| Tannase as an acyl hydrolase, which is produced by certain microorganisms with inducers, for example tannic acid, has been widely applied in such areas as food and beverages, cosmetics and feed industries. Gallic acid and its ester derivatives are important pharmaceutical and chemical raw materials, which are manufactured by the Tala acid and tannic acid hydrolysis. For acid hydrolysis causes environmental pollution, corrosion problems, the replacement of acid method by tannase method to produce gallic acid and its esters has been explored for many years. But the current low yield of tannase method limited its application in industrial production, therefore to undertake research to raise the acticity of tannase then enzymatic conversion yield of gallic acid and gallic acid esters have positive practical significance.This thesis uses Aspergillus strains preserved in laboratory as original strains to screen by UV and nitrogen ion injection. And the conditions of fermentation of tannase from Aspergillus niger, Aspergillus niger fermentation kinetics, tannase immobilization and enzyme properties were studied. Tannase enzymatic conversion conditions for gallic acid and its ester were also optimized. Main findings are as follows:Ten tannase-producing strains were selected as test strains from Aspergillus strains preserved in laboratory. Strain N5with86U/ml of enzymic activity was screened out. Through3min UV mutation, a mutant strain named N5-U5-3was obtained. The tannase activity of which reached158U/ml,83.7%higher than that of original strain. Though the genetic stability verification, its enzyme performaned relatively stable.N5-U5-3was treated with10KeV and3×1015N+/cm2. After prescreening and rescreening experiments in rotation-flask, a high-yield strain Aspergillus nigerlll was obtained. The tannase activity of which reached198U/mL, almost25.3%higher than that of N5-U5-3. The genetic stability of Aspergillus niger111was comparative stable.Single factor experiments, central composite design and response surface method(RSD) were used for optimization on Aspergillus niger tannase producing conditions. The most three important factors of determining the tannase production were tannic acid concentration, temperature and incubation time. The optimum conditions for enzyme production are as follows:30℃; speed,180r/min; tannic acid concentration,1.76%; initial pH,6.0; inoculum,10%; bottling volume,20%and culture time,82.2h. In these conditions, the fermentation tannase activity reached380.34U/ml.Based on the data gotten from Aspergillus niger111batch fermentation in the5L fermenter, kinetic models of cell growth, synthesis of tannase and substrate consumption were established.Compared with various carriers, LX-1000HA were chosen to immobilize tannase. Then Box-Behnken experimental design and RSD were used to optimize the conditions for immobilizing tannase by LX-1000HA resin. The results showed that the best conditions were17.6(v/m) of the enzyme solution/carrier ratio, pH6.9,24h and36.5℃. Under these conditions, the activity of the immobilized enzyme was (18495±200) U/g with activity recovery of45.25%. The optimal reaction temperature, pH for immobilized enzyme were50℃,6.5, respectively.Immobilized tannase by LX-1000HA was applied to hydrolyse tannic acid to produce gallic acid. The best conditions were got by the monofactorial experiment which were pH,6.2-6.8; temperature range,48℃-52℃; immobilized enzyme,3.5-4.5g; time range,25-35h. Conditions were optimized by orthogonal experimental design and the best converting conditions were:pH6.5,50℃,4.5g of immobilized tannse and30h. Under these conditions, the concentration of gallic acid could reach27.93mg/ml and the conversion of tannin could reached93.1%.Immobilized tannase by LX-1000HA was applied to synthetize propyl gallate in the system of organic reaction. Firstly, cyclohexane was chosen as the best organic reaction media, then the best synthesize conditions were obtained through monofactorial experiment and orthogonal experimental design as follow:52℃,4:1of cyclohexane to propanol,1.8%of gallic acid and40h. Under these conditions, the production of propyl gallate could reach90.64%.
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