State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.
International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, China.
J Sci Food Agric. 2021 Sep;101(12):5154-5162. doi: 10.1002/jsfa.11161. Epub 2021 Mar 5.
Alginate lyases (EC 4.4.2.3/4.4.2.11) have been applied to produce alginate oligosaccharides, which have physiological advantages such as prebiotic and antidiabetic effects, and are of benefit in the food and pharmaceutical industries. Extracellular production of recombinant proteins in Escherichia coli presents advantages including simplified downstream processing and high productivity; however, the presence of certain signal peptides does not always ensure successful secretion, which make the extracellular production of alginate lyase in E. coli rarely reported but of great significance.
A PL7 family alginate lyase, Aly01, with its native signal peptide from Vibrio natriegens SK42.001, was identified, characterized, and extracellularly expressed in E. coli. The enzyme specifically released trisaccharide from alginate and was strictly NaCl activated. Green fluorescent protein (GFP) was fused with the Aly01 signal peptide and successfully secreted in E. coli to expand the feasibility of using this signal peptide to produce other heterologous proteins extracellularly. Through a synergistic strategy of utilizing Terrific Broth (TB) medium supplemented with 120 mmol L glycine and 10 mmol L calcium, the lag phase of protein secretion was reduced to 3 h from 12 h; meanwhile calcium remedied glycine-related cell growth impairment, leading to further enhancement of overall enzyme productivity, reaching a maximum of 4.55 U mL .
A new salt-activated alginate lyase, Aly01, was identified and characterized. E. coli employed its signal peptide and extracellularly expressed both Aly01 and a GFP, which indicated the signal peptide of Aly01 could be a powerful tool for extracellular production of other heterologous proteins in E. coli. © 2021 Society of Chemical Industry.
褐藻胶裂解酶（EC 4.4.2.3/4.4.2.11）已被应用于生产褐藻胶寡糖，这些寡糖具有益生元和抗糖尿病等生理优势，在食品和制药行业具有益处。在大肠杆菌中进行重组蛋白的胞外生产具有简化下游加工和高生产力等优势；然而，某些信号肽的存在并不总是能确保成功分泌，这使得褐藻胶裂解酶在大肠杆菌中的胞外生产很少有报道，但却具有重要意义。
从纳氏弧菌 SK42.001 中鉴定出一种 PL7 家族褐藻胶裂解酶 Aly01，其具有天然的信号肽。该酶能特异性地从褐藻酸盐中释放三糖，且严格依赖 NaCl 激活。绿色荧光蛋白（GFP）与 Aly01 信号肽融合，并在大肠杆菌中成功分泌，从而扩大了使用该信号肽胞外生产其他异源蛋白的可行性。通过利用补充有 120 mmol L 甘氨酸和 10 mmol L 钙的 Terrific Broth（TB）培养基的协同策略，将蛋白分泌的迟滞期从 12 小时缩短至 3 小时；同时，钙补救了甘氨酸引起的细胞生长损伤，进一步提高了整体酶的生产能力，最大达到 4.55 U mL。
鉴定并表征了一种新的盐激活褐藻胶裂解酶 Aly01。大肠杆菌利用其信号肽在胞外表达了 Aly01 和 GFP，这表明 Aly01 的信号肽可以成为大肠杆菌胞外生产其他异源蛋白的有力工具。© 2021 化学工业协会。
