Resolutions in Branched Alkanes using Lipase in Beads

描述（由申请人提供）：本研究的长期目标是研究和表征用于化学反应的新鲜和冷冻干燥的大分子负载多糖基水凝胶珠，以及它们在用于治疗目的的大分子递送中的应用。这些小珠将用于治疗性肽和蛋白质的递送，特别是在口服给药后将这些药物特定部位递送到结肠。利用包被酶可以研究各种化学反应。长期目标包括将这些微球应用于催化性能良好的反应中，这样就可以研究微球的性能（而不是反应）。提出的研究将脂肪酶纳入这些微球中，以便于回收和随后的递送。溶剂为直链烷烃和甲基支链烷烃，以验证支链能提高脂肪酶活性和对映选择性的假设。短期目标是通过游离的或包裹在壳聚糖珠中的脂肪酶的对映选择性酯化反应，分离一种外消旋混合物布洛芬的药理学活性对映体。分离活性对映体将成为一项监管要求，因为只给药活性对映体可以降低毒性和剂量。使用包封的脂肪酶进行分离，提供了快速的对映选择性反应；重复应用，因为酶可以回收；并且在合成序列中具有经济的终端分辨率。在第一项研究中，由已知活度的烷烃水组成的溶剂体系将被优化为最快的反应和最高的对映体选择性。Michaelis-Menten参数将被评估和比较。在第二项研究中，制备了脂酶负载的壳聚糖水凝胶珠。成功的坚固的球形基质可以捕获酶，对活性的干扰最小，并允许底物和产物扩散。通过测量衬底在衬底内的扩散系数来研究衬底的渗透率。在第三项研究中，将包被酶的反应参数与游离酶的反应参数进行比较。在第三项研究中，还将评估微球的稳定性、脂肪酶包埋的效率以及作为重复应用函数的脂肪酶活性降低。

DESCRIPTION (provided by applicant): The long-term goals of this research are to investigate and characterize fresh and freeze-dried macromolecule-loaded polysaccharide-based hydrogel beads for applications in chemical reactions, and their application in macromolecule delivery for therapeutic purposes. The use of these beads for therapeutic peptide and protein delivery will be pursued, in particular for site specific delivery of these agents to the colon following oral administration. Various chemical reactions can be investigated using entrapped enzymes. The long-term goals include applications of these beads in reactions where the catalysis is well characterized, such that the bead performance (and not the reaction) is studied. The proposed research incorporates lipase into these beads for easy recovery and subsequent delivery. The solvents are straight chain and methyl-branched alkanes to test the hypothesis that branching can enhance the activity and enantioselectivity of lipase. The short-term goal is the resolution of the pharmacologically active enantiomer of a drug that is a racemic mixture, ibuprofen, by enantioselective esterification by a lipase that is free or entrapped in the chitosan bead. Isolation of the active, enantiomer will become a regulatory requirement because administration of only the active enantiomer can reduce the toxicity and dose. Resolution using an entrapped lipase offers a rapid, enantioselective reaction; repeated applications since the enzyme can be recovered; and economical terminal resolution in the synthesis sequence. In the first study, the solvent system, consisting of water at a known activity in an alkane, will be optimized for the fastest reactions and the highest enantioselectivity. Michaelis-Menten parameters will be evaluated and compared. In the second study, lipase-loaded chitosan hydrogel beads are fabricated. The successful rugged, spherical matrix can entrap the enzyme, minimally interfere with activity, and allow substrate and product diffusion. Permeability of the bead will be investigated by measuring the substrate diffusion coefficient within the bead. Reaction parameters of the entrapped enzyme will be compared to those of the free enzyme in the third study. The stability of the bead, the efficiency of the lipase entrapment, and the reduction in lipase activity as a function of repeated applications will also be evaluated in the third study.