Production of recombinant glycosidases fused with Usp45 and SpaX to avoid the purification and immobilization stages

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Enzyme and Microbial Technology Pub Date : 2024-04-04 DOI:10.1016/j.enzmictec.2024.110445

José Antonio Curiel , Estela de Vega , Susana Langa , Ángela Peirotén , José María Landete

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Abstract

The elucidation of the physicochemical properties of glycosidases is essential for their subsequent technological application, which may include saccharide hydrolysis processes and oligosaccharide synthesis. As the application of cloning, purification and enzymatic immobilization methods can be time consuming and require a heavy financial investment, this study has validated the recombinant production of the set of Lacticaseibacillus rhamnosus fucosidases fused with Usp45 and SpaX anchored to the cell wall of Lacticaseibacillus cremoris subsp cremoris MG1363, with the aim of avoiding the purification and stabilization steps. The cell debris harboring the anchored AlfA, AlfB and AlfC fucosidases showed activity against p-nitrophenyl α-L-fucopyranoside of 6.11 ± 0.36, 5.81 ± 0.29 and 9.90 ± 0.58 U/mL, respectively, and exhibited better thermal stability at 50 °C than the same enzymes in their soluble state. Furthermore, the anchored AlfC fucosidase transfucosylated different acceptor sugars, achieving fucose equivalent concentrations of 0.94 ± 0.09 mg/mL, 4.11 ± 0.21 mg/mL, and 4.08 ± 0.15 mg/mL of fucosylgalatose, fucosylglucose and fucosylsucrose, respectively.

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生产与 Usp45 和 SpaX 融合的重组糖苷酶，以避免纯化和固定阶段

阐明糖苷酶的理化性质对其后续的技术应用至关重要，包括糖的水解过程和寡糖的合成。由于应用克隆、纯化和酶固定等方法耗时长且需要大量资金投入，本研究验证了将一组融合了 Usp45 和 SpaX 的鼠李糖乳杆菌岩藻糖苷酶锚定在 cremoris subsp cremoris MG1363 的细胞壁上进行重组生产的方法，目的是避免纯化和稳定化步骤。锚定AlfA、AlfB和AlfC岩藻糖苷酶的细胞碎片对对硝基苯α-L-岩藻糖苷的活性分别为6.11 ± 0.36、5.81 ± 0.29和9.90 ± 0.58 U/mL，在50 °C下的热稳定性优于可溶状态下的同种酶。此外，锚定的 AlfC 岩藻糖苷酶还能对不同的受体糖进行岩藻糖基化，使岩藻糖基加拉塔糖、岩藻糖基葡萄糖和岩藻糖基蔗糖的岩藻糖当量浓度分别达到 0.94 ± 0.09 mg/mL、4.11 ± 0.21 mg/mL 和 4.08 ± 0.15 mg/mL。

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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.