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From Secretion in Pichia pastoris to Application in Apple Juice Processing: Exo-Polygalacturonase from Sporothrix schenckii 1099-18
Protein & Peptide Letters ( IF 1.6 ) Pub Date : 2021-06-30 , DOI: 10.2174/1871530321666210106110400
Ersin Karataş 1 , Ahmet Tülek 1 , Mehmet Mervan Çakar 1 , Faruk Tamtürk 2 , Fatih Aktaş 3 , Barış Binay 4
Affiliation  

Background: 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.

Objectives: 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.

Methods: 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.

Results: 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 KM and kcat values for the degradation of polygalacturonic acid (PGA) by the purified enzyme were found to be 0.5868 μM and 179 s-1, respectively. Cu2+ was found to enhance SsExo-PG activity while Ag2+ and Fe2+ 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.

Conclusion: 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.



中文翻译:

从毕赤酵母的分泌到在苹果汁加工中的应用:申克孢子丝菌的外聚半乳糖醛酸酶 1099-18

背景:多聚半乳糖醛酸酶是果胶分解酶下的一组酶,与水解果胶物质的酶有关。聚半乳糖醛酸酶已用于各种工业应用,例如果汁澄清、植物纤维沤制、废水处理饮料发酵和油提取。

目标:该研究在来自毕赤酵母的申克孢子丝菌 1099-18 (SsExo-PG) 的新型外切多聚半乳糖醛酸酶的异源表达、纯化、生化表征、计算模型和苹果汁澄清性能方面进行了评估。

方法:本研究采用重组DNA技术。分别用天然信号序列-ssexo-pg和α信号序列-ssexo-pg构建了两种不同的pPIC9K质粒。质粒转化到毕赤酵母后进行蛋白质表达和纯化。使用纯 SsExo-PG 进行生化和结构分析。

结果:使用Ni-NTA色谱系统实现了SsExo-PG的纯化。发现该酶的分子量约为 52 kDa。SsExo-PG 在很宽的温度和 pH 值范围内表现稳定,并且比其他商业果胶分解酶混合物更稳定。结构分析表明,SsExo-PG 的催化残基与其他 Exo-PG 有点相似。发现纯化酶降解聚半乳糖醛酸 (PGA)的 K M和 k cat值分别为 0.5868 μM 和 179 s-1。发现Cu 2+增强 SsExo-PG 活性,而 Ag 2+和 Fe 2+几乎完全抑制酶活性。该酶将浊度降低了 80%,从而增强了苹果汁的澄清度。与其他商业果胶分解酶混合物相比,SsExo-PG 显示出有希望的性能。

结论: SsExo-PG 的澄清潜力是通过与商业果胶分解酶的比较来揭示的。以下苹果汁澄清过程的工艺参数表明,SsExo-PG 高度稳定且具有新颖的性能。

更新日期:2021-08-05
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