Hyperthermophilic alpha-L-arabinofuranosidase from Thermotoga maritima MSB8: molecular cloning, gene expression, and characterization of the recombinant protein

摘要

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 similar to 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 similar to 50% in 20 min; thereafter, inactivation occurred very slowly exhibiting a half-life of similar to 2.7 h, characterizing the enzyme to be the most thermophilic AFase reported thus far.