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Purification and properties of S-hydroxymethylglutathione dehydrogenase of Paecilomyces variotii no. 5, a formaldehyde-degrading fungus
S-hydroxymethylglutathione dehydrogenase from Paecilomyces variotii No. 5 strain (NBRC 109023), isolated as a formaldehyde-degrading fungus, was purified by a procedure that included ammonium sulfate precipitation, DEAE-Sepharose and hydroxyapatite chromatography and isoelectrofocusing. Approximately 122-fold purification was achieved with a yield of 10.5%. The enzyme preparation was homogeneous as judged by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the purified enzyme was estimated to be 49 kDa by SDS-PAGE and gel filtration, suggesting that it is a monomer. Enzyme activity was optimal at pH 8.0 and was stable in the range of pH 7.0–10. The optimum temperature for activity was 40°C and the enzyme was stable up to 40°C. The isoelectric point was pH 5.8. Substrate specificity was very high for formaldehyde. Besides formaldehyde, the only aldehyde or alcohol tested that served as a substrate was pyruvaldehyde. Enzyme activity was enhanced by several divalent cations such as Mn2+ (179%), Ba2+ (132%), and Ca2+ (112%) but was completely inhibited by Ni2+, Fe3+, Hg2+, p-chloromercuribenzoate (PCMB) and cuprizone. Inactivation of the enzyme by sulfhydryl reagents (Hg2+ and PCMB) indicated that the sulfhydryl group of the enzyme is essential for catalytic activity.
S-hydroxymethylglutathione dehydrogenase; Enzyme purification; Formaldehyde metabolism; Paecilomyces variotii; SH enzyme
Establishment of a Pure Culture of the Hitherto Uncultured Unicellular Cyanobacterium Aphanothece sacrum, and Phylogenetic Position of the Organism
Applied and Environmental Microbiology
Aphanothece sacrum, an edible freshwater unicellular cyanobacterium, was isolated by using novel synthetic media (designated AST and AST-5xNP). The media were designed on the basis of the ratio of inorganic elements contained in A. sacrum cells cultured in a natural pond. The isolated strain exhibits unicellular rod-shaped cells ∼6 μm in length that are scattered in an exopolysaccharide matrix, a feature similar to that of natural A. sacrum. DNA analysis of the isolated strain revealed that it carried two ferredoxin genes whose deduced amino acid sequences were almost identical to previously published sequences of ferredoxins from natural A. sacrum. Analysis of the 16S rRNA gene and ferredoxin genes revealed that A. sacrum occupies a phylogenetically unique position among the cyanobacteria.
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