离子液体双水相提取木瓜蛋白酶及条件优化

Extraction of Papain by Ionic Liquid/Aqueous Two-phase System and Optimization of Process Conditions

采用[C4mim]Br/K2HPO4双水相体系提取木瓜蛋白酶。首先考察各成相试剂对木瓜蛋白酶活性的影响,其次考察了离子液体侧烷基链长度、离子液体浓度、酶添加量、pH、温度对木瓜蛋白酶分配行为的影响;并采用响应面法(CCD)优化[C4mim]Br/K2HPO4双水相萃取木瓜蛋白酶的条件。结果表明:离子液体对木瓜蛋白酶的活性影响较小,低浓度时还能促进酶活性,而无机盐对木瓜蛋白酶活性的影响较大;高温不利于离子液体双水相萃取木瓜蛋白酶;各因素对木瓜蛋白酶萃取的影响从大到小依次为K2HPO4的浓度、[C4mim]Br的浓度、酶添加量、pH。响应面优化得到的最佳萃取条件:0.30 g/mL的[C4mim]Br,0.30 g/mL的K2HPO4,pH 6.0,酶添加量3.0 mg/mL,温度30℃。在此条件下木瓜蛋白酶的酶活性回收率达到91.20%,纯化因子可以达到1.73。为该体系的放大实验或规模化生产提供理论指导。

Papain was extracted by a [C4mim] Br/K2HPO4 ionic liquid/aqueous two-phase system. First, the influence of reagents on the activity of papain was investigated. Second, the effects of alkyl chain length and concentration of the ionic liquid, dosage of papain, pH, and temperature on the partitioning behavior of papain were discussed concretely, and the extraction conditions were optimized using the central composite design （CCD）. The results showed that ionic liquids had little impact on the activity of papain and could promote the activity at low concentrations, while the inorganic salt had a larger influence on the activity of papain. There was a negative influence on the extraction of papain in the ionic liquid/aqueous two-phase system at high temperature. The influence of the various parameters was in the following order： concentration of K2HPO4 〉 concentration of [C4mim]Br〉dosage of papain〉 pH. The optimized extraction conditions were as follows： [C4mim] Br content, 0.30 g/mL; KEHPO4 content, 0.30 g/mL; pH, 6.0; dosage of papain, 3.0 mg/mL; reaction temperature, 30.0 ℃. Under the optimum conditions, the activity recovery of the enzyme and purification factor reached 91.20% and 1.73, respectively. The result provided the experimental basis for further research and large-scale production.