DEVELOPMENT OF AMIDASES FOR B-LACTAM MODIFICATION

生产了几种临床上重要的β-内酰胺类抗生素 通过D-（-）-α-氨基苯乙酸与 多步化学过程中的青霉烯和头孢烯核。 一个 用于合成这些抗生素的有效酶促方法 将导致增加的产品产量和降低的成本 生产 先前描述的酶催化这些化合物的合成。 抗生素的使用率不足以进行商业化生产。 第一阶段研究的目的是证明酶能够 以高速率催化这些抗生素的合成， 由青霉素酰胺酶衍生而来。 为了实现这一目标，变种人将 在对携带克隆基因的细胞进行诱变后选择 对于青霉素酰胺酶，其基于改进的水解 具有D-（-）-α-氨基苯乙酰基部分的模型底物。 从这些突变体中纯化的酰胺酶催化 模型底物的水解和D-（- ）-α-氨基苯乙酸与6-氨基青霉烷酸反应， 生产氨苄青霉素将被确定和比较的比率 观察野生型酰胺酶。 第二阶段研究的目标是重复使用 在第一阶段开发的方法，以获得能够 以商业上有用的速率合成氨苄青霉素和头孢氨苄。

Several clinically important beta-lactam antibiotics are produce through condensation of D-(-)-alpha-aminophenylacetic acid with penam and cephem nuclei in multi-step chemical processes. An efficient enzymatic process for the synthesis of these antibiotics would lead to increased product yields and decreased cost of production. Previously described enzymes catalyze the synthesis of these antibiotics at rates that are inadequate for commercial processes. The aim of Phase I research is to demonstrate that enzymes able to catalyze the synthesis of these antibiotics at high rates can be derived from penicillin amidase. To achieve this, mutants will be selected following mutagenesis of cells that carry the cloned gene for penicillin amidase based on an improved ability to hydrolyze a model substrate with a D-(-)-alpha-aminophenylacetyl moiety. The rates at which amidase purified from these mutants catalyze the hydrolysis of the model substrate and the condensation of D-(- )-alpha-aminophenylacetic acid with 6-aminopenicillanic acid to produce ampicillin will be determined and compared to the rates observed with the wild-type amidase. The aim of Phase II research will be the reiterative use of the methods developed in Phase I to obtain enzymes able to synthesize ampicillin and cephalexin at commercially useful rates.