Please cite this article in press as: A. Mitrovic, et al., J. Mol. Catal. B: Enzym. (2014), http://dx.doi.org/10.1016/j.molcatb.2013.12.009 ARTICLE IN PRESS G Model MOLCAB-2845; No. of Pages 8 Journal of Molecular Catalysis B: Enzymatic xxx (2014) xxx–xxx Contents lists available at ScienceDirect Journal of Molecular Catalysis B: Enzymatic jo ur nal home p age: www.elsevier.com/locate/molcatb Thermostability improvement of endoglucanase Cel7B from Hypocrea pseudokoningii Aleksandra Mitrovic a,1 , Karlheinz Flicker a,1 , Georg Steinkellner a , Karl Gruber b , Christoph Reisinger c , Georg Schirrmacher c , Andrea Camattari d , Anton Glieder a,∗ a Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria b Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/3, 8010 Graz, Austria c Clariant, Biotech & Renewables Center, Staffelseestrasse 6, 81477 Munich, Germany d Institute for Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria a r t i c l e i n f o Article history: Available online xxx Keywords: Endoglucanase I Thermostability Enzyme engineering Pichia pastoris a b s t r a c t Exploiting enzymes for industrial purposes often requires engineering of these enzymes to adapt them to the industrial requirements. In order to meet industrial demands, we improved the thermostability of endoglucanase Cel7B from Hypocrea pseudokoningii (HpCel7B), which was heterologously expressed in the yeast Pichia pastoris. Random mutants showing higher activity at elevated temperature have been selected and sequenced. In addition a model structure of our target enzyme was compared to structures of homologous but more thermostabile endoglucanases. This comparison pointed out several potential hot spots that were recognized as important for thermostability. The most promising mutations from both rational and non-rational approaches were randomly recom- bined by gene synthesis to evaluate potential additive effects for thermostability. This recombination library yielded a number of improved variants, of which the best ones were sequenced and charac- terized. Compared to the starting variant, recombination mutants showed up to 10 ◦ C higher melting temperatures and can be used at higher temperatures than the natural enzyme. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Lignocellulose, as the most abundant carbohydrate polymer in nature [1], has great potential as a sustainable raw material for bio- chemical and biofuel production. Despite many advantages [2,3], commercial processes for lignocellulose utilization still seem to be too expensive due to its extreme recalcitrance [4,5]. However this is currently changing due to new tools and technologies. The biotechnological lignocellulose exploitation via total hydrolysis consists of three stages: (I) physical and chemical pre- treatment and fractionation of raw plant biomass, (II) enzymatic degradation of the pretreated fibres, and (III) biotransformation of released sugars. Among these three steps, a key-challenge is the enzymatic degradation of cellulose fibres and the release of ∗ Corresponding author. Tel.: +43 316 873 9300. E-mail addresses: aleksandra.mitrovic@acib.at (A. Mitrovic), karlheinzf@miltenyibiotec.de (K. Flicker), georg.steinkellner@acib.at (G. Steinkellner), karl.gruber@acib.at (K. Gruber), christoph.reisinger@clariant.com (C. Reisinger), georg.schirrmacher@clariant.com (G. Schirrmacher), andrea.camattari@acib.at (A. Camattari), glieder@glieder.com, anton.glieder@acib.at (A. Glieder). 1 These authors contributed equally to the work described in this paper. fermentable sugars in a cost effective way [6]. Hydrolytic enzymes degrading cellulose and hemicelluloses are required for this step and are supported by redox enzymes and esterases. Hydrolases are major components of efficient cellulose degrading enzyme cock- tails and they belong to three different families: endoglucanases, which cut randomly at internal amorphous sites in the polysaccha- ride chains; then cellobiohydrolases and -glucosidases, degrading cellulose to sugar dimers, and finally to single glucose molecules [7]. In many cases, natural enzymes are poorly suited for industrial applications and often need to be adapted to withstand extreme conditions, such as high temperature [8]. Elevated temperatures of industrial processes allow higher reaction rates, better solubility of reactants, and solve microbial contamination issues. Therefore, the thermostability is a highly desired property for enzymes in industrial biorefining [9]. We have been engineering the ther- mostability of an endoglucanase Cel7B originating from Hypocrea pseudokoningii (HpCel7B). HpCel7B belongs to glycosyl hydrolase family 7 (GH7), and it is a single module enzyme. GH7 enzymes con- sist of two anti-parallel -sheets forming a -sandwich. In addition to GH7, this fold is also characteristic for family 12 enzymes. To achieve functional expression of HpCel7B, we opted for Pichia pastoris as a host. Ability to perform necessary posttransla- tional modifications such as N-glycosylation and disulphide bond 1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molcatb.2013.12.009