Abstract
The cost-efficient degradation of xylan to fermentable sugars is of particular interest in second generation bioethanol production, feed, food, and pulp and paper industries. Multiple potentially secreted enzymes involved in polysaccharide deconstruction are encoded in the genome of Paenibacillus sp. A59, a xylanolytic soil bacterium, such as three endoxylanases, seven GH43 β-xylosidases, and two GH30 glucuronoxylanases. In secretome analysis of xylan cultures, ten glycoside hydrolases were identified, including the three predicted endoxylanases, confirming their active role. The two uni-modular xylanases, a 32-KDa GH10 and a 20-KDa GH11, were recombinantly expressed and their activity on xylan was confirmed (106 and 85 IU/mg, respectively), with differences in their activity pattern. Both endoxylanases released mainly xylobiose (X2) and xylotriose (X3) from xylan and pre-treated biomasses (wheat straw, barley straw, and sweet corn cob), although only rGH10XynA released xylose (X1). rGH10XynA presented optimal conditions at pH 6, with thermal stability at 45–50 °C, while rGH11XynB showed activity in a wider range of pH, from 5 to 9, and was thermostable only at 45 °C. Moreover, GH11XynB presented sigmoidal kinetics on xylan, indicating possible cooperative binding, which was further supported by the structural model. This study provides a detailed analysis of the complete set of carbohydrate-active enzymes encoded in Paenibacillus sp. A59 genome and those effectively implicated in hemicellulose hydrolysis, contributing to understanding the mechanisms necessary for the bioconversion of this polysaccharide. Moreover, the two main free secreted xylanases, rGH10XynA and rGH11XynB, were fully characterized, supporting their potential application in industrial bioprocesses on lignocellulosic biomass.
Highlights
Increasing the need for renewable fuels highlights the requirement of suitable technology development from sustainableElectronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Lignocellulose is the complex structural material of all plant cell walls, composed mainly from cellulose (40–50%), hemicellulose (25–30%), and lignin (15–20%) [4]
Most frequently one domain was assigned to a single protein, in some cases, a combination of a catalytic domain with one or more carbohydrate-binding domains (CBMs) or S-layer homology domains (SLHs) domains was predicted in the same protein
In order to define the potential extracellular glycoside hydrolases involved in cellulose or hemicelluloses hydrolysis, the output results obtained from dbCAN were subjected to BLASTP homology search and signal peptide prediction analysis
Summary
Lignocellulose is the complex structural material of all plant cell walls, composed mainly from cellulose (40–50%), hemicellulose (25–30%), and lignin (15–20%) [4] The recalcitrance of these structural components is based on a complex polymeric network that provides plants with strengh and resistance. GH10 includes a few enzymes with endo-β-1,3-xylanase activity (EC 3.2.1.32), the majority are endo-β-1,4-xylanases (EC 3.2.1.8) Some of the latter display limited activity on aryl cellobiosides, but not on cellulose. They exhibit greater catalytic versatility and lower substrate specificity than enzymes from family GH11, and they are generally able to act on the xylan backbone, close to substituted regions. While family GH10 generally includes xylanases of high molecular weight/low isoelectric point, family GH11 includes mainly low molecular weight/ high isoelectric point enzymes [11, 12]
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