Auto-folding lipases for enantio-specific hydrolysis

描述（由申请人提供）：本阶段SBIR项目的目标是 开发一种广泛适用的工艺，以创造改进的类型， 1.1和1.2脂肪酶用于医药和精细化学品生产。 脂肪酶可用于化学合成，因为它们能够分解 重要化学中间体的外消旋混合物。它们的用途有限 因为它们的成本很高。与这些酶相关的大部分成本是 由于独特的生产和纯化困难， 这些酶的表达和折叠要求。不寻常的存在 折叠催化剂，在异源宿主中表达差， 这些脂肪酶在周质中获得活性构象。最 当前脂肪酶生产方案的有效性需要生产这些 脂肪酶特异性伴侣在单独的发酵，然后用于 变性脂肪酶的体外复性，增加了相当大的成本。我们将DNA 将确定自动折叠突变体的序列，使得类似的 在其他商业上重要的脂肪酶中重建突变。 此外，我们将创建基因融合，将亲和标签， 改善脂肪酶纯化，并允许脂肪酶和 伴侣蛋白作为单一多肽。 拟定商业应用： 在该项目执行过程中开发的新型酶和工艺将作为生物催化剂立即应用于生产对映体纯化学中间体和其他工业过程。

DESCRIPTION (provided by applicant): The goal of this Phase I SBIR project is to develop a broadly applicable process for purpose of creating improved type 1.1 and 1.2 lipases used in pharmaceuticals and fine chemicals production. Lipases are useful for chemical synthesis because of their ability to resolve racemic mixtures of important chemical intermediates. Their use is limited because of their high cost. Much of cost associated with these enzymes is attributed to production and purification difficulties caused by the unique expression and folding requirements of these enzymes. The presence of unusual folding catalysts, poorly expressed in heterologous hosts, is required for these lipases to attain active conformations in the periplasm. The most efficient of current lipase production protocols require production of these lipase specific chaperones in separate fermentations, which are then used to refold the denatured lipase in vitro, adding a considerable cost. We will DNA sequences of the auto-folding mutants will be determined, enabling similar mutations to be recreated in other commercially important lipases. Additionally, we will create gene fusions that incorporate affinity tags to improve lipase purification, and allow the production of both the lipase and chaperone as a single polypeptide. PROPOSED COMMERCIAL APPLICATION: The novel enzymes and processes developed during the performance of this project will find immediate applications as biocatalysts for the production of enantio-pure chemical intermediates, and other industrial processes.