噬琼胶菌产新琼二糖热稳定琼胶酶的酶学性质及酶解产物的抗氧化活性

Characterization of A Neoagarobiose-Producing and Thermostableβ-agarase from Agarivorans sp.AL1 and Antioxidant Activity of the Enzymatic Hydrolysates

利用PCR从噬琼胶菌AL1中扩增琼胶酶基因,将其克隆至表达载体p ET-28a;表达产物用亲和层析纯化,研究重组酶的酶学性质;利用薄层色谱和质谱鉴定酶解产物,并测定酶解产物的还原能力和清除DPPH、ABTS+和·OH自由基的能力,分析酶解产物的抗氧化活性。结果表明,来自噬琼胶菌AL1重组琼胶酶的分子质量为105 ku。该琼胶酶能专一地降解琼脂,为β-琼胶酶。重组琼胶酶在50℃和p H 7.0表现出最大酶活力,在50℃以下,p H 5.00~11.00宽p H范围内稳定性良好,说明该重组酶具有良好的热稳定性和p H稳定性。经薄层色谱和质谱鉴定酶解产物为新琼二糖,该酶解产物对·OH、ABTS和DPPH自由基的半数抑制剂量IC50分别为1.28 mg/mL、3.46 mg/mL和9.87 mg/mL,还原能力较强,说明该酶解产物具有良好的抗氧化活性。

The agarase gene AL1 was amplified by PCR and cloned into pET-28 a expression vector.The expression product was purified by affinity chromatography to study the enzymatic properties of the recombinant enzyme.The enzymatic hydrolysate was identified by TLC and LC-MS,and its reducing ability and scavenging ability of DPPH,ABTS+·and·OH radicals were determined,and the antioxidant activity of the hydrolysate was analyzed.The molecular weight of the recombinant agarase from Agarivorans sp.AL1 was 105 ku.The agarase could specifically degrade agar as aβ-agarase.The recombinant agarase showed the maximum activity at 50℃and p H 7.00.The recombinant agarase was stable in a wide pH range from 5.00 to 11.00 below 50℃,indicating that the recombinant enzyme had good thermal stability and p H stability.The enzymatic hydrolysate product was identified as neoagarobiose by TLC and LC-MS.The enzymatic hydrolysates had the reducing power.The half-inhibition concentration IC50 of the enzymatic hydrolysate to·OH,ABTS and DPPH free radicals are 1.28 mg/mL,3.46 mg/m L and 9.87 mg/m L,respectively,indicating that the hydrolysate had good antioxidant activity.