Voronezh State University, Universitetskaya sq. 1, Voronezh 394018, Russian Federation; Sevastopol State University, Universitetskaya st. 33, Sevastopol 299053, Russian Federation.
Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of RAS, 2/31, Lobachevsky st., Kazan 420111, Russian Federation.
Int J Biol Macromol. 2021 Jun 1;180:161-176. doi: 10.1016/j.ijbiomac.2021.03.016. Epub 2021 Mar 5.
Bromelain, papain, and ficin are studied the most for meat tenderization, but have limited application due to their short lifetime. The aim of this work is to identify the adsorption mechanisms of these cysteine proteases on chitosan to improve the enzymes' stability. It is known that immobilization can lead to a significant loss of enzyme activity, which we observed during the sorption of bromelain (protease activity compared to soluble enzyme is 49% for medium and 64% for high molecular weight chitosan), papain (34 and 28% respectively) and ficin (69 and 70% respectively). Immobilization on the chitosan matrix leads to a partial destruction of protein helical structure (from 5 to 19%). Using computer modelling, we have shown that the sorption of cysteine proteases on chitosan is carried out by molecule regions located on the border of domains L and R, including active cites of the enzymes, which explains the decrease in their catalytic activity upon immobilization. The immobilization on chitosan does not shift the optimal range of pH (7.5) and temperature values (60 °C for bromelain and papain, 37-60 °C for ficin), but significantly increases the stability of biocatalysts (from 5.8 times for bromelain to 7.6 times for papain).
菠萝蛋白酶、木瓜蛋白酶和无花果蛋白酶在嫩化肉类方面的研究最多，但由于它们的半衰期短，应用有限。本工作的目的是确定这些半胱氨酸蛋白酶在壳聚糖上的吸附机制，以提高酶的稳定性。众所周知，固定化会导致酶活性的显著损失，我们在菠萝蛋白酶（与可溶性酶相比，中分子量和高分子量壳聚糖的酶活性分别为 49%和 64%）、木瓜蛋白酶（分别为 34%和 28%）和无花果蛋白酶（分别为 69%和 70%）的吸附过程中观察到了这种情况。在壳聚糖基质上的固定化会导致蛋白质螺旋结构的部分破坏（从 5%到 19%）。通过计算机建模，我们已经表明，半胱氨酸蛋白酶在壳聚糖上的吸附是通过位于结构域 L 和 R 边界的分子区域进行的，包括酶的活性位点，这解释了固定化后其催化活性的下降。固定化不会改变最适 pH 值范围（7.5）和温度值（菠萝蛋白酶和木瓜蛋白酶为 60°C，无花果蛋白酶为 37-60°C），但显著提高了生物催化剂的稳定性（从菠萝蛋白酶的 5.8 倍到木瓜蛋白酶的 7.6 倍）。
