XYLAN UTILIZATION SYSTEM OF Paenibacillus xylanivorans

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

VIRTUAL

Congreso; Congreso conjunto SAIB-SAMIGE 2021; 2021

Sociedad Argentina de Investigación Bioquímica

The bioconversion of residual lignocellulosic biomass into fermentable sugars and/or prebiotic oligosaccharides is animportant process for the sustainability of biorefineries. Thus, it is necessary to improve the efficiency of the enzymaticdeconstruction of cellulose and hemicelluloses, the structural polysaccharides of plant cell walls. Cellulose is a linear polymerof 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 enzymesunder appropriate culture conditions. The objective of this work was the optimization of the extracellular xylanase activity ofP. xylanivorans. Based on previous results, different culture conditions were tested using minimal media (MM) with wheatbran (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 centrifugationand had a xylanase activity of 6.15±0.45 IU/ml (approximately 14 IUxyn/mgprot). The xylanase activity in the extracellularextract maintained more than 85% relative activity for at least 4 weeks at 4°C, -20°C, and -80°C. The concentration of theextract was successfully achieved by dry- freeze, resulting in an extract of 48.0±2.8 IU/ml. By mass spectrometry analysis, weidentified the main enzymes responsible for the observed activity and estimated their relative abundance (% emPAI). Fourteenpolysaccharide degrading enzymes were identified with high confidence. Among these, we identified 3 xylanases, 1 betaxylosidase, 3 cellulases, 2 chitinases, 1 alpha-amylase, 1 beta-glucanase, 1 galactanse, 1 beta-glucosidase, and 1 LPMO. Inaddition, several extracellular components of ABC transporters were identified, which could be involved in the transport ofmono- and small oligo-saccharides into the cell. The most abundant proteins were a substrate-binding component of an ABCtransporter, a GH13 alpha-amylase, and a GH10 xylanase. Some of the coding sequences for the extracellular enzymes wereorganized in polysaccharide utilization loci, in which we identified regulatory regions and putative intracellular glycosidehydrolases, 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 betaglucanase. These results allowed us to build a model for polysaccharide utilization in P. xylanivorans and demonstrated theviability of obtaining extracellular enzymatic extracts with high xylanase activity, for their application in xylan bioprocessing.