Mobayed Francielle Herrmann, Nunes Juliane Carraro, Gennari Adriano, de Andrade Bruna Coelho, Ferreira Matheus Loch Velvites, Pauli Paolla, Renard Gaby, Chies Jocelei Maria, Volpato Giandra, Volken de Souza Claucia Fernanda
Laboratório de Biotecnologia de Alimentos, Universidade do Vale do Taquari - Univates, Av. Avelino Tallini, 171, ZC, Lajeado, RS, 95914-014, Brazil.
Curso de Biotecnologia, Instituto Federal de Educação, Ciência e Tecnologia do Rio Grande do Sul - IFRS, Campus Porto Alegre, Porto Alegre, RS, Brazil.
Biotechnol Lett. 2021 Mar;43(3):589-599. doi: 10.1007/s10529-020-03028-3. Epub 2020 Oct 14.
The aim of the present study was to evaluate the efficiency of lactose derived from cheese whey and cheese whey permeate as inducer of recombinant Kluyveromyces sp. β-galactosidase enzyme produced in Escherichia coli. Two E. coli strains, BL21(DE3) and Rosetta (DE3), were used in order to produce the recombinant enzyme. Samples were evaluated for enzyme activity, total protein content, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis after induction with isopropyl-β-D-1-thiogalactoside (IPTG) (0.05 and 1 mM) and lactose, cheese whey, and cheese whey permeate solutions (1, 10, and 20 g/L lactose) at shake-flask cultivation, and whey permeate solution (10 g/L lactose) at bioreactor scale.
The highest specific activities obtained with IPTG as inducer (0.05 mM) after 9 h of induction, were 23 and 33 U/mg with BL21(DE3) and Rosetta(DE3) strains, respectively. Inductions performed with lactose and cheese whey permeate (10 and 20 g/L lactose) showed the highest specific activities at the evaluated hours, exhibiting better results than those obtained with IPTG. Specific activity of recombinant β-galactosidase using whey permeate (10 g/L lactose) showed values of approximately 46 U/mg after 24-h induction at shake-flask study, and approximately 26 U/mg after 16-h induction at bench bioreactor.
The induction with cheese whey permeate was more efficient for recombinant β-galactosidase expression than the other inducers tested, and thus, represents an alternative form to reduce costs in recombinant protein production.
本研究旨在评估源自奶酪乳清和奶酪乳清渗透液的乳糖作为重组克鲁维酵母属β-半乳糖苷酶在大肠杆菌中产生的诱导剂的效率。使用了两种大肠杆菌菌株，BL21(DE3)和Rosetta (DE3)来生产重组酶。在用异丙基-β-D-1-硫代半乳糖苷（IPTG）（0.05和1 mM）以及乳糖、奶酪乳清和奶酪乳清渗透液溶液（1、10和20 g/L乳糖）诱导后，在摇瓶培养中对样品进行酶活性、总蛋白含量和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析，并在生物反应器规模下对乳清渗透液溶液（10 g/L乳糖）进行分析。
以IPTG作为诱导剂（0.05 mM）诱导9小时后，BL21(DE3)和Rosetta(DE3)菌株获得的最高比活性分别为23和33 U/mg。用乳糖和奶酪乳清渗透液（10和20 g/L乳糖）进行的诱导在评估时间显示出最高比活性，比用IPTG获得的结果更好。在摇瓶研究中，使用乳清渗透液（10 g/L乳糖）的重组β-半乳糖苷酶在诱导24小时后的比活性约为46 U/mg，在台式生物反应器中诱导16小时后的比活性约为26 U/mg。
用奶酪乳清渗透液诱导重组β-半乳糖苷酶表达比测试的其他诱导剂更有效，因此，代表了一种降低重组蛋白生产成本的替代形式。
