厌氧菌Sunxiuqinia sp. SH-52外切型海藻酸裂解酶的异源表达及酶学特征

Heterologous expression and characterization of exo-type alginate lyase from anaerobic bacterium Sunxiuqinia sp. SH-52

摘要:

【背景】目前已报道的海藻酸分解菌多数为好氧菌，未见有关厌氧菌的报道。从分离的海藻酸分解菌中表征的海藻酸裂解酶大多为内切型海藻酸裂解酶，外切型较少。【目的】研究来自厌氧海藻酸分解菌的海藻酸裂解酶基因，表征新型海藻酸裂解酶并阐明其酶学性质，为海藻酸裂解酶的多样性和微生物降解海藻酸机制提供理论依据。【方法】对来自厌氧海藻酸分解菌Sunxiuqinia sp. SH-52的海藻酸裂解酶SHA-4编码基因进行克隆，分析基因序列，构建重组质粒PGEX-4T-1-SHA-4并在大肠杆菌中实现异源表达，经纯化后对其酶学性质及降解特征进行研究。【结果】该酶在28 °C、用0.1 mmol/L IPTG (异丙基-β-D-硫代半乳糖苷)条件下诱导6 h达到最大表达量，纯化后酶的比活力达到21 U/mg。酶学性质分析表明SHA-4的最适温度为37 °C；最适pH为7.5；对PolyMG (杂聚古罗糖醛酸-甘露糖醛酸嵌段)具有底物偏好性；Na+对该酶的活性具有抑制作用，而金属离子Cu2+具有明显促进作用，使活性提高了约168%；SHA-4催化海藻酸的Km值为2.5 mg/mL，Vmax为8.7 mg/(mL·min)；SHA-4为外切型海藻酸裂解酶，降解海藻酸终产物为单糖。【结论】异源表达了来自一株厌氧海藻酸分解菌Sunxiuqinia sp. SH-52的海藻酸裂解酶SHA-4，该酶是PL6家族中第一个对PolyMG有底物偏好性的外切型海藻酸裂解酶，而且活性较高，作为工具酶有很好的应用前景，为海藻酸降解机制的探索提供了新的线索。

Abstract:

[Background] Most alginolytic bacteria currently reported are aerobic bacteria and studies on anaerobic ones have not been reported yet. Characterization of alginate lyases isolated from alginolytic bacteria are mainly focused on the endo-type alginate lyases and rarely on exo-type ones. [Objective] To investigate the genes encoding alginate lyases isolated from anaerobic alginolytic bacteria, to characterize the novel alginate lyase, and to elucidate its enzymatic properties, providing a theoretical basis for the diversity of alginate lyases and the mechanism of microbial degradation of alginate. [Methods] The gene encoding SHA-4, an alginate lyase isolated from an anaerobic alginolytic bacterium Sunxiuqinia sp. SH-52, was cloned and sequenced. Recombinant plasmid PGEX-4T-1-SHA-4 was constructed and heterologously expressed in E. coli. The expressed enzyme was purified and the enzymatic and degradation characteristics were analyzed. [Results] Maximum expression was achieved when induced by 0.1 mmol/L IPTG (Isopropyl-β-D-Thiogalactoside) at 28 °C for 6 hours and the specific activity of the purified enzyme was 21 U/mg. Characterization of the enzyme showed that the optimal condition for SHA-4 includes the temperature at 37 °C, pH at 7.5, and PolyMG (heteropolymeric MG blocks) as a preferred substrate. The activity of this enzyme was inhibited by Na+ and significantly elevated to about 168% by Cu2+. Km and Vmax of SHA-4 when used to catalyze alginate were 2.5 mg/mL and 8.7 mg/(mL·min), respectively. SHA-4 was an exo-type alginate lyase with monosaccharides as the final degradation products. [Conclusion] An alginate lyase SHA-4 isolated from an anaerobic alginolytic bacterium Sunxiuqinia sp. SH-52 is successfully expressed. Among other PL6 family members, SHA-4 is the first exo-type alginate lyase with PolyMG as a preferred substrate. Along with its relatively high enzymatic activity, SHA-4 may serve as a tool enzyme with promising application and provide new clues for further exploration of the mechanism of alginate degradation.