Laboratório de Microbiologia, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
Laboratório de Microbiologia, Universidade Federal da Fronteira Sul (UFFS), Cerro Largo, Brazil.
Prep Biochem Biotechnol. 2023;53(1):12-21. doi: 10.1080/10826068.2022.2033997. Epub 2022 Feb 14.
sp. CL14 crude protease was partially characterized and applied to obtain antioxidant whey protein isolate (WPI) hydrolysates. Optimal activity occurred at pH 9.0 and 60 °C. Ca, Mg, and Mn (5 mM) enhanced activity (12-26%), whereas Co, Cu, Fe, and Zn inhibited it (50-94%). At 1% (v/v), Tween 20 and Triton X-100 enhanced activities (21-27%), β-mercaptoethanol decreased it (15%), and dimethyl sulfoxide (DMSO) had no effect. Sodium dodecyl sulfate (SDS; 0.1%, w/v) increased activity by 36%. Complete inhibition by phenylmethylsulfonyl fluoride (PMSF), and 85% inhibition by ethylenediaminotetraacetic acid, indicates its serine protease character and the importance of cations for activity/stability. With 5 mM Ca, protease was optimally active at 65 °C and completely stable after 20 min at 40-55 °C. Crude protease preferentially hydrolyzed WPI and soy protein, followed by casein. WPI hydrolysis was then performed (55 °C, pH 9.0, 5 mM Ca) for 0-180 min. Contents of trichloroacetic acid (TCA)-soluble proteins in WPI hydrolysates (HWPI) increased from 29% (0 min) to 50-52% (60-180 min), accompanied by enhanced radical scavenging activity (14%, 0 min; ∼34%, 60-180 min) and Fe-chelating ability (56%, 0 min; ∼74%, 45-180 min). CL14 protease might represent an alternative biocatalyst to obtain antioxidant hydrolysates from WPI and, potentially, from other food proteins.
sp. CL14 粗蛋白酶的部分特性得到了鉴定，并被应用于获得具有抗氧化活性的乳清蛋白分离物（WPI）水解产物。该酶在 pH9.0 和 60°C 时活性最高。Ca、Mg 和 Mn（5mM）能提高其活性（12-26%），而 Co、Cu、Fe 和 Zn（50-94%）则抑制其活性。在 1%（v/v）时，Tween 20 和 Triton X-100 能提高其活性（21-27%），β-巯基乙醇使其降低（15%），二甲基亚砜（DMSO）则没有影响。十二烷基硫酸钠（SDS；0.1%，w/v）能使其活性提高 36%。苯甲基磺酰氟（PMSF）完全抑制，乙二胺四乙酸（EDTA）抑制 85%，表明该酶具有丝氨酸蛋白酶的特性，且阳离子对其活性/稳定性非常重要。在有 5mM Ca 的情况下，该酶在 65°C 时活性最佳，在 40-55°C 下 20 分钟后完全稳定。粗蛋白酶优先水解 WPI 和大豆蛋白，然后是酪蛋白。然后在 55°C、pH9.0、5mM Ca 条件下对 WPI 进行水解（0-180min）。WPI 水解产物（HWPI）中三氯乙酸（TCA）可溶性蛋白的含量从 29%（0min）增加到 50-52%（60-180min），同时自由基清除活性（14%，0min；34%，60-180min）和 Fe 螯合能力（56%，0min；74%，45-180min）也得到了增强。CL14 蛋白酶可能代表了一种替代生物催化剂，可用于从 WPI 甚至其他食物蛋白中获得具有抗氧化活性的水解产物。
