title: Discovery of pectin-degrading enzymes and directed evolution of a novel pectate lyase for processing cotton fabric.
abstract: There is a growing need in the textile industry for more economical and environmentally responsible approaches to improve the scouring process as part of the pretreatment of cotton fabric. Enzymatic methods using pectin-degrading enzymes are potentially valuable candidates in this effort because they could reduce the amount of toxic alkaline chemicals currently used. Using high throughput screening of complex environmental DNA libraries more than 40 novel microbial pectate lyases were discovered, and their enzymatic properties were characterized. Several candidate enzymes were found that possessed pH optima and specific activities on pectic material in cotton fibers compatible with their use in the scouring process. However, none exhibited the desired temperature characteristics. Therefore, a candidate enzyme was selected for evolution. Using Gene Site Saturation Mutagenesistrade mark technology, 36 single site mutants exhibiting improved thermotolerance were produced. A combinatorial library derived from the 12 best performing single site mutants was then generated by using Gene Reassemblytrade mark technology. Nineteen variants with further improved thermotolerance were produced. These variants were tested for both improved thermotolerance and performance in the bioscouring application. The best performing variant (CO14) contained eight mutations and had a melting temperature 16 degrees C higher than the wild type enzyme while retaining the same specific activity at 50 degrees C. Optimal temperature of the evolved enzyme was 70 degrees C, which is 20 degrees C higher than the wild type. Scouring results obtained with the evolved enzyme were significantly better than the results obtained with chemical scouring, making it possible to replace the conventional and environmentally harmful chemical scouring process.
authors.affiliation: Diversa Corporation, San Diego, California 92121, USA.
authors.forename: Arne I
authors.initials: AI
authors.lastname: Solbak
authors.forename: Toby H
authors.initials: TH
authors.lastname: Richardson
authors.forename: Ryan T
authors.initials: RT
authors.lastname: McCann
authors.forename: Katie A
authors.initials: KA
authors.lastname: Kline
authors.forename: Flash
authors.initials: F
authors.lastname: Bartnek
authors.forename: Geoff
authors.initials: G
authors.lastname: Tomlinson
authors.forename: Xuqiu
authors.initials: X
authors.lastname: Tan
authors.forename: Lilian
authors.initials: L
authors.lastname: Parra-Gessert
authors.forename: Gerhard J
authors.initials: GJ
authors.lastname: Frey
authors.forename: Mircea
authors.initials: M
authors.lastname: Podar
authors.forename: Peter
authors.initials: P
authors.lastname: Luginbühl
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Gray
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Mathur
authors.forename: Dan E
authors.initials: DE
authors.lastname: Robertson
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Burk
authors.forename: Geoffrey P
authors.initials: GP
authors.lastname: Hazlewood
authors.forename: Jay M
authors.initials: JM
authors.lastname: Short
authors.forename: Janne
authors.initials: J
authors.lastname: Kerovuo
mesh_terms: D001419:Bacteria / Q000145:classification / Q000201:enzymology; D032561:Cotton Fiber; D019020:Directed Molecular Evolution; D015723:Gene Library; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; C023911:pectate lyase