Screening Of Tannase Genes In Aspergillus Niger And Tannase Protein Expression

Tannin acyl hydrolase?tannase,EC3.1.1.20?catalyzes the hydrolysis of hydrolyzable tannins.It is widely used in the leather,pharmaceutical,beverage and food industries.So far,the main applications of the tannase are instant tea,acorn liquor,as well gallic acid production from plant materials high in gallotannins.Tannase can be used as clarifying agent in juices and flavored coffee soft drinks.It helps to reduce the adverse effects of tannins in beverages and foods.Tannae is also used as an additive in animal food preparation.Finally,this enzyme has been proposed for use in environmental biotechnology,e.g.,for the treatment of tannery effluents that are rich in poylphenols.Tannase is now know to be an ubiquitous enzyme of the microbial word and has widespread occurrence in various fungi,bacteria and yeast.Among all know microbial producers of tannase,strains of some of the Aspergillus spp.are commercially the most efficient producers of this enzyme.In this work,a Aspergillus niger strain with low-background of secreted proteins was used as a host to overexpression the tannase genes in Aspergillus niger.The specific contents of this study are as follows:?1?According to AspGD database,there are 17 genes related to tannase expression.And a phylogenetic tree was builded by blasting the amino acid sequences.In this work,each of tannase genes was overexpressed,respectively,in A.niger Bdel4 which is low-background of secreted proteins.?2?Then,screening the highest enzyme activity strain Bdel4_Tan7,enzyme activity achieved 111.9 U/m L at 168 h in the shake fermentation,and the purity of the enzyme in the broth supernatant was estimated to be over 70%.In the tank fermentation enzyme activity achieved 1385.5 U/mL at 138 h,which increasing more than ten times comparing with the shake fermentation.It is indicated that this recombinant tannase can be used for mass production of tannase in industrial.?3?The recombinant tannase Bdel4_Tan7 was purified by desalting,followed by gel filtration for characterization.The purity of the purified enzyme estimated to be over 90%.The transformant Bdel4_Tan7 produced an approximately 85 kDa protein.And a protein band?approximately 60⁷0 kDa?was produced after the deglycosylation by PNGase F,suggesting that the recombinant tannase is a glycoprotein.The optimum temperature and pH of the recombinant tannase was 40°C and 7.0,respectively.The tannase activity was inhibited by Mg²⁺,Ca²⁺,Cu²⁺,Ba²⁺,Ni²⁺and EDTA,and was enhanced by Mn²⁺and Co²⁺about 30%enzyme activity.