title: A high-throughput screening system for the evaluation of biomass-hydrolyzing glycoside hydrolases.
abstract: To implement a protein engineering strategy for the improvement of enzyme performance on biomass, a straightforward, robust high-throughput method was devised and tested with recombinant GH11 xylanase as acting on wheat straw. The method requires automated liquid handling equipment, but avoids the need for specialized milling and powder weighing devices and the use of labour intensive steps such as manual cutting of pipette tips. After expression in Escherichia coli cells grown in microtiter plates, recombinant xylanase was released into the culture medium and used directly for biomass hydrolysis. Reactions were monitored using a micro-3,5-dinitrosalicylic acid assay. The cumulative error of the method was less than 15%. To validate the method, randomly generated xylanase mutants were analyzed. This allowed the detection of one mutant, which produced a 74% increase in hydrolysis compared to the parental enzyme. Closer analysis revealed that this increase in activity was correlated with a twofold increase in xylanase expression.
authors.affiliation: Université de Toulouse, INSA, UPS, INP, France.
authors.forename: Letian
authors.initials: L
authors.lastname: Song
authors.forename: Sandrine
authors.initials: S
authors.lastname: Laguerre
authors.forename: Claire
authors.initials: C
authors.lastname: Dumon
authors.forename: Sophie
authors.initials: S
authors.lastname: Bozonnet
authors.forename: Michael J
authors.initials: MJ
authors.lastname: O'Donohue
mesh_terms: D001407:Bacillus / Q000201:enzymology; D001483:Base Sequence; D001681:Biological Assay / Q000295:instrumentation; D018533:Biomass; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D012459:Salicylates / Q000378:metabolism; D014908:Triticum / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D012459:Salicylates; C027011:3,5-dinitrosalicylic acid; D006026:Glycoside Hydrolases; D043364:Endo-1,4-beta Xylanases