Nagai Keisuke, Iida Kazuki, Shimizu Kimiaki, Kinugasa Ryo, Izumi Motoki, Kato Dai-Ichiro, Takeo Masahiro, Mochiji Kozo, Negoro Seiji
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo, 671-2280, Japan.
Appl Microbiol Biotechnol. 2014 Oct;98(20):8751-61. doi: 10.1007/s00253-014-5885-2. Epub 2014 Jun 25.
Nylon hydrolase degrades various aliphatic nylons, including nylon-6 and nylon-66. We synthesized a nylon-66 copolymer (M w = 22,900, M n = 7,400), in which a part of an adipoyl unit (32 % molar ratio) of nylon-66 was replaced with a succinyl unit by interfacial polymerization. To quantify the reaction rate of the enzymatic hydrolysis of nylons at the surface of solid polymers, we prepared a thin layer of nylons on the bottom surface of each well in a polystyrene-based micro-assay plate. The thickness of the nylon layer was monitored by imaging analysis of the photographic data. More than 99 % of the copolymer with thicknesses of 260 nm (approximately 600 layers of polymer strands) were converted to water-soluble oligomers by nylon hydrolase (3 mg enzyme ml(-1)) at 30 °C within 60 h. These results were further confirmed by TLC analysis of the reaction products and by assay of liberated amino groups in the soluble fractions. The degradation rate of the thin-layered nylon-6 was similarly analyzed. We demonstrate that this assay enables a quantitative evaluation of the reaction rate of hydrolysis at the interface between the solid and aqueous phases and a quantitative comparison of the degradability for various polyamides.
尼龙水解酶可降解各种脂肪族尼龙，包括尼龙6和尼龙66。我们通过界面聚合反应合成了一种尼龙66共聚物（Mw = 22,900，Mn = 7,400），其中尼龙66的部分己二酰单元（摩尔比32%）被琥珀酰单元取代。为了量化固体聚合物表面尼龙酶促水解的反应速率，我们在聚苯乙烯基微量分析板的每个孔底部制备了一层尼龙薄层。通过对照片数据的成像分析监测尼龙层的厚度。在30℃下，厚度为260nm（约600层聚合物链）的共聚物中超过99%在60小时内被尼龙水解酶（3mg酶ml(-1)）转化为水溶性低聚物。反应产物的TLC分析以及可溶性部分中游离氨基的测定进一步证实了这些结果。对薄层尼龙6的降解速率进行了类似分析。我们证明，该分析方法能够对固液界面的水解反应速率进行定量评估，并对各种聚酰胺的降解性进行定量比较。
