Fuel From Self-Degrading Bioengineering Packaging
Abstract
Research focused on developing an enzyme that could be embedded within a single-use plastic and convert the plastic after use to products of value such as fuel to troops in the field. Polylactic acid (PLA) was selected for this work since it is commercially available, degradable by an enzyme (Proteinase K), and is known to have properties compatible with those required by the military for many single-use materials. Engineering of Proteinase K for PLA degradation required a low throughput assay that accurately selects variants that offer improvements in a wide range of enzyme characteristics including the structure of products formed, activity at low pH, and thermal stability. This required a new technological approach to protein engineering. Using an integrated substitution choice method and sequence-activity models we obtained dramatic improvements in enzyme performance while screening only 100 variants. The small number of variants required allowed us to accurately assess the distribution of products formed and enzyme behavior while varying different physical parameters of importance for final process design such as temperature and pH. A new LC-MS method was developed that provided information on degradation product chain length, concentration, and stereochemical composition. Improvements in enzyme performance relative to the wild-type achieved by this new protein engineering method include identification of variants with: i) 12-fold improvement at low pH (5.5), ii) 5-fold improvement in dimer yield, and iii) 2-fold improvement in heat stability at 650C. This contrasts to other methods such as directed evolution that requires screening of thousands of variants per cycle to obtain improvement factors normally 1.5 to 2-fold.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 01, 2005
- Accession Number
- ADA430101
Entities
People
- Richard A. Gross