Hyperthermophilic alpha-L: -arabinofuranosidase from Thermotoga maritima MSB8: molecular cloning, gene expression, and characterization of the recombinant protein.
A putative alpha-L: -arabinofuranosidase (AFase) gene belonging to family 51 of glycosyl hydrolases of a hyperthermophilic bacterium Thermotoga maritima MSB8 was cloned, sequenced, and overexpressed in Escherichia coli. The recombinant protein (Tm-AFase) was purified to apparent homogeneity by heat treatment (80 degrees C, 30 min), followed by hydrophobic interaction, anion-exchange, and gel permeation column chromatography. Tm-AFase had a molecular mass of 55,284 Da on matrix assisted laser desorption ionization time-of-flight mass spectrometry and approximately 332 kDa on gel permeation column chromatography. Therefore, Tm-AFase comprised six identical subunits as in the case of homologous AFase from Geobacillus stearothermophilus. Regarding substrate specificity, Tm-AFase was active with p-nitrophenyl alpha-L: -arabinofuranoside but not with p-nitrophenyl alpha-L: -arabinopyranoside. Regarding polysaccharides, Tm-AFase hydrolyzed arabinan and debranched arabinan but not arabinoxylan, arabinogalactan, and carboxymethyl cellulose. Tm-AFase was extremely thermophilic, displaying an optimal reaction temperature of 90 degrees C in a 10 min assay. When Tm-AFase was heated at 90 degrees C, no loss of activity was observed for at least 24 h. At 100 degrees C, the activity dropped to approximately 50% in 20 min; thereafter, inactivation occurred very slowly exhibiting a half-life of approximately 2.7 h, characterizing the enzyme to be the most thermophilic AFase reported thus far.
Miyazaki
Kentaro
K
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. miyazaki-kentaro@aist.go.jp
D000595:Amino Acid Sequence; D001483:Base Sequence; D002413:Cations, Divalent / Q000737:chemistry; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression / Q000235:genetics*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism; D019216:Metals, Heavy / Q000737:chemistry / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013696:Temperature; D020124:Thermotoga maritima / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*
D002413:Cations, Divalent; D006027:Glycosides; D019216:Metals, Heavy; D011134:Polysaccharides; D011994:Recombinant Proteins; D018698:Glutamic Acid; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase