| Autor: |
Hadi T; GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania, 19426, USA., Nozzi N; GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania, 19426, USA., Melby JO; GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania, 19426, USA., Gao W; GE Global Research, One Research Circle, Niskayuna, NY, 12309, USA., Fuerst DE; GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania, 19426, USA., Kvam E; GE Global Research, One Research Circle, Niskayuna, NY, 12309, USA. kvame@ge.com. |
| Abstrakt: |
The ability to quickly and easily assess the activity of large collections of enzymes for a desired substrate holds great promise in the field of biocatalysis. Cell-free synthesis, although not practically amenable for large-scale enzyme production, provides a way to accelerate the timeline for screening enzyme candidates using small-scale reactions. However, because cell-free enzyme synthesis requires a considerable amount of template DNA, the preparation of high-quality DNA "parts" in large quantities represents a costly and rate-limiting prerequisite for high throughput screening. Based on time-cost analysis and comparative activity data, a cell-free workflow using synthetic DNA minicircles and rolling circle amplification enables comparable biocatalytic activity to cell-based workflows in almost half the time. We demonstrate this capability using a panel of sequences from the carbon-nitrogen hydrolase superfamily that represent possible green catalysts for synthesizing small molecules with less waste compared to traditional industrial chemistry. This method provides a new alternative to more cumbersome plasmid- or PCR-based protein expression workflows and should be amenable to automation for accelerating enzyme screening in industrial applications. |
