Efficient activity screening of new glucuronoyl esterases using a pNP-based assay

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology Pub Date : 2024-04-03 DOI:10.1016/j.enzmictec.2024.110444

Michael S. Madsen , Pedro A. Martins , Jane W. Agger

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引用次数: 0

Abstract

Glucuronoyl esterases (CE15, EC 3.1.1.117) catalyze the hydrolysis of ester bonds between lignin and carbohydrates in lignocellulose. They are widespread within fungi and bacteria, and are subjects to research interest due to their potential applicability in lignocellulose processing. Identifying new and relevant glucuronoyl esterase candidates is challenging because available model substrates poorly represent the natural substrate, which leads to inefficient screening for the activity. In this study, we demonstrate how fifteen novel, fungal, putative glucuronoyl esterases from family CE15 were expressed and screened for activity towards a commercially available, colorimetric assay based on the methyl-ester of 4-O-methyl-aldotriuronic acid linked to para-nitrophenol (methyl ester-UX-β-pNP) and coupled with the activity of GH67 (α-glucuronidase) and GH43 (β-xylosidase) activity. The assay provides easy means for accurately establishing activity and determining specific activity of glucuronoyl esterases. Out of the fifteen expressed CE15 proteins, seven are active and were purified to determine their specific activity. The seven active enzymes originate from Auricularia subglabra (3 proteins), Ganoderma sinensis (2 proteins) and Neocallimastix californiae (2 proteins). Among the CE15 proteins not active towards the screening substrate (methyl ester-UX-β-pNP) were proteins originating from Schizophyllum commune, Podospora anserina, Trametes versicolor, and Coprinopsis cinerea. It is unexpected that CE15 proteins from such canonical lignocellulose degraders do not have the anticipated activity, and these observations call for deeper investigations.

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利用基于 pNP 的检测方法高效筛选新型葡萄糖醛酸酯酶的活性

葡萄糖醛酸酯酶（CE15，EC 3.1.1.117）可催化水解木质纤维素中木质素与碳水化合物之间的酯键。它们广泛存在于真菌和细菌中，因其在木质纤维素加工中的潜在适用性而成为研究兴趣的主题。鉴定新的相关葡萄糖醛酸酯酶候选物具有挑战性，因为现有的模型底物不能很好地代表天然底物，从而导致活性筛选效率低下。在本研究中，我们展示了如何表达 CE15 家族的 15 种新型真菌推定葡萄糖醛酸酯酶，并通过一种市售的比色测定法筛选其活性，该测定法基于与对硝基苯酚（甲酯-UX-β-pNP）相连的 4-O-甲基-醛三尿酸的甲酯，并与 GH67（α-葡萄糖醛酸酶）和 GH43（β-木糖苷酶）的活性相结合。该测定法为准确确定葡萄糖醛酸酯酶的活性和特异性活性提供了简便的方法。在表达的 15 个 CE15 蛋白中，有 7 个具有活性，并通过纯化确定了它们的特异性活性。这七种有活性的酶分别来自 Auricularia subglabra（3 种蛋白）、Ganoderma sinensis（2 种蛋白）和 Neocallimastix californiae（2 种蛋白）。在对筛选底物（甲酯-UX-β-pNP）没有活性的 CE15 蛋白质中，有来自五味子（Schizophyllum commune）、Podospora anserina、Trametes versicolor 和 Coprinopsis cinerea 的蛋白质。出乎意料的是，来自这些典型木质纤维素降解物的 CE15 蛋白并不具有预期的活性，这些观察结果需要进行更深入的研究。

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来源期刊

Enzyme and Microbial Technology 生物-生物工程与应用微生物

CiteScore

7.60

自引率

5.90%

发文量

142

审稿时长

38 days

期刊介绍： Enzyme and Microbial Technology is an international, peer-reviewed journal publishing original research and reviews, of biotechnological significance and novelty, on basic and applied aspects of the science and technology of processes involving the use of enzymes, micro-organisms, animal cells and plant cells. We especially encourage submissions on: Biocatalysis and the use of Directed Evolution in Synthetic Biology and Biotechnology Biotechnological Production of New Bioactive Molecules, Biomaterials, Biopharmaceuticals, and Biofuels New Imaging Techniques and Biosensors, especially as applicable to Healthcare and Systems Biology New Biotechnological Approaches in Genomics, Proteomics and Metabolomics Metabolic Engineering, Biomolecular Engineering and Nanobiotechnology Manuscripts which report isolation, purification, immobilization or utilization of organisms or enzymes which are already well-described in the literature are not suitable for publication in EMT, unless their primary purpose is to report significant new findings or approaches which are of broad biotechnological importance. Similarly, manuscripts which report optimization studies on well-established processes are inappropriate. EMT does not accept papers dealing with mathematical modeling unless they report significant, new experimental data.