Screening Of High Temperature Resistance Tannase Strain And Study On The Characterization And Application Of Its Extracellular Tannase

Tannin acyl hydrolase, commonly referred as tannase (EC.3.1.20), is an inducible enzyme produced by microorganisms. It catalyzes the hydrolysis of ester and depside bonds in hydrolysable tannins and gallic acid ester. Tannase can catalyze the astringency source of food——tannic acid to the intermediates1,2,3,4,6-pentagalloyglucose and2,3,4,6-tetragalloylglucose, and further catalyzes the hydrolysis of ester to get final products such as gallic acid and glucose. Tannase has been confirmed as safety food additive by FDA of USA. Tannase is a catalytic enzyme in enzymatic preparation of gallic acid, an intermediate of the antibacterial agent trimethoprim and the food additives propyl gallate. As an additive the enzyme is widely used in the preparation of instant tea, coffee flavor soft drink, beer and wine, in order to solve the muddy, astringency and other quality problems caused by tannic acid. Furthermore, it has been used in the leather industry as tanning agent, immobilized tannase has been extensively used for the treatment of wastewater contaminated with polyphenolic compounds such as tannins. In the cosmetics industry, turbidity or agglomeration caused by the addition of plant extracts riched with tannin can also be solved by tannase.Now the main problems of tannase industrialization are the low enzyme activity and the weak stability of high-temperature, which restricts the industrial application of tannase. In this paper, a tannase producing strain Aspergillus awamori FCYN206was obtained from YunNan black tea, and the conditions for its tannase production was optimized. Preliminary study was done on the properties of the tannase produced from Aspergillus awamori FCYN206. And Aspergillus awamori FCYN206was used in the preparation silage of banana stems and leaves. The main results obtained in this research are summarized as fllows:1. Screening and identify the high-yield-tannase strain. Three tannase producing strains were obtained from YunNan black tea, and the highest tannase activity (50.24U/mL) in medium of strain FCYN206, which was identified as fungi according to the morphology. Extracting DNA genome and18S rRNA gene sequence analysis,1333bp genome of18S rRNA was cloned. Sequence analyses and homologous comparisons of the sequence in GenBank shows that its sequence homology is99%with strain Aspergillus awamori PSFW1RH-1. So the strain was identified as Aspergillus awamori, and applied for a new strain sequence in NCBI, and gained the GenBank strain serial number:JQ728449.2. The optimum condition of extracellular tannase production from Aspergillus awamori FCYN206was investigated with Response Surface Methodology. Firstly, the influence factors of extracellular tannase production such as tannic acid concentration, carbon sources and concentration, different nitrogen sources and concentration intial pH were invested. Then the response surface experiments were designed with using the Box-Behnkens by the software Design-Expert8.05to establish the secondary many mathematics model of producing tannin enzyme by Aspergillus awamori FCYN206liquid decompressing bottle fermenting. The optimal conditions for the enzyme production are:2.18%of tannins,1.88%of sugar,0.12%of NaNO3and initial pH is4.01. Under this condition, the tannin enzyme activity of fermentation broth is improved by3.43times than before optimization.3. The extracellular tannase stability of Aspergillus awamori FCYN206was studied. Experiments showed that the tannin enzyme has good stability. Firstly it has good storage stability. After stored at4℃for4and12weeks, the activity of extracellular tannase still survived98.02%and90.49%. Secondly it has the superior stability under the high temperature. Enzymatic activity remained above90%at20～80℃for1h, and can still keep88%at90℃for1h. However the optimal enzyme reaction temperature is40℃. Thirdly, it can work in a wide pH field. The enzymatic activity remained is above90%on the condition of, stored at4℃, pH3.0～6.5for24h, The most suitable pH of enzymatic reaction is5.5.4. The influence of NaCl concentration to the extracellular tannase from Aspergillus awamori FCYN206was studied. The results showed that extracellular tannase risen slightly when the NaCl concentration less than lmmol/L, Whereas the concentration of NaCl more than lmmol/L, the extracellular tannase decreased with increasing with the NaCl concentration, when the NaCl concentration was5mmol/L, the enzyme activity of tannin reduced to66.32%.5. The influence of mental ions to the extracellular tannase activity of Aspergillus awamori FCYN206was studied. The experimental results showed that2mmol/L of K+, Ag+, Zn2+metal ion can promote the enzyme activity by2.34%,6.27%,26.35%. While2mmol/L Na+, Mg2+, Ca2+, Fe2+, Fe3+, Hg2+, Co2+, Mn2+, Cu2+, and Ba2+all can inhibit the enzyme activity in different extents. Among them, the Co2+, Cu2+have the more stronger enzyme inhibition, and the enzyme activity decrease31.01%,35.04%respectively.6. The influence of organic solvents to the extracellular tannase activity of Aspergillus awamori FCYN206was studied. The results showed that adding1%(v/v) of methanol, isoamyl alcohol, glycerin and n-butanol can promote tannase reaction, while ethanol with no effect. Isopropyl alcohol can improve the tannase activity by12.16%.7. The influence of surfactant and chelating agent to the extracellular tannase activity of Aspergillus awamori FCYN206was studied. The results showed that Tween20, Tween80, Triton X-100, EDTA and SDS can inhibit the enzyme reaction in different degrees. The longer the time is, the stronger the inhibition rate is. While0.2%(v/v) Tween20was add to the reaction agent, tannin enzyme activity is reduced to65.32%after5min at30℃. After1h, it is reduced to43.23%.8. The Km and Vmax of tannase was studied using propyl gallate as the substrate. The optimum substrate concentration is2.5mmol/L at30℃and pH5.0. Through the Linewear-Burk double bottom gragh, the Km of tannin is calculated as0.673mmol/L, and Vmax0.065mmol/L·min.9. The different effects of Aspergillus awamori FCYN206, extracellular tannase from Aspergillus awamori FCYN206, lactic acid bacteria, saccharomyces cerevisiae and beneficial microbe(EM) on banana stems and leaves fermented feed characteristics was studied. The results showed that all the strains and extracellular tannase can degrade of tannins in the banana stems and leaves. The fermented feed experimental groups with Aspergillus awamori FCYN206and extracellular tannase can reduce the content of tannin in banana leaves and stems16.9%. In addition, the crude protein content of fermented feed group with strain increased7.5%, indicating that the nutritional value of banana stems and leaves was improved.