Date Published: June 25, 2012
Author(s): Ryohei Fukuda, Kazuhiro Nagahama, Kohsai Fukuda, Keisuke Ekino, Takuji Oka, Yoshiyuki Nomura.
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.
Formaldehyde is a ubiquitous compound that is a product of biological sources (from photooxidation of atmospheric hydrocarbons) ([Levy 1971]; [Zimmerman et al. 1978]) and environmental sources (emissions from industrial processes) ([Ando 1998]). An advanced technology for potable water pretreatment includes ozonation, during which formaldehyde is generated as a result of the reaction of ozone with traces of humus ([Schechter and Singer 1995]). Formaldehyde acts as disinfectant at concentrations as low as 0.1%. Therefore, it is used for room sterilization, viscosity stabilization and preservation of adhesives made from starch and for preservation of experimental specimens. Formaldehyde is a highly toxic compound due to nonspecific reactivity with proteins and nucleic acids ([Grafstrom et al. 1983]), so it is an environmental pollutant.
We isolated a fungus that can degrade a concentration of formaldehyde as high as 2.4%. Based on the DNA sequence of 18S the ribosomal RNA gene of this fungus, we named it P. variotii NBRC 109023. To investigate the mechanism of degradation of high concentrations of formaldehyde by P. variotii NBRC 109023, we attempted to purify S-HMGSH dehydrogenase, a key enzyme of detoxification in eukaryotic organisms. S-HMGSH dehydrogenase was purified from a cell-free extract of P. variotii NBRC 109023 by ammonium sulfate precipitation, DEAE-Sepharose chromatography, hydroxyapatite chromatography and isoelectrofocusing. The purity of the purified enzyme preparation was checked by SDS-PAGE. The purified enzyme gave a single band after electrophoresis. The molecular weight of the purified enzyme was estimated to be approximately 49 kDa by SDS-PAGE and chromatography on TSK-gel G2000SW, suggesting that it is a monomer with an isoelectric point of 5.8.
The authors declare that they have no competing interests.