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Title: Engineering glycoside hydrolase stability by the introduction of zinc binding

Abstract

The development of robust enzymes, in particular cellulases, is a key step in the success of biological routes to `second-generation' biofuels. The typical sources of the enzymes used to degrade biomass include mesophilic and thermophilic organisms. The endoglucanase J30 from glycoside hydrolase family 9 was originally identified through metagenomic analyses of compost-derived bacterial consortia. These studies, which were tailored to favor growth on targeted feedstocks, have already been shown to identify cellulases with considerable thermal tolerance. The amino-acid sequence of J30 shows comparably low identity to those of previously analyzed enzymes. As an enzyme that combines a well measurable activity with a relatively low optimal temperature (50°C) and a modest thermal tolerance, it offers the potential for structural optimization aimed at increased stability. Here, the crystal structure of wild-type J30 is presented along with that of a designed triple-mutant variant with improved characteristics for industrial applications. Through the introduction of a structural Zn 2+ site, the thermal tolerance was increased by more than 10°C and was paralleled by an increase in the catalytic optimum temperature by more than 5°C.

Authors:
; ; ; ORCiD logo; ; ; ; ; ; ;
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1457487
Grant/Contract Number:
AC02-76SF00515
Resource Type:
Journal Article: Published Article
Journal Name:
Acta Crystallographica Section D Structural Biology
Additional Journal Information:
Journal Volume: 74; Journal Issue: 7; Related Information: CHORUS Timestamp: 2018-06-29 11:40:12; Journal ID: ISSN 2059-7983
Publisher:
International Union of Crystallography (IUCr)
Country of Publication:
United Kingdom
Language:
English

Citation Formats

Ellinghaus, Thomas L., Pereira, Jose H., McAndrew, Ryan P., Welner, Ditte H., DeGiovanni, Andy M., Guenther, Joel M., Tran, Huu M., Feldman, Taya, Simmons, Blake A., Sale, Kenneth L., and Adams, Paul D. Engineering glycoside hydrolase stability by the introduction of zinc binding. United Kingdom: N. p., 2018. Web. doi:10.1107/S2059798318006678.
Ellinghaus, Thomas L., Pereira, Jose H., McAndrew, Ryan P., Welner, Ditte H., DeGiovanni, Andy M., Guenther, Joel M., Tran, Huu M., Feldman, Taya, Simmons, Blake A., Sale, Kenneth L., & Adams, Paul D. Engineering glycoside hydrolase stability by the introduction of zinc binding. United Kingdom. doi:10.1107/S2059798318006678.
Ellinghaus, Thomas L., Pereira, Jose H., McAndrew, Ryan P., Welner, Ditte H., DeGiovanni, Andy M., Guenther, Joel M., Tran, Huu M., Feldman, Taya, Simmons, Blake A., Sale, Kenneth L., and Adams, Paul D. Wed . "Engineering glycoside hydrolase stability by the introduction of zinc binding". United Kingdom. doi:10.1107/S2059798318006678.
@article{osti_1457487,
title = {Engineering glycoside hydrolase stability by the introduction of zinc binding},
author = {Ellinghaus, Thomas L. and Pereira, Jose H. and McAndrew, Ryan P. and Welner, Ditte H. and DeGiovanni, Andy M. and Guenther, Joel M. and Tran, Huu M. and Feldman, Taya and Simmons, Blake A. and Sale, Kenneth L. and Adams, Paul D.},
abstractNote = {The development of robust enzymes, in particular cellulases, is a key step in the success of biological routes to `second-generation' biofuels. The typical sources of the enzymes used to degrade biomass include mesophilic and thermophilic organisms. The endoglucanase J30 from glycoside hydrolase family 9 was originally identified through metagenomic analyses of compost-derived bacterial consortia. These studies, which were tailored to favor growth on targeted feedstocks, have already been shown to identify cellulases with considerable thermal tolerance. The amino-acid sequence of J30 shows comparably low identity to those of previously analyzed enzymes. As an enzyme that combines a well measurable activity with a relatively low optimal temperature (50°C) and a modest thermal tolerance, it offers the potential for structural optimization aimed at increased stability. Here, the crystal structure of wild-type J30 is presented along with that of a designed triple-mutant variant with improved characteristics for industrial applications. Through the introduction of a structural Zn 2+ site, the thermal tolerance was increased by more than 10°C and was paralleled by an increase in the catalytic optimum temperature by more than 5°C.},
doi = {10.1107/S2059798318006678},
journal = {Acta Crystallographica Section D Structural Biology},
number = 7,
volume = 74,
place = {United Kingdom},
year = {Wed Jun 27 00:00:00 EDT 2018},
month = {Wed Jun 27 00:00:00 EDT 2018}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1107/S2059798318006678

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