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

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:
 [1] ;  [2] ;  [1] ;  [2] ;  [1] ;  [2] ;  [1] ;  [2] ;  [1] ;  [2] ;  [1] ;  [3] ;  [1] ;  [3] ;  [1] ;  [3] ;  [1] ;  [2] ;  [1] ;  [3] more »;  [1] ;  [2] ;  [4] « less
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States)
  2. (LBNL), Berkeley, CA (United States)
  3. (SNL-CA), Livermore, CA (United States)
  4. (United States)
Publication Date:
Report Number(s):
SAND2018-9901J
Journal ID: ISSN 2059-7983; ACSDAD; 667734
Grant/Contract Number:
AC04-94AL85000; AC02-76SF00515
Type:
Published Article
Journal Name:
Acta Crystallographica. Section D. Structural Biology
Additional Journal Information:
Journal Volume: 74; Journal Issue: 7; Journal ID: ISSN 2059-7983
Publisher:
IUCr
Research Org:
Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES
OSTI Identifier:
1457487
Alternate Identifier(s):
OSTI ID: 1476933

Ellinghaus, Thomas L., Lawrence Berkeley National Lab., Pereira, Jose H., Lawrence Berkeley National Lab., McAndrew, Ryan P., Lawrence Berkeley National Lab., Welner, Ditte H., Lawrence Berkeley National Lab., DeGiovanni, Andy M., Lawrence Berkeley National Lab., Guenther, Joel M., Sandia National Lab., Tran, Huu M., Sandia National Lab., Feldman, Taya, Sandia National Lab., Simmons, Blake A., Lawrence Berkeley National Lab., Sale, Kenneth L., Sandia National Lab., Adams, Paul D., Lawrence Berkeley National Lab., and Univ. of California, Berkeley, CA. Engineering glycoside hydrolase stability by the introduction of zinc binding. United States: N. p., Web. doi:10.1107/S2059798318006678.
Ellinghaus, Thomas L., Lawrence Berkeley National Lab., Pereira, Jose H., Lawrence Berkeley National Lab., McAndrew, Ryan P., Lawrence Berkeley National Lab., Welner, Ditte H., Lawrence Berkeley National Lab., DeGiovanni, Andy M., Lawrence Berkeley National Lab., Guenther, Joel M., Sandia National Lab., Tran, Huu M., Sandia National Lab., Feldman, Taya, Sandia National Lab., Simmons, Blake A., Lawrence Berkeley National Lab., Sale, Kenneth L., Sandia National Lab., Adams, Paul D., Lawrence Berkeley National Lab., & Univ. of California, Berkeley, CA. Engineering glycoside hydrolase stability by the introduction of zinc binding. United States. doi:10.1107/S2059798318006678.
Ellinghaus, Thomas L., Lawrence Berkeley National Lab., Pereira, Jose H., Lawrence Berkeley National Lab., McAndrew, Ryan P., Lawrence Berkeley National Lab., Welner, Ditte H., Lawrence Berkeley National Lab., DeGiovanni, Andy M., Lawrence Berkeley National Lab., Guenther, Joel M., Sandia National Lab., Tran, Huu M., Sandia National Lab., Feldman, Taya, Sandia National Lab., Simmons, Blake A., Lawrence Berkeley National Lab., Sale, Kenneth L., Sandia National Lab., Adams, Paul D., Lawrence Berkeley National Lab., and Univ. of California, Berkeley, CA. 2018. "Engineering glycoside hydrolase stability by the introduction of zinc binding". United States. doi:10.1107/S2059798318006678.
@article{osti_1457487,
title = {Engineering glycoside hydrolase stability by the introduction of zinc binding},
author = {Ellinghaus, Thomas L. and Lawrence Berkeley National Lab. and Pereira, Jose H. and Lawrence Berkeley National Lab. and McAndrew, Ryan P. and Lawrence Berkeley National Lab. and Welner, Ditte H. and Lawrence Berkeley National Lab. and DeGiovanni, Andy M. and Lawrence Berkeley National Lab. and Guenther, Joel M. and Sandia National Lab. and Tran, Huu M. and Sandia National Lab. and Feldman, Taya and Sandia National Lab. and Simmons, Blake A. and Lawrence Berkeley National Lab. and Sale, Kenneth L. and Sandia National Lab. and Adams, Paul D. and Lawrence Berkeley National Lab. and Univ. of California, Berkeley, CA},
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 States},
year = {2018},
month = {6}
}