Graham Centre (an Alliance between Charles Sturt University and the NSW Department of Primary Industries), Boorooma Street, Wagga Wagga, NSW 2678, Australia.
ARC Industrial Transformation Training Centre for Functional Grains, School of Biomedical Sciences, Charles Sturt University, Boorooma Street, Wagga Wagga, NSW 2678, Australia.
Molecules. 2021 Apr 4;26(7):2078. doi: 10.3390/molecules26072078.
Valorization of vegetable oil waste residues is gaining importance due to their high protein and polyphenol contents. Protease inhibitors (PIs), proteins from these abundantly available waste residues, have recently gained importance in treating chronic diseases. This research aimed to use canola meal of genetically diverse genotypes, BLN-3347 and Rivette, to identify PIs with diverse functionalities in therapeutic and pharmacological applications. The canola meal PI purification steps involved: native PAGE and trypsin inhibition activity, followed by ammonium sulfate fractionation, anion exchange, gel filtration, and reverse-phase chromatography. The purified PI preparations were characterized using SDS-PAGE, isoelectric focusing (IEF), and N terminal sequencing. SDS-PAGE analysis of PI preparations under native reducing and nonreducing conditions revealed three polymorphic PIs in each genotype. The corresponding IEF of the genotype BLN-3347, exhibited three acidic isoforms with isoelectric points (p) of 4.6, 4.0, and 3.9, while Rivette possessed three isoforms, exhibiting two basic forms of p 8.65 and 9.9, and one acidic of p 6.55. Purified PI preparations from both the genotypes displayed dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE) inhibition activities; the BLN-3347 PI preparation exhibited a strong inhibitory effect with lower IC values (DPP-IV 37.42 µg/mL; ACE 129 µg/mL) than that from Rivette (DPP-IV 67.97 µg/mL; ACE 376.2 µg/mL). In addition to potential human therapy, these highly polymorphic PIs, which can inhibit damaging serine proteases secreted by canola plant pathogens, have the potential to be used by canola plant breeders to seek qualitative trait locus (QTLs) linked to genes conferring resistance to canola diseases.
由于植物油废料中含有丰富的蛋白质和多酚，因此其再利用变得越来越重要。蛋白酶抑制剂（PI）是这些大量存在的废料中的蛋白质，最近在治疗慢性疾病方面受到了重视。本研究旨在使用遗传多样性基因型 BLN-3347 和 Rivette 的油菜籽饼来鉴定具有治疗和药理学应用多样性功能的 PI。油菜籽饼 PI 纯化步骤包括：天然 PAGE 和胰蛋白酶抑制活性，然后进行硫酸铵分级、阴离子交换、凝胶过滤和反相色谱。使用 SDS-PAGE、等电聚焦（IEF）和 N 端测序对 PI 制剂进行了表征。PI 制剂在天然还原和非还原条件下的 SDS-PAGE 分析表明，每个基因型中存在三种多态性 PI。基因型 BLN-3347 的相应 IEF 显示三个酸性同工型，等电点（pI）分别为 4.6、4.0 和 3.9，而 Rivette 则具有三个同工型，表现为两种碱性形式 pI 为 8.65 和 9.9，以及一种酸性形式 pI 为 6.55。两种基因型的纯化 PI 制剂均显示二肽基肽酶-IV（DPP-IV）和血管紧张素转化酶（ACE）抑制活性；BLN-3347 PI 制剂具有较强的抑制作用，IC 值较低（DPP-IV 为 37.42μg/mL；ACE 为 129μg/mL），而 Rivette 的则较低（DPP-IV 为 67.97μg/mL；ACE 为 376.2μg/mL）。除了潜在的人类治疗外，这些高度多态性的 PI 可以抑制油菜植物病原体分泌的破坏性丝氨酸蛋白酶，有望被油菜植物育种者用于寻找与赋予油菜疾病抗性相关的基因的质量性状基因座（QTL）。
