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.
Subject(s)
Cotton Fiber , Polysaccharide-Lyases/metabolism , Bacteria/classification , Bacteria/enzymology , Directed Molecular Evolution , Gene Library , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Phylogeny , Polysaccharide-Lyases/chemistry , Polysaccharide-Lyases/genetics , Protein Conformation , Recombinant Proteins/metabolismABSTRACT
Gene site saturation mutagenesis (GSSM) technology is applied for the directed evolution of a nitrilase. The nitrilase effectively catalyzes the desymmetrization of the prochiral substrate 3-hydroxyglutaronitrile to afford (R)-4-cyano-3-hydroxybutyric acid, a precursor to the valuable cholesterol-lowering drug Lipitor. The discovered wild-type enzyme effectively performs the reaction at the industrially relevant 3 M substrate concentration but affords a product enantiomeric excess of only 87.6% ee. Through GSSM, a mutagenesis technique that effects the combinatorial saturation of each amino acid in the protein to each of the other 19 amino acids, combined with a novel high-throughput mass spectroscopy assay, a number of improved variants were identified, the best of which is the Ala190His mutant that yields product enantiomeric excess of 98.5% at 3 M substrate loading and a volumetric productivity of 619 g L-1 d-1.
Subject(s)
Aminohydrolases/chemistry , Aminohydrolases/genetics , Amino Acid Substitution , Aminohydrolases/metabolism , Hydroxybutyrates/chemical synthesis , Mutagenesis, Site-Directed , StereoisomerismABSTRACT
Forty medical device companies have been surveyed to find out their attitudes, practices and expectations when working with contract manufacturers. This article reports on the findings and their plans for outsourcing in the future.
Subject(s)
Benchmarking , Equipment and Supplies , Interinstitutional Relations , Outsourced Services/organization & administration , Outsourced Services/statistics & numerical data , Commerce , Cooperative Behavior , Data Collection , Europe , Outsourced Services/economics , Outsourced Services/methods , Quality Control , United StatesABSTRACT
Medical device manufacturers and diagnostics companies have significantly increased their use of contract manufacturers to outsource production of components. This, the first of a two-part article, reviews strategic benefits and best practices in outsourcing.
