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Title: Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules

Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed. CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolytic activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexosemore » and pentose fractions of ionic-liquid pretreated switchgrass. In conclusion, we have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.« less
Authors:
 [1] ;  [2] ;  [3] ;  [4] ;  [1] ;  [1] ;  [1] ;  [5] ;  [6] ;  [1]
  1. Univ. of Wisconsin, Madison, WI (United States)
  2. Univ. of Wisconsin, Madison, WI (United States); Hokkaido Univ., Sapporo (Japan)
  3. US Dept. of Energy Joint BioEnergy Institute, Emeryville, CA (United State); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
  4. Univ. of Wisconsin, Madison, WI (United States); Univ. of Wisconsin-Oshkosh, Oshkosh, WI (United States)
  5. US Dept. of Energy Joint BioEnergy Institute, Emeryville, CA (United State); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
  6. US Dept. of Energy Joint BioEnergy Institute, Emeryville, CA (United State); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Publication Date:
Report Number(s):
SAND-2016-6409J
Journal ID: ISSN 1754-6834; PII: 402
Grant/Contract Number:
FC02-07ER64494; AC02-05CH11231; DGE-1256259; AC04-94AL85000
Type:
Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 8; Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); National Science Foundation (NSF); National Institutes of Health (NIH)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; cellulase; xylanase; hemicellulase; mannanase; carbohydrate binding module; Ruminoclostridium thermocellum; enzyme engineering; biofuels; mass spectrometry; kinetic analysis
OSTI Identifier:
1257273
Alternate Identifier(s):
OSTI ID: 1262308; OSTI ID: 1378704

Walker, Johnnie A., Takasuka, Taichi E., Deng, Kai, Bianchetti, Christopher M., Udell, Hannah S., Prom, Ben M., Kim, Hyunkee, Adams, Paul D., Northen, Trent R., and Fox, Brian G.. Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules. United States: N. p., Web. doi:10.1186/s13068-015-0402-0.
Walker, Johnnie A., Takasuka, Taichi E., Deng, Kai, Bianchetti, Christopher M., Udell, Hannah S., Prom, Ben M., Kim, Hyunkee, Adams, Paul D., Northen, Trent R., & Fox, Brian G.. Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules. United States. doi:10.1186/s13068-015-0402-0.
Walker, Johnnie A., Takasuka, Taichi E., Deng, Kai, Bianchetti, Christopher M., Udell, Hannah S., Prom, Ben M., Kim, Hyunkee, Adams, Paul D., Northen, Trent R., and Fox, Brian G.. 2015. "Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules". United States. doi:10.1186/s13068-015-0402-0. https://www.osti.gov/servlets/purl/1257273.
@article{osti_1257273,
title = {Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules},
author = {Walker, Johnnie A. and Takasuka, Taichi E. and Deng, Kai and Bianchetti, Christopher M. and Udell, Hannah S. and Prom, Ben M. and Kim, Hyunkee and Adams, Paul D. and Northen, Trent R. and Fox, Brian G.},
abstractNote = {Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed. CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolytic activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass. In conclusion, we have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.},
doi = {10.1186/s13068-015-0402-0},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 8,
place = {United States},
year = {2015},
month = {12}
}

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