天然低共熔溶剂提取米糠酯酶工艺优化及其纯化与酶学性质研究

Natural Deep Eutectic Solvents Extraction and Purification of Rice Bran Esterase and Its Characteristics

目的:采用天然低共熔溶剂(Natural deep eutectic solvents,NADES)提取米糠酯酶并优化提取工艺参数,纯化米糠酯酶获得高纯度米糠酯酶,并探究其酶学特性。方法:合成了10种不同的NADES、选用了水浴搅拌和超声两种处理方式进行筛选,然后对提取效果最佳的NADES和方法进行单因素和响应面优化,参数包括料液比、含水量、提取温度和提取时间;将最优提取工艺得到的粗酶液通过阴离子交换树脂DEAE纯化,然后进行酶学性质的探究。结果:最佳提取米糠酯酶的工艺为在含有5%水分的脯氨酸-甘油(摩尔比1:2)溶剂体系中,水浴搅拌3.0 h,温度74.0℃,米糠和溶剂料液比为9:30,此时米糠酯酶活力为2.96 U。通过阴离子交换树脂DEAE纯化,获得了纯度较高的米糠酯酶,纯化倍数为1.74倍,回收率为69.40%,分子量约为35 kDa,对硝基苯酚乙酯为最适底物,最适温度和pH分别是40.0℃、pH8.0,在30.0~40.0℃和pH7.0~9.0时米糠酯酶均保持良好的稳定性。氯化胆碱-甘油、脯氨酸-甘油两种NADES对米糠酯酶的活力有较好的维持作用。结论:天然低共熔溶剂提取方式是一种简单、绿色的提取方式,可为生物活性化合物的绿色提取研究提供一些参考。

Objective: Rice bran esterase was extracted from rice bran using natural deep eutectic solvents(NADES) as media and the extraction processing of parameters was systematically optimized. The higher purity of rice bran esterase was obtained after purified with DEAE column and its enzyme properties were also investigated. Methods: Ten kinds of NADES including organic acids-based, polyols-based, amine-based and amino acids-based NADES were prepared. The extraction procedures including water bath agitation and ultrasonication were carried out. The effects of single factor such as the solid-to-liquid ratio, water content, extraction temperature and time on enzyme activity was studied. And then response surface methodology was used for optimization of the extraction processing. The crude enzyme solution was purified using DEAE column, as well as its enzyme characteristics were investigated. Results: The rice bran esterase activity of 2.96 U was obtained under the extraction conditions of proline-glycerol(molar ratio 1:2, contained 5% of water), 74.0 ℃, 3 h and a rice bran to NADES ratio of 9:30. The higher purity of rice bran esterase was obtained after purification with DEAE column. The purification efficiency and the recovery of rice bran esterase were 1.74 times, 69.40%,respectively. The molecular weight of rice bran esterase was approximately 35 kDa. The optimal substrate was a pnitrophenyl acetate, and the optimal temperature and pH were 40.0 ℃, 8.0, respectively. The better stability of rice bran esterase could be maintained under 30.0~40.0 ℃ and pH7.0~9.0, as well as the similar performance in choline chlorideglycerol and proline-glycerol. Conclusion: Natural deep eutectic solvents used as a medium for extraction procedure is a simple and green extraction route, which would provide the understanding of extraction strategies for preparation of function compounds.