Study On Biocatalytic Synthesis Of Propyl Gallate By Transesterification In Organic Medium

Biocatalysis in anhydrous medium is an important subdivision of industrial biocatalysis. Presently, to improve biocatalytic efficiency of enzyme is one of key challenges in biocatalytic technology. Propyl gallate (PG) is an excellent antioxidant and is also considered as important pharmaceutical intermediates. In this paper, the study on biocatalytic process in which tannase catalyzed the direct synthesis PG from tannic acid and propanol by transesterification was carried out.In view of the flexibility of enzymatic conformation in aqueous phase and its rigidity in organic medium, imprinting technique is competent to hyper-activate enzyme in anhydrous phase. Imprinting, cryogenic protection, and immobilization techniques were complexly applied to improve the transesterification-catalyzing performance of tannase in organic media. The results showed that such treatments as pH tunning, substrate imprinting, and interfacial imprinting all hyper activate tannase and improve its biocatalytic performance by123folds relative to the original control. Furthermore, immobilizing the imprinted tannase remarkably relieves its aggregation caused by the imprinting, and thus the immobilization increases the apparent activity of the imprinted enzyme and makes its conversion rate of substrate (CR) reach40%. On the other hand, combinational application of Triton X-100, mannose, and magnesium ions reduces the damage of enzyme activity resulted from lyophilization in the protocol of imprinting and promotes its CR up to49%. Not only does the study broaden the application fields of imprinting technique but also presents a reference for modification of enzyme in the future.The analysis on thermostability of the imprinted tannase found that its activation energy of irreversible thermal inactivation (Ed) and half-time are85.54kJ mol-1and1710h, respectively, which are more than the previous reports. The thermodynamic analysis of the imprinted tannase indicated that the free energy of Gibbs (ΔG) and enthalpy (ΔH) in enzyme-catalyzing transesterification at40-60℃are97.1～98.4kJ mol-1and82.77～82.94kJ mol-1, respectively, which are lower than the free energy stack from hydrolysis and esterification both catalyzed by tannase. This reveals the biocatalytic efficiency of transesterification (direct synthesis) is higher than that of the two-step biocatalysis composed of hydrolysis and esterification. Entropy of activation of denaturation (ΔS) in the direct synthesis is-0.047～-0.045kJmol-1K-1at40-60℃. It indicates that the reaction cannot run spontaneously. The kinetic analysis of the imprinted tannase showed the Km at40℃is0.054mM, which is less than that in tannic acid-hydrolyzing reaction by tannase. It is inferred that the imprinting enhances the biocatalytic performance of enzyme by improving its affinity to substrate.The organic reaction system was optimized further to increase the biocatalytic efficiency of the imprinted tannase. The results showed that the polarity of organic solvent has a significant effect on its biocatalytic efficiency. The organic solvent with a lower polarity, in particular, is more suitable for transesterification. Otherwise, water and propanol contents in the reaction mixture also affect the efficicency. The study on the reaction mode showed that semicontinuous catalysis gives rise to the biocatalytic efficiency2.5-fold increment and achieves a CR of75%. It manifests that to control the process of the enzyme-catalyzed transesterification in organic media is capable of improving its catalytic efficiency. The resultant data can be used as the base for PG production in commercial scale.Direct utilization of mycelium as a biocatalyst instead of pure enzyme avails in diminishing the cost in separation and purification of enzyme. In this study, Aspergillus niger mycelium was applied to catalyze the transesterification from tannic acid to PG. The CR of43%was obtained through optimizations of the reaction system and mode. Consequently, it is concluded that mycelium catalyzing the transesterification in organic media is feasible.The conclusion in this paper will provide a technical support to ecofriendly synthesize PG with agriculture and forestry residues as raw materials. Moreover, such strengthened measures as imprinting and cryogenic protection not only is widespreadly suitable to biocatalysis in organic media, but also gives a experience means to biosynthesize a few chemicals in organic media.