Department of Microbiology, Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan.
Laboratory of Microbiology-Protein Research Unit, Ghent University, Karel Lodewijk Ghent, Belgium.
PLoS One. 2023 Aug 16;18(8):e0269717. doi: 10.1371/journal.pone.0269717. eCollection 2023.
Carlsberg subtilisin from Bacillus licheniformis PB1 was investigated as a potential feed supplement, through immobilizing on bentonite for improving the growth rate of broilers. Initially, the pre-optimized and partially-purified protease was extracted and characterized using SDS-PAGE with MW 27.0 KDa. The MALDI-TOF-MS/MS spectrum confirmed a tryptic peptide peak with m/z 1108.496 referring to the Carlsberg subtilisin as a protein-digesting enzyme with alkaline nature. The highest free enzyme activity (30 U/mg) was observed at 50°C, 1 M potassium phosphate, and pH 8.0. the enhanced stability was observed when the enzyme was adsorbed to an inert solid support with 86.39 ± 4.36% activity retention under 20 optimized conditions. Additionally, the dried immobilized enzyme exhibited only a 5% activity loss after two-week storage at room temperature. Structural modeling (Docking) revealed that hydrophobic interactions between bentonite and amino acids surrounding the catalytic triad keep the enzyme structure intact upon drying at RT. The prominent hygroscopic nature of bentonite facilitated protein structure retention upon drying. During a 46-days study, supplementation of boilers' feed with the subtilisin-bentonite complex promoted significant weight gain i.e. 15.03% in contrast to positive control (p = 0.001).
从地衣芽孢杆菌中提取的卡尔伯格枯草杆菌蛋白酶被研究作为一种潜在的饲料添加剂，通过固定化在膨润土上来提高肉鸡的生长速度。最初，使用 SDS-PAGE 提取和表征了预优化和部分纯化的蛋白酶，MW 为 27.0 kDa。MALDI-TOF-MS/MS 谱证实了一种胰蛋白酶肽峰，m/z 为 1108.496，指的是卡尔伯格枯草杆菌蛋白酶是一种具有碱性的蛋白水解酶。在 50°C、1 M 磷酸钾和 pH 8.0 的条件下，游离酶的活性最高（30 U/mg）。当酶吸附到惰性固体载体上时，观察到增强的稳定性，在 20 个优化条件下，酶的活性保留率为 86.39±4.36%。此外，干燥的固定化酶在室温下储存两周后，仅损失 5%的活性。结构建模（对接）表明，膨润土与催化三联体周围氨基酸之间的疏水相互作用在 RT 下干燥时保持酶结构完整。膨润土的显著吸湿性有助于干燥时保持蛋白质结构。在 46 天的研究中，在饲料中添加枯草杆菌蛋白酶-膨润土复合物显著促进了肉鸡的体重增加，即 15.03%，与阳性对照组相比（p = 0.001）。
