Using morpholine as sole source of carbon, nitrogen and energy, strain HE5 (DSM 44238) was isolated from forest soil. The isolated strain was identified as a member of the subgroup of fast-growing Mycobacterium species as revealed by 16S rDNA analysis. An identity of 994 % was obtained to Mycobacterium gilvum ; however, the type strain was unable to utilize morpholine. A maximal growth rate of 017 h N1 was observed at a morpholine concentration of 30 mM, 30 SC and pH 72. The substrate was tolerated at concentrations up to 100 mM. Besides morpholine, the strain utilized pyrrolidine, piperidine and proposed intermediates in morpholine metabolism such as glycolate, glyoxylate and ethanolamine. Degradation of morpholine, piperidine and pyrrolidine by resting or permeabilized cells was strictly dependent on the presence of oxygen. Addition of the cytochrome-P450-specific inhibitor metyrapone to the growth medium resulted in a significantly decreased growth rate if these cyclic amines were used as a substrate. Carbon monoxide difference spectra of crude extracts from cells grown on these substrates compared to spectra obtained for extracts of succinate-grown cells indicated that cytochrome P450 is specifically expressed during growth on the cyclic amines. These data indicated that a cytochrome-P450-dependent monooxygenase is involved in the degradation of the three cyclic amines.
An NAD-dependent, morpholine-stimulated L-alanine dehydrogenase activity was detected in crude extracts from morpholine-, pyrrolidine-, and piperidine-grown cells of Mycobacterium strain HE5. Addition of morpholine to the assay mixture resulted in an up to 4. 6-fold increase of L-alanine dehydrogenase activity when L-alanine was supplied at suboptimal concentration. L-alanine dehydrogenase was purified to near homogeneity using a four-step purification procedure. The native enzyme had a molecular mass of 160 kDa and contained one type of subunit with a molecular mass of 41 kDa, indicating a tetrameric structure. The sequence of 30 N-terminal amino acids was determined and showed a similarity of up to 81% to that of various alanine dehydrogenases. The pH optimum for the oxidative deamination of L-alanine, the only amino acid converted by the enzyme, was determined to be pH 10.1, and apparent Km values for L-alanine and NAD were 1.0 and 0.2 mM, respectively. Km values of 0. 6, 0.02, and 72 mM for pyruvate, NADH, and NH4+, respectively, were estimated at pH 8.7 for the reductive amination reaction.
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