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Purification and properties of methanol dehydrogenase from Methylocystis sp. GB 25
Author(s) -
Grosse Stephan,
Wendlandt KarinDagmar,
Kleber HansPeter
Publication year - 1997
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/jobm.3620370406
Subject(s) - methanol dehydrogenase , methylotroph , chemistry , chromatography , molecular mass , formaldehyde dehydrogenase , isoelectric point , biochemistry , methanol , formate dehydrogenase , enzyme , size exclusion chromatography , formaldehyde , nad+ kinase , cofactor , organic chemistry
Methanol dehydrogenase (MDH) from Methylocystis sp. GB 25, which belongs to the group II of methanotrophic bacteria, is able to catalyse the oxidation of methanol to formate directly. The enzyme was purified 20‐fold by a 5 step procedure to electrophoretic homogeneity. After cell disruption by French press, about 95% of MDH‐activity was found in the soluble fraction. The relative molecular mass of the native enzyme has been estimated to be 122 kDa by gel filtration and 115 kDa by the method of H EDRICK and S MITH (1968). It seems to be composed of two identical subunits with a relative molecular mass of 62 kDa (estimated by SDS gel electrophoresis). The isoelectric point was found to be about 8.3. The amino terminal sequence shows a strong similarity to the α‐chain of MDH from the facultative methylotrophic bacterium Methylobacterium extorquens AM1. PQQ, the probable prosthetic group of MDH, could be detected in the supernatant of the culture by using the apoenzyme of a membrane‐bound glucose dehydrogenase from Pseudomonas aeruginosa but not absolutely in the absorption spectra of the enzyme after DEAE‐chromatography. The purified MDH has an optimum activity at pH 9.0 and at 45 °C. MDH of Methylocystis sp. GB 25 oxidises only primary alcohols from methanol to heptanol and aldehydes from formaldehyde to propionaldehyde and the glutaraldehyde, respectively. The estimated K m ‐values show no dependence upon the chain length of substrates.