Characterization of a Family GH5 Xylanase with Activity on Neutral Oligosaccharides and Evaluation as a Pulp Bleaching Aid

A new bacterial xylanase belonging to family 5 of glycosyl hydrolases was identified and characterized. The xylanase, Xyn5B from Bacillus sp. strain BP-7, was active on neutral, nonsubstituted xylooligosaccharides, showing a clear difference from other GH5 xylanases characterized to date that show a requirement for methyl-glucuronic acid side chains for catalysis. The enzyme was evaluated on Eucalyptus kraft pulp, showing its effectiveness as a bleaching aid.The catabolic breakdown of xylan is a critical step in the recycling of carbon in nature and has been targeted as a subject of intense research as a renewable energy resource as well as for bioconversion of plant biomass into high-added-value products (21, 29, 37, 40). Biodegradation of xylan is a complex process that requires the coordinate action of several enzymes, among which xylanases (1,4-β-D-xylan xylanohydrolase; EC 3.2.1.8), cleaving internal linkages on the β-1,4-xylose backbone, play a key role.Most known xylanases are grouped into glycoside hydrolase (GH) families 10 and 11 (CAZy Carbohydrate-Active enZYmes] database) (17), although a few xylanases have recently been ascribed to glycoside hydrolase families 5, 7, 8, and 43 (8, 9, 24). Among xylanases not grouped in the typical families GH10 and GH11, only two xylanases belonging to family GH5 have been biochemically characterized in detail. The enzymes XynA from Erwinia chrysanthemi (18, 39) and XynC from Bacillus subtilis (32) hydrolyze glucuronoxylan to branched xylooligosaccharides. The activity of Erwinia chrysanthemi XynA has also been evaluated on other substrates containing xylose, showing an absolute requirement for methyl-glucuronic substitutions. In this way, only methyl-glucuronic acid-branched oligosaccharides can be cleaved by XynA, whereas linear xylooligosaccharides or arabinoxylans are not cleaved by this enzyme (39). This type of xylanases must play an important role in complementing the action of GH10 and GH11 enzymes during depolymerization of glucuronoxylans in lignocellulosic fibers.Xylanases are widely used in the pulp industry to enhance the effectiveness of bleaching agents, thereby reducing the generation of toxic wastes (adsorbable organic halogens; AOX) (1, 38). Several reports have evaluated the effectiveness of family GH10 and GH11 xylanases on the bleaching process, showing that GH11 xylanases usually display better performance (7, 12), although there are many other factors that contribute to the bleach-boosting effect of a xylanase, such as the source of the pulp and the pulping process itself (6, 11). Besides their contribution to the increase in brightness, an innovative aspect of the application of xylanases is their contribution to the removal of hexenuronic acids (HexA) produced during the kraft cooking process, which can accelerate the brightness reversion (yellowing tendency) of paper (35). However, it remains to be known if all xylanases are capable of removing HexAs and/or enhancing bleachability.Bacillus sp. strain BP-7 is a xylanolytic strain isolated from agricultural soils (25). It shows a multiple enzymatic system for xylan degradation, including a GH11 xylanase cloned and characterized previously (13). In this work, we describe the identification and cloning of a second xylanase from the strain, belonging to the GH5 family. The enzyme hydrolyzes linear xylooligosaccharides, clearly differing from the two GH5 xylanases characterized up to date. The new enzyme has been tested on Eucalyptus pulps, showing good performance as a bleaching aid. The results obtained suggest an important role for the enzyme in xylan degradation and indicate the potential of this xylanase for biotechnological applications in the bioconversion of glucuronoxylan-containing biomass.