微泡菌属ALW5褐藻胶裂解酶的酶学性质及酶解产物的抗氧化活性

Characterization of Alginate Lyase from Microbulbifer sp. ALW5 and Antioxidant Activity of Its Hydrolysates

利用褐藻胶为唯一碳源的选择性培养基,从腐烂海带分离产褐藻胶裂解酶菌株。采用16S r RNA基因序列分析,对其进行分类鉴定;采用DNS-还原糖法测定褐藻胶裂解酶活性,对其酶学性质进行研究;通过测定酶解产物清除DPPH自由基、·OH自由基和还原能力来评价其抗氧化活性。结果表明:获得1株产褐藻胶裂解酶菌株ALW5,16S rRNA基因序列分析显示该菌株归属于微泡菌属。菌株ALW5产胞外褐藻胶裂解酶,酶的最适反应温度45℃,最适pH 7.0;在50℃下处理30 min,可保留63%的酶活性,在55℃时酶的稳定性差;在pH 4.0~11.0条件下处理30 min,可保留60%以上的酶活性,pH稳定性范围宽;10 mmol/L的K^+、Na^+和Mg^(2+)对酶有明显的激活作用,而Ca^(2+)、Co^(2+)、Cu^(2+)、Ni^(2+)、Ba^(2+)、Zn^(2+)、Fe^(2+)、Fe3+和Al3+均对酶有不同程度的抑制作用;除了EDTA和SDS,酶对其余抑制剂和去污剂具有好的抗性。褐藻胶裂解酶酶解产物对DPPH自由基和·OH自由基的半数抑制剂量IC50分别为10.3 mg/m L和2.9 mg/m L,还原能力较强,具有良好的抗氧化能力。

An alginate lyase-producing marine bacterial strain was isolated from decayed Laminaria that would utilize alginate as a sole carbon source. The strain was identified by analysis of its 16 S r RNA gene sequence, phenotype and biochemical reactions. Alginate lyase activity was determined by dinitrosalicylic acid method, and the characteristics of alginate lyase were determined. The antioxidant activity of hydrolysate was studied by DPPH radical scavenging, ·OH radical scavenging and reducing capacity. The results showed that strain ALW5 could produce extracellular alginate lyase,which belonged to polymannuronic-specific lyase. 16 S r RNA phylogenetic analysis showed that this strain was assigned to the genus Microbulbifer sp.. The alginate lyase was with an optimal temperature and pH of 45 ℃ and pH 7.0, respectively. It retained 63% residual activity after incubation at 50 ℃ for 30 min, and had poor stability at 55 ℃. The enzyme showed stability over a wide pH range of 4.0-11.0, maintaining over 60% residual activities after incubation for 30 min.10 mmol/L of K^＋, Na^＋, and Mg^2＋were found to significantly enhance the enzyme activity, while Ca^2＋, Co^2＋, Cu^2＋, Ni^2＋,Ba^2＋, Zn^2＋, Fe^2＋, Fe3 ＋, and Al3 ＋suppressed the enzymatic activity. Except for EDTA and SDS, strain ALW5 alginate lyase had good resistance to other detected inhibitors and detergents. The hydrolysates had reducing power. The half inhibitory concentration（IC50） values of scavenging DPPH and ·OH activity were 10.3 mg/m L and 2.9 mg/m L, individually.Therefore, the hydrolysates had an obvious antioxidant activity.