Development of a novel biotechnology for decomposing of plastic using PET (polyethylene terephthalate)-decomposing microorganisms

开发利用PET（聚对苯二甲酸乙二醇酯）分解微生物分解塑料的新型生物技术

• 批准号: 11555249
• 负责人: KIMURA Yoshiharu
• 金额: $ 8.83万
• 依托单位: Kyoto Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11555249/
• 关键词: polyethylene terephthalate; terephthalate; plastic; PET-decomposing microorganism; polyester; bio-degradation; 酵母; 分解機構; 分解酵素; 細菌; バイオリアクター; 生分解

The results in this research are as follows.1.Screening, isolation, and identification of PET-decomposing microorganisms : We have succeeded in getting six strains, which could decompose the non-crystalline PET fibers. Some of them were identified as Trichosporon sp.No.85, Sphingomonas paucimobilis No.77-2, and Arthrobacter sp.No.97-2, respectively.2.Purification of PET-decomposing enzyme from Arthrobacter sp.No, 97-2 : A 35-kDa protein was purified as a candidate for PET-decomposing enzymes from the culture filtrate of Arthrobacter sp.No.97-2. The amino-terminal sequence of the purified enzyme showed high similarity with that of flagellin (identity ; 63% and similarity ; 75%). We could not elucidate enzymatic properties, because of low specific activity.3.Development of new assay methods : Observations of the surface of PET fiber by a scanning electron microscopy and measurements of tensile strength of PET fiber have been used for assay of PET-decomposing activities. However, these assay methods take a lot of time, and also their data are not accurate. To improve assay methods for PET-decomposing enzymes, measurements of viscosity, gel permeation chromatography, and NMR spectroscopy were introduced. Four kinds of substrates were used : PET fiber, PET film, PET oligomer, and OEST (oligo (ethylene succinate-co-terephthalate). These assay methods turned out to be useful for assay of PET-decomposing activities.Some of our researches were not carried. The reasons are (1) it was very difficult to keep such PET-decomposing strains with high activities. These enzymes might be coded on plasmids. (2) There were no any adequate assay methods, which are reliable.

主要研究结果如下：1. PET纤维降解菌的筛选、分离和鉴定：成功地筛选出6株能降解非晶PET纤维的菌株。部分菌株经鉴定分别为丝孢酵母菌（Trichosporon sp.No.85）、少动鞘氨醇单胞菌（Sphingomonaspaucimobilis No.77 - 2）和节杆菌（Arthrobacter sp.No.97 - 2）。纯化酶的氨基端序列与鞭毛蛋白的氨基酸序列具有很高的相似性（同一性：63%和相似性：75%）。由于酶的比活力较低，无法阐明酶的性质。3.新的测定方法的开发：通过扫描电镜观察PET纤维的表面和测量PET纤维的拉伸强度来测定PET分解活性。然而，这些测定方法花费大量时间，并且它们的数据也不准确。为了改进PET分解酶的测定方法，引入了粘度测量、凝胶渗透色谱和NMR光谱。使用了四种基材：PET纤维、PET膜、PET低聚物和OEST（低聚（乙二醇琥珀酸酯-共-对苯二甲酸酯））。这些测定方法对PET降解活性的测定有一定的参考价值，但我们的一些研究工作尚未开展。其原因是（1）很难保持这种高活性的PET分解菌株。这些酶可能编码在质粒上。(2)没有任何适当的可靠的测定方法。

项目成果

A.El Salmawy and Y.Kimura: "Structure and Properties of Bicomponent Core-Sheath Fibers of Poly (ethylene terephthalate) and Biodegradable Aliphatic Polyesters."Text.Res.J.. 71 (2). 145-152 (2001)

A.El Salmawy 和 Y.Kimura：“聚对苯二甲酸乙二醇酯和可生物降解脂肪族聚酯双组分芯鞘纤维的结构和性能”Text.Res.J. 71 (2)。

A.El Salmawy, et al.: "Properties of a Core-Sheath Conjugate Fiber Composed of Poly (butylene terephthalate) and Poly (L-lactic acid)"Sen-I Gakkaishi. 56(5). 241-248 (2000)

A.El Salmawy等人：“由聚(对苯二甲酸丁二醇酯)和聚(L-乳酸)组成的芯鞘共轭纤维的特性”Sen-I Gakkaishi。

京都工芸繊維大学学長: "含芳香族ポリエステルの分解方法、含芳香族ポリエステル繊維の減量加工法、含芳香族ポリエステル繊維および含芳香族ポリエステルの分解菌"公開特許公報(A)特開2000-143863. (2000)

京都工业大学校长：“芳香族聚酯的分解方法、芳香族聚酯纤维的减重加工方法、芳香族聚酯纤维及芳香族聚酯降解菌” 公开专利公报(A)2000-143863(2000)

A.El Salmawy, et al.: "Structure and Properties of Bicomponent Core-Sheath Fibers of Poly (ethylene terephthalate) and Biodegradable Aliphatic Polyesters"Text.Res.J.. 71(2). 145-152. (2001)

A.El Salmawy 等人：“聚对苯二甲酸乙二醇酯和可生物降解脂肪族聚酯的双组分芯鞘纤维的结构和性能”Text.Res.J. 71(2)。

President of Kyoto Institute of Technology: "A method for the composting polyesters containing aromatic moieties, a denier reduction method of fiber, and microorganisms having activity of decomposing the polyester"Japan Kokai Tokkyo Koho. No.2000-143863.

京都工业大学校长：“含有芳香族部分的聚酯的堆肥方法、纤维纤度降低方法以及具有分解聚酯活性的微生物”日本Kokai Tokkyo Koho。