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KMID : 1094720110160010114
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Biotechnology and Bioprocess Engineering
2011 Volume.16 No. 1 p.114 ~ p.119
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Effects of various inhibitors on β-galactosidase purified from the thermoacidophilic Alicyclobacillus acidocaldarius subsp. Rittmannii isolated from Antarctica
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Guven Reyhan Gul
Kaplan Alevcan
Guven Kemal
Matpan Fatma
Dogru Mehmet
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Abstract
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β-Galactosidase purified from the thermoacidophilic Alicyclobacillus acidocaldarius subsp. rittmannii isolated from Antarctica is a member of the GH42 family. The enzyme was not effected by various concentrations of its reaction product glucose, but was greatly inhibited by the other reaction product galactose using both substrates, ONPG and lactose. Linewever-Burk plot analysis derived from both ONPG and lactose hydrolysis results showed that galactose is a mixed-type inhibitor of the purified β-galactosidase. The enzyme was slightly activated by Mg2+ (13% at 20 mM), while inhibited at higher concentrations of Ca+2 (33% at 10 mM), Zn+2 (86% at 8 mM) and Cu+2 (87% at 4 mM). The enzyme activity was not significantly altered by the metal ion chelators EDTA and 1,10-phenanthroline up to 20 mM, indicating that this enzyme is not a metalloenzyme. 2-Mercaptoethanol and DTT were found to enhance β-galactosidase activity, while p-chloromercuribenzoic acid (PCMB) completely inhibited enzymatic activity (97% at 1 mM; 99.7% at 2 mM), indicating at least one essential Cys residue modified by the reagents in the active site of β-galactosidase. Iodoacetamide and Nethylmaleimide had little effect on the β-galactosidase. Phenylmethylsulfonyl fluoride (PMSF) inhibited the enzyme strongly (19.8% at 1 mM; 71.9% at 10 mM), also showing the participation of serine for enzyme activity.
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KEYWORD
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β-Galactosidase, metal ion chelators, inhibition, galactose, PMCB and PMSF
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