微泡菌昆布多糖酶Lam128A的酶学性质及酶解产物抗氧化活性

Enzymatic Properties of Laminarinase Lam128A from Microbulbifer sp. ALW1 and Antioxidant Activity of Its Enzymatic Hydrolysates

摘要:

该研究探讨微泡菌ALW1来源的昆布多糖酶Lam128A的酶学性质及其酶解产物的抗氧化活性。利用毕赤酵母异源表达重组蛋白rLam128A，该昆布多糖酶的分子量为70 ku，最适反应温度和最适反应pH值分别为45 ℃和pH值5.5；温度低于45 ℃时稳定，分别在pH值3.0、5.0和11.0条件下处理96 h后，残余酶活力不低于60%。还原试剂二硫苏糖醇（DTT）对昆布多糖酶活力有促进作用，rLam128A对螯合剂乙二胺四乙酸（EDTA）表现出良好的稳定性，对去垢剂Tween 20和Tween 80、变性剂尿素表现出一定的稳定性。昆布多糖经rLam128A水解后，酶解产物对DPPH、ABTS和OH·自由基的半数抑制剂量IC50分别为7.12、1.01和 2.40 mg/mL，相比未经酶解处理的昆布多糖表现出更显著的抗氧化活性。微泡菌昆布多糖酶Lam128A的酶学性质为该酶开发利用昆布多糖生物资源提供了理论基础。

Abstract:

The enzymatic properties of laminarinase Lam128A derived from Microbulbifer sp. ALW1 and antioxidant activity of its enzymatic hydrolysates were investigated. Recombinant laminarinase (rLam128A) with a molecular mass of 70 ku was heterologously expressed in Pichia pastoris. The optimal reaction temperature and pH were 45°C and 5.5, respectively. The enzyme was stable at temperatures below 45 °C, and the residual enzyme activity did not drop below 60% following treatment at pH 3.0, 5.0, and 11.0 for 96 h. The reducing reagent dithiothreitol promoted laminarinase activity. rLam128A showed good stability in the presence of the chelating agent ethylenediaminetetraacetic acid as well as certain stability in the presence of the detergents Tween 20, Tween 80, and denaturant urea. Following laminarin hydrolysis by rLam128A, the IC50 values of the hydrolysates against DPPH, ABTS, and OH free radicals were 7.12, 1.01, and 2.40 mg/mL, respectively. These results indicate that the enzymatic hydrolysates exhibited more significant antioxidant activity than the unhydrolyzed laminarin. The above enzymatic properties of laminarinase Lam128A from Microbulbifer sp. ALW1 provide a theoretical basis for its application in the development and utilization of biological resources of laminarin.