Article

BioEnergy Research

, Volume 3, Issue 1, pp 67-81

Strategy for Identification of Novel Fungal and Bacterial Glycosyl Hydrolase Hybrid Mixtures that can Efficiently Saccharify Pretreated Lignocellulosic Biomass

  • Dahai GaoAffiliated withBiomass Conversion Research Laboratory (BCRL), Department of Chemical Engineering and Materials Science, Michigan State UniversityGreat Lakes Bioenergy Research Center (GLBRC), Michigan State UniversityMichigan State University Email author 
  • , Shishir P. S. ChundawatAffiliated withBiomass Conversion Research Laboratory (BCRL), Department of Chemical Engineering and Materials Science, Michigan State UniversityGreat Lakes Bioenergy Research Center (GLBRC), Michigan State University
  • , Tongjun LiuAffiliated withBiomass Conversion Research Laboratory (BCRL), Department of Chemical Engineering and Materials Science, Michigan State UniversityCollege of Food and Bioengineering, Shandong Institute of Light Industry
  • , Spencer HermansonAffiliated withLucigen CorporationGreat Lakes Bioenergy Research Center (GLBRC), Michigan State University
  • , Krishne GowdaAffiliated withLucigen CorporationGreat Lakes Bioenergy Research Center (GLBRC), Michigan State University
  • , Phillip BrummAffiliated withLucigen CorporationGreat Lakes Bioenergy Research Center (GLBRC), Michigan State University
  • , Bruce E. DaleAffiliated withBiomass Conversion Research Laboratory (BCRL), Department of Chemical Engineering and Materials Science, Michigan State UniversityGreat Lakes Bioenergy Research Center (GLBRC), Michigan State University
  • , Venkatesh BalanAffiliated withBiomass Conversion Research Laboratory (BCRL), Department of Chemical Engineering and Materials Science, Michigan State UniversityGreat Lakes Bioenergy Research Center (GLBRC), Michigan State University

Abstract

A rational four-step strategy to identify novel bacterial glycosyl hydrolases (GH), in combination with various fungal enzymes, was applied in order to develop tailored enzyme cocktails to efficiently hydrolyze pretreated lignocellulosic biomass. The fungal cellulases include cellobiohydrolase I (CBH I; GH family 7A), cellobiohydrolase II (CBH II; GH family 6A), endoglucanase I (EG I; GH family 7B), and β-glucosidase (βG; GH family 3). Bacterial endocellulases (LC1 and LC2; GH family 5), β-glucosidase (LβG; GH family 1), endoxylanases (LX1 and LX2; GH family 10), and β-xylosidase (LβX; GH family 52) from multiple sources were cloned, expressed, and purified. Enzymatic hydrolysis for varying enzyme combinations was carried out on ammonia fiber expansion (AFEX)-treated corn stover at three total protein loadings (i.e., 33, 16.5, and 11 mg enzyme/g glucan). The optimal mass ratio of enzymes necessary to maximize both glucan and xylan yields was determined using a suitable design of experiments. The optimal hybrid enzyme mixtures contained fungal cellulases (78% of total protein loading), which included CBH I (loading ranging between 9-51% of total enzyme), CBH II (9-51%), EG I (10-50%), and bacterial hemicellulases (22% of total protein loading) comprising of LX1 (13%) and LβX (9%). The hybrid mixture was effective at 50°C, pH 4.5 to maximize saccharification of AFEX-treated corn stover resulting in 95% glucan and 65% xylan conversion. This strategy of screening novel enzyme mixtures on pretreated lignocellulose would ultimately lead to the development of tailored enzyme cocktails that can hydrolyze plant cell walls efficiently and economically to produce cellulosic ethanol.

Keywords

AFEX Enzymatic hydrolysis Ethanol Glycosyl hydrolases Lignocellulose