Purification and Characterization of Thermostable Endo-1,5-α- l -Arabinase from a Strain of Bacillus thermodenitrificans

Abstract

A strain of a thermophilic bacterium, tentatively designated Bacillus thermodenitrificans TS-3, with arabinandegrading activity was isolated. It produced an endo-arabinase (ABN) (EC 3.2.1.99) and two arabinofuranosidases (EC 3.2.1.55) extracellularly when grown at 60°C on a medium containing sugar beet arabinan. The ABN (tentatively called an ABN-TS) was purified 7,417-fold by anion-exchange, hydrophobic, size exclusion, and hydroxyapatite chromatographies. The molecular mass of ABN-TS was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was pH 4.5. The enzyme was observed to be more thermostable than known ABNs; it had a half-life of 4 h at 75°C. The enzyme had optimal activity at 70°C and pH 6.0. The enzyme had apparent K m values of 8.5 and 45 mg/ml and apparent V max values of 1.6 and 1.1 mmol/min/mg of protein against debranched arabinan (␣-1,5-arabinan) and arabinan, respectively. The enzyme had no pectin-releasing activity (protopectinase activity) from sugar beet protopectin, differing from an ABN (protopectinase-C) from mesophilic Bacillus subtilis IFO 3134. The pattern of degradation of debranched arabinan by ABN-TS indicated that the enzyme was an endo-acting enzyme and the main end products were arabinobiose and arabinose. The results of preliminary experiments indicated that the culture filtrate of strain TS-3 is suitable for L-arabinose production from sugar beet pulp at high temperature.Hemicelluloses compose a large fraction of plant cell walls and are a heterogeneous mixture of polysaccharides that include xylans, glucans, mannans, galactans, and arabinans. Arabinans consists of a backbone of ␣-1,5-linked L-arabinofuranosyl residues, some of which are substituted with ␣-1,2-and ␣-1,3-linked side chains of L-arabinose in the furanose conformation (1). There is some evidence to suggest that in plant cell walls arabinan is generally linked to the rhamnopyranosyl units of rhamnogalacturonan (13,14). Sakai and Sakamoto described a protopectinase (protopectinase-C [PPase-C]) from Bacillus subtilis IFO 3134 that did not catalyze polygalacturonic acid degradation (11). By endo-1,5-␣-L-arabinase (ABN) activity, PPase-C splits the ␣-1,5-L-arabinofuranoside linkage of the arabinan region in arabinogalactan, which attaches pectin to the cell wall constituents, so that PPase-C releases pectin (12). In addition to endo-ABN, ␣-L-arabinofuranosidase (␣-L-ABF) also is involved in degradation of arabinan. The enzyme cleaves the arabinose side chains, allowing endo-ABN to attack the arabinan backbone. These enzymes act synergistically in degrading branched arabinan to generate L-arabinose (21).It has been proved that L-arabinose selectively inhibits intestinal sucrase in a noncompetitive manner and reduces the glycemic response after sucrose ingestion in animals (16, 17). Based on these observations, L-arabinose can be used as a physiologically functional sugar that inhibits sucrose digestion. Effective L-arabinose production is, therefore, important in t...

Purification and Functional Characterization of a Novel α- l -Arabinofuranosidase from Bifidobacterium longum B667

“…The multimeric quaternary structures of several arabinofuranosidases have been reported previously (9,32). The optimal temperature for AbfB activity (45°C) was higher than the optimal temperatures for the arabinosidases AXH-d3 (30°C) and AXH-m23 (37°C) from Bifidobacterium adolescentis (43,44) but lower than the optimal temperatures for most arabinofuranosidases from other bacteria and fungi (8,9,11,16,31,39). The optimal pH of AbfB (pH 6.0) fell in the same range as the optimal pHs found for AXH-d3 and AXH-m2 (43,44).…”

“…This suggests that B. longum is effectively involved in polysaccharide assimilation in this environment. The activity of AbfB was completely inhibited by Hg 2ϩ and Cu 2ϩ and was partially inhibited by Zn 2ϩ ; these are known inhibitors of other enzymes of this family (8,14,17,39,47). Hg 2ϩ is known to react with sulfhydryl groups of proteins, as well as with histidine and tryptophan residues.…”

“…Several arabinosidases from different microorganisms, including yeast (47), fungi (11,13,16,31,46), and bacteria (8,9,14,17,21,24,34,39,43,44), have been documented and characterized to date. Among the bifidobacteria, two different arabinosidases from B. adolescentis, AXH-d3 and AXH-m23, have been partially characterized (43,44).…”

“…These enzymes are part of a set of glycosidases required for complete degradation of polymeric substrates such as arabinan, arabinoxylan, and other polysaccharides, which are major components of plant cell walls (32,39). Such hydrolases are endo-and exoenzymes that cleave the glycosidic linkages of the polymeric backbone and the various side chains, providing many microorganisms, both prokaryotic and eukaryotic, with soluble saccharides that can be used as carbon or energy sources (29,32).…”

Characterization of abn2 ( yxiA ), Encoding a Bacillus subtilis GH43 Arabinanase, Abn2, and Its Role in Arabino-Polysaccharide Degradation

“…Substrate specificity analysis indicated that Abn2 is an endo-␣-1,5-L-arabinanase, active toward linear ␣-1,5-L-arabinan, sugar beet arabinan, and pectin from apple (Table 4). The biochemical properties of Abn2 resemble those of AbnA and other purified endo-␣-1,5-L-arabinanases from other bacteria and fungi (19,27,38; reviewed in reference 2). The temperature for Abn2 maximum activity (50°C) was lower than that for AbnA (60°C); however, Abn2 appeared to be a more thermostable enzyme at its optimal temperature and in the absence of substrate than AbnA was (19).…”