In this work, the effect of adding different quaternary ammonium ionic liquids (ILs) on the phase formation behaviour of a PEG400+(NH₄)₂SO₄+H₂O aqueous two-phase system (ATPS) and separation and purification of crude papain extract from papaya latex in different ATPSs was studied. Phase diagrams were established for different systems: experimental phase diagram data were fitted by the Merchuk equation, and the tie line (TL), composition and tie line length (TLL) were determined. The papain purification effect of ATPSs with various ILs was systematically scrutinized. The absorption spectrum of papain before and after extraction was characterized by UV-vis absorption spectroscopy. SDS-PAGE electrophoresis was used to verify the purity of papain extracted by the ATPSs. Quaternary ammonium ILs as adjuvants did not extend the biphasic area in the phase diagram, but the partition behaviour of crude papain extract in ATPSs changed, resulting in additional IL and papain interactions that promoted papain purification. The highest papain purity was obtained when tetramethyl ammonium bromide ([N₁₁₁₁]Br) was added to the ATPS, and with 20 wt% (NH₄)₂SO₄ and 20 wt% PEG400 at pH 7.0 and 60 °C, the purification factor of crude papain extract was 2.13 and 13.51 and the papain recovery rate was 64.2% and 96.46% for ATPSs with no IL and 4 wt% [N₁₁₁₁] Br, respectively. The UV spectra of papain before and after extraction were the same. The SDS-PAGE analysis revealed that the ATPS containing 4 wt% [N₁₁₁₁] Br enhanced papain purification relative to the ATPS without IL. The addition of 4 wt% IL greatly improved the purification factor of crude papain extract enriched in the PEG phase, providing a potential method for large-scale industrial production of high-purity papain.

在这项工作中，添加不同的季铵离子液体（ILs）对PEG400 +（NH ₄）₂ SO ₄ + H ₂的相形成行为的影响研究了水两相系统（ATPS）和木瓜乳胶粗提物在不同ATPS中的分离纯化。建立了针对不同系统的相图：通过Merchuk方程拟合实验相图数据，并确定联系线（TL），组成和联系线长度（TLL）。系统地研究了具有各种IL的ATPS的木瓜蛋白酶纯化效果。用紫外可见吸收光谱法对木瓜蛋白酶提取前后的吸收光谱进行表征。SDS-PAGE电泳用于验证ATPS提取的木瓜蛋白酶的纯度。季铵ILs作为佐剂并未扩展相图中的双相区，但粗木瓜提取物在ATPS中的分配行为发生了变化，导致额外的IL和木瓜蛋白酶相互作用，促进木瓜蛋白酶的纯化。当四甲基溴化铵（[N_将1111 ] Br）添加到ATPS中，并在pH 7.0和60°C下使用20 wt％（NH ₄）₂ SO ₄和20 wt％PEG400，粗木瓜提取物的纯化因子为2.13和13.51，木瓜蛋白酶的回收率没有IL和4 wt％[N ₁₁₁₁ ] Br的ATPS的产率分别为64.2％和96.46％。提取前后木瓜蛋白酶的紫外光谱相同。SDS-PAGE分析显示，相对于不含IL的ATPS，包含4 wt％[N ₁₁₁₁ ] Br的ATPS增强了木瓜蛋白酶的纯化。添加4 wt％IL大大提高了PEG相中富集的粗木瓜提取物的提纯率，为大规模工业生产高纯度木瓜蛋白酶提供了一种潜在的方法。