title: Engineering highly thermostable xylanase variants using an enhanced combinatorial library method.
abstract: A new directed evolution method was used to enhance the thermostability of the wild-type GH11 xylanase 2 (known as BD-11) from Hypocrea jecorina (Trichoderma reesei). Both Look-Through Mutagenesis (LTM™), which is a method for rapidly screening selected positions in the protein sequence for amino acids that introduce favorable properties, and Combinatorial Beneficial Mutagenesis (CBM™), which is a method for identifying the best ensemble of individual mutations, were employed to enhance the stability of an enzyme that has been thoroughly engineered by various means during the past 20 years. A diverse set of novel mutations was discovered, including N71D, Y73G, T95G and Y96Q. When these mutations were combined into a single construct (Hjx-81), the purified protein was active even after heating at 100°C for 20 min. This time-effective method should be generally applicable for quickly improving the physico-chemical properties of other industrial and therapeutic enzymes in only several months time.
authors.affiliation: Protelica 26136 Research Road, Hayward, CA 94545, USA. chokanson@protelix.com
authors.forename: Craig A
authors.initials: CA
authors.lastname: Hokanson
authors.forename: Guido
authors.initials: G
authors.lastname: Cappuccilli
authors.forename: Tatjana
authors.initials: T
authors.lastname: Odineca
authors.forename: Marino
authors.initials: M
authors.lastname: Bozic
authors.forename: Craig A
authors.initials: CA
authors.lastname: Behnke
authors.forename: Michael
authors.initials: M
authors.lastname: Mendez
authors.forename: William J
authors.initials: WJ
authors.lastname: Coleman
authors.forename: Roberto
authors.initials: R
authors.lastname: Crea
mesh_terms: D000595:Amino Acid Sequence; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D019020:Directed Molecular Evolution / Q000379:methods; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010843:Pichia; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases