游离和固定化壳聚糖基质蛋白酶的紫外敏感性研究

S. Pankova, M. Holyavka, V. Artyukhov
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引用次数: 0

摘要

:在现代环境条件下,紫外线辐射是自然和人工气候的重要因素,对生命系统具有持续的影响。胶原酶、菠萝蛋白酶、无花果蛋白酶、木瓜蛋白酶(Sigma-Aldrich: St. Louis, MO, USA)和胰蛋白酶(MP biomedicals: Santa Ana, CA, USA)是本研究的对象。水解底物为BSA (Sigma-Aldrich: St. Louis, MO, USA),固定化载体为壳聚糖(< 100,200和350 kDa)和壳聚糖丁二酸盐(Bioprogress: Shchyolkovo, Russia)。采用吸附法固定化蛋白酶。采用改进的Lowry法测定样品中的蛋白质量及其催化活性。对蛋白酶进行剂量为151 ~ 6040 J/ m2的紫外线照射。水解酶按光敏程度依次排列为:胶原酶→菠萝蛋白酶→无花果蛋白酶→木瓜蛋白酶→胰蛋白酶。与游离酶相比,壳聚糖和壳聚糖琥珀酸盐对多相(固定化)生物催化剂的紫外稳定性有所提高。壳聚糖的光保护作用可能是由于以下原因:酶与壳聚糖相互作用形成抗光复合物;木聚糖屏蔽活性自由基,防止某些氨基酸的光氧化,包括在所研究的酶的活性中心在紫外线照射的影响下。
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UV-Sensitivity of Free and Immobilized on Chitosan Matrix Proteases
: UV irradiation is an essential factor in natural and artificial climate in modern environmental conditions, which has a constant effect on living systems. Collagenase, bromelain, ficin, papain (Sigma-Aldrich: St. Louis, MO, USA) and trypsin (MP biomedicals: Santa Ana, CA, USA) were the objects of this study. The substrate for hydrolysis was BSA (Sigma-Aldrich: St. Louis, MO, USA), the carriers for immobilization were chitosans (<100, 200 and 350 kDa) and chitosan succinate (Bioprogress: Shchyolkovo, Russia). The protease immobilization was carried out by the adsorption. The determination of the protein amount in samples and their catalytic activity was carried out by the modified Lowry method. UV irradiation of proteases was performed using doses 151–6040 J/m 2 . By the degree of photosensitivity, hydrolases can be arranged in the next row: collagenase → bromelain → ficin → papain → trypsin. Adsorption on a chitosan and succinate of chitosan leads to an increase in the stability to ultraviolet light of heterogeneous (immobilized) biocatalysts compared to free enzymes. Photoprotective effect of the chitosan may be due to the following reasons: enzyme interact with the chitosan to form photo resistant complexes; с hitosan screens active free-radicals, preventing the photooxidation of a certain number of amino acids, including the active centers of the studied enzymes under the influence of UV irradiation.
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