title: Engineered thermostable fungal Cel6A and Cel7A cellobiohydrolases hydrolyze cellulose efficiently at elevated temperatures.
abstract: Thermostability is an important feature in industrial enzymes: it increases biocatalyst lifetime and enables reactions at higher temperatures, where faster rates and other advantages ultimately reduce the cost of biocatalysis. Here we report the thermostabilization of a chimeric fungal family 6 cellobiohydrolase (HJPlus) by directed evolution using random mutagenesis and recombination of beneficial mutations. Thermostable variant 3C6P has a half-life of 280 min at 75°C and a T(50) of 80.1°C, a ~15°C increase over the thermostable Cel6A from Humicola insolens (HiCel6A) and a ~20°C increase over that from Hypocrea jecorina (HjCel6A). Most of the mutations also stabilize the less-stable HjCel6A, the wild-type Cel6A closest in sequence to 3C6P. During a 60-h Avicel hydrolysis, 3C6P released 2.4 times more cellobiose equivalents at its optimum temperature (T(opt)) of 75°C than HiCel6A at its T(opt) of 60°C. The total cellobiose equivalents released by HiCel6A at 60°C after 60 h is equivalent to the total released by 3C6P at 75°C after ~6 h, a 10-fold reduction in hydrolysis time. A binary mixture of thermostable Cel6A and Cel7A hydrolyzes Avicel synergistically and released 1.8 times more cellobiose equivalents than the wild-type mixture, both mixtures assessed at their respective T(opt). Crystal structures of HJPlus and 3C6P, determined at 1.5 and 1.2 Å resolution, indicate that the stabilization comes from improved hydrophobic interactions and restricted loop conformations by introduced proline residues.
authors: Division of Chemistry and Chemical Engineering, California Institute of Technology 210-41, Pasadena, CA 91125, USA.
authors: Indira
authors: I
authors: Wu
authors: Frances H
authors: FH
authors: Arnold
mesh_terms: D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution / Q000379:methods; D004795:Enzyme Stability; D006868:Hydrolysis; D035901:Hypocrea / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D020037:Sordariales / Q000201:enzymology / Q000235:genetics; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase