GH10 XynA is the main xylanase identified in the crude enzymatic extract of Paenibacillus sp. A59 when grown on xylan or lignocellulosic biomass

Abstract

A novel bacterial isolate with polysaccharides degrading activity was identified as Paenibacillus sp., and named Paenibacillus sp. A59. Even though it is a strict mesophile, optimal xylanase activity of the crude enzymatic extract was achieved between 50°C and 70°C and more than 60% of the activity was retained after incubation for 48h at 50°C, indicating thermotolerance of the enzymes involved. The extract was also active on pre-treated sugarcane residue (SCR) and wheat straw, releasing xylobiose and xylose as the main products, therefore confirming its predominantly xylanolytic activity. By zymograms and mass spectrometry of crude enzymatic extracts of xylan or SCR cultures, a 32kDa GH10 beta- 1,4- endoxylanase with xylanase and no CMCase activity was identified. We named this enzyme XynA and it was the only xylanase identified under both conditions assayed, suggesting that it is a good candidate for recombinant expression and evaluation in hemicelluloses deconstruction applications. Also, a protein with two S-layer homology domains (SLH) and a large uncharacterized C-terminal domain as well as an ABC substrate binding protein were identified in crude extracts of SCR cultures. We propose that Paenibacillus sp. A59 uses a system similar to anaerobic and other Gram positive bacteria, with SLH-domain proteins anchoring polysaccharide-degrading enzymes close to the membrane and the substrate binding protein assisting translocation of simple sugars to the cell interior.

Paenibacillus sp. A59 GH10 and GH11 Extracellular Endoxylanases: Application in Biomass Bioconversion

“…Reducing sugars released from xylan hydrolysis were measured using the 3,5dinitrosalicylic acid (DNS) assay [30] with xylose as standard. The optimal pH (from 3 to 10) and temperature (from 30 to 70°C) of xylanase activity were assayed as previously described [21]. Thermal stability was performed by preincubating the enzymes at 45, 50, and 60°C from 0 to 20 min.…”

“…Paenibacillus sp. A59, a novel hemicellulolytic strain, was isolated from decaying forest soil [21]. In order to study the xylanolytic and cellulolytic system of this isolate, its genome was sequenced by Illumina MiSeq [24] and coding sequences for proteins potentially involved in biomass deconstruction were identified.…”

“…Maximal xylanase activity was achieved in culture supernatant when Paenibacillus sp. A59 was grown on xylan, compared to lignocellulosic biomass or CMC [21]. By mass…”

“…In this context, we aimed to describe the predicted carbohydrate-active enzymes of the recently isolated hemicellulolytic Paenibacillus sp. A59 strain [21] based on genome sequence analysis, along with the study of the main secreted glycoside hydrolases by bacterial growth in xylan. Also, we have recombinantly expressed two of the major xylanases identified, predicted to belong to GH10 and GH11 families, and we have characterized their activity on xylan and lignocellulosic biomass, for further application in industrial bioconversion processes.…”

Isolation and characterization of α‐(1→3)‐glucan‐degrading bacteria from the gut of Diaperis boleti feeding on Laetiporus sulphureus

“…For example, Paenibacillus sp. A59 is capable of degrading cellulose and xylan (Ghio et al 2016), whereas P. taichungensis CHE N1 and P. pabuli BM17 produce chitinolytic enzymes (Juarez-Jimenez et al 2008;Chen et al 2010).…”