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

当真菌生长在涂有蜡聚酯以防止病原体的植物或昆虫表面上时，真菌通常形成气生菌丝以接触表面。气生结构如菌丝和分生孢子梗被疏水蛋白包被，疏水蛋白是参与粘附到疏水表面的表面活性蛋白。当在含有可生物降解的聚酯聚丁二酸丁二醇酯-共己二酸酯（PBSA）的液体培养基中培养工业真菌黑曲霉时，编码I型疏水蛋白RolA的RolA基因被高度转录。高水平的RolA定位于细胞表面，并且也分泌到液体培养基中。在此条件下，A.同时产生角质酶CutL 1，其水解PBSA。RolA吸附到培养基中PBSA颗粒的疏水表面募集CutL 1，导致CutL 1在PBSA表面上的缩合，并随后刺激PBSA水解（1，2）。RolA附着在PBSA表面募集酯酶CutL 1的能力是疏水蛋白中新发现的功能。在本研究中，我们研究了参与RolA-CutL 1相互作用的氨基酸残基通过定点突变和化学修饰。用RolA及其衍生物涂覆Teflon颗粒（平均直径100-200 nm）。将涂有RolA或其衍生物的Teflon颗粒与可溶性CutL 1一起孵育，并通过离心分离上清液和颗粒。用SDS从离心的颗粒中提取募集的CutL 1，并通过SDS-PAGE分析定量测量。我们发现RolA的His 32是RolA-CutL 1相互作用所需的重要氨基酸残基。

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.