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AMB Express. 2012; 2: 32.
Published online Jun 25, 2012. doi:  10.1186/2191-0855-2-32
PMCID: PMC3439253
Purification and properties of S-hydroxymethylglutathione dehydrogenase of Paecilomyces variotii no. 5, a formaldehyde-degrading fungus
Ryohei Fukuda,1 Kazuhiro Nagahama,1 Kohsai Fukuda,1 Keisuke Ekino,1 Takuji Oka,1 and Yoshiyuki Nomuracorresponding author1
1Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Ikeda 4-22-1, Nishi-ku, Kumamoto City, 860-0082, Japan
corresponding authorCorresponding author.
Ryohei Fukuda: o199786/at/starzen.jp; Kazuhiro Nagahama: kazuhiro/at/bio.sojo-u.ac.jp; Kohsai Fukuda: kohsai/at/bio.sojo-u.ac.jp; Keisuke Ekino: ekino/at/bio.sojo-u.ac.jp; Takuji Oka: oka/at/bio.sojo-u.ac.jp; Yoshiyuki Nomura: nomura/at/bio.sojo-u.ac.jp
Received May 22, 2012; Accepted June 9, 2012.
Abstract
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.
Keywords: S-hydroxymethylglutathione dehydrogenase, Enzyme purification, Formaldehyde metabolism, Paecilomyces variotii, SH enzyme
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