Martínez-Corona Ricardo, Canizal-García Renato, Madrigal-Perez Luis Alberto, Cortés-Penagos Carlos, de la Riva de la Riva Gustavo Alberto, González-Hernández Juan Carlos
Tecnológico Nacional de México / Instituto Tecnológico de Morelia, Av. Tecnológico No. 1500, Morelia, Michoacán 58120, Mexico.
Tecnológico Nacional de México / Instituto Tecnológico Superior de Ciudad Hidalgo, Av. Ing. Carlos Rojas Gutiérrez 2120, Ciudad Hidalgo, Michoacán 61100, Mexico.
J Genet Eng Biotechnol. 2024 Sep;22(3):100396. doi: 10.1016/j.jgeb.2024.100396. Epub 2024 Jun 21.
Lipases are used in many food, energy, and pharmaceutical processes. Thus, new systems have been sought to synthesize alternative lipases with potential biotechnological applications. Kluyveromyces marxianus is a yeast with recognized lipase activity; at least ten putative lipases/esterases in its genome have been detected, and two of them possess a signal peptide for extracellular secretion. The study of extracellular lipases becomes more relevant since they usually have higher activity rates than intracellular lipases and simpler purification mechanisms. For these reasons, this study aimed to characterize the production and lipase activity of the putative extracellular lipases of the K. marxianus L-2029 strain, encoded in the genes LIP3 and YJR107W. Both genes were heterologously expressed in Saccharomyces cerevisiae BY4742 (yeast strain without extracellular lipase activity) using a pYES2.1/V5-His-TOPO® plasmid. Herein, we show evidence that the strain transformed with the LIP3 gene did not show lipase activity during flask galactose induction. On the other hand, the strain transformed with the YJR107W gene showed a specific activity of 0.397 U/mg, with an optimum temperature of 37 °C and pH 6. For maximum cell production, glucose and yeast extract concentrations were evaluated by a 2 factorial design, followed by the validation of the best concentrations predicted by a statistical model; a 2 factorial design was also carried out to evaluate the concentration of the inducer galactose on the transformed strains, and the intracellular and extracellular lipase specific activities were quantified. Finally, the biomass and lipase production were determined for each strain, which was grown in a stirred tank bioreactor with a working volume of 1.5 L. The specific activities of the transformed strains obtained in the bioreactor were 1.36 U/mg for the LIP3 transformant and 1.25 U/mg for the YJR107W transformant, respectively.
脂肪酶被用于许多食品、能源和制药过程。因此，人们一直在寻找新的系统来合成具有潜在生物技术应用价值的替代脂肪酶。马克斯克鲁维酵母是一种具有公认脂肪酶活性的酵母；在其基因组中已检测到至少十种假定的脂肪酶/酯酶，其中两种具有用于细胞外分泌的信号肽。细胞外脂肪酶的研究变得更加重要，因为它们通常比细胞内脂肪酶具有更高的活性速率和更简单的纯化机制。基于这些原因，本研究旨在表征马克斯克鲁维酵母L-2029菌株中由LIP3和YJR107W基因编码的假定细胞外脂肪酶的产生及脂肪酶活性。使用pYES2.1/V5-His-TOPO®质粒，将这两个基因在酿酒酵母BY4742（无细胞外脂肪酶活性的酵母菌株）中进行异源表达。在此，我们有证据表明，用LIP3基因转化的菌株在摇瓶半乳糖诱导过程中未显示出脂肪酶活性。另一方面，用YJR107W基因转化的菌株显示出比活性为0.397 U/mg，最适温度为37°C，最适pH为6。为了实现最大细胞产量，通过二因素设计评估葡萄糖和酵母提取物的浓度，随后验证统计模型预测的最佳浓度；还进行了二因素设计以评估诱导剂半乳糖对转化菌株的浓度，并对细胞内和细胞外脂肪酶的比活性进行定量。最后，测定了在工作体积为1.5 L的搅拌罐生物反应器中生长的每个菌株的生物量和脂肪酶产量。在生物反应器中获得的转化菌株的比活性分别为：LIP3转化体为1.36 U/mg，YJR107W转化体为1.25 U/mg。
