Resolutions in Branched Alkanes using Lipase in Beads

描述(申请人提供)：这项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微珠，以及它们在治疗目的的大分子输送中的应用。将继续使用这些珠子进行治疗性多肽和蛋白质递送，特别是在口服给药后将这些药物定点递送到结肠。可以用包埋的酶来研究各种化学反应。长期目标包括将这些小球应用于催化性能良好的反应，这样就可以研究小球的性能(而不是反应)。这项拟议的研究将脂肪酶加入到这些珠子中，以便于回收和随后的传递。以直链和甲基支链烷烃为溶剂，验证了支化可以提高脂肪酶活性和对映体选择性的假设。短期目标是通过游离或包裹在壳聚糖珠中的脂肪酶进行对映选择性酯化，拆分外消旋混合物布洛芬的药理活性对映体。分离活性对映体将成为一项法规要求，因为只给药活性对映体可以减少毒性和剂量。使用包埋脂肪酶的拆分提供了快速的对映选择性反应；由于酶可以回收而重复使用；在合成序列中提供了经济的终端拆分。在第一个研究中，由烷烃中已知活度的水组成的溶剂体系将进行优化，以实现最快的反应和最高的对映选择性。将评估和比较Michaelis-Menten参数。在第二项研究中，制备了脂肪酶负载的壳聚糖水凝胶微珠。成功的坚固的球形基质可以包埋酶，最大限度地减少对活性的干扰，并允许底物和产品扩散。微珠的渗透性将通过测量微珠内的基材扩散系数来进行研究。在第三项研究中，将比较包埋酶和游离酶的反应参数。在第三项研究中，还将评估珠子的稳定性、脂肪酶包埋的效率以及作为重复使用的函数的脂肪酶活性的降低。

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.