Thermostability improvement of endoglucanase Cel7B from Hypocrea pseudokoningii

• Published in: Journal of molecular catalysis. B, Enzymatic Vol. 103; pp. 16 - 23
• Main Authors: Mitrovic, Aleksandra, Flicker, Karlheinz, Steinkellner, Georg, Gruber, Karl, Reisinger, Christoph, Schirrmacher, Georg, Camattari, Andrea, Glieder, Anton
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.05.2014
• Subjects: Endoglucanase I; Enzyme engineering; Pichia pastoris; Thermostability; Thermostability; Pichia pastoris; Enzyme engineering; Endoglucanase I
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/j.molcatb.2013.12.009

•Random mutagenesis improved thermostability of endoglucanase.•Additional mutations by structural comparison with homologues enzymes.•Some beneficial random mutations and from structure-based approach were additive.•Increased melting temperature of up to 10°C.•Increased hydrophobicity of a surface cavity might explain stability effect. 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 recombined 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 characterized. 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.