Enhancing the Thermostability of Papain by Immobilizing on Deep Eutectic Solvents-Treated Chitosan With Optimal Microporous Structure and Catalytic Microenvironment

Abstract

Deep eutectic solvents (DESs) have attracted an increasing attention in the fields of biocatalysis and biopolymer processing. In this study, papain immobilized on choline chloride- lactic acid (ChCl-Lac) DES-treated chitosan exhibited excellent thermostability as compared to the free enzyme. The properties of native or DES-treated chitosan and immobilized enzyme were characterized by FT-IR, SEM, surface area and pore property analysis. Like the common enzyme immobilization, papain immobilized on DES-treated chitosan resulted in a lower catalytic efficiency and a higher thermostability than the free enzyme due to the restricted diffusion. The results also revealed that DES could control the active group content, thus achieving the appropriate microporous structure of immobilized enzyme. Meanwhile, it could also help to construct the optimal microenvironment by hydrogen-bonding interaction between enzyme, chitosan, and residual DES, which are benefit for maintaining an active conformation and subsequently a high thermostability of papain. Moreover, it was found that trace DES (10 mM) significantly promoted the activity of free papain (145%). Deactivation thermodynamics study showed that the DES could enhance the thermostability of papain especially at high temperature (half-life of 7.4 vs. 3.5 h) because of the increased Gibbs free energy of denaturation. Secondary structure analysis by circular dichroism spectroscopy (CD) agreed well with the activity and thermostability data, further confirming the formation of rigid conformation induced by a specific amount of DES. This work provides a new way of enzyme immobilization synergistically intensified by solvents and supporting materials to achieve better microporous structure and catalytic microenvironment.

Modifying Thermostability and Reusability of Hyperthermophilic Mannanase by Immobilization on Glutaraldehyde Cross-Linked Chitosan Beads

“…The possible reason for increased optimum temperature of immobilized enzyme may be because of the synergic effect be tween enzyme and chitosan beads. In addition, the immobilized matrix may be able to provide a suitable micro-environment at an active center for enzyme and substrate inter action and keep its rigid confirmation and function, especially at higher temperatures [38] However, the extent of effect varies for different support materials used and with the type of interaction established between enzyme and the solid support. The temperature dependence of free and immobilized Man/Cel5B was studied between 60 and 95 • C. The optimum temperatures of free and immobilized Man/Cel5B were found to be 85 • C and 95 • C, respectively (Figure 2).…”

Application of Plant Proteases in Meat Tenderization: Recent Trends and Future Prospects

“…Papain is traditionally extracted from papaya using precipitation methods; however, this method is not able to achieve a desirable purification percentage (below 40%) [ 58 ]. Papain thermostability as compared to the free enzyme could be further enhanced by immobilization on choline-lactic acid (ChCl-Lac) deep eutectic solvent treated with chitosan [ 59 ]. Aqueous two-phase system (ATPS) extraction is used to clarify, concentrate, and purify the enzyme extracted simultaneously in a single process.…”

Natural multi-osmolyte cocktails form deep eutectic systems of unprecedented complexity: discovery, affordances and perspectives

“…In addition, versatile enzymes (e.g. laccases, 83 lipases, 84 proteases 85 ) show excellent thermal stability in synthetic DESs composed of osmolytes such as betaine, polyols and sugar alcohols. Delorme et al 83 showed that laccase is much less thermostabilized in the presence of betaine and xylitol alone than in a DES formed by those two osmolytes.…”