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

A putative α-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°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 ~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 α-L-arabinofuranoside but not with p-nitrophenyl α-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°C in a 10 min assay. When Tm-AFase was heated at 90°C, no loss of activity was observed for at least 24 h. At 100°C, the activity dropped to ~50% in 20 min; thereafter, inactivation occurred very slowly exhibiting a half-life of ~2.7 h, characterizing the enzyme to be the most thermophilic AFase reported thus far.

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AFase:

α-L-Arabinofuranosidase

DTT:

Dithiothreitol

GH-51:

Glycosyl hydrolase family 51

Gs-AFase:

α-L-Arabinofuranosidase from Geobacillus stearothermophilus

pNP:

p-nitrophenyl

Tm-AFase:

α-L-Arabinofuranosidase from Thermotoga maritima MSB8

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1. 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

   Kentaro Miyazaki

Correspondence to Kentaro Miyazaki.

Communicated by K. Horikoshi

Miyazaki, K. Hyperthermophilic α-L-arabinofuranosidase from Thermotoga maritima MSB8: molecular cloning, gene expression, and characterization of the recombinant protein. Extremophiles 9, 399–406 (2005). https://doi.org/10.1007/s00792-005-0455-2

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• Received: 06 February 2005

• Accepted: 08 April 2005

• Published: 18 June 2005

• Issue Date: October 2005

• DOI: https://doi.org/10.1007/s00792-005-0455-2

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