Heterologous Expression Of Tannase From Aspergillus Niger And Its Application In Simultaneous Extraction Of Green Tea

Tannase is a multifunctional enzyme,which not only can catalyze the cleavage of ester and acetal acid bonds in hydrolyzed tannins,but also can be used to synthesize gallates in organic reaction media.Therefore,tannase has a wide range of applications,especially in the food and pharmaceutical industries.In order to excavate the tannase gene of Aspergillus niger to enrich the gene library of tannase,and to develop novel applications of tannase,this research has studied from the following three aspects:?1?Heterologous expression of Aspergillus niger tannase in Pichia pastorisTwo tannase genes,An-Tan1 and An-Tan2,were cloned from Aspergillus niger FJ0118,which contained 1713 bp and 1752 bp fulllength gene sequence length,encoding 570 and 583amino acids,respectively.Bioinformatics analysis showed that both An-Tan1 and An-Tan2 were hydrophilic proteins with molecular weights of about 62.96 kDa and 63.74 kDa,respectively.Then,these two gene were ligated to pPIC9K expression vector and successfully expressed in Pichia pastoris.After these two recombinant strains were cultured in 500 mL flasks for 168 h,the tannase activity reached the peak?0.093 U/mL and 0.094 U/mL?,respectively.Furthermore,through using5 L fermentation tanks,GS115-An-Tan1 and GS115-An-Tan2 reached the maximum enzyme activity of 4.97 U/mL and 7.07 U/mL,which was increased by 53-fold and 75-fold,respectively.?2?Enzymatic properties of the recombinant tannaseThese two recombinant tannase from Aspergillus niger FJ0118 were purified and characterized.The optimum temperatures of An-Tan1 and An-Tan2 were 50oC and 35oC,respectively.The calculated half-life values of An-Tan1 at 50oC and 60oC were 97.1 h and 2.8 h,exhibited the excellent thermal stability.The residual enzyme activity of An-Tan2 was basically unchanged at 4oC after 120 min incubation,and the relative enzyme activities at 4oC and 10oC were 49%and 60%,respectively,indicating that An-Tan2 was a cold-adapted enzyme.Both the optimal pH of An-Tan1 and An-Tan2 were 5.0,and the residual enzyme activities of them were more than 60%after 24 h of treatment at pH 2.0-7.0.These acidophilic tannase had important industrial application value.The polar solvent had significant inhibitory effects on An-Tan1 and An-Tan2,low concentration of formaldehyde and tetrahydrofuran completely inactivated the tannase activity.Compared with An-Tan1,An-Tan2 was more tolerant to high concentrations of SDS and EDTA.An-Tan1 had better degradation activity on catechins,while An-Tan2 was more inclined to degrade tannin acid?TA?and epicatechin gallate?ECG?.The K_m values of An-Tan1and An-Tan2 for the propyl gallate and TA implied that An-Tan1 and An-Tan2 had higher affinity for TA.?3?Novel application of thermophilic tannase for simultaneous extraction of green teaThe thermophilic tannase was used for simultaneous extraction of green tea.The optimal extraction conditions were 20 U,70oC and 40 min.Furthermore,comparing with the properties of green tea infusion obtained by different extraction methods,the results showed that both simultaneous extraction and stepwise extraction degraded the catechins effectively.But the green tea infusion obtained by the simultaneous extraction has better low-temperature storage stability,and the content of tea polyphenols were 1.57-fold of that obtain using the stepwise extraction.Moreover,the green tea infusion obtained by the simultaneous extraction displayed better reducing power and the scavenging abilities towards hydroxyl radicals,DPPH·and ABTS?,which EC₅₀decreased by 0.43 mg/mL,0.02 mg/mL,and 0.03 mg/mL,respectively.The green tea infusion obtained using simultaneous extraction has the better inhibitory effect on pancreatic lipase,and the IC₅₀ decreased by 15.8 mg/mL compared to that using stepwise extraction.These results indicated that the simultaneous extraction of green tea method had good application potential in tea beverage industry.