Gene cloning of the maoA gene and overproduction of a soluble monoamine oxidase from Klebsiella aerogenes

Abstract

We cloned the structural gene for monoamine oxidase (maoA) from Klebsiella aerogenes into a pKI212 vector in an maoA mutant strain of K. aerogenes. Deletion analysis and complementation tests of the recombinant plasmid showed that the maoA gene was located entirely within a 4.1-kb segment. In an maoA mutant strain harbouring the cloned maoA gene, synthesis of monoamine oxidase was induced by addition of tyramine and related compounds. Transfer of a plasmid containing the maoA gene into a monoamine oxidase-producing strain of K. aerogenes W70 resulted in about a 30- to 40-fold increase in total production of the enzyme. When cells of K. aerogenes carrying the plasmid containing the maoA gene were grown with tyramine, more than 85% of the monoamine oxidase was produced in soluble form, whereas the parent strain W70 produced most monoamine oxidase as the membrane-bound form.

A monoamine-regulated Klebsiella aerogenes operon containing the monoamine oxidase structural gene (maoA) and the maoC gene

“…The effect of tyramine on the synthesis of monoamine oxidase in the mutant strain MKN63 (maoA) with or without plasmid pTO58, which contains the monoamine oxidase gene (28) (Fig. 1), was tested.…”

“…The bacterial strains used in this study are listed in Table 1. pKI212 (18), a plasmid conferring resistance to kanamycin (Kmr), and pTO58, a plasmid containing the maoA gene from K aerogenes W70, were constructed previously (28). pMC1403 (4), a plasmid containing the lacZ gene without the promoter region, was provided by A. Nakazawa (Yamaguchi University).…”

“…as described previously (21) or by a colorimetric assay coupled with peroxidase and o-phenylenediamine (28). One unit of monoamine oxidase activity was defined as the amount metabolizing 1 pumol of hydrogen peroxide per min at 30°C.…”

“…Recently, we cloned the gene for tyramine oxidase from Kiebsiella aerogenes into a multicopy plasmid; this cloning resulted in the high production of the soluble form of monoamine oxidase (28). This overproduction made possible the purification and characterization of monoamine oxidase.…”

Distribution of amine oxidases and amine dehydrogenases in bacteria grown on primary amines and characterization of the amine oxidase from Klebsiella oxytoca

“…With respect to the N-terminal region, this is not surprising since a stretch of about 100 amino acids is lacking in the N-terminal region in the amine oxidases of Gram-positive bacteria . Okamura et al (1976) and Sugino et al (1991) reported an amine oxidase in K . aerogenes ( K . pneumoniae) which was a membrane-integrated, hydrophobic enzyme, but they were unable to isolate it.…”