Production And Application Of Epigallocatechin Gallate Hydrolase

Catechins are antioxidants known to exhibit beneficial biological activities,such as antioxidative, antitumoral,and antimutagenic effects.Catechins may function as scavengers of active oxygen species in biological systems.They seem to play an important role in reducing the risk of the formation of a number of diseases like cancer,coronary heart diseases,arteriosclerosis,ischaemia events and inflammatory diseases.Five main catechins are found in green tea.Each catechin has its own biological features.In order to fully exploit prominent biological activities of specific catechins and to develop new Pharmaceuticals based on catechins,it is necessary to obtain pure catechin preparations by isolation from natural sources or by chemical synthesis or biotransformation reactions with high yield and specificity.So far,most experiments had been just conducted to isolate and purify catechins from tea extract.The production of epigallocatechin(EGC)is limited by its low content in tea.Attempts to produce pure EGC by chemical synthesis have resulted in low product yields.As epigallocatechin gallate(EGCG),which is abundant in green tea,is an ester formed by EGC and gallic acid(GA) structurally,many studies aimed at EGCG hydrolysis using regular chemical methods,for instance acidic or alkaline hydrolysis.However,the hydrolytic efficiency is rather low under acidic condition and the hydrolysate is so unstable that it could be oxidized very easily under alkaline condition.By now there have been very few reports about biotransformation of catechins by using enzymes and microbial systems and with low efficiency.It is meaningful to develop new approaches to produce pure catechins. In our studies,we developed a new method to produce EGC by enzymatic EGCG hydrolysis.Aspergillus niger,Aspergillus oryzae and Trichoderma reesei Rut C30 were used in this studies.A sterile EGCG solution or olive oil was added to the autoclaved culture media to induce the production of EGCG hydrolase.Enzymatic hydrolytic systems were analyzed by HPLC and TLC in order to select the strains with the capability to produce EGCG hydrolase.The enzyme activity and features were also studied in this study.The result showed that Aspergillus niger and Aspergillus oryzae could utilize EGCG as carbon source to maintain their growth while the growth of Trichoderma reesei Rut C30 was inhibited by EGCG Olive oil could remarkably increase the biomass of the three strains.The higher the concentration of olive oil was,the more the biomass.The enzyme induced by EGCG was able to catalyze EGCG hydrolysis,but the enzyme without induction or induced with olive oil didn't have the capacity to transform EGCG to EGC.The effects of EGCG concentrations on the growth and enzyme production by Aspergillus niger and Aspergillus oryzae were also studied,respectively.The results showed that the pH values were decreased with the increase of EGCG concentration,while the glucose concentrations and biomass were increased with the increase of EGCG concentration.In the changing trends of protein concentration and hydrolase activities,there was difference between Aspergillus niger and Aspergillus oryzae.According to the result of volume activities,specific activities and productivities of enzyme,the optimal EGCG concentration of Aspergillus niger was 2.5-7.5 mg/mL,and Aspergillus oryzae was 10.0 mg/mL. Aspergillus niger was easer to be induced to produce EGCG hydrolysis than Aspergillus oryzae.