Xylan utilization system of Paenibacillus xylanivorans

TOPALIAN JULIANA; GARRIDO MERCEDES M; VALACCO MARIA PIA; BLASCO MARTÍN; CAMPOS ELEONORA

Congreso; SAIB-SAMIGE 2021: LVII Reunión Anual de SAIB y el XVI Congreso Anual de la Asociación Civil de Microbiología General (SAMIGE); 2021

The bioconversion of residual lignocellulosic biomass into fermentable sugars and/or prebiotic oligosaccharides is an important process for the sustainability of biorefineries. Thus, it is necessary to improve the efficiency of the enzymatic deconstruction of cellulose and hemicelluloses, the structural polysaccharides of plant cell walls. Cellulose is a linear polymer of beta 1,4 linked glucopyranose molecules while hemicellulose is a branched heteropolysaccahride of variable composition. Xylan is the main hemicellulose of secondary plant cell walls.. Paenibacillus xylanivorans secretes xylan degrading enzymes under appropriate culture conditions. The objective of this work was the optimization of the extracellular xylanase activity of P. xylanivorans. Based on previous results, different culture conditions were tested using minimal media (MM) with wheat bran (WB). The highest extracellular xylanase activity was achieved by supplementing the MM with 0.1% yeast extract, 1% WB, for 48 hours at 28 °C, 200 rpm in baffled shake flasks. The enzymatic extracellular extract was obtained by centrifugation and had a xylanase activity of 6.15±0.45 IU/ml (approximately 14 IUxyn/mgprot). The xylanase activity in the extracellular extract maintained more than 85% relative activity for at least 4 weeks at 4°C, -20°C and -80°C. Concentration of the extract was successfully achieved by dry- freeze, resulting in an extract of 48.0±2.8 IU/ml. By mass spectrometry analysis we identified the main enzymes responsible for the observed activity and estimated their relative abundance % emPAI). Fourteen polysaccharide degrading enzymes were identified with high confidence. Among these, we identified 3 xylanases, 1 beta-xylosidase, 3 cellulases, 2 chitinases, 1 alpha-amylase, 1 beta-glucanase, 1 galactanse, 1 beta-glucosidase and 1 LPMO. 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. The most abundant proteins were a substrate binding component of an ABC transporter, a GH13 alpha- amylase and a GH10 -xylanase. Some of the coding sequences for the extracellular enzymes were organized in polysaccharide utilization loci, in which we identified regulatory regions and putative intracellular glycoside hydrolases, which could be involved in the final degradation of short oligosaccharides. Extracellular extracts from sucrose (SAC) cultures were analyzed as control and only two enzymes were identified: the GH13 alpha- amylase and a GH16 beta-glucanase.. 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.