PRODUCTION, PURIFICATION AND CHARACTERIZATION OF GENETICALLY ENGINEERED PAPAIN FROM CARICA PAPAYA

Abstract

Background:
Commercially available papain is prepared by performing a tedious and costly purification method that yields papain at different degrees of purity.
Methods:
The expressed ~43 kDa active papain was purified by single-step affinity chromatography and confirmed by SDS-PAGE analysis and Western blotting. A 3.7-fold lab-scale purification and 64 % recovery yield of the enzyme were achieved.
Results:
In this research work, the recombinant stem papain has been expressed in E. coli BL21. The purified enzyme exhibited maximum activity at a pH range of 5-8 on synthetic substrates studied with an optimum temperature of 45 ºC. It was inhibited by E-64 (10µM) but only slightly inhibited by non-cysteine protease inhibitors and activated by all the Sulphur-containing reducing reagents studied. Kinetic studies on the enzyme yielded lower values of Ki and Km coupled with a higher kcat/Km ratio for recombinant papain; implying that it had more affinities towards the inhibitor used and all the substrates than commercial papain.
Conclusion:
This study successfully developed expression, characterization, and cultivation conditions for better production of recombinant papain from Carica papaya in E.coli (BL21-AI). Drying technologies such as spray drying and freeze-drying could be explored to establish the best means of recombinant papain technology