| Alkyl gallate is a very important kind of gallic acid derivatives. They have been widely used within food, cosmetology and feed industries because of their excellent antioxidant activity and anti-microbe activity .At present, alkyl gallates are produced by chemical synthesis with single product and low conversion, demanding huge energy , eliminating seriously environment and wasting natural resource. Biocatalyst or biosynthesis is the direction of industry in future and the necessary pathway to establishing harmonious relationship between human economic activities and nature.In the paper, on the base of studies on screening mutant Asper.niger strain by UV and preparing tannase from it, A series of alkyl gallates were synthesized in reverse micelle. At first, the method of determined tannase activity was established, catalysis kinetics, catalysis characteristcs of tannase and solubility of it in RM were also studied. At the same time, various of factors effecting tannase activity were investigated . The optimum conditions for PG,BG and AG synthesis were determined . Tannase were immobilized in microemulsion-based gels and three kind of gallate esters were produced successfully , the technology of controlling water in three reaction systems were studied. At last, the anti-microbe and anti-oxidation abilities of the three gallates synthesized by tannase were compared and discussed., the results were as followed:1 Mutagenesis Breeding of High Tannase Activity Strains and preparing of TannaseAs a original strain, Asp.niger CY was mutated by UV treatment. One strain was obtained from the mutants. Its activity was higher than that of original strain by nearly 2. A comparatively suitable liquid culture medium for producing tannase was mainly composed of 3.0% soybean cake powder and tannin 2%, The relatively suitable culture condition are as followed: to put 50ml culture in a 250ml cone bottle with 2.5ml seed culture liquid(48h), rolling rate was 150r/min, and keep it being oscillated for 24h at temperature of 28CThe tannase was separated and purified by (NH4)2SO4 fractional precipitation, DEAE-cellulose ion-exchange chromatography and gel filtration . It was determined by SDS-PAGE that the tannase has two kinds subunits; their molecular weight is 75.9KD and 60.3KD respectively, the content of sugar is 12.3%. Its optimum pH and temperature are 5 and 40C respectively.2 Solubility and Kinetics Characteristics of Tannase in RM System2.1 A method of HPLC was set up to assay the content of gallic acid and alkyl gallate at the same time. Chromatographic conditions were : using Lichrospher C18 colume(250mm X 4.6mm), 70%A(80%methanol)and 30%B(5ml aceticgallate at the same time. Chromatographic conditions were : using Lichrospher C18 colume(250mm X 4.6mm), 70%A(80%methanol)and 30%B(5ml acetic acid/1000mLH2O )as mobile phase with 1ml min-1 of flow rate, detecting at 274nm. The column temperature was at 40C and injection volume was 20ul. The tannase activity in RM reaction system was assayed by this method.2.2 Tannase in RM and aqueous have been investigated by UV absorption spectrum and fluorescence spectrum ,the results showed that the spectrum of tannase in RM were significant different from that in aqueous. Tannase was possibly solubilized with interfacial location or adhering to micelle wall.2.3 Reverse micelle system consists of sodium bis(2-ethylhexyl) sulfosuccinate and iso-octane is established, in which n-propyl gallate i- propyl gallate n-butyl gallate i- butyl gallate n-amyl gallate and i- amyl gallate were synthesized .2.4 The kinetics parameters are different because of different alcohol in various reaction system. According to the kinetics parameters , the conclusions were drawed: tannase in RM had no high specificity for alcohol (C3-C5); catalysis efficiency for esterification of straight alcohol was higher than that for branch alcohol; the affinity of tannase to gallic acid is the strongest and catalysis efficiency is the highest when C4 alcohol e...
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