Catalyzation Of Gallic Acid Ester Bond By Tannase In Organic Solvents

Tannase (tannin acyl hydrolase, EC 3.1.1.20) hydrolyses the ester and depside bonds of gallotannins and gallic acid esters. For industrial application, the ability to make high cost enzymes re-usable and stable has meant that immobilized enzymes have attracted a great deal of attention. Tannases have significant potential to synthesize gallic acid esters with a variety of alcohols and diols in organic media. Although several synthetic studies of gallic acid esters have been performed in organic medium, very little is known about the effect of nonaqueous media on tannase. In this work, Aspergillus niger tannase was employed to investigate the synthesis of gallic acid esters in organic solvents. Aspergillus oryzae tannase gene was cloned and expressed in Pichia pastoris and the recombinant Pichia pastoris was employed to synthesize propyl gallate in nonaqueous media.1. Tannase from Aspergillus niger was microcapsulated with a coacervate calcium alginate membrane surrounding a liquid core. The optimum pH and temperature for free and immobilized tannase were 5.0 and 30 ℃, 6.0 and 40 ℃ respectively. The pH stability, as well as thermal stability, was improved significantly after microencapsulation. The immobilized tannase was stable enough to be used for up to 15 runs. On the other hand, tannase was microencapsulated by a chitosan-alginate complex coacervate membrane with the yield of propyl gallate 34.59%. Microencapsulated tannase showed higher synthetic activity than free enzyme. The water content of the system could significantly affect the equilibrium shift. The effects of various organic solvents on the enzymatic reactions were investigated and the higher yields were 44.3% in benzene and 35.7% in hexane.2. The mechanism of synthesis of propyl gallate with mycelium-bound tannase was investigated, such as the influence of organic solvents, water, mass transfer limitations, protonation state of mycelium-bound enzyme, the catalyst concentration, the amount of substrates, and enzyme specificity. The results showed that the direct use of mycelium-bound tannase achieved more promising results than immobilized free tannase or microencapsulated mycelium-bound tannase. The effects of various organic solvents on the enzymatic reactions also showed that benzene was the suitable solvent. The maximum molar conversion (65%) was achieved with 7.3% (V/V) 1-propanol and 5.56 mmol L^-1 gallic acid at stirring speeds of 200 rpm, 40 ℃ in presence ofPEG-10000. After the mycelium-bound tannase was reused three times, the yield of propyl gallate began to decrease gradually. Enzyme specificity for the alcohol portion (C1-C8) of the ester showed that the optimum synthesis was observed with alcohols ranging from C3 to C5.3. Kinetics and thermodynamics of synthesis of propyl gallate by mycelium-bound tannase from Aspergillus niger in organic solvent was studied. The experimental results confirmed that the deactivation process of mycelium-bound tannase follows the first-order kinetics pattern, and the mycelium-bound enzyme showed improved stability in organic solvent. At 40 °C the first-order kinetics pattern was shown as follows.[Ea] = [E0]x exp(-V) ^=0.0028 h"1A kinetic model of esterification by mycelium-bound tannase was developed based on the Ping-Pong Bi-Bi kinetic mechanism, considering not only the effects by substrates and products, but also tannase denaturation. The kinetic model was shown as follows.=_<sub><sub><sub><sub><sub><sub><sub><sub><sub>0.0056 exp(-0.0028Q[A]_<sub><sub><sub><sub><sub><sub><sub><sub><sub>V⁰.0041(l+[Q]/(9.81xl0-*))+[A](l+[A]/0.0103)4. Aspergillus oryzae tannase gene was cloned from Aspergillus oryzae (Ahlb.) Cohn and expressed in Pichia pastoris. Recombinant tannase consisted of two subunits (30kDa and 33kDa) linked by a disulfide bond. Expression of tannase in vitro by GS115-pPIC9k-Tan Muts and GS115-pPIC9k-Tan Mut+ showed that the highest amount of tannase was 159mg L"1. Expression of tannase in vivo by GS115-pPIC3.5k-Tan Muts showed that the highest amount of tannase was 178mg L'1. GS115-pPIC9k-Tan muts gained the highest activity of 1118 U L'1, followed by GS115-pPIC3.5k-Tan muts (960 U L'1) and GS115-pPlC9k-Tan mut+ (845 U U1), which were two to three times higher than that of Aspergillus niger tannase. GS115-pPIC3.5k-Tan muts was employed to synthesize propyl gallate in nonaqueous media and the yield of propyl gallate was 53.6%.