BIOSYNTHESIS OF PHAS, BIODEGRADABLE POLYMERS

生物可降解聚合物相的生物合成

• 批准号: 2186718
• 负责人: JOANNE STUBBE
• 金额: $ 20.25万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-03-01 至 1998-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2186718
• 关键词: acyltransferase; bacterial proteins; biotransformation; enzyme structure; enzyme substrate analog; microorganism metabolism; polymers; protein purification

The potential for utilizing biological systems as a source of biodegradable thermoplastics is becoming increasingly attractive given the problems with disposal of traditional oil based polymers. Biodegradable polymers are now being investigated as potential vehicles for controlled release of drugs, artificial skin and sutures. Polyhydroxyalkanoates (PHAs) are natural products which can constitute up to 80% of the dry cell weight of bacteria, when the bacteria are subjected to deprivation of O2, nitrogen, phosphates. These biological polyesters are composed of a variety of 3R hydroxyfatty acids (from C4 to C12) resulting in polymers of a wide range of molecular weights, depending on the organism and the C- source available for growth. We have recently cloned, sequenced, overexpressed, and purified the PHA synthase from Alcaligenes eutrophus. This enzyme catalyzes formation in vivo of polyhydroxybutyrates (PHBs) and polyhydroxyvalerates (PHVs). We have also cloned and sequenced the synthase from Pseudomonas oleovorans. Preliminary results suggest that we have been successful in expression of this protein as well. This enzyme polymerizes (C6-C12) 3R-hydroxyfatty acids. The present proposal seeks support for characterization of the A eutrophus synthase as well as purification of the P oleovorans synthase. The substrate specificity of these proteins will be investigated in detail as will their unique mechanisms of initiation, elongation and termination. Both protein analogs (site directed mutants) and substrate analogs (mechanism based inhibitors) will be used as probes. The mechanism of processivity will also be investigated. These studies should provide new insight into the mechanisms of synthases that catalyze formation of homopolymers. The information learned is crucial to the design, using biotechnological advances, of synthases which can make homo and co.polymers which will be useful, due to their biodegradability and unique thermoplastic and elastic properties, to the community in general. Recently these polymers have been found in eucaryotic membranes as well as in human plasma. The function and biosynthesis of these polymers promises to be interesting.

利用生物系统作为来源的潜力 鉴于 处置传统石油聚合物的问题。可生物降解 现在正在研究聚合物作为受控的潜在工具 释放药物，人造皮肤和缝合线。多羟基烷烃 （PHA）是天然产品，最多可以构成干细胞的80％ 细菌的重量，当细菌被剥夺O2时 氮，磷酸盐。这些生物聚酯由A组成 各种3R羟基脂肪酸（从C4到C12）导致聚合物的聚合物 取决于生物体和C-的分子量范围很广 可用于增长的来源。 我们最近克隆，测序， 过表达，并从Alcaligenes Eutrophus中纯化PHA合酶。 这种酶在多羟基丁酸（PHB）和 多羟基化（PHV）。我们还克隆并测序了 假单胞菌的合酶。初步结果表明我们 也成功地表达了该蛋白质。这种酶 聚合（C6-C12）3R-羟基脂肪酸。目前的提议寻求 支持A鲁aphus综合酶的表征以及 纯化的合酶的纯化。底物特异性 这些蛋白质将详细研究，它们的独特 启动，伸长和终止的机制。两个蛋白类似物 （位于位置的突变体）和底物类似物（基于机理的抑制剂） 将用作探针。 加工性机理也将是 调查。这些研究应提供有关机制的新见解 催化均聚物形成的合成酶。信息 学到的对于使用生物技术进步的设计至关重要 可以使Homo and Co.聚合物的合成酶由于 它们的生物降解性以及独特的热塑性和弹性特性， 整个社区。最近发现了这些聚合物 桉树膜以及人血浆中。功能和 这些聚合物的生物合成有望有趣。