Purification and Properties of ArfI, an α- l -Arabinofuranosidase from Cytophaga xylanolytica

Abstract

An α-l-arabinofuranosidase (α-l-arabinofuranoside arabinofuranohydrolase [EC3.2.1.55 ]; referred to below as ArfI) from Cytophaga xylanolytica XM3 was purified 85-fold by anion-exchange and hydrophobic interaction column chromatography. The native enzyme had a pI of 6.1 and an apparent molecular mass of 160 to 210 kDa, and it appeared to be a trimer or tetramer consisting of 56-kDa subunits. Withp-nitrophenyl-α-l-arabinofuranoside as the substrate, the enzyme exhibited a Km of 0.504 mM and a V max of 319 μmol · min−1 · mg of protein−1, and it had optimum activity at pH 5.8 and 45°C. ArfI was relatively stable over a pH range of 4 to 10 and at temperatures up to 45°C, and it retained nearly full activity when stored at 4°C for periods as long as 24 months. The enzyme also released arabinose from 4-methylumbelliferyl-α-l-arabinofuranoside, as well as from rye, wheat, corn cob, and oat spelt arabinoxylans and sugar beet arabinan, but not from arabinogalactan. ArfI showed no hydrolytic activity toward a range of p-nitrophenyl- or 4-methylumbelliferyl-glycosides other than arabinoside, for which it was entirely specific for the α-l-furanoside configuration. ArfI interacted synergistically with three partially purified endoxylanase fractions from C. xylanolytica in hydrolyzing rye arabinoxylan. However, cell fractionation studies revealed that ArfI was largely, if not entirely, cytoplasmic, so its activity in vivo is probably most relevant to hydrolysis of arabinose-containing oligosaccharides small enough to pass through the cytoplasmic membrane. Antibodies prepared against purified ArfI also cross-reacted with arabinofuranosidases from other freshwater and marine strains of C. xylanolytica, as well as with some proteins that did not possess arabinofuranosidase activity. To our knowledge, this is the first α-l-arabinofuranosidase to be purified and characterized from any gliding bacterium.

Production of L‐Arabinose from Corn Hull Arabinoxylan by Arthrobacter aurescens MK5 α‐L‐Arabinofuranosidase

“…The enzyme from A. aurescens strain MK5 corresponds to this 3rd group of enzymes because it showed little α‐L‐arabinofuranosidase activity to p ‐nitrophenyl α‐L‐arabinofuranoside (see Table 1). Although many α‐L‐arabinofuranosidases show synergism with β‐xylanase during hydrolysis of arabinoxylan (Kormelink and others 1991; Schyn and others 1994; Renner and Breznak 1998), this was not observed with the MK5 enzyme in experiments using β‐xylanase contained within commercial cellulase, Meicelase (of Trichoderma viride origin), or β‐xylanase from A. awamori (data not shown).…”

“…With respect to their activity against arabinoxylan, α‐L‐arabinofuranosidases can be further classified into 3 types. (1) The α‐L‐arabinofuranosidases from Cytophaga xylanolytica , Trichoderma reesei , and Streptomyces daiastaticus have broad substrate specificity and can attack p ‐nitrophenyl α‐L‐arabinofuranoside, arabinoxylooligosaccharide, arabinoxylan, or arabinan (Tajana and others 1992; Kaneko and others 1998; Renner and Breznak 1998). (2) The A. niger 5–16, B. subtilis 3–6, and Bacteroides xylanolyticus X5–1 enzymes attack p ‐nitrophenyl α‐L‐arabinofuranoside or arabinoxylooligosaccharide, but not arabinoxylan (Kaneko and others 1993, 1994; Schyn and others 1994).…”

Research and Application of Marine Microbial Enzymes: Status and Prospects

“…Sarwar et al , using carrageenan containing medium, cultured cytophaga lk-C783, and obtained extracellular κ-carrageenase with a molecular weight of 10 kD [ 69 ]. In 2004, Mou et al isolated an extracellular κ-carrageenase with a molecular weight of 30 kD from marine Cytophaga MCA-2 [ 71 ]. A distinct λ-Carrageenan-degrading Pseudoalteromonas bacterium (CL19)was isolated from a deep -sea sediment sample in 2006 by Yukari and Yuji; the molecular mass of this purified enzyme was approximately 100 kD[ 72 ].…”