Abstract
The gene encoding family 8 glycoside hydrolases from Bacillus halodurans C-125 (BH2105), an alkalophilic bacterium with a known genomic sequence, was expressed in Escherichia coli. The protein was expressed with the intact N-terminal sequence, suggesting that it did not possess a signal peptide and that it was an intracellular enzyme. The recombinant enzyme showed no hydrolytic activity on xylan, whereas it had been annotated as xylanase Y. It hydrolyzed xylooligosaccharide whose degree of polymerization is greater than or equal to 3 in an exo-splitting manner with anomeric inversion, releasing the xylose unit at the reducing end. Judging from its substrate specificity and reaction mechanism, we named the enzyme reducing end xylose-releasing exo-oligoxylanase (Rex). Rex was found to utilize only the beta-anomer of the substrate to form beta-xylose and alpha-xylooligosaccharide. The optimum pH of the enzymatic reaction (6.2-7.3) was found in the neutral range, a range beneficial for intracellular enzymes. The genomic sequence suggests that B. halodurans secretes two endoxylanases and possesses two alpha-arabinofuranosidases, one alpha-glucuronidase, and three beta-xylosidases intracellularly in addition to Rex. The extracellular enzymes supposedly hydrolyze xylan into arabino/glucurono-xylooligosaccharides that are then transported into the cells. Rex may play a role as a key enzyme in intracellular xylan metabolism in B. halodurans by cleaving xylooligosaccharides that were produced by the action of other intracellular enzymes from the arabino/glucurono-xylooligosaccharides.
Highlights
Endo--1,4-xylanases (EC 3.2.1.8) are glycoside hydrolases (GHs)1 that catalyze the degradation of xylan, a main component of hemicelluloses
releasing exo-oligoxylanase (Rex) may play a role as a key enzyme in intracellular xylan metabolism in B. halodurans by cleaving xylooligosaccharides that were produced by the action of other intracellular enzymes from the arabino/glucurono-xylooligosaccharides
The GH8 endo--1,4-xylanase from P. haloplanktis has the highest amino acid identity (32.6%) with the protein encoded by the BH2105 gene (GenBankTM accession number BAB05824) of Bacillus halodurans C-125 [18], which is annotated as “xylanase Y.”
Summary
Materials—The B. halodurans C-125 (9153) strain was obtained from the Japan Collection of Microorganisms (Wako, Japan). Purification of Recombinant BH2105—The cell-free extract was loaded onto a nickel-nitrilotriacetic acid-agarose (Qiagen) column (1 ϫ 3 cm), and the enzyme was eluted with a stepwise gradient of imidazole (1, 10 mM; 2, 20 mM; 3, 250 mM) in 50 mM sodium phosphate buffer (pH 8.0) containing 0.3 M NaCl. The fraction containing the BH2105 protein was desalted using a PD-10 column (Amersham Biosciences). The thermostability was determined by incubating the enzyme at each temperature for 30 min in 50 mM sodium phosphate buffer (pH 7.1) followed by measuring the activity under the standard conditions at 40 °C. After incubation for 1 and 25 min, an aliquot (10 l) of the reaction solution was immediately loaded onto a TSK-GEL Amide-80 column (4.6 ϫ 250 mm, Tosoh, Japan), and eluted with acetonitrile:water (7:3, v/v) at a flow rate of 1.5 ml/min at 25 °C, separating the xylooligosaccharides anomers. The mutated enzymes were prepared and purified as described above
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