Changes in protein substrate specificity during inactivation of different Alcalase formulations depends on inactivation conditions

IF 4.4 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry Pub Date : 2026-03-01 Epub Date: 2026-01-07 DOI:10.1016/j.procbio.2026.01.003

Alan Portal D'Almeida , Pedro Abellanas- Perez , Tiago Lima de Albuquerque , Luciana Rocha Barros Gonçalves , Ivanildo José da Silva Junior , Roberto Fernandez-Lafuente

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Abstract

Free and carrageenan immobilized Alcalase preparations were incubated at 70ºC and pHs 5, 7, and 10, and their activities were monitored with the synthetic substrate Boc-L-Ala-4-nitrophenyl ester (NPA), casein, bovine serum albumin and hemoglobin. The inactivation profiles were fully different when followed using the different substrates. Stability was much lower when using NPA compared to the results obtained when using the substrate proteins. A great diversity could also be observed using the different protein substrates, however, the highest stability for the free and immobilized Alcalase was observed using different substrate proteins depending on the inactivation conditions, thus affecting the stabilization factors achieved by the immobilization (e.g., stabilization caused by the immobilization was negligible at pH 10 using HG, but very relevant at pH 5 using NPA). This was explained because the new enzyme conformations generated during inactivation, which depend on the inactivation conditions and enzyme formulations, have new protein substrate specificities.

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不同Alcalase制剂失活过程中蛋白质底物特异性的变化取决于失活条件

游离Alcalase和卡拉胶固定化Alcalase制剂分别在70℃和ph值5、7和10下孵育，并与合成底物boc - l - ala -4-硝基苯基酯（NPA）、酪蛋白、牛血清白蛋白和血红蛋白一起监测其活性。不同底物的失活谱完全不同。与使用底物蛋白时获得的结果相比，使用NPA时的稳定性要低得多。使用不同的蛋白质底物也可以观察到很大的多样性，然而，根据失活条件的不同，使用不同的底物蛋白质可以观察到自由和固定化Alcalase的最高稳定性，从而影响固定化所达到的稳定因子（例如，使用HG在pH 10时固定化所引起的稳定性可以忽略不计，但使用NPA在pH 5时则非常相关）。这是因为在失活过程中产生的新酶构象取决于失活条件和酶制剂，具有新的蛋白质底物特异性。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Process Biochemistry 生物-工程：化工

CiteScore

8.30

自引率

4.50%

发文量

374

审稿时长

53 days

期刊介绍： Process Biochemistry is an application-orientated research journal devoted to reporting advances with originality and novelty, in the science and technology of the processes involving bioactive molecules and living organisms. These processes concern the production of useful metabolites or materials, or the removal of toxic compounds using tools and methods of current biology and engineering. Its main areas of interest include novel bioprocesses and enabling technologies (such as nanobiotechnology, tissue engineering, directed evolution, metabolic engineering, systems biology, and synthetic biology) applicable in food (nutraceutical), healthcare (medical, pharmaceutical, cosmetic), energy (biofuels), environmental, and biorefinery industries and their underlying biological and engineering principles.