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Recruitment of plastic-degrading enzymes onto the surface of plastics via fungal hydrophobin RolA

通过真菌疏水蛋白 RolA 将塑料降解酶招募到塑料表面

基本信息

批准号：
18580324
负责人：
ABE Keietsu
金额：
$ 2.53万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18580324/
关键词：
hydrophobin RolA Aspergillus oryzae interaction solid-liquind interface reaction biodegradable plastic FRAP

项目摘要

When fungi grow on plant or insect surfaces coated with wax polyesters that protect against pathogens, the fungi generally form aerial hyphae to contact the surfaces. Aerial structures such as hyphae and conidiophores are coated with hydrophobins, which are surface-active proteins involved in adhesion to hydrophobic surfaces. When the industrial fungus Aspergillus oryzae is cultivated in a liquid medium containing the biodegradable polyester polybutylene succinate-coadipate (PBSA), the rolA gene encoding Type I hydrophobin RolA is highly transcribed. High levels of RolA are localized on the cell surface and also secreted into the liquid medium. Under these conditions, A. oryzae simultaneously produces the cutinase CutL1, which hydrolyzes PBSA. RolA adsorbed to the hydrophobic surface of PBSA particles in the medium recruits CutL1, resulting in condensation of CutL1 on the PBSA surface and consequent stimulation of PBSA hydrolysis (1, 2). The ability that RolA attached to the PBSA surfaces recruits esterase CutL1 is a newly discovered function in hydrophobin. In the present study, we studied amino acid residues involved in the RolA-CutL1 interaction by means of site-directed mutagenesis and chemical modification. Teflon particles (average diameter 100-200 nm) were coated with RolA and its derivatives. The Teflon particles coated with RolA or its derivatives were incubated with soluble CutL1 and the supernatant and particles were separated by centrifugation. Recruited CutL1 was extracted from the centrifuged particles with SDS and quantitatively measured by SDS-PAGE analyses. We found that His32 of RolA is an important amino acid residue required for RolA-CutL1 interaction.

当真菌生长在涂有蜡聚酯的植物或昆虫表面上时，真菌通常会形成气生菌丝来接触表面。菌丝和分生孢子梗等空中结构涂有疏水蛋白，疏水蛋白是参与疏水表面粘附的表面活性蛋白。当工业真菌米曲霉在含有可生物降解的聚酯聚丁二酸己二酸丁二醇酯(PBSA)的液体培养基中培养时，编码I型疏水蛋白RolA的rolA基因被高度转录。高水平的 RolA 位于细胞表面，也分泌到液体培养基中。在这些条件下，米曲霉同时产生角质酶 CutL1，该酶可水解 PBSA。 RolA 吸附到培养基中 PBSA 颗粒的疏水表面，招募 CutL1，导致 CutL1 在 PBSA 表面凝结，从而刺激 PBSA 水解 (1, 2)。 RolA 附着在 PBSA 表面上招募酯酶 CutL1 的能力是疏水蛋白中新发现的功能。在本研究中，我们通过定点诱变和化学修饰研究了参与 RolA-CutL1 相互作用的氨基酸残基。聚四氟乙烯颗粒（平均直径100-200 nm）涂有RolA及其衍生物。将涂有RolA或其衍生物的聚四氟乙烯颗粒与可溶性CutL1一起孵育，并通过离心分离上清液和颗粒。用 SDS 从离心颗粒中提取招募的 CutL1，并通过 SDS-PAGE 分析进行定量测量。我们发现RolA的His32是RolA-CutL1相互作用所需的重要氨基酸残基。