Study On Multi-stage Aqueous Two-phase Extraction Of Papain

Papain is one of the important commercialized enzymes in the world and is widely used in food and pharmaceutical industries,but the traditional method of extracting and isolating papain has the defects of low recovery of enzyme activity.The aqueous two-phase system（ATPS）is widely used for the recovery and purification of biochemical materials due to its mild extraction environment and simple,efficient method.The application of ATPS technology to the extraction of papain is important for obtaining high quality papain.In industrial large-scale production,higher extractant concentrations and volume ratios are required to obtain the higher recovery,but this leads to lower purity and concentration of the target.In this paper,the transfer of ATPS between multiple centrifuge tubes is used to simulate the multi-stage crosscurrent extraction and multi-stage countercurrent extraction processes commonly used in industry and will obtain the relevant process parameters to apply to large-scale production,which can reduce the limitations of industrial large-scale ATPS production of papain.The ATPS extraction method can only extract papain into one single phase,which has the problem of difficult separation of polyethylene glycol（PEG）and papain,limiting the wide application of ATPS technology in the extraction of papain.This paper uses ultrafiltration and centrifugation to separate PEG and papain,both for separation purposes and to recover PEG for reuse.The contents are as follows:（1）Determination of the ATPS extraction system for papain and optimization of extraction conditionsFirstly,several PEG/salt ATPS were constructed to investigate the effects of reagents and pH on the enzymatic activity of papain,and to study the effects of PEG molecular weight,mass percent concentration of salt solution and PEG,pH and other factors on the partitioning behavior of papain in ATPS.The optimized conditions were pH 6.97,Na₃C₆H₅O₇ mass concentration of 18.86%and PEG6000 mass concentration of 18.18%,and the partition coefficient was 2.69 and the enzyme activity recovery was76.88%.（2）Multi-stage ATPS extraction of papainPEG6000/Na₃C₆H₅O₇ multi-stage ATPS extraction was used to improve the recovery of papain enzyme activity.Under laboratory conditions,the transfer of top and bottom phases between multiple centrifuge tubes was used to simulate the multi-stage crosscurrent extraction（Cro）and multi-stage countercurrent（Coun）extraction processes commonly used in industry.Comparing the advantages and disadvantages of the two multi-stage extraction methods under fixed volume ratio conditions,the results showed that both methods could achieve high recoveries after multi-stage extraction,and the amount of PEG consumed by multi-stage countercurrent extraction was lower than that consumed by multi-stage crosscurrent extraction.The McCabe Thiele diagram was used to obtain the theoretical number of stages for the multi-stage countercurrent ATPE.The results showed that the recovery of papain activity using the three-stage countercurrent ATPE achieved 96.75%when the the volume radio was 0.5 and the initial concentration of papain in the bottom phase of the feedstock was 0.12 mg/m L.（3）Application of ultrafiltration centrifugation in ATPSThe effects of ultrafiltration centrifugation speed,ultrafiltration centrifugation time and dilution multiple on the ultrafiltration centrifugation separation of papain and PEG6000 were investigated,and the Box-Behnken experimental design was used to optimize the optimal conditions for the ultrafiltration centrifugation separation of PEG6000 and papain.The optimal conditions obtained after optimization were:centrifugation speed of 12000 rpm,centrifugation time of 19.84 min and dilution of4.84,and the distribution coefficient enzyme activity recovery obtained under this condition was 91.81%,and the recovery of PEG was 92.43%.UV-vis spectral scanning and FTIR of papain from multi-stage countercurrent extraction and ultrafiltration centrifugation showed that no structural changes occurred in papain.