School of Food Science and Engineering, Key Laboratory for Agricultural Products Processing of Anhui Province, Hefei University of Technology, Hefei, China.
Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, College of Life Sciences, Nankai University, Tianjin, China.
Appl Microbiol Biotechnol. 2018 Jul;102(13):5533-5543. doi: 10.1007/s00253-018-9000-y. Epub 2018 Apr 25.
Microbial transglutaminase (MTG) from Streptomyces mobaraensis has been widely used for crosslinking proteins in order to acquire products with improved properties. To improve the yield and enable a facile and efficient purification process, recombinant vectors, harboring various heterologous signal peptide-encoding fragments fused to the mtg gene, were constructed in Escherichia coli and then expressed in Bacillus subtilis. Signal peptides of both WapA and AmyQ (SP and SP ) were able to direct the secretion of pre-pro-MTG into the medium. A constitutive promoter (P ) was used for the expression of SP -mtg, while an inducible promoter (P ) was used for SP -mtg. After purification from the supernatant of the culture by immobilized metal affinity chromatography and proteolysis by trypsin, 63.0 ± 0.6 mg/L mature MTG was released, demonstrated to have 29.6 ± 0.9 U/mg enzymatic activity and shown to crosslink soy protein properly. This is the first report on secretion of S. mobaraensis MTG from B. subtilis, with similar enzymatic activities and yields to that produced from Escherichia coli, but enabling a much easier purification process.
来自藤野氏链霉菌的微生物转谷氨酰胺酶（MTG）已被广泛用于交联蛋白质，以获得具有改善性能的产品。为了提高产量并实现简便高效的纯化过程，构建了携带各种异源信号肽编码片段融合到 mtg 基因的重组载体，并在枯草芽孢杆菌中表达。WapA 和 AmyQ 的信号肽（SP 和 SP ）都能够将预-pro-MTG 分泌到培养基中。组成型启动子（P ）用于表达 SP -mtg，而诱导型启动子（P ）用于表达 SP -mtg。通过固定化金属亲和层析从培养物上清液中纯化并通过胰蛋白酶进行蛋白水解后，释放出 63.0±0.6 mg/L 的成熟 MTG，证明其具有 29.6±0.9 U/mg 的酶活，并能正确交联大豆蛋白。这是首次报道藤野氏链霉菌 MTG 从枯草芽孢杆菌中的分泌，其酶活和产量与从大肠杆菌中获得的相似，但可实现更简单的纯化过程。
