title: Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: US Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706 USA.|Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 USA.
authors.forename: Johnnie A
authors.initials: JA
authors.lastname: Walker
authors.affiliation: US Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706 USA.|Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 USA.|Research Faculty of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan.
authors.forename: Taichi E
authors.initials: TE
authors.lastname: Takasuka
authors.affiliation: US Department of Energy Joint BioEnergy Institute, Emeryville, CA 94608 USA.|Sandia National Laboratories, Livermore, CA 94551 USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Deng
authors.affiliation: US Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706 USA.|Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 USA.|Department of Chemistry, University of Wisconsin-Oshkosh, Oshkosh, WI 54901 USA.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Bianchetti
authors.affiliation: US Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706 USA.
authors.forename: Hannah S
authors.initials: HS
authors.lastname: Udell
authors.affiliation: US Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706 USA.
authors.forename: Ben M
authors.initials: BM
authors.lastname: Prom
authors.affiliation: US Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706 USA.
authors.forename: Hyunkee
authors.initials: H
authors.lastname: Kim
authors.affiliation: US Department of Energy Joint BioEnergy Institute, Emeryville, CA 94608 USA.|Lawrence Berkeley National Laboratory, Berkeley, CA 94720 USA.|Department of Bioengineering, University of California, Berkeley, CA 94720 USA.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
authors.affiliation: US Department of Energy Joint BioEnergy Institute, Emeryville, CA 94608 USA.|Lawrence Berkeley National Laboratory, Berkeley, CA 94720 USA.
authors.forename: Trent R
authors.initials: TR
authors.lastname: Northen
authors.affiliation: US Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706 USA.|Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 USA.
authors.forename: Brian G
authors.initials: BG
authors.lastname: Fox
keywords: Biofuels; Carbohydrate binding module; Cellulase; Enzyme engineering; Hemicellulase; Kinetic analysis; Mannanase; Mass spectrometry; Ruminoclostridium thermocellum; Xylanase