9561829 Stein This Small Business Innovation Research Phase I project will determine the feasibility of using a novel cloning and automated screening approach to assemble a library of genes encoding cold-adapted lipase. The approach is based on the construction of "environmental libraries" which represent the collective genomes of naturally occurring prokaryotes archived in cloning vectors that can be propagated in E. coli or other suitable host. Because the cloned DNA is initially extracted directly from environmental samples, the libraries are not limited to the small fraction of prokaryotes that can be grown in pure culture nor are they biased towards a few rapidly growing species. These libraries are then screened using fluor-linked substrates and high throughput robotics to identify clones expressing the activity of interest. In addition, specialized cloning vectors will be employed that allow the rapid identification of cloned DNA fragments containing signal sequences which target the gene product for export. Such signals frequently precede genes encoding hydrolytic enzymes such as lipases. These approaches will be used to screen environmental libraries constructed from DNA extracted from a unique environment: the bacterial mats encrusting lipid-rich whale skeletons in deep sea basins. Preliminary work suggests that these sites harbor a diverse bacterial community that subsist on the copious amounts of complex lipids that exude from the whale bones over a period of many years. By coupling the environmental library approach to high throughput automated screening, hundreds of thousands of recombinant clones from the whale bone bacteria will be screened for lipase activity. Candidate genes encoding lipases that display high activity at low temperatures yet remain stable at moderate temperatures will be overexpressed for evaluation as detergent additives. In addition, other unique lipases found will be evaluated for use in other industrial processes which include thos e in food and beverage, pharmaceutical, waste systems, chemical and medical industries.
