设计量身定制的立体物质,提高固定化菲辛对小蛋白和大蛋白的特异性

IF 4.1 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
El Hocine Siar , Pedro Abellanas-Perez , Roberto Morellon-Sterling , Juan M. Bolivar , Javier Rocha-Martin , Roberto Fernandez-Lafuente
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引用次数: 0

摘要

开发能够通过产生立体阻碍来调整固定蛋白酶大小特异性的策略可能会扩大其应用范围。以固定在乙醛琼脂糖上的 ficin 为模型,我们采用了两种策略来产生量身定制的立体障碍。首先,在涂有大分子蛋白质(血红蛋白或牛血清白蛋白(BSA))的载体上共同固定了 ficin。与 BSA 共同固定的 ficin 对任何检测底物的活性都没有影响,而与血红蛋白共同固定的 ficin 对酪蛋白和血红蛋白的特异性有所提高,但对血红蛋白的活性仍然很强。其次,还采用了醛葡聚糖来修饰固定化的 ficin,试图产生立体阻碍,以避免大蛋白(血红蛋白)进入,同时使小蛋白(酪蛋白)能够进入。这也提高了飞蓟素的大小特异性,但仍不能抑制其对血红蛋白的活性。将这两种策略结合起来,并在蛋白水解过程中使用 37ºC 温度,几乎完全消除了对血红蛋白的水解活性,同时保留了很高比例的对酪蛋白的水解活性。这些改性提高了酶的稳定性,生物催化剂可重复使用 5 个周期而不会改变其特性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Designing tailor-made steric matters to improve the immobilized ficin specificity for small versus large proteins

The development of strategies that can permit to adjust the size specificity of immobilized proteases by the generation of steric hindrances may enlarge its applicability. Using as a model ficin immobilized on glyoxyl agarose, two strategies were assayed to generate tailor made steric hindrances. First, ficin has been coimmobilized on supports coated with large proteins (hemoglobin or bovine serum albumin (BSA)). While coimmobilization of ficin with BSA presented no effect on the activity versus any of the assayed substrates, coimmobilization with hemoglobin permitted to improve the immobilized ficin specificity for casein versus hemoglobin, but still significant activity versus hemoglobin remained. Second, aldehyde-dextran has been employed to modify the immobilized ficin, trying to generate steric hindrances to avoid the entry of large proteins (hemoglobin) while enabling the entry of small ones (casein). This also increased the size specificity of ficin, but still did not suppress the activity versus hemoglobin. The combination of both strategies and the use of 37ºC during the proteolysis enabled to almost fully nullify the hydrolytic activity versus hemoglobin while preserving a high percentage of the activity versus casein. The modifications improved enzyme stability and the biocatalyst could be reused for 5 cycles without alteration of its properties.

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来源期刊
Journal of biotechnology
Journal of biotechnology 工程技术-生物工程与应用微生物
CiteScore
8.90
自引率
2.40%
发文量
190
审稿时长
45 days
期刊介绍: The Journal of Biotechnology has an open access mirror journal, the Journal of Biotechnology: X, sharing the same aims and scope, editorial team, submission system and rigorous peer review. The Journal provides a medium for the rapid publication of both full-length articles and short communications on novel and innovative aspects of biotechnology. The Journal will accept papers ranging from genetic or molecular biological positions to those covering biochemical, chemical or bioprocess engineering aspects as well as computer application of new software concepts, provided that in each case the material is directly relevant to biotechnological systems. Papers presenting information of a multidisciplinary nature that would not be suitable for publication in a journal devoted to a single discipline, are particularly welcome.
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