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首页> 外文期刊>Biotechnology Progress >Protein Hydrolysis by Immobilized and Stabilized Trypsin
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Protein Hydrolysis by Immobilized and Stabilized Trypsin

机译：固定化和稳定化胰蛋白酶的蛋白质水解

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摘要

The preparation of novel immobilized and stabilized derivatives of trypsin is reported here. The new derivatives preserved 80% of the initial catalytic activity toward synthetic substrates [benzoyl-arginine p-nitroanilide (BAPNA)] and were 50,000-fold more thermally stable than the diluted soluble enzyme in the absence of autolysis. Trypsin was immobilized on highly activated glyoxyl-Sepharose following a two-step immobilization strategy: (a) first, a multipoint covalent immobilization at pH 8.5 that only involves low pK_a amino groups (e.g., those derived from the activation of trypsin from trypsinogen) is performed and (b) next, an additional alkaline incubation at pH 10 is performed to favor an intense, additional multipoint immobilization between the high concentration of proximate aldehyde groups on the support surface and the high pK_a amino groups at the enzyme surface region that participated in the first immobilization step. Interestingly, the new, highly stable trypsin derivatives were also much more active in the proteolysis of high molecular weight proteins when compared with a nonstabilized derivative prepared on CNBr-activated Sepharose. In fact, all the proteins contained a cheese whey extract had been completely proteolyzed after 6 h at pH 9 and 50℃, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under these experimental conditions, the immobilized biocatalysts preserve more than 90% of their initial activity after 20 days. Analysis of the three-dimensional (3D) structure of the best immobilized trypsin derivative showed a surface region containing two amino terminal groups and five lysine (Lys) residues that may be responsible for this novel and interesting immobilization and stabilization. Moreover, this region is relatively far from the active site of the enzyme, which could explain the good results obtained for the hydrolysis of high-molecular weight proteins.
机译：此处报道了胰蛋白酶的新型固定化和稳定化衍生物的制备。在没有自溶作用的情况下，新衍生物保留了对合成底物[苯甲酰基-精氨酸对硝基苯胺（BAPNA）]的80％的初始催化活性，并且热稳定性比稀释的可溶性酶高50,000倍。遵循两步固定策略，将胰蛋白酶固定在高度活化的乙二醛-琼脂糖上：（a）首先，在pH 8.5的多点共价固定仅涉及低pK_a氨基（例如，源自胰蛋白酶原的胰蛋白酶活化的氨基）。进行（b），然后在pH 10进行额外的碱性孵育，以促进在支持物表面高浓度的近醛基团和参与该酶作用的酶表面区域的高pK_a氨基之间进行强烈的多点固定化。第一步固定。有趣的是，与在CNBr活化的Sepharose上制备的不稳定化合物相比，新的高度稳定的胰蛋白酶衍生物在高分子量蛋白质的蛋白水解中也更具活性。实际上，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定，pH 9和50℃下6 h后，所有含有干酪乳清提取物的蛋白质都已被完全蛋白水解。在这些实验条件下，固定的生物催化剂在20天后保留了超过90％的初始活性。最佳固定化胰蛋白酶衍生物的三维（3D）结构分析显示，含有两个氨基末端基团和五个赖氨酸（Lys）残基的表面区域可能是这种新颖而有趣的固定化和稳定作用的原因。此外，该区域距离酶的活性位点相对较远，这可以解释高分子量蛋白水解获得的良好结果。

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