A novel β-xylosidase from Anoxybacillus sp. 3M towards an improved agro-industrial residues saccharification

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Abstract

An intracellular β-xylosidase (AbXyl), from the thermoalkaline Anoxybacillus sp. 3M, was purified and characterized. The homodimeric enzyme (140 kDa) was optimally active at 65 °C and pH 5.5, exhibited half life of 10 h at 60 °C, 78 and 88% residual activity after 24 h, at pH 4.5 and 8.0, respectively. Fe2+, Cu2+, Al3+, Ag+ and Hg2+ inhibited the enzyme; the activity was moderately stimulated by SDS and not influenced by β-mercaptoethanol. In the presence of p-nitrophenyl-β-d-xylopyranoside, AbXyl exhibited Km of 0.19 mM, Kcat of 453.29 s−1, Kcat Km−1 of 2322 s−1 mM and was moderately influenced by xylose (Ki 21.25 mM). The enzyme hydrolyzed xylo-oligomers into xylose and catalyzed transxylosilation reactions also in presence of alcohols as acceptors, producing xylo-oligosaccharides and alkyl-xylosides. Finally AbXyl was applied towards a statistically optimized process of brewery's spent grain bioconversion, highlighting the important role of this biocatalyst in reaching high yields of fermentable sugars.

Introduction

Xylan is, after cellulose, the most abundant renewable carbon source present in wood and agricultural residues. Xylans consist of β-(1,4)-linked d-xylopyranose residues substituted with α-l-arabinofuranose, 4-O-d-methyl-glucuronic acids and acetyl groups [1]. In nature the complete hydrolysis of xylan occurs by the synergistic action of several enzymes, among which the endo-β-1,4-xylanases and 1,4-β-xylosidases play the major role. The xylan deconstruction starts with the action of debranching enzymes such as α-l-arabinofuranosidases, α-d-glucuronidases and acetyl xylan esterases which remove the side chains that hinder the xylanases attack onto the polysaccharide. The internal β-1,4-xylosidic linkages in the xylan backbone are cleaved by the endo-β-1,4-xylanases, yielding soluble xylo-oligosaccharides which are hydrolyzed to d-xylose by 1,4-β-xylosidases that proceed from the non-reducing ends [2,3]. These biocatalysts are also necessary to reduce the end product inhibition that usually affects the endo-β-1,4-xylanases and for this reason are rate-limiting in xylan degradation [4]. Currently, growing interest in xylan bioconversion has arisen due its potential applications in several agro-industrial processes. These include the conversion of hemicellulosic materials for second generation biofuel production in which the enzymatic depolimerization of xylan represents an eco-friendly alternative to the physicochemical extraction of lignin and hemicellulose [5]. Moreover the recovery of xylose in a free form from lignocellulosic wastes as substrates is significant for the overall efficiency of bio-ethanol production process [6]. Hemicellulases have also many other applications on industrial scale. A complete biodegradation of xylans is one of the goals for the paper industry [7] and the exploitation of xylanolytic activities is also relevant in feed industries to hydrolyze the hemicelluloses in cereals for enhancing the availability of nutrients and promoting their absorption [2]. Xylanases are employed also in food sectors to improve bread dough quality [8], to promote the release of aroma during wine making process [9] and simultaneous recovery of free D-xylose [10]. Another industrial application of particular interest for β-xylosidases is the synthesis of xylo-oligosaccharides, valuable compounds with prebiotic and drug functions [11]. New xylosidic linkages can be catalyzed by retaining-xylosidases in transxylosilation reactions and the alkyl-xylosides, that can be obtained by this process, are non-ionic surfactants endowed with relevant properties to be utilized in pharmaceutical, cosmetic and food industries [12].

Anoxybacillus has currently been studied as a genus comprising important species from the perspective of biomass saccharification. A cellulose degrading strain A. flavithermus BTN7B, isolated from Egyptian soils [13] was reported to reach an high cellulase production in presence of sucrose as carbon source. Recently, sequenced genomes of Anoxybacillus spp. revealed the presence of several genes coding for key enzymes involved in lignocelluloses deconstruction [14]. In the genomes from Anoxybacillus sp. DT3–1 and A. flavithermus subsp. yunnanensis E13T, two β-glucosidases with very low sensitivity to glucose and xylose were investigated as crucial activities for the production of fermentable sugars from biomass [15,16]. In particular the enzyme from A. flavithermus subsp. yunnanensis E13T was able to enhance the sugar cane bagasse conversion respect to the commercial enzyme preparation also in presence of high end products concentrations [16].

The investigations on xylanolytic enzymes produced by Anoxybacillus sp. are relatively novel and limited. Xylanolytic activity was observed in A. flavithermus BC and TWXYL3 [17,18], A. pushchinoensis A8 [19], Anoxybacillus sp. E2 [20]. The presence of xylanase, β-xylosidase, α-L-arabinofuranosidase and acetyl esterase in cultures of Anoxybacillus sp. JT-12, was recently discovered [21] but they were not purified and characterized. To our knowledge this is the first identified, purified and characterized β-xylosidase from Anoxybacillus species. The existence of hemicellulolytic activity in Anoxybacillus sp. 3M (formerly Bacillus 3M) was firstly reported by Marques et al. [22] that isolated the microorganism from terrestrial hot springs (90 °C) in São Miguel, Azores. In the present work, the main goal consisted in purification and characterization of a novel β-xylosidase from the thermoalkaline Anoxybacillus sp. 3M (AbXyl), and in the evaluation of its robustness as a tool towards biotechnological applications.

In order to apply this novel biocatalyst in agro-industrial residues recycling and valorization, the xylosidase was utilized for the hydrolysis of brewery's spent grain (BSG), a feedstock generated during the beer-brewing process, that has stimulated great scientific interest [23]. Herein, a set of experiments at different operating conditions were performed accordingly to a Plackett-Burman statistical design, aiming to optimize the key parameters for BSG hydrolysis process.

Section snippets

Chemicals

Yeast extract and peptone were purchased from Oxoid Ltd. (Oxford, UK). Salts, substrates and other reagents were obtained from Sigma-Aldrich (St. Louis MO). Azo-Oat Spelt Xylan was from Megazyme Co. (Bray, Ireland). All chemicals were of reagent grade.

Media, growth conditions and induction of β-xylosidase

Anoxybacillus sp. 3M is kept at the national culture collection of microorganisms CCM at Laboratorio Nacional de Energia e Geologia, Unidade de Bioenergia Lisbon, Portugal, (strain number UB/#557).

The microorganism was grown aerobically at 55 °C

Production and localization of β-xylosidase

A first set of preliminary experiments to evaluate the potential of Anoxybacillus sp. 3M to produce β-xylosidase was performed by growing this bacterium on the AM medium supplemented with 1% (w/v) beechwood xylan or oat spelts xylan as inducers. The microorganism growth was prolonged until the late stationary phase, where the highest intracellular enzyme concentration was detected as already well established [33], highlighting that the enzyme production was not growth-linked. Moreover, the

Discussion

At our knowledge this is the first report on the purification and characterization of a β-xylosidase from Anoxybacillus spp. The novel enzyme was efficiently produced by using xylan from beechwood as inducer, was purified to homogeneity and showed, in the native state, a molecular mass of 140 kDa, indicating a homo dimeric structure for the protein, composed by two subunits of 70 kDa. The multimeric β-xylosidases reported in literature have usually a dimeric structure [34,35], except for the

Conflict of interest

All authors declare that they have no competing interests.

Acknowledgements

The authors are grateful to Dr. Gabriella Pocsfalvi and Ms. Immacolata Fiume (Institute of Biosciences and Bioresources, CNR-Naples) for the nano-HPLC-ESI-MS/MS analysis and information regarding the enzyme identification.

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