title: Hemicellulases and auxiliary enzymes for improved conversion of lignocellulosic biomass to monosaccharides.
abstract: BACKGROUND
  High enzyme loading is a major economic bottleneck for the commercial processing of pretreated lignocellulosic biomass to produce fermentable sugars. Optimizing the enzyme cocktail for specific types of pretreated biomass allows for a significant reduction in enzyme loading without sacrificing hydrolysis yield. This is especially important for alkaline pretreatments such as Ammonia fiber expansion (AFEX) pretreated corn stover. Hence, a diverse set of hemicellulases supplemented along with cellulases is necessary for high recovery of monosaccharides.
  
  
  RESULTS
  The core fungal cellulases in the optimal cocktail include cellobiohydrolase I [CBH I; glycoside hydrolase (GH) family 7A], cellobiohydrolase II (CBH II; GH family 6A), endoglucanase I (EG I; GH family 7B) and β-glucosidase (βG; GH family 3). Hemicellulases tested along with the core cellulases include xylanases (LX1, GH family 10; LX2, GH family 10; LX3, GH family 10; LX4, GH family 11; LX5, GH family 10; LX6, GH family 10), β-xylosidase (LβX; GH family 52), α-arabinofuranosidase (LArb, GH family 51) and α-glucuronidase (LαGl, GH family 67) that were cloned, expressed and/or purified from different bacterial sources. Different combinations of these enzymes were tested using a high-throughput microplate based 24 h hydrolysis assay. Both family 10 (LX3) and family 11 (LX4) xylanases were found to most efficiently hydrolyze AFEX pretreated corn stover in a synergistic manner. The optimal mass ratio of xylanases (LX3 and LX4) to cellulases (CBH I, CBH II and EG I) is 25:75. LβX (0.6 mg/g glucan) is crucial to obtaining monomeric xylose (54% xylose yield), while LArb (0.6 mg/g glucan) and LαGl (0.8 mg/g glucan) can both further increase xylose yield by an additional 20%. Compared with Accellerase 1000, a purified cocktail of cellulases supplemented with accessory hemicellulases will not only increase both glucose and xylose yields but will also decrease the total enzyme loading needed for equivalent yields.
  
  
  CONCLUSIONS
  A diverse set of accessory hemicellulases was found necessary to enhance the synergistic action of cellulases hydrolysing AFEX pretreated corn stover. High glucose (around 80%) and xylose (around 70%) yields were achieved with a moderate enzyme loading (~20 mg protein/g glucan) using an in-house developed cocktail compared to commercial enzymes.
authors.affiliation: Biomass Conversion Research Lab (BCRL), Department of Chemical Engineering and Materials Science, Michigan State University, MBI Building, 3900 Collins Road, Lansing, Michigan 48910, USA. gaodahai@msu.edu.
authors.forename: Dahai
authors.initials: D
authors.lastname: Gao
authors.forename: Nirmal
authors.initials: N
authors.lastname: Uppugundla
authors.forename: Shishir Ps
authors.initials: SP
authors.lastname: Chundawat
authors.forename: Xiurong
authors.initials: X
authors.lastname: Yu
authors.forename: Spencer
authors.initials: S
authors.lastname: Hermanson
authors.forename: Krishne
authors.initials: K
authors.lastname: Gowda
authors.forename: Phillip
authors.initials: P
authors.lastname: Brumm
authors.forename: David
authors.initials: D
authors.lastname: Mead
authors.forename: Venkatesh
authors.initials: V
authors.lastname: Balan
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Dale