嗜热菌蛋白酶(TLN)是一种微生物高价的热稳定金属内切蛋白酶,具有与科学和工业中广泛用于蛋白质消化和凝乳的蛋白酶互补的底物特异性。本研究首次将TLN固定在胺化超顺磁性纳米粒子(Fe3O4@silica-NH2)上,旨在提高稳定性,可恢复性,可重用性,以及在蛋白水解和作为微生物凝乳酶样凝乳酶中的适用性。开发的纳米生物催化剂(Fe3O4@silica-TLN)在合成TLN基底上显示水解活性,显然,通过磁倾析从反应介质中完全回收。更重要的是,Fe3O4@silica-TLN在钙离子存在下保持TLN催化性能,即使暴露于60°C48小时,在4°C下储存80天,在室温下储存42天,用于蛋白水解,和牛奶凝固长达11个周期。它在24小时内对牛乳酪蛋白的蛋白水解活性提供了84个肽,其中29具有潜在的生物活性。此外,Fe3O4@二氧化硅-TLN催化消化牛血清白蛋白.总之,Fe3O4@silica-TLN是一种新的,更少的自溶,热稳定,无毒,磁性可分离,和可重复使用的纳米生物催化剂,具有对科学(肽/蛋白质化学和结构,蛋白质组学研究,以及寻找新的生物活性肽)和食品工业(奶酪制造)。
Thermolysin (TLN) is a microbial highly-priced thermostable metallo-endoprotease with complementary substrate specificity to those of proteases widely used in science and industry for protein digestion and milk-clotting. This study is the first to immobilize TLN on aminated superparamagnetic nanoparticles (Fe3O4@silica-NH2) aiming for higher stability, recoverability, reusability, and applicability in proteolysis and as a microbial rennet-like milk-clotting enzyme. The nanobiocatalyst developed (Fe3O4@silica-TLN) displays hydrolytic activity on a synthetic TLN substrate and, apparently, was fully recovered from reaction media by magnetic decantation. More importantly, Fe3O4@silica-TLN retains TLN catalytic properties in the presence of calcium ions even after exposure to 60 °C for 48 h, storage at 4 °C for 80 days and room temperature for 42 days, use in proteolyses, and in milk-clotting for up to 11 cycles. Its proteolytic activity on bovine milk casein in 24 h furnished 84 peptides, of which 29 are potentially bioactive. Also, Fe3O4@silica-TLN catalyzed the digestion of bovine serum albumin. In conclusion, Fe3O4@silica-TLN showed to be a new, less autolytic, thermostable, non-toxic, magnetically-separable, and reusable nanobiocatalyst with highly attractive properties for both science (peptide/protein chemistry and structure, proteomic studies, and the search for new bioactive peptides) and food industry (cheese manufacture).