Hyperthermostable Thermotoga maritima xylanase XYN10B shows high activity at high temperatures in the presence of biomass-dissolving hydrophilic ionic liquids.

Tianyi Yu,Sasikala Anbarasan,Sami Havukainen,Baris Binay,Piia Iivonen,Aşkın Sevinç Aslan,Tero Mentunen,Ossi Turunen,Michael Hummel,Yin Zhou,Zhengding Su,Hairong Xiong,Kübra Telli,Yawei Wang,Li Zhang,Herbert Sixta

doi:10.1007/s00792-016-0841-y

Abstract

The gene of Thermotoga maritima GH10 xylanase (TmXYN10B) was synthesised to study the extreme limits of this hyperthermostable enzyme at high temperatures in the presence of biomass-dissolving hydrophilic ionic liquids (ILs). TmXYN10B expressed from Pichia pastoris showed maximal activity at 100 °C and retained 92 % of maximal activity at 105 °C in a 30-min assay. Although the temperature optimum of activity was lowered by 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc), TmXYN10B retained partial activity in 15–35 % hydrophilic ILs, even at 75–90 °C. TmXYN10B retained over 80 % of its activity at 90 °C in 15 % [EMIM]OAc and 15–25 % 1-ethyl-3-methylimidazolium dimethylphosphate ([EMIM]DMP) during 22-h reactions. [EMIM]OAc may rigidify the enzyme and lower Vmax. However, only minor changes in kinetic parameter Km showed that competitive inhibition by [EMIM]OAc of TmXYN10B is minimal. In conclusion, when extended enzymatic reactions under extreme conditions are required, TmXYN10B shows extraordinary potential.Electronic supplementary materialThe online version of this article (doi:10.1007/s00792-016-0841-y) contains supplementary material, which is available to authorized users.

Highlights

Xylanases, members of glycosidases (O-glycoside hydrolases, EC 3.2.1.x), catalyse the endohydrolysis of 1,4-betaD-xylosidic linkages in xylan (Collins et al 2005)
The prokaryotic expression of TmXYN10B was induced by IPTG in E. coli BL21 (DE3) under the control of the T7 promoter, and the eukaryotic expression was induced by methanol in P. pastoris GS115 under the control of the AOX promoter
The protein produced in E. coli had a size of 38 kDa, and the protein produced in P. pastoris had a size of 42 kDa, indicating that TmXYN10B produced in P. pastoris was glycosylated

Summary

Introduction

Members of glycosidases (O-glycoside hydrolases, EC 3.2.1.x), catalyse the endohydrolysis of 1,4-betaD-xylosidic linkages in xylan (Collins et al 2005). Thermostable xylanases are widely used in biotechnological processes that take place at high temperatures, such as those in the feed and pulp and paper industry (Vieille and Zeikus 2001; Kumar et al 2016). Xylanases are used in industrial applications for the deconstruction of plant cell walls to facilitate biofuel production from lignocellulose. Lignocellulosic materials from agricultural and forestry waste can serve as key substrates for second-generation biofuels due to their low cost and abundance (Yeoman et al 2010; Bhalla et al 2013). The application of thermostable enzymes in industrial processes, which are carried out at high temperatures, have several advantages, including extended reaction times and stability against denaturing conditions other than

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