Breeding And Application Of High-yield Tannase Strains

The enzyme tannase(tannin acyl hydrolyase, EC 3.1.1.20)also called ester acyl radical hydrostatic enzyme, and is also a kind of reducible enzyme. It can catalyzes the hydrolysis of ester and depside bonds in hydrolysable tannins, releasing glucose and gallic acid. Aspergillus niger B1401 strain producing tannase highly was selected from an Aspergillus niger B0201 strain to atmospheric and Room Temperature Plasma mutafacient method in this paper. Study of the enzyme production stains liquid fermention conditions, the strains producing enzymes directly into tannins generated gallic acid process condition were studied, and to explore the experimental conditions for formation of gallic acidin 5 L fermentor. The following conclusions are obtained.By atmospheric and room temperature plasma mutagenesis systematization of Aspergillus niger B0201, screening culture medium adding trace amount of bromophenol blue in the plate, the growth of Aspergillus niger to produce gallic acid degradation of tannic acid to form color ring strain on the screen. Then, according to the ratio of the color circle diameter and colony diameter were screened. Again to rescreening by fermenting enzyme production. The successful slate of high yield strains by Aspergillus niger B1401 strain reached 17.6 U?g-1 live production of tannase enzyme. 2 times the activity of tannase activity was the original strain 8.94 U?g-1 enzyme activity.Study on the enzyme production of Aspergillus niger B1401 in liquid fermentation, the fermentation medium and culture conditions of enzyme production conditions were preliminary inquiry, and through the orthogonal experiment, to determine the most optimize concentration of tannic acid was 5%, glucose 2.5%, medium volume 90 m L, inoculation amount 2%.To explore the process of preparing for gallic acid tannic acid added in the optimal fermentation conditions, the single factor and the conversion time, the conversion of substrate concentration, p H value,temperature, conversion speed and so on.The optimum reaction conditions were: 48 h total transformation time, tannic acid concentration in 20.4% p H conversion solution was 4, the transformation temperature of 30℃, conversion speed of 160 r·min-1.Gallic acid the concentration of transformation reached 128 mg·m L-1. The yield was more than 70%.Amplification experiments in 5 L fermentor, bacteria culture and enzyme production stage to adjust the p H above 4 in the medium was beneficial to the cell growth and enzyme production ahead of time to 60 h-65 h, the preparation of gallic acid in 44 h-48 h, the concentration reached 161 mg·m L-1, the yield of gallic acid can reach 74.04%, and the crude product of gallic acid the yield reached 73.9%.