Study on the promotion of cellulase hydrolysis of oleuropein by natural eutectic solvents
Olea europaea L. is a plant belonging to the Oleaceae family and the Oleaceae genus. It is an important oil crop in the Mediterranean region, and Gansu and Yunnan are the main planting areas for olives in China. Olive leaves are a cheap byproduct of olive tree cultivation. Each olive tree needs to trim approximately 25kg of leaves each year, and the total weight of harvested leaves accounts for 10% of the total weight of olives. Olive leaves contain various bioactive compounds. Such as oleanolic acid, hawthorn acid, ursolic acid, oleuropein, hydroxytyrosol, etc.
Research has shown that oleuropein is distributed in all parts of olive trees, with the highest content found in olive leaves. In dry olive leaves, the content of oleuropein can reach 10% to 17%. As shown in Figure 1, oleuropein hydrolyzes to produce hydroxytyrosol, glucose, and olenic acid. Among them, hydroxytyrosol exhibits better biological activity than oleuropein. At present, the hydrolysis methods of oleuropein mainly include acid hydrolysis, alkaline hydrolysis, and enzymatic hydrolysis. Acid hydrolysis and alkaline hydrolysis require strong acids, strong bases, and high temperature environments, with strict reaction conditions. In recent years, the use of enzymes as catalysts for the biotransformation of by-products has received attention. Compared with traditional acid-base hydrolysis methods, enzymatic hydrolysis of oleuropein has the advantages of mild reaction conditions and less environmental pollution. Liu et al. used cellulase as a food additive to break glycosidic bonds and hydrolyze oleuropein to obtain hydroxytyrosol. Due to the thermal instability of cellulase, it cannot maintain high activity continuously at the optimal temperature of the enzyme, resulting in low enzymatic hydrolysis efficiency. Therefore, we are trying to find a new green solvent to replace the buffer solution, which can enhance the solubility of oleuropein and improve the stability of cellulase in long-term reactions, thereby improving the enzymatic hydrolysis efficiency of oleuropein.
Deep eutectic solvents (DES) are homogeneous and transparent mixtures formed by high-temperature stirring of hydrogen bond acceptors (HBA) and hydrogen bond donors (HBD), with lower melting points compared to individual components. Due to its excellent physical and chemical properties, including low flammability, low volatility, easy preparation, high solubility, and high compatibility with enzymes, DES is increasingly being used as a non-toxic extractant and solubilizer. The most important thing is that enzymes are prone to deactivation in organic solvents, and DES as a solvent can prevent this phenomenon from occurring. This also makes it possible for DES to replace organic solvents and incubate cellulases. The DES composed of natural HBA and HBD is called NADES, which is safer and greener than conventional DES. NADES has received widespread attention as a green solvent in the field of enzyme catalyzed reactions. NADES has been successfully applied in specific enzymatic reactions, exerting favorable effects on both enzyme activity and stability. The above research results demonstrate that NADES has the potential to become a good solvent for cellulase hydrolysis of oleuropein.
This study prepared six types of NADES using choline chloride or betaine as HBA and different types of polyols as HBD, and investigated their effects on cellulase hydrolysis of oleuropein. By exploring the effects of different types and concentrations of NADES and their monomers on enzymatic hydrolysis rate, a new efficient and green method for cellulase hydrolysis of oleuropein was constructed.

This study investigated the effect of NADES on the hydrolysis rate of oleuropeinase by adding different types and concentrations of NADES to the buffer solution. It was found that selecting the appropriate type and concentration of NADES can increase the stability of cellulase and promote the hydrolysis of oleuropeinase. Under the conditions of a temperature of 50 ℃ and pH=5, the hydrolysis rate of oleuropeinase in a 10% DES-5 (V/V) solvent system was 1.7 times that of the buffer solution after hydrolysis for 3 hours; The enzymatic hydrolysis rate after 36 hours of hydrolysis is 1.5 times that of the buffer solution; On the basis of mild, efficient, and environmentally friendly enzymatic hydrolysis conditions, this method makes it possible for cellulase to efficiently hydrolyze oleuropein for a long time. This study used choline chloride or betaine as HBA and polyols as HBD in NADES to enhance the stability of cellulase and promote the hydrolysis of oleuropeinase, providing a scientific basis for the efficient preparation of oleuropeinase hydrolysis products.