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

本研究的结果如下：1。PET分解微生物的筛选、分离与鉴定：我们成功获得了6株能够分解非结晶PET纤维的菌株。其中一些被鉴定为Trichosporon sp.。85；少动鞘单胞菌编号77-2；97 - 2, respectively.2。从节肢杆菌sp.No.97-2中纯化pet分解酶：从节肢杆菌sp.No.97-2的培养滤液中纯化了一个35 kda的蛋白作为pet分解酶的候选蛋白。纯化酶的氨基末端序列与鞭毛蛋白具有较高的相似性（同源性63%，相似性75%）。由于比活性低，我们无法阐明酶的性质。新分析方法的发展：利用扫描电子显微镜观察PET纤维表面和测量PET纤维的拉伸强度已被用于分析PET分解活性。然而，这些分析方法需要花费大量时间，而且它们的数据也不准确。为了改进pet分解酶的测定方法，介绍了粘度测定法、凝胶渗透色谱法和核磁共振波谱法。采用四种衬底：PET纤维、PET薄膜、PET低聚物和OEST（低聚丁二甲酸乙酯-对苯二甲酸乙酯）。这些分析方法对pet分解活性的测定是有用的。我们的一些研究没有进行。原因是：(1)保持这种高活性的pet分解菌株非常困难。这些酶可能被编码在质粒上。(2)目前尚无可靠的测定方法。

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.