DEVELOPMENT OF AMIDASES FOR B-LACTAM MODIFICATION

用于 B-内酰胺修饰的酰胺酶的开发

• 批准号: 3498043
• 负责人: LARRY J FORNEY
• 金额: $ 5万
• 依托单位: SYNERGEN, INC.
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-03-01 至 1988-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3498043
• 关键词: Escherichia coli; acid base balance; amidohydrolases; ampicillin; catalyst; chemical condensation; drug design /synthesis /production; enzyme mechanism; enzyme substrate; genetic manipulation; high performance liquid chromatography; hydrolysis; molecular cloning; mutant; plasmids

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.

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 多步骤化学过程中的脑南和头孢子核。一个 这些抗生素的高效酶促合成工艺 将导致产品产量的增加和成本的降低 制作。 前面描述的酶催化合成这些 抗生素的价格不足以满足商业流程的需求。 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 来源于青霉素酰胺酶。为了实现这一点，变种人将被 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，基于提高的水解酶A的能力 含有D-(-)-α-氨基苯乙酰部分的模型底物。 从这些突变体中提纯的酰胺酶催化的速率 模型底物的水解和D-(-)的缩合 )-α-氨基苯乙酸与6-氨基青霉烷酸 生产的氨苄西林将被测定并与 用野生型酰胺酶观察。 第二阶段研究的目标将是反复使用 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西林和头孢氨苄。