Engineering highly thermostable xylanase variants using an enhanced combinatorial library method

• Published in: Protein engineering, design and selection Vol. 24; no. 8; pp. 597 - 605
• Main Authors: Hokanson, Craig A., Cappuccilli, Guido, Odineca, Tatjana, Bozic, Marino, Behnke, Craig A., Mendez, Michael, Coleman, William J., Crea, Roberto
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-08-2011
• Subjects: Amino Acid Sequence; Combinatorial Chemistry Techniques - methods; Combinatorial libraries; directed evolution; Directed Molecular Evolution - methods; Endo-1,4-beta Xylanases - chemistry; Endo-1,4-beta Xylanases - genetics; Endo-1,4-beta Xylanases - metabolism; Enzyme Stability; Enzymes; Hot Temperature; Hypocrea jecorina; Kinetics; Molecular Sequence Data; Mutagenesis; Mutation; Physicochemical properties; Pichia; Protein Engineering - methods; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Alignment; Thermal stability; Trichoderma - enzymology; Trichoderma - genetics; Xylan endo-1,3- beta -xylosidase; thermostability; Look-Through Mutagenesis; Combinatorial beneficial mutagenesis; directed evolution
• ISSN: 1741-0126; 1741-0134
• DOI: 10.1093/protein/gzr028

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.