Morpholine-induced formation of l-alanine dehydrogenase activity in Mycobacterium strain HE5 |
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Authors: | Grit Schuffenhauer Thomas Schräder J R Andreesen |
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Institution: | Institut für Mikrobiologie, Martin-Luther-Universit?t Halle, Kurt-Mothes-Stra?e 3, D-06099 Halle, Germany e-mail: j.andreesen@mikrobiologie.uni-halle.de, Tel.: +49-345-5526350, Fax: +49-345-5527010, DE
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Abstract: | 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 K
m values for l-alanine and NAD were 1.0 and 0.2 mM, respectively. K
m 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.
Received: 25 September 1998 / Accepted: 11 March 1999 |
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Keywords: | Alanine dehydrogenase Ammonia assimilation Mycobacterium Morpholine degradation |
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