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.
Highlights
Before cotton fabric or yarn can be efficiently dyed it needs to be pretreated to remove materials that inhibit dye binding
The majority of the non-cellulosic compounds are found in the primary cell wall, which is a complex lattice of pectin, protein, cellulose, hemicellulose, and waxes
The ability to rapidly access and screen DNA derived from discrete biotopes to discover novel enzymes was key to this study; the targeted discovery effort yielded over 50 novel pectinolytic clones
Summary
Fied DNA was used to make highly complex expression libraries This methodology was developed to circumvent the inefficiency encountered in trying to grow all of the organisms present in the environmental samples. All possible single site mutants of the wild type enzyme were created using the Gene Site Saturation Mutagenesis (GSSM) technology [9]. This comprehensive technique introduced point mutations into every position within the target gene, using degenerate primer sets containing 32 codons to generate a complete library of variants. Acid substitutions, a library representing all possible combinations of the mutations was created using the Gene Reassembly technology that could be screened to identify the optimal enzyme for this application
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