Cardoso Samuel Leite, Souza Paula Monteiro, Rodrigues Kelly, Mota Isabella de Souza, Filho Edivaldo Ferreira, Fávaro Léia Cecilia de Lima, Saldanha-Araujo Felipe, Homem-de-Mello Mauricio, Pessoa Adalberto, Silveira Dâmaris, Fonseca-Bazzo Yris Maria, Magalhães Pérola Oliveira
Health Science School, University of Brasilia, Brasilia 70910-900, Brazil.
Brazilian Agricultural Research Corporation-EMBRAPA Agroenergia, Brasilia 70770-901, Brazil.
Pharmaceutics. 2023 Sep 20;15(9):2352. doi: 10.3390/pharmaceutics15092352.
The search for new drug-producing microorganisms is one of the most promising situations in current world scientific scenarios. The use of molecular biology as well as the cloning of protein and compound genes is already well established as the gold standard method of increasing productivity. Aiming at this increase in productivity, this work aims at the cloning, purification and in silico analysis of l-asparaginase from in Komagataella phaffii ( protein expression systems. The l-asparaginase gene (NCBI OQ439985) has been cloned into strains. Enzyme production was analyzed via the quantification of aspartic B-hydroxamate, followed by purification on a DEAE FF ion exchange column. The in silico analysis was proposed based on the combined use of various technological tools. The enzymatic activity found intracellularly was 2.84 IU/g. A purification factor of 1.18 was observed. The in silico analysis revealed the position of five important amino acid residues for enzymatic activity, and likewise, it was possible to predict a monomeric structure with a C-score of 1.59. The production of the enzyme l-asparaginase from in was demonstrated in this work, being of great importance for the analysis of new methodologies in search of the production of important drugs in therapy.
寻找新的产药微生物是当前世界科学领域最具前景的情况之一。分子生物学的应用以及蛋白质和化合物基因的克隆已成为提高生产力的金标准方法。为了提高生产力，本研究旨在对法夫酵母（Komagataella phaffii）中的L-天冬酰胺酶进行克隆、纯化及计算机模拟分析（蛋白质表达系统）。L-天冬酰胺酶基因（NCBI OQ439985）已被克隆到菌株中。通过定量分析天冬氨酸β-异羟肟酸来分析酶的产生，随后在DEAE FF离子交换柱上进行纯化。基于多种技术工具的联合使用进行计算机模拟分析。细胞内发现的酶活性为2.84 IU/g。观察到纯化因子为1.18。计算机模拟分析揭示了酶活性的五个重要氨基酸残基的位置，同样，还可以预测出C分数为1.59的单体结构。本研究证明了在法夫酵母（Komagataella phaffii）中产生L-天冬酰胺酶，这对于寻找治疗中重要药物生产的新方法的分析具有重要意义。
