SBIR-STTR Award

Enzyme catalyzed processes offer significant advantages over traditional chemical methods in organic syntheses, including superior efficiency, stereoselectivity, and specificity. However, soluble and conventionally immobilized enzymes are often inactive under typical laboratory or manufacturing conditions, which may include high temperatures, extremes of pH, near-anhydrous organic solvents, and mixed aqueous-organic solvents. Enzyme crystals, cross-linked using a bifunctional reagent such as glutaraidehyde, constitute a novel form of enzyme immobilization. Such cross-linked enzyme crystals (CLECS) are superior to conventionally immobilized enzymes in many respects. For example, the crystal lattice interactions, supplemented by chemical cross-links, provide the individual enzyme molecules within a CLEC with an enhanced stability that enables CLECs to function in harsh environments (such as elevated temperatures and organic solvents) that would not otherwise be compatible with catalytic activity. These features make CLECS valuable as stereospecific catalytic reagents. CLECs of pig liver esterase (PLE) and pig pancreatic lipase (PPL) are being investigated to demonstrate the practical feasibility of CLECs as laboratory catalysts in organic syntheses. Esterase and lipase enzymes were chosen for their superior catalytic characteristics compared with current chemical methods for stereospecific hydrolysis and reduction.The potential commercial application as described by the awardee: CLECs could replace the complex chemical methods now used to produce chiral compounds, and be employed as stereospecific catalysts in laboratory synthesis. CLECs may also be employed in high volume, industrial scale preparation of chiral compounds for use in the pharmaceutical, food, agrochemical, and specialty chemical industries, including aspartame, acrylamide, and the synthetic penicillins and cephalosporins.

The research described here focuses on human cDNAs and genes that encode FKBP (FK506 binding protein) and FKBP-related proteins. FKBP is an abundant cytosolic protein that binds with high affinity to the immunosuppressants FK506 and rapamycin, compounds that inhibit T lymphocyte signal transduction pathways. FKBP is an attractive candidate for the functional in vivo receptor of these drugs, an obvious key modulator of T cell activation. This work draws strongly upon techniques of nucleic acid hybridization, DNA sequencing and PCR analysis. Specifically we aim to: (1) search for additional classes of human FKBP transcripts, (2) sequence all FKBP exons within the human gene(s) and determine the splicing patterns that produce the FKBP transcript family, and (3) search for human cDNAs that encode functional and structural analogs of FKBP. These goals address molecular and biochemical aspects of FKBP function, could define diagnostic tools to monitor patient response to FKS06 or FK506-like immunosuppressants, and could provide marketable probes for identifying additional proteins involved in regulating T cell activation. The research also could identify transcripts that encode cellular receptors responsible for FKSO6/rapamycin-like immunomodulatory actions or receptors that modulate otheractions in a similar biochemical manner, thereby defining additional targets for novel therapeutics.Awardee's statement of the potential commercial applications of the research:Identifying all classes of FKBP transcripts should provide diagnostic molecules for localizing tissue and cell-specific FKBP messenger RNA'S. This would provide technology to determine whether the emerging spectrum of FK506-induced effects correlates with the presence or absence of specific FKBP transcripts and could provide a basis for monitoring patient response to FK506 or FKS06-like immunosuppressants. Marketable probes to identify other proteins involved in T cell activation and additional therapeutic targets for immunomodulation also could result.National Institute of Allergy and Infectious Diseases (NIAID)