even higher activities. Chemical mutagenesis of strain MM8 followed by growth in acrylamlde-limited continuous culture led to the selection of a strain (MM15) which continued to overexpress amidase, but which exhibited a low amidase activity and a greatly decreased Km for acrylamide. Determination of the concentrations of the enzyme revealed that amidase comprised approximately 20-25% of the total cell protein in each case, and these values were confirmed following purification of the enzyme from each of the different strains. The hyperactivity of strain MM6 was due predominantly to the production of more wild-type enzyme, whilst in strain MM8 it was due to production of approximately the same amount of enzyme, but with a substantially enhanced Kcat. The low amidase activity of strain MM15 was due to its increased thermolability since approximately four-fifths of the enzyme was present as inactive monomers and dimers. Growth of strain MM15 at lower temperatures was found to have a profound effect upon the subsequent Kcat of the enzyme. The conditions under which switch-off occurs have been investigated, and amidase was purified in its switched-off form and compared for the presence of modifying groups in parallel with purified active amidase. A protein involved in the reactivation of inactive amidase was isolated and purified, and the physico-chemical properties of both inactive amidase and the activation protein have been investigated.