title: SCHEMA recombination of a fungal cellulase uncovers a single mutation that contributes markedly to stability.
abstract: A quantitative linear model accurately (R(2) = 0.88) describes the thermostabilities of 54 characterized members of a family of fungal cellobiohydrolase class II (CBH II) cellulase chimeras made by SCHEMA recombination of three fungal enzymes, demonstrating that the contributions of SCHEMA sequence blocks to stability are predominantly additive. Thirty-one of 31 predicted thermostable CBH II chimeras have thermal inactivation temperatures higher than the most thermostable parent CBH II, from Humicola insolens, and the model predicts that hundreds more CBH II chimeras share this superior thermostability. Eight of eight thermostable chimeras assayed hydrolyze the solid cellulosic substrate Avicel at temperatures at least 5 degrees C above the most stable parent, and seven of these showed superior activity in 16-h Avicel hydrolysis assays. The sequence-stability model identified a single block of sequence that adds 8.5 degrees C to chimera thermostability. Mutating individual residues in this block identified the C313S substitution as responsible for the entire thermostabilizing effect. Introducing this mutation into the two recombination parent CBH IIs not featuring it (Hypocrea jecorina and H. insolens) decreased inactivation, increased maximum Avicel hydrolysis temperature, and improved long time hydrolysis performance. This mutation also stabilized and improved Avicel hydrolysis by Phanerochaete chrysosporium CBH II, which is only 55-56% identical to recombination parent CBH IIs. Furthermore, the C313S mutation increased total H. jecorina CBH II activity secreted by the Saccharomyces cerevisiae expression host more than 10-fold. Our results show that SCHEMA structure-guided recombination enables quantitative prediction of cellulase chimera thermostability and efficient identification of stabilizing mutations.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: Pete
authors.initials: P
authors.lastname: Heinzelman
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Snow
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Smith
authors.forename: Xinlin
authors.initials: X
authors.lastname: Yu
authors.forename: Arvind
authors.initials: A
authors.lastname: Kannan
authors.forename: Kevin
authors.initials: K
authors.lastname: Boulware
authors.forename: Alan
authors.initials: A
authors.lastname: Villalobos
authors.forename: Sridhar
authors.initials: S
authors.lastname: Govindarajan
authors.forename: Jeremy
authors.initials: J
authors.lastname: Minshull
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology; D001665:Binding Sites / Q000235:genetics; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019295:Computational Biology / Q000379:methods; D004795:Enzyme Stability / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D035901:Hypocrea / Q000201:enzymology; D016014:Linear Models; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase