PAENIBACILLUS XYLANIVORANS EXTRACELLULAR XYLANASES AND THEIR USE FOR XYLAN VALORIZATION

TOPALIAN, JULIANA; GARRIDO, MERCEDES MARÍA; NAVAS, LAURA; VALACCO, MARIA PIA; BLASCO, M; CAMPOS, ELEONORA

Rio de Janeiro

Simposio; XXIII SIMPÓSIO NACIONAL DE BIOPROCESSOS; 2022

SINAFERM SHEB ENZITEC

Lignocellulosic biomass provides a renewable source for the production of different chemicals, platform molecules, fuels and energy to enhance the development of a sustainable bio-economy. It consists of plants secondary cell-wall mainly composed of cellulose (40–60%), hemicellulose (20–40%) and lignin (10–24%). Enzymatic deconstruction of the structural polysaccharides, cellulose and hemicellulose, into easily fermentable sugars requires multiple enzymes with different specificities, which act synergistically. Cellulose is a linear polymer of β 1,4 linked glucopyranose molecules while hemicellulose is a branched heteropolysaccahride of variable composition. Xylan is the main hemicellulose of terrestrial secondary plant cell walls and it consists of a backbone of β 1,4 xylose residues taht can be highly modified with decorations of arabinose, acetyl and (methyl)glucuronic acid side groups, depending on the source of biomass (DE BHOWMICK et al. 2018; PUTRO et al., 2016).The hydrolysis of cellulose requires the core enzymatic activities: β-1,4-endo-glucanases (EC 3.2.1.4); β-1,4-exoglucanases (acting from the non-reducing end, EC 3.2.1.91, and from the reducing end, EC 3.2.1.176), and β- β-1,4-glucosidases (EC.3.2.1.21). On the other hand, the hydrolytic deconstruction of hemicellulose requires a higher number of different enzymatic activities due to the variability of its composition and structure. β-1,4 xylanases (EC 3.2.1.8) and β -xylosidases (EC 3.2.1.37) are required to degrade the xylan backbone. In turn, the debranching enzymes such as α- L-arabinofuranosidases (EC 3.2.1.55) become relevant depending on the composition of the biomass (ÁLVAREZ et al., 2016). These enzymes are classified as Carbohydrate Active Enzymes (CAZYmes) and are grouped into discrete families based on their hydrolysis mechanism and structural characteristics (http://www.cazy.org) (LOMBARD et al., 2013).Bacteria belonging to the Paenibacillus genus secrete a complex array of polysaccharide-degrading enzymes under appropriate culture conditions. Paenibacillus xylanivorans A59 is a bacterial strain with capacity of degrading polysaccharides (xylan, cellulose, chitin and starch) and high tolerance to salt (growth up to 7% NaCl), previously isolated from decaying soil of a pristine forest (GHIO et al., 2015 and 2016). Under appropriate culture conditions P. xylanivorans A59 can secrete different CAZymes (GHIO et al., 2016 and 2018). In this work, we studied the optimization of the extracellular xylanase activity of Paenibacillus xylanivorans A59 strain by culture on several substrates and upscaled the process for production of the enzymatic extract in bioreactors. Wheat bran and sugar cane straw were the most suitable carbon sources for secretion of enzymes with xylanase activity. Upscaling from flasks to bench scale bioreactor, using wheat bran 1% as culture substrate, resulted in approximately five-fold increase of extracellular xylanolytic activity, with a yield of 27,640 IU/L of culture, determined as xylose equivalent reducing sugars, by DNS assay. A 3.5-fold concentration of activity was further achieved by ultrafiltration (3 KDa cut off). Freeze-drying of the extracellular enzymatic extract was a successful method for long term conservation, as well as concentration, reaching up to 10 fold concentration factor, although there was a loss of approximately 50% in the overall yield. By mass spectrometry analysis we identified the main enzymes responsible for the observed activity and estimated their relative abundance by the exponentially modified protein abundance index (%emPAI). Fourteen polysaccharide degrading enzymes were identified with high confidence in P. xylanivorans wheat bran culture supernatant. PxXyn10A, a GH10 xylanase, and a GH13 α-amylase were identified as the most abundant enzymes (10.37±3.47% and 24.27±12.22% of total proteins, respectively). In addition, several extracellular components of ABC transporters were identified, which could be involved in the transport of mono- and small oligo-saccharides into the cell. Extracellular extracts from sucrose (SAC) cultures were analyzed as control and only three enzymes were identified: the G13 α-amylase, a GH16 β-glucanase and a GH5 endoglucanase. These results allowed us to build a model for polysaccharide utilization in P. xylanivorans and demonstrated the viability of obtaining extracellular enzymatic extracts with high xylanase activity, for their application in xylan bioprocessing.