从海洋假交替单胞菌 SN-1009 中过表达和生化表征一种截短的内切-α(1→3)-岩藻聚糖酶。

Overexpression and biochemical characterization of a truncated endo-α (1 → 3)-fucoidanase from alteromonas sp. SN-1009.

机构信息

College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, China; National Key Laboratory of Biochemical Engineering, National Engineering Research Center for Biotechnology (Beijing), Key Laboratory of Biopharmaceutical Production & Formulation Engineering, PLA, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.

College of Life Sciences, Capital Normal University, Beijing 10048, China.

出版信息

Food Chem. 2021 Aug 15;353:129460. doi: 10.1016/j.foodchem.2021.129460. Epub 2021 Mar 7.

DOI:10.1016/j.foodchem.2021.129460

PMID:33725543

Abstract

Endo-fucoidanases are important in structural analysis of fucoidans and preparation of fuco-oligosaccharides. However their enzymological properties and analysis of degradation products are scarcely investigated. Truncated endo-α (1 → 3)-fucoidanase Fda1 (tFda1B from Alteromonas sp. was overexpressed and characterized, showing highest activity at pH 7.0, 35 °C, and 1.0 M NaCl. Its K and k were 3.88 ± 0.81 mg/mL and 0.82 ± 0.17 min. Fe and Mn enhanced activity by 100% and 19.5% respectively. Co and Cu completely inactivated tFda1B, whereas Ni, Mg, Zn, Pb, Ca, Ba and Li decreased activity by 58.8%, 56.0%, 50.6%, 47.7%, 28.9%, 15.6% and 37.5%, respectively. Catalytic residues were identified through structure and sequence alignment, and confirmed by mutagenesis. Degradation products of Kjellmaniella crassifolia fucoidan by tFda1B were characterized by LC-ESI-MS/MS, confirming tFda1B belongs to endo-(1 → 3)-fucoidanases, and backbone of K. crassifolia fucoidan is 1 → 3 fucoside linkage. This endo-α (1 → 3)-fucoidanase would be useful for elucidating fucoidan structures, and be used as a food enzyme.

摘要

内切岩藻聚糖酶在岩藻聚糖的结构分析和岩藻寡糖的制备中具有重要作用。然而，它们的酶学性质和降解产物的分析研究甚少。本研究从交替单胞菌中过表达并表征了截短的内切α(1→3)-岩藻聚糖酶 Fda1(tFda1B)，该酶在 pH7.0、35°C 和 1.0M NaCl 下表现出最高活性。其 K 和 k 分别为 3.88±0.81mg/mL 和 0.82±0.17min。Fe 和 Mn 分别使酶活提高了 100%和 19.5%。Co 和 Cu 完全使 tFda1B 失活，而 Ni、Mg、Zn、Pb、Ca、Ba 和 Li 分别使酶活降低了 58.8%、56.0%、50.6%、47.7%、28.9%、15.6%和 37.5%。通过结构和序列比对鉴定了催化残基，并通过突变证实了这些残基。利用 LC-ESI-MS/MS 对 Kjellmaniella crassifolia 岩藻聚糖的降解产物进行了表征，证实 tFda1B 属于内切-(1→3)-岩藻聚糖酶，K. crassifolia 岩藻聚糖的骨架为 1→3 岩藻糖苷键。该内切α(1→3)-岩藻聚糖酶将有助于阐明岩藻聚糖的结构，并可作为一种食品酶使用。