Paludibacter propionicigenes GH10 xylanase as a tool for enzymatic xylooligosaccharides production from heteroxylans

Highlights

• P. propionicigenes GH10 xylanase is a halotolerant and ethanol resistant enzyme.

• PpXyn10A produces high yields of xylooligosaccharides from heteroxylans.

• PpXyn10A greatly enhances short XOS yields from hydrothermally pretreated corn cobs.

• Aromatic interactions in the +2 subsite of the enzyme may explain XOS profile.

• PpXyn10A-produced XOS stimulate B. adolescentis growth.

Abstract

Bioconversion of lignocellulosic biomass into value-added products relies on polysaccharides depolymerization by carbohydrate active enzymes. This work reports biochemical characterization of Paludibacter propionicigenes xylanase from GH10 (PpXyn10A) and its application for enzymatic xylooligosaccharides (XOS) production from commercial heteroxylans and liquor of hydrothermally pretreated corn cobs (PCC). PpXyn10A is tolerant to ethanol and NaCl, and releases xylobiose (X2) and xylotriose (X3) as the main hydrolytic products. The conversion rate of complex substrates into short XOS was approximately 30% for glucuronoxylan and 8.8% for rye arabinoxylan, after only 4 h; while for PCC, PpXyn10A greatly increased unbranched XOS yields. B. adolescentis fermentation with XOS from beechwood glucuronoxylan produced mainly acetic and lactic acids. Structural analysis shows that while the glycone region of PpXyn10A active site is well preserved, the aglycone region has aromatic interactions in the +2 subsite that may explain why PpXyn10A does not release xylose.

Introduction

Lignocellulosic biomass is one of the most abundant sources of renewable carbon on Earth. It holds a promise of sustainable production of second generation biofuels and green chemicals, with simultaneous alleviation of environmental impacts (Bhatia et al., 2020; Lorenci Woiciechowski et al., 2020). About 120 × 10⁹ tons of biomass are produced annually, which energetically corresponds to 2.2 × 10²¹ J. This is almost 300 times the amount of energy consumed in the world (Abraham et al., 2020). Plant biomass is mainly composed of carbohydrate biopolymers (cellulose and hemicellulose) and lignin. Conversion of these compounds in the most diverse products relies on their fractionation and depolymerization, which can be achieved via thermochemical pretreatments and enzymatic hydrolysis (Alokika & Singh, 2019; Evangelista et al., 2019; Lian et al., 2020).

Xylanases (endo-β-1,4-xylanases, EC 3.2.1.8) are a group of enzymes that act on xylan, the main constituent of hemicellulose and second major component of plant cell walls. Xylan is composed of a linear β-1,4-xylopyranosyl backbone decorated with α-arabinofuranosyl, 4-O-methyl-d-glucuronosyl, and acetyl residues (Alokika & Singh, 2019; Li et al., 2020; Liu et al., 2020).

Endo-1,4-β-xylanases are classified within a number of glycoside hydrolase (GH) families, according to CAZy database (http://www.cazy.org) with a vast majority of characterized xylanases belonging to GH10 and GH11 families (Bouiche et al., 2020; Nguyen, Freund, Kasanjian, & Berlemont, 2018).

In nature, xylanases are produced by a range of microorganisms, including fungi and bacteria. The enzymes have wide biotechnological applications in a number of industrial sectors such as pulp and paper production, pharmaceuticals, beverages, food and feed (Juturu & Wu, 2012). They are also used in second generation bioethanol technologies, for enhancing digestibility of animal feed and production of xylooligosaccharides (XOS) (Ariaeenejad et al., 2019; Bouiche et al., 2020; Gowdhaman & Ponnusami, 2015; Juturu & Wu, 2012). Therefore, discovery of new enzymes with elevated activity in a broad pH range, thermal stability and desired specificity is of significant importance for modern biotechnological applications (Sepulchro et al., 2020).

One of the most important value-added products generated by xylanases are xylooligosaccharides (XOS), non-digestible oligosaccharides composed of 2–10 xylose units linked by β-1,4 linkages. They are capable of stimulating growth of intestinal probiotic bacteria such as Bifidobacterium sp. and Lactobacillus sp., in addition to having antioxidant and anti-inflammatory properties (Liu et al., 2020).

There is an increasing demand for value-added products that are environmentally friendly and have health-promoting characteristics. In fact, recent studies indicate that consumers are increasingly willing to pay more for such goods (Amorim, Silvério, Prather, & Rodrigues, 2019; Liu et al., 2020; Samanta et al., 2015). The global prebiotic market was valued at US$ 3.65 billions in 2016 and is estimated to reach US$ 7.37 billions until 2023 (Amorim et al., 2019; MarketsanMarkets, 2018).

In this context, “green” and cost-effective pretreatment strategies for lignocellulosic wastes, such as, for example, hydrothermal pretreatment, have been assessed and optimized (Brar et al., 2020). This pretreatment, in particular associated with a consequent enzymatic step, is particularly suitable for prebiotic XOS production (Santibáñez et al., 2021). Furthermore, xylanases with low xylosidase activity are preferred for enzymatic production of XOS since their use for XOS production results in smaller amounts of xylose, which is desirable for production of prebiotics (Bouiche et al., 2020; Gowdhaman & Ponnusami, 2015).

GH10 xylanase from Paludibacter propionicigenes (PpXyn10A), a strictly anaerobic, Gram-negative, non-spore-forming and non-motile bacteria (Ueki, Akasaka, Suzuki, & Ueki, 2006) was identified and cloned previously (Camilo & Polikarpov, 2014). Here, we report PpXyn10A biochemical characterization, production of XOS from commercial heteroxylans and hydrothermally pretreated corn cobs liquor via enzymatic hydrolysis and prebiotic activity of produced XOS on B. adolescentis. We also performed a phylogenetic analysis and molecular modeling of the enzyme, aiming to explain possible structural reasons for the experimentally observed low xylose titers during XOS production by PpXyn10A.