Summary: The obligately methylotrophic bacterium methylotrophus hydrolyses acetamide and acrylamide using a cytoplasmic amidase. In previous work, continuous culture was used to isolate spontaneous mutants which overexpressed either the wild-type amidase (strain MM6) or a mutant amidase with an apparently higher (strain MM8). We now report that NTG mutagenesis of strain MM8 followed by acrylamide-limited growth at low dilution rate ( 0·025 h; 37 °C) led to the selection of a strain which continued to overexpress the amidase, but which exhibited an unexpectedly low amidase activity and a greatly decreased for acrylamide (strain MM15). Amidases from the wild-type and mutant strains were purified and shown to be homotetramers (subunit 38000, pI 4·1). The N-terminal amino acid sequence of the wild-type enzyme was 90% homologous with the aliphatic amidase from , and Southern blotting using an oligonucleotide probe for this region showed that overexpression of the enzyme in the mutant strains was not due to gene amplification. Compared with the wild-type and MM6 enzymes, the MM8 enzyme exhibited a threefold higher and a slightly lower for acrylamide, whereas the MM15 enzyme exhibited a similar and an eightfold lower for acrylamide. The MM15 enzyme also reacted more extensively with the thiol group reagent DTNB, had a significantly lower sedimentation coefficient and exhibited a more relaxed substrate specificity, all of which were compatible with a looser tetrameric structure. It was also much more susceptible than the other three enzymes to inactivation by high temperature or by freezing and thawing (MM15“MM8>MM6/wild-type), both of which variably dissociated the enzyme into inactive dimers and monomers. The amidase activity of strain MM15 was almost 15-fold higher following growth at 25 °C than at 37 °C, since at this lower temperature the enzyme exhibited a similar to the MM8 enzyme and was not significantly dissociated. However, as strain MM15 readily outgrew the organism from which it was derived (strain MM8) during acrylamide-limited continuous culture at 37 °C, it is clear that under these conditions a low was a greater selective advantage than a high .


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