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The importance of pyroglutamate in cellulase Cel7A
Author(s) -
Dana Craig M.,
DotsonFagerstrom Alexandra,
Roche Christine M.,
Kal Sarala M.,
Chokhawala Harshal A.,
Blanch Harvey W.,
Clark Douglas S.
Publication year - 2014
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.25178
Subject(s) - neurospora crassa , saccharomyces cerevisiae , cellulase , biochemistry , yeast , glycoside hydrolase , biology , chemistry , enzyme , gene , mutant
The commercialization of lignocellulosic biofuels relies in part on the ability to engineer cellulase enzymes to have properties compatible with practical processing conditions. The cellulase Cel7A has been a common engineering target because it is present in very high concentrations in commercial cellulase cocktails. Significant effort has thus been focused on its recombinant expression. In particular, the yeast Saccharomyces cerevisiae has often been used both in the engineering and basic study of Cel7A. However, the expression titer and extent of glycosylation of Cel7A expressed in S. cerevisiae vary widely for Cel7A genes from different organisms, and the recombinant enzymes tend to be less active and less stable than their native counterparts. These observations motivate further study of recombinant expression of Cel7A in S. cerevisiae . Here, we compare the properties of Cel7A from Talaromyces emersonii expressed in both the budding yeast S. cerevisiae and the filamentous fungus Neurospora crassa . The Cel7A expressed in N. crassa had a higher melting temperature (by 10°C) and higher specific activity (twofold at 65°C) than the Cel7A expressed in S. cerevisiae . We examined several post‐translational modifications and found that the underlying cause of this disparity was the lack of N‐terminal glutamine cyclization in the Cel7A expressed in S. cerevisiae . Treating the enzyme in vitro with glutaminyl cyclase improved the properties of Cel7A expressed in S. cerevisiae to match those of Cel7A expressed in N. crassa . Biotechnol. Bioeng. 2014;111: 842–847. © 2013 Wiley Periodicals, Inc.