Purification and characterization of an α-l-arabinofuranosidase from Streptomyces lividans 66 and DNA sequence of the gene (abfA)

Abstract

The gene encoding an alpha-L-arabinofuranosidase (abfA) was homologously cloned in Streptomyces lividans and its DNA sequence was determined. The enzyme was purified from the cytoplasm of the hyperproducing clone S. lividans IAF116. Its M(r) was estimated by gel filtration and found to be approx. 380,000. Since SDS/PAGE indicated a native protein of M(r) 69,000, it can be concluded that the native protein consists of several subunits of that size. The pI value was 4.6. The kinetic constants determined with p-nitrophenyl alpha-L-arabinofuranoside as substrate were a Vmax of 180 units/mg of protein and a Km of 0.6 mM. The specific activity of the purified enzyme on this substrate was 153 units/mg of protein. Optimal enzyme activity was obtained at 60 degrees C and pH 6.0. The enzyme cleaved p-nitrophenyl alpha-L-arabinofuranoside, but had no activity on a variety of other p-nitrophenyl glycosides, except on p-nitrophenyl beta-D-xylopyranoside. The enzyme showed no activity on oat-spelts (Avena sativa) xylan or arabinogalactan, but acted on beet (Beta) arabinan or arabinoxylan. Hydrolysis occurred on arabino-oligoxylosides obtained from oat-splets xylan after digestion with xylanases. Since S. lividans normally does not secrete arabinofuranosidase, this enzyme may play a role in the assimilation of arabinose moieties from arabinose-containing xylo-oligosaccharides generated by beta-xylosidases or xylanases.

Pseudomonas cellulosa expresses a single membrane-bound glycoside hydrolase family 51 arabinofuranosidase

“…The location of Abf51A on the outer membrane of P. cellulosa is in contrast to arabinofuranosidases from other aerobic microorganisms, which are either in the cytoplasm [7,8] or in the culture supernatant [6]. It is believed that the primary target of intracellular and extracellular arabinofuranosidases are oligosaccharides and polysaccharides, respectively.…”

“…In addition to differences in substrate specificity, the cellular location of these enzymes will also influence their biological role. For example, arabinofuranosidases that are secreted into the culture media are likely to attack polysaccharides [6], while the targets for arabinofuranosidases that lack signal peptides and are retained within the cytoplasm [7,8] are presumably oligosaccharides.…”

Production and characterisation of recombinant α-l-arabinofuranosidase for production of xylan hydrogels

“…Consequently, the secreted AbfBs are often contaminated with endo-β-xylanase activities and therefore are not suitable for production of xylan hydrogels. Although the α-L-arabinofuranosidase B gene (abfB) of Aspergillus niger, which was first described by Tagawa and Kaji (1988), was cloned in various microbial production systems (Flipphi et al 1993;Manin et al 1994;Crous et al 1996;Gouka et al 1996;Van Den Hombergh et al 1997;Wang et al 2005), such systems failed to secrete the AbfBs free of endo-β-xylanase activities probably due to complexity of the secretion path. The AbfBs can be secreted selectively by specifically designed recombinant microbial enzyme systems and in specifically designed cultivation media.…”

Distinct Actions by Paenibacillus sp. Strain E18 α- l -Arabinofuranosidases and Xylanase in Xylan Degradation

“…␣-L-Arabinofuranosidase activity was assayed as described previously (19,20), the dinitrosalicylic acid (DNS) method (15,21) was used to assay xylanase activity, and ␤-xylosidase activity was determined as reported in reference22. Each reaction solution contained an appropriately diluted enzyme sample and 1 mM 4-nitrophenyl ␣-L-arabinofuranoside or p-nitrophenyl-␤-D-xylopyranoside or 1.0% (wt/vol) xylan and was incubated at 40°C (pH 6.0) for 10 min in 100 mM Na 2 HPO 4 -citric acid.…”