Department of Molecular Biology and Genetics, Gebze Technical University, Gebze 41400, Kocaeli, Turkey.
Dohler Food & Beverage Ingredients, 70100 Merkez, Karaman, Turkey.
Protein Pept Lett. 2021;28(7):817-830. doi: 10.2174/1871530321666210106110400.
Polygalacturonases are a group of enzymes under pectinolytic enzymes related to enzymes that hydrolyse pectic substances. Polygalacturonases have been used in various industrial applications such as fruit juice clarification, retting of plant fibers, wastewater treatment drinks fermentation, and oil extraction.
The study was evaluated at the heterologous expression, purification, biochemical characterization, computational modeling, and performance in apple juice clarification of a new exo-polygalacturonase from Sporothrix schenckii 1099-18 (SsExo-PG) in Pichia pastoris.
Recombinant DNA technology was used in this study. Two different pPIC9K plasmids were constructed with native signal sequence-ssexo-pg and alpha signal sequence-ssexo-pg separately. Protein expression and purification performed after plasmids transformed into the Pichia pastoris. Biochemical and structural analyses were performed by using pure SsExo-PG.
The purification of SsExo-PG was achieved using a Ni-NTA chromatography system. The enzyme was found to have a molecular mass of approximately 52 kDa. SsExo-PG presented as stable at a wide range of temperature and pH values, and to be more storage stable than other commercial pectinolytic enzyme mixtures. Structural analysis revealed that the catalytic residues of SsExo- PG are somewhat similar to other Exo-PGs. The K and k values for the degradation of polygalacturonic acid (PGA) by the purified enzyme were found to be 0.5868 μM and 179 , respectively. Cu was found to enhance SsExo-PG activity while Ag and Fe almost completely inhibited enzyme activity. The enzyme reduced turbidity up to 80% thus enhanced the clarification of apple juice. SsExo-PG showed promising performance when compared with other commercial pectinolytic enzyme mixtures.
The clarification potential of SsExo-PG was revealed by comparing it with commercial pectinolytic enzymes. The following parameters of the process of apple juice clarification processes showed that SsExo-PG is highly stable and has a novel performance.
聚半乳糖醛酸酶是果胶酶相关酶系中的一组酶，与水解果胶物质的酶有关。聚半乳糖醛酸酶已在各种工业应用中得到应用，例如果汁澄清、植物纤维沤制、废水处理、饮料发酵和石油提取。
本研究评估了来自嗜热丝孢菌 1099-18（SsExo-PG）的新型外切聚半乳糖醛酸酶在毕赤酵母中的异源表达、纯化、生化特性、计算建模和在苹果汁澄清中的性能。
本研究使用重组 DNA 技术。使用带有天然信号序列-ssexo-pg 和α信号序列-ssexo-pg 的两个不同 pPIC9K 质粒分别构建。将质粒转化入毕赤酵母后进行蛋白表达和纯化。通过使用纯 SsExo-PG 进行生化和结构分析。
使用 Ni-NTA 色谱系统实现了 SsExo-PG 的纯化。该酶的分子量约为 52 kDa。SsExo-PG 在广泛的温度和 pH 值范围内表现稳定，并且比其他商业果胶酶混合物更稳定。结构分析表明，SsExo-PG 的催化残基与其他 Exo-PGs 有些相似。纯化酶对聚半乳糖醛酸（PGA）的降解的 K 和 k 值分别为 0.5868 μM 和 179 。Cu 被发现增强了 SsExo-PG 的活性，而 Ag 和 Fe 几乎完全抑制了酶的活性。该酶可将浊度降低高达 80%，从而增强了苹果汁的澄清度。与其他商业果胶酶混合物相比，SsExo-PG 表现出了有前景的性能。
通过将 SsExo-PG 与商业果胶酶进行比较，揭示了其澄清潜力。苹果汁澄清过程的以下参数表明 SsExo-PG 高度稳定且具有新颖的性能。
