Discovery of Pectin-degrading Enzymes and Directed Evolution of a Novel Pectate Lyase for Processing Cotton Fabric

• Published in: The Journal of biological chemistry 2005-03, Vol.280 (10), p.9431-9438
• Main Authors: Solbak, Arne I., Richardson, Toby H., McCann, Ryan T., Kline, Katie A., Bartnek, Flash, Tomlinson, Geoff, Tan, Xuqiu, Parra-Gessert, Lilian, Frey, Gerhard J., Podar, Mircea, Luginbühl, Peter, Gray, Kevin A., Mathur, Eric J., Robertson, Dan E., Burk, Mark J., Hazlewood, Geoffrey P., Short, Jay M., Kerovuo, Janne
• Format: Article
• Language: English
• Subjects: Bacteria - classification; Bacteria - enzymology; Cotton Fiber; Directed Molecular Evolution; Gene Library; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Phylogeny; Polysaccharide-Lyases - chemistry; Polysaccharide-Lyases - genetics; Polysaccharide-Lyases - metabolism; Protein Conformation; Recombinant Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M411838200

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 Mutagenesis™ 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 Reassembly™ 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 °C higher than the wild type enzyme while retaining the same specific activity at 50 °C. Optimal temperature of the evolved enzyme was 70 °C, which is 20 °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.