title: Cloning, expression, and characterization of a thermostable GH51 α-L-arabinofuranosidase from Paenibacillus sp. DG-22.
abstract: The gene encoding α-L-arabinofuranosidase (AFase) from Paenibacillus sp. DG-22 was cloned, sequenced, and expressed in Escherichia coli. The AFase gene (abfA) comprises a 1,509 bp open reading frame encoding 502 amino acids with a molecular mass of 56,520 daltons. The deduced amino acid sequence of the gene shows that AbfA is an enzyme consisting of only a catalytic domain, and that the enzyme has significant similarity to AFases classified into the family 51 of the glycosyl hydrolases. abfA was subcloned into the pQE60 expression vector to fuse it with a six-histidine tag and the recombinant AFase (rAbfA) was purified to homogeneity. The specific activity of the recombinant enzyme was 96.7 U/mg protein. Determination of the apparent molecular mass by gel-filtration chromatography indicated that AbfA has a tetrameric structure. The optimal pH and temperature of the enzyme were 6.0 and 60°C, respectively. The enzyme activity was completely inhibited by 1 mM HgCl2. rAbfA was active only towards p-nitrophephenyl α-L-arabinofuranoside and exhibited Km and Vmax values of 3.5 mM and 306.1 U/mg, respectively. rAbfA showed a synergistic effect in combination with endoxylanase on the degradation of oat spelt xylan and wheat arabinoxylan.
authors.affiliation: Department of Biotechnology, Dongguk University, Gyeongju 780-714, Republic of Korea.
authors.forename: Sun Hwa
authors.initials: SH
authors.lastname: Lee
authors.forename: Yong-Eok
authors.initials: YE
authors.lastname: Lee
mesh_terms: D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008627:Mercuric Chloride / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D008627:Mercuric Chloride; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase