Synergistic function of four novel thermostable glycoside hydrolases from a long-term enriched thermophilic methanogenic digester.

Meng Wang,Xiao-Lei Wu,Liming Liu,Shuang Geng,Baoli Zhu,Zhongping Shi,Guo-Li Lai,Yong Nie

doi:10.3389/fmicb.2015.00509

Abstract

In biofuel production from lignocellulose, low thermostability and product inhibition strongly restrict the enzyme activities and production process. Application of multiple thermostable glycoside hydrolases, forming an enzyme “cocktail”, can result in a synergistic action and therefore improve production efficiency and reduce operational costs. Therefore, increasing enzyme thermostabilities and compatibility are important for the biofuel industry. In this study, we reported the screening, cloning and biochemical characterization of four novel thermostable lignocellulose hydrolases from a metagenomic library of a long-term dry thermophilic methanogenic digester community, which were highly compatible with optimal conditions and specific activities. The optimal temperatures of the four enzymes, β-xylosidase, xylanase, β-glucosidase, and cellulase ranged from 60 to 75°C, and over 80% residual activities were observed after 2 h incubation at 50°C. Mixtures of these hydrolases retained high residual synergistic activities after incubation with cellulose, xylan, and steam-exploded corncob at 50°C for 72 h. In addition, about 55% dry weight of steam-exploded corncob was hydrolyzed to glucose and xylose by the synergistic action of the four enzymes at 50°C for 48 h. This work suggested that since different enzymes from a same ecosystem could be more compatible, screening enzymes from a long-term enriching community could be a favorable strategy.

Highlights

Enzymes catalyzing the hydrolysis of cellulose and hemicellulose are a source of worldwide interest for alternative bio-energy development and other environmental applications, such as treating wastewater from the dying, textile and pulping industries
According to the average insert size of fosmid clones, the hit rates of cellulase, xylanase and βglucosidase were 1/8.1, 1/10.8, and 1/4.4, respectively, which were higher than those metagenomes from a 1.5 year enriched biogas digester (Yan et al, 2013), from buffalo rumen (Duan et al, 2009) and from soil (Kim et al, 2008), suggesting that long-term enrichment could increase the possibility of identifying the target genes
From the inserted DNA fragments in fosmids F52, F85, and F175, putative genes coding for β-xylosidase, xylanase, β-glucosidase, and cellulase and their glycoside hydrolase (GH) family were annotated (Supplementary Table 2) and designated as Xyl522, Xyn526, Bgl8520, and Cel1753, respectively

Summary

Introduction

Enzymes catalyzing the hydrolysis of cellulose and hemicellulose are a source of worldwide interest for alternative bio-energy development and other environmental applications, such as treating wastewater from the dying, textile and pulping industries. Synergism of metagenomic thermostable hydrolases processes, including exoglucanase (EC 3.2.1.91), endoglucanase (EC 3.2.1.4), and β-glucosidase (EC 3.2.1.21) for cellulose hydrolysis; xylanase (EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37) to hydrolyze xylan; and α-L-arabinofuranosidase (EC 3.2.1.55) and α-glucuronidase (EC 3.2.1.39) to hydrolyze side chains in hemicellulose (Beg et al, 2001) To utilize these enzymes in biofuel production, two processes are generally used: separate hydrolysis and fermentation (SHF), and simultaneous saccharification and fermentation (SSF). The combination of exoglucanase, endoglucanase, and β-glucosidase is used to increase cellulose degradation efficiency (Gusakov et al, 2007), and the combination of xylanase and β-xylosidase has been shown to enhance hemicellulose hydrolysis (Fan et al, 2009) These cocktails are formed from widely used commercial enzymes, such as the cellobiohydrolases I and II and endoglucanases I and II from the fungus Trichoderma reesei (Henrissat et al, 1985). Since the host microorganisms from which these enzymes were originally isolated are from different habitats and have different optimal growth conditions, the isolated enzymes generally have different optimal conditions, and may not be compatible in the same cocktail

Methods

Results

Conclusion