Auto-folding lipases for enantio-specific hydrolysis

描述(申请人提供)：该SBIR第一期项目的目标是 开发一个广泛适用的流程，以创建改进的类型 1.1和1.2用于制药和精细化学品生产的脂肪酶。 脂肪酶对化学合成很有用，因为它们能够分解 重要化学中间体的外消旋混合物。它们的使用是有限的 因为它们的成本很高。与这些酶相关的大部分成本是 归因于生产和提纯困难造成的独特 这些酶的表达和折叠要求。不同寻常的存在 折叠催化剂，在异源宿主中表达很差，需要 这些脂肪酶在周质中获得活跃的构象。最多的 当前脂肪酶生产方案的效率要求生产这些 脂肪酶特定的伴侣在单独的发酵中，然后被用来 变性脂肪酶在体外重新折叠，增加了相当大的成本。我们会用DNA 将确定自动折叠突变体的序列，使类似的 突变将在其他商业上重要的脂肪酶中重新产生。 此外，我们将创造融合了亲和力标签的基因融合 提高了脂肪酶的纯度，使脂肪酶和 伴侣蛋白是一种单一的多肽。 建议的商业应用： 在该项目实施期间开发的新型酶和工艺将立即被用作生产对映体纯化学中间体和其他工业工艺的生物催化剂。

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.