Contribution of a family 1 carbohydrate-binding module in thermostable glycoside hydrolase 10 xylanase from Talaromyces cellulolyticus toward synergistic enzymatic hydrolysis of lignocellulose.

Hiroyuki Inoue,Seiichiro Kishishita,Akio Kumagai,Kazuhiko Ishikawa,Tatsuya Fujii,Misumi Kataoka

doi:10.1186/s13068-015-0259-2

Hiroyuki Inoue, Seiichiro Kishishita + Show 4 more

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https://doi.org/10.1186/s13068-015-0259-2

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Abstract

BackgroundEnzymatic removal of hemicellulose components such as xylan is an important factor for maintaining high glucose conversion from lignocelluloses subjected to low-severity pretreatment. Supplementation of xylanase in the cellulase mixture enhances glucose release from pretreated lignocellulose. Filamentous fungi produce multiple xylanases in their cellulase system, and some of them have modular structures consisting of a catalytic domain and a family 1 carbohydrate-binding module (CBM1). However, the role of CBM1 in xylanase in the synergistic hydrolysis of lignocellulose has not been investigated in depth.ResultsThermostable endo-β-1,4-xylanase (Xyl10A) from Talaromyces cellulolyticus, which is recognized as one of the core enzymes in the fungal cellulase system, has a modular structure consisting of a glycoside hydrolase family 10 catalytic domain and CBM1 at the C-terminus separated by a linker region. Three recombinant Xyl10A variants, that is, intact Xyl10A (Xyl10Awt), CBM1-deleted Xyl10A (Xyl10AdC), and CBM1 and linker region-deleted Xyl10A (Xyl10AdLC), were constructed and overexpressed in T. cellulolyticus. Cellulose-binding ability of Xyl10A CBM1 was demonstrated using quartz crystal microbalance with dissipation monitoring. Xyl10AdC and Xyl10AdLC showed relatively high catalytic activities for soluble and insoluble xylan substrates, whereas Xyl10Awt was more effective in xylan hydrolysis of wet disc-mill treated rice straw (WDM-RS). The enzyme mixture of cellulase monocomponents and intact or mutant Xyl10A enhanced the hydrolysis of WDM-RS glucan, with the most efficient synergism found in the interactions with Xyl10Awt. The increased glucan hydrolysis yield exhibited a linear relationship with the xylan hydrolysis yield by each enzyme. This relationship revealed significant hydrolysis of WDM-RS glucan with lower supplementation of Xyl10Awt.ConclusionsOur results suggest that Xyl10A CBM1 has the following two roles in synergistic hydrolysis of lignocellulose by Xyl10A and cellulases: enhancement of lignocellulosic xylan hydrolysis by binding to cellulose, and the efficient removal of xylan obstacles that interrupt the cellulase activity (because of similar binding target of CBM1). The combination of CBM-containing cellulases and xylanases in a fugal cellulase system could contribute to reduction of the enzyme loading in the hydrolysis of pretreated lignocelluloses.

Highlights

Enzymatic removal of hemicellulose components such as xylan is an important factor for maintaining high glucose conversion from lignocelluloses subjected to low-severity pretreatment
Expression and purification of recombinant enzymes: Xyl10Awt, Xyl10AdC, and Xyl10AdLC To evaluate the role of CBM1 in Xyl10A from T. cellulolyticus, three recombinant forms of Xyl10A were designed, that is, Xyl10Awt, an intact enzyme; Xyl10AdC, a CBM1deleted form that has Gly372 as the C-terminal residue; and Xyl10AdLC, a CBM1 and linker region-deleted form that has Leu334 as the C-terminal residue (Figure 1)
We demonstrated that cellulose binding by Xyl10A CBM1 serves two roles in the synergistic hydrolysis of lignocellulose using Xyl10A and cellulases

Summary

Introduction

Enzymatic removal of hemicellulose components such as xylan is an important factor for maintaining high glucose conversion from lignocelluloses subjected to low-severity pretreatment. An efficient enzymatic hydrolysis of the cellulose and hemicellulose components to fermentable sugars is a key step in the bioconversion of lignocellulose [1,2]. The digestibility of these components is increased by pretreatment processes such as dilute acid, alkali, hot compressed water, and milling treatments that disrupt the rigid structural network consisting of cellulose-hemicellulose-lignin [3]. An efficient enzymatic removal of hemicellulose is an important factor to maintain high glucose conversion from lignocellulose subjected to the low-severity pretreatment

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