Characterization of a Paenibacillus sp. A59 endo-xylanase for hemicellulose bioconversion

GHIO, S; GRASSO D; CAMPOS E

San Martin, Prov. Buenos Aires

Simposio; 5th International Symposium on Environmental Biotechnology and Engineering (5ISEBE); 2016

ISEBE

Xylan, the main hemicellulose in plants, is a branched and heterogeneous polymer.Its bioconversion requires the synergistic action of multiple xylanolytic enzymes, mainlyendo- -xylosidases. Endo-xylanases (1,4- -D-xylan xylohydrolase, EC3.2.1.8) are considered one of the most important enzymes as they initiate thedegradation of xylan into xylose and xylo-oligosaccharides of different sizes. The costefficientdegradation of xylan to fermentable sugars is of particular interest in secondgeneration bioethanol production and in pulp and paper industries. The aim of this workwas the cloning, heterologous expression and activity characterization of a potentialendoxylanase from Paenibacillus sp. A59 hemicellulolytic strain, isolated from forest soil.The complete xylanase coding sequence was identified in the recently sequencedgenome of Paenibacillus sp. A59. The amino acid sequence analysis by NCBI-BLAST,resulted in high identity (98%) with a GH11 endoxylanase of Paenibacillus sp. HY8,isolated from an herbivorous beetle. Conserved domains related to family GH11xylanase were identified and confirmed by PROSITE. Based on the nucleotidesequence, specific primers were designed for amplification, cloning and recombinantexpression. The complete coding gene was amplified from Paenibacillus sp. A59 totalDNA. The amplification product was cloned and the recombinant protein was expressedwith an N-terminal 6xHis fusion tag. Purification was performed with IMAC using Ni-NTAagarose resin.Xylanase activity was confirmed by hydrolysis of beechwood xylan, measuring thereducing sugars released by DNS as well as identifying and quantifying the main solubleproducts (xylotriose, xylobiose and xylose) by HPLC. Zimography was also performed inSDS-PAGE gels co-polymerized with xylan from beechwood. The recombinant enzymeshowed true endoxylanase activity with no activity on carboxymethyl cellulose, Avicel,PNP-cellobioside, xylo or glucopyranoside. We therefore named it Xyn11.Profiles of enzymatic activity at pH 3 to 10 and at 30°C to 70°C, as well as thermaltolerance were studied. Xyn11 showed optimal activity at pH 9 (retaining more than 60% activity from pH 4 to 8) and at 45°C (with more than 80 % activity from 40°C to 55°C).It showed good thermal stability, retaining at least 90% of xylanase activity after 120minutes at optimal conditions (45°C, pH 9). Michaelis-Menten enzyme kineticparameters were also determined resulting in a Kcat/KM constant of 3.11x105 seg-1 mgml-1.These results indicate that Xyn11 is an alkaline cellulase free-xylanase. It has thepotential to be used in bioconversion of lignocellulosic biomass as well as biobleachingin the pulp and paper industry.