Extraction Of Papain With Fe³⁺-chelated Affinity Aqueous Two-phase System And Building The Partition Model

Papain is a proteolytic enzyme which extracted from Carica papaya. Papain was widely used in medicine, food, textile, leather, animal feed and other industries as its strong protein hydrolysis ability, wide temperature and pH adaptable. Therefore, it is of great importance to study the separation and purification methods and mechanisms for high-quality papain. In this study, the metal-chelated affinity ligands were introduced into aqueous two-phase system, formed a novel affinity aqueous two-phase system to papain extraction. The synthetic routes of metal chelate affinity ligands were investigated. The papain extraction conditions were optimized using BBD (one of response surface methodology). Papain affinity partition model in aqueous two-phase system was established. Here are the main results:1. Synthesis of metal chelate affinity ligands. Epichlorohydrin method was used to activate the hydroxyl group of PEG4000and adopted iminodiacetic acid (IDA) as the coupling agent. Under the optimal reaction conditions, the different kinds of PEG-IDA-Metals'metal ion content are basically the same which can reach at0.97mol/mol PEG4000. The result showed that binding forces between IDA and different metal ions (Cu2+?Fe3+?Zn2+?Ni2+?Mg2+?Ca2+) are similar.2. Extraction of papain use affinity aqueous two-phase system. After comparing the different affinity effect on papain offered by different affinity ligands, chose PEG-IDA-Fe3+as affinity ligand, using BBD to optimizes the affinity extraction conditions of papain in aqueous two-phase system. The results revealed that:(NH4)2SO4mass fraction significantly affect the papain partition behavior in the affinity aqueous two-phase system, followed by PEG-IDA-Fe3+content and PEG content, pH has little effect on papain behavior. On the basis of statistical models that optimized conditions for partitioning occurred when pH6.9, PEG (16%, w/w),(NH4)2SO4(20%, w/w) and PEG-IDA-Fe3+(3%, w/w), and the highest values of Y (%) and Kp can be predicted as90.50%and1.85respectively. UV-vis and FT-IR spectra of papain before and after the extraction revealed that the change of characteristic absorption peak did not occur. The affinity aqueous two-phase system provides a mild environment for the extraction of papain, also supply the theory basis for the affinity aqueous two-phase systems' application.3. Through the study of a large number of basic experimental data, regress the model use Mathematic software, fit the parameters use the least square method, a papain affinity partition model in aqueous two phase system was built. The model was applied to correlate the papain affinity partitioning coefficient K, the concentration difference of the components and the metal ion concentration. The model equation: Through validation, the relative errors of experimental and predicted values of partition coefficient are less than15%, could provide a good representation of papain affinity partition behavior.In this paper, PEG-IDA-Fe3+was selected as the affinity ligand, explored the papain partition behaviors in metal chelate affinity aqueous two-phase system, established the papain affinity partition model. The research is innovative and has some reference value.This work was funded by National Science Foundation (No.31260401).