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The thermostable α‐ l ‐rhamnosidase RamA of Clostridium stercorarium : biochemical characterization and primary structure of a bacterial α‐ l ‐rhamnoside hydrolase, a new type of inverting glycoside hydrolase
Author(s) -
Zverlov Vladimir V.,
Hertel Christian,
Bronnenmeier Karin,
Hroch Angelika,
Kellermann J.,
Schwarz Wolfgang H.
Publication year - 2000
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2000.01691.x
Subject(s) - glycoside hydrolase , biochemistry , biology , hydrolase , escherichia coli , hydrolysis , glycosyl , molecular mass , cellulase , microbiology and biotechnology , enzyme , gene
An α‐ l ‐rhamnosidase clone was isolated from a genomic library of the thermophilic anaerobic bacterium Clostridium stercorarium and its primary structure was determined. The recombinant gene product, RamA, was expressed in Escherichia coli , purified to homogeneity and characterized. It is a dimer of two identical subunits with a monomeric molecular mass of 95 kDa in SDS polyacrylamide gel electrophoresis. At pH 7.5 it is optimally active at 60°C and insensitive to moderate concentrations of Triton X100, ethanol and EDTA. It hydrolysed p ‐nitrophenyl‐α‐ l ‐rhamnopyranoside, naringin and hesperidin with a specific activity of 82, 1.5 and 0.46 U mg −1 respectively. Hydrolysis occurs by inversion of the anomeric configuration as detected using 1 H‐NMR, indicating a single displacement mechanism. Naringin was hydrolysed to rhamnose and prunin, which could further be degraded by incubation with a thermostable β‐glucosidase. The secondary structure of RamA consists of 27% α‐helices and 50% β‐sheets, as detected by circular dichroism. The primary structure of the ramA gene has no similarity to other glycoside hydrolase sequences and possibly is the first member of a new enzyme family.