TERMINUS WP4 Enzyme immobilization, protection, and triggering. TASK 4.1 Experimental data – Biocatalytic red-ox systems based on laccases

Abstract

Laccases (EC 1.10.3.2) are multi-copper oxidoreductase metalloenzymes found in various organisms, including bacteria, fungi, and plants. Laccases typically catalyze the oxidation of phenolic compounds along with the reduction of molecular oxygen to water. In TERMINUS WP4, task 4.1, we strive to improve the thermostability of the laccase enzyme from Pleurotus ostreatus (received from Biopox Srl). Our enzyme protection strategy is based on a method developed by FHNW that consists of shielding enzymes immobilized at the surface of silica-based nanoparticles within a soft organosilica layer. The components of this protective shield, establishing non-covalent interactions with the protein's surface, contribute to a remarkable stabilization of the enzyme. We used tetraethyl-orthosilicate (TEOS) and aminopropyl-triethoxysilane (APTES) as building blocks of the organosilica layer. Considering the dimension of the enzyme (7 nm), we used a 7-hour incubation time to fully cover (9 nm layer thickness) the enzyme inside the protection layer. For example, with this method, we could increase the half-life (𝑡50) of Biopox EV4 at 50°C from 18 minutes for the soluble enzyme to over 60 minutes for the protected enzyme. This will enable the enzyme to be active for a longer time in a harsh environment like high temperature used in the lamination process of multilayer packaging to degrade the adhesive polymer layer, which is the aim of the TERMINUS project.

Dataset

This dataset contains all the UV-kinetic and immobilization endpoint raw data, SEM images, SDS-PAGE images, and Zeta potential to characterize and calculate the laccase immobilization, protection, and activity on silica nanoparticles used in Deliverable 1.5. Data are available in a compressed .zip file with one folder (Laccase-Biocatalytic-Redox-Systems_v1.0_TER_WP4_D4-1) containing seven files:

Immobilization-BCA_D4-1-Laccase-Biocatalytic-Redox-Systems.xlsx contains all the BCA assay raw data for the concentration, immobilization, and immobilization optimization measurements.

Kinetics_D4-1-Laccase-Biocatalytic-Redox-Systems.xlsx contains all the UV-kinetic activity assay raw data for soluble, immobilized, and protected enzymes in thermostability experiments.

SDS-PAGE_D4-1-Laccase-Biocatalytic-Redox-Systems.pdf contains SDS-PAGE images obtained before and after dialysis for all the enzymes received from Biopox.

SEM-images_D4-1-Laccase-Biocatalytic-Redox-Systems.pdf includes the SEM images used for characterization of silica nanoparticles before and after layer growth.

Zeta-potential_D4-1-Laccase-Biocatalytic-Redox-Systems.pdf contains the raw data regarding the zeta potential measurements for silica nanoparticles and amino-modified silica nanoparticles.

Materials_and_experimental_methods_D4-1-Laccase-Biocatalytic-Redox-Systems.pdf details the experimental method and conditions for the data acquisition presented in the data set. Guidance is also provided on using the data to calculate the laccase immobilization, activity, and thermostability.

Metadata_information_D4-1-Laccase-Biocatalytic-Redox-Systems.pdf includes more detailed metadata information for the dataset represented in the excel files.

摘要
漆酶（Laccase，EC 1.10.3.2）是一类广泛存在于细菌、真菌及植物等多种生物体内的多铜氧化还原金属酶，其典型功能为催化酚类化合物氧化，同时将分子氧还原为水。在TERMINUS项目WP4的任务4.1中，我们致力于提升源自糙皮侧耳（Pleurotus ostreatus，由Biopox Srl提供）的漆酶热稳定性。本研究的酶保护策略基于FHNW开发的方法：对固定于二氧化硅基纳米颗粒表面的酶包裹一层柔性有机硅层，该保护层组分可与蛋白质表面形成非共价相互作用，从而显著提升酶的稳定性。我们选用正硅酸乙酯（tetraethyl-orthosilicate，TEOS）与氨丙基三乙氧基硅烷（aminopropyl-triethoxysilane，APTES）作为有机硅层的构筑单元；考虑到目标酶尺寸为7纳米，我们采用7小时孵育时间以实现对酶的完全包覆（涂层厚度达9纳米）。例如，通过该方法可将Biopox EV4酶在50℃下的半衰期（t50）从可溶性酶的18分钟提升至保护酶的60分钟以上。这可使该酶在多层包装层压工艺所需的高温等严苛环境中维持更长时间的活性，进而降解粘合剂聚合物层，这正是TERMINUS项目的核心目标。

数据集
本数据集涵盖所有用于表征与计算本研究中二氧化硅纳米颗粒负载漆酶的固定化效率、保护效果及活性的紫外动力学与固定化终点原始数据、扫描电子显微镜（SEM）图像、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）图像及Zeta电位数据，对应交付件1.5的相关研究内容。数据以压缩ZIP文件形式提供，其中包含一个名为Laccase-Biocatalytic-Redox-Systems_v1.0_TER_WP4_D4-1的文件夹，内含7个文件：
1. Immobilization-BCA_D4-1-Laccase-Biocatalytic-Redox-Systems.xlsx：包含用于浓度测定、固定化及固定化优化检测的所有BCA法原始实验数据。
2. Kinetics_D4-1-Laccase-Biocatalytic-Redox-Systems.xlsx：包含可溶性酶、固定化酶及保护酶在热稳定性实验中的所有紫外动力学活性检测原始数据。
3. SDS-PAGE_D4-1-Laccase-Biocatalytic-Redox-Systems.pdf：包含从Biopox获取的所有酶在透析前后的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）图像。
4. SEM-images_D4-1-Laccase-Biocatalytic-Redox-Systems.pdf：包含用于表征涂层生长前后二氧化硅纳米颗粒的扫描电子显微镜（SEM）图像。
5. Zeta-potential_D4-1-Laccase-Biocatalytic-Redox-Systems.pdf：包含二氧化硅纳米颗粒及氨基改性二氧化硅纳米颗粒的Zeta电位检测原始数据。
6. Materials_and_experimental_methods_D4-1-Laccase-Biocatalytic-Redox-Systems.pdf：详细阐述本数据集所呈现数据的实验方法与条件，并提供了利用该数据计算漆酶固定化效率、活性及热稳定性的操作指南。
7. Metadata_information_D4-1-Laccase-Biocatalytic-Redox-Systems.pdf：包含本数据集Excel文件中相关数据的更详细元数据信息。