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Stabilization of Glycosylated β-Glucosidase by Intramolecular Crosslinking Between Oxidized Glycosidic Chains and Lysine Residues.
Applied Biochemistry and Biotechnology ( IF 3 ) Pub Date : 2020-05-07 , DOI: 10.1007/s12010-020-03321-x
Laura Marina Pinotti 1 , Paulo Waldir Tardioli 2 , Cristiane Sanchez Farinas 2, 3 , Gloria Fernández-Lorente 4 , Alejandro H Orrego 5 , Jose M Guisan 5 , Benevides C Pessela 4
Affiliation  

Many industrial enzymes can be highly glycosylated, including the β-glucosidase enzymes. Although glycosylation plays an important role in many biological processes, such chains can cause problems in the multipoint immobilization techniques of the enzymes, since the glycosylated chains can cover the reactive groups of the protein (e.g., Lys) and do not allow those groups to react with reactive groups of the support (e.g., aldehyde and epoxy groups). Nevertheless, the activated glycosylated chains can be used as excellent crosslinking agents. The glycosylated chains when oxidized with periodate can generate aldehyde groups capable of reacting with the amino groups of the protein itself. Such intramolecular crosslinks may have significant stabilizing effects. In this study, we investigated if the intramolecular crosslinking occurs in the oxidized β-glucosidase and its effect on the stability of the enzyme. For this, the oxidation of glycosidic chains of β-glucosidase was carried out, allowing to demonstrate the formation of aldehyde groups and subsequent interaction with the amine groups and to verify the stability of the different forms of free enzyme (glycosylated and oxidized). Furthermore, we verified the influence of the glycosidic chains on the immobilization of β-glucosidase from Aspergillus niger and on the consequent stabilization. The results suggest that intramolecular crosslinking occurred and consequently the oxidized enzyme showed a much greater stabilization than the native enzyme (glycosylated). When the multipoint immobilization was performed in amino-epoxy-agarose supports, the stabilization of the oxidized enzyme increases by a 6-fold factor. The overall stabilization strategy was capable to promote an enzyme stabilization of 120-fold regarding to the soluble unmodified enzyme.

中文翻译:

通过氧化的糖苷链和赖氨酸残基之间的分子内交联来稳定糖基化的β-葡萄糖苷酶。

许多工业酶可以高度糖基化,包括β-葡萄糖苷酶。尽管糖基化在许多生物学过程中起着重要作用,但是由于糖基化的链可以覆盖蛋白质的反应性基团(例如Lys),并且不允许这些基团发生反应,因此此类链会在酶的多点固定化技术中引起问题。具有载体的反应性基团(例如,醛基和环氧基)。然而,活化的糖基化链可以用作优异的交联剂。当用高碘酸盐氧化时,糖基化的链可以产生能够与蛋白质本身的氨基反应的醛基。这样的分子内交联可以具有显着的稳定作用。在这个研究中,我们研究了在氧化的β-葡萄糖苷酶中是否发生了分子内交联及其对酶稳定性的影响。为此,进行了β-葡糖苷酶的糖苷链的氧化,从而证明了醛基的形成以及随后与胺基的相互作用,并验证了不同形式的游离酶(糖基化和氧化)的稳定性。此外,我们验证了糖苷链对黑曲霉固定化β-葡萄糖苷酶的影响以及对随后稳定性的影响。结果表明发生了分子内交联,因此氧化酶显示出比天然酶(糖基化的)更大的稳定性。当在氨基-环氧-琼脂糖支持物上进行多点固定时,氧化酶的稳定性提高了6倍。总体稳定策略能够促进相对于可溶性未修饰酶的酶稳定120倍。
更新日期:2020-05-07
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