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微泡菌QZHA1褐藻胶裂解酶MAAL1的酶学性质研究
闫康路,邵宗泽,王万鹏,谢珍玉,周梅先
0
(海南大学海洋生物与水产学院,海南 海口570000;自然资源部第三海洋研究所、自然资源部海洋生物遗传资源重点实验室,福建 厦门 361005)
摘要:
为探究Microbulbifer sp. QZHA1褐藻胶裂解(Escherichia coli)酶MAAL1的酶学性质,将褐藻胶裂解酶基因maal构建至pET-28a表达载体并利用大肠杆菌BL21(DE3)进行异源表达。研究发现:重组酶MAAL1与来源于Microbulbifer sp. ALW1菌株的褐藻胶裂解酶(WP_23625014.1)同源性最高,为93.69%,且与PL7家族蛋白聚为一支;重组酶MAAL1最适温度为35 ℃,最适pH为7.5,在pH为 5.5~10.5范围内保存24 h仍能保持60%以上的酶活力;MAAL1具备良好的耐有机溶剂特性,在测试的9种有机溶剂中,除异丙醇外,其他有机溶剂在添加量达到30%(体积分数)后,酶活力依然保持在59%以上;重组酶MAAL1最适条件下酶活力为4.3 U/mg,米氏常数(Km)值为1.08 mg/mL,最大反应速率(Vmax)为4.75 mg/(mL·min),催化常数(Kcat)值为4.52 s-1;重组酶MAAL1对聚β-D-甘露糖醛酸(polymannuronic acid, PolyM)具有底物特异性,对聚α-L-古罗糖醛酸(polyguluronic acid, PolyG)无降解能力;薄层层析分析显示,MAAL1降解海藻酸钠的主要终产物是单糖、二糖和三糖,降解PolyM的主要终产物是二糖和三糖,降解MG杂合片段(heteropolymeric MG blocks, PolyMG)的主要终产物为单糖和二糖,故MAAL1是一种内切型聚甘露糖醛酸裂解酶。重组酶MAAL1具有良好的pH和有机溶剂耐受性,对PolyM具有底物特异性且不降解PolyG,该特性褐藻胶裂解酶首次在微泡菌属中被发现,该酶可为制备甘露糖醛酸寡糖(mannuronate oligosaccharides, MAOS)提供新的候选用酶。
关键词:  海洋生物学  微泡菌属  褐藻胶裂解酶  克隆表达  聚甘露糖醛酸  耐有机溶剂
DOI:10.3969/J.ISSN.2095-4972.20230224001
基金项目:国家自然科学基金(42030412);中国大洋协会资助项目(DY135-B2-01)
Enzymatic properties of alginate lyase MAAL1 from Microbulbifer sp. QZHA1
YAN Kanglu,SHAO Zongze,WANG Wanpeng,XIE Zhenyu,ZHOU Meixian
(School of Marine Biology and Fisheries, Hainan University, Haikou 570228, China;Key Laboratory of Marine Genetic Resources, Third Institute of Oceanography, MNR, Xiamen 361005, China)
Abstract:
To investigate the enzymatic properties of MAAL1, an alginate lyase fromMicrobulbifer sp. QZHA1, the alginate lyase gene maal was constructed into the pET-28a expression vector and heterologously expressed using Escherichia coli BL21 (DE3). The recombinant enzyme MAAL1 has the highest homology of 93.69% with the alginate lyase (WP_23625014.1) from Microbulbifer sp. ALW1 and is clustered in the Polysaccharide Lyase family 7 (PL7). The optimum temperature for the recombinant enzyme MAAL1 is 35 °C and the optimum pH is 7.5, and it maintains more than 60% of its enzymatic activity when stored at pH 5.5 to 10.5 for 24 hours. MAAL1 has good resistance to organic solvents. Among the nine organic solvents tested, the enzyme activity remained above 59% after the addition of 30% (v/v) of all organic solvents except isopropyl alcohol. The specific activity of recombinant enzyme MAAL1 was 4.3 U/mg under the optimum conditions, the Km value of the constant was 1.08 mg/mL, the maximum reaction rate Vmax was 4.75 mg/(mL·min), and the catalytic constantKcat was 4.52 s-1. The recombinant enzyme MAAL1 had a substrate preference for polymannuronic acid (PolyM), and no degradation ability to polyguluronic acid (PolyG). Thin-layer chromatography analysis showed that the main end-products of degradation of sodium alginate by MAAL1 were monosaccharides, disaccharides and trisaccharides, the main end-products of degradation of PolyM were disaccharides and trisaccharides, and the main end-products of degradation of heteropolymeric MG blocks (PolyMG) were monosaccharides and disaccharides. It suggests that MAAL1 is an endolytic polymannuronic acid specific lyase. The recombinant enzyme MAAL1 has good pH and organic solvent tolerance, is substrate specific for PolyM and does not degrade PolyG. This characteristic alginate lyase was first identified in Microbulbifer, and this enzyme may provide a new candidate for use in the preparation of mannuronate oligosaccharides.
Key words:  marine biology  Microbulbifer  alginate lyase  cloning and expression  polymanuronic acid  organic solvent resistance

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