Cloning,Expression And Relative Gene Deletion Research Of Tannase From Aspergillus Niger

Tannin acyl hydrolase,commonly referred as tannase,is a attractive biocatalysts for biodegradation of ester bond and depside bonds in tannins.Tannase has comprehensive applications in food,beverage,cosmetics,leather and feed industry.In the present work,tannase from Aspergillus niger CICC41034 was cloned and expressed through Pichia pastoris expression system.The obtained tannase was isolated and purified for investigating its enzymatic activity properties and application in tea stalks extract.In order to study its biosynthetic mechanism,the method of deleting Tan and CreA gene was establishmented to lay the foundation for follow-up researches.Firstly,this tannase which cloned from A.niger CICC41034 was 1725 bp in length,and encoded a polypeptide of 575 amino acid residues.The prediction of signal peptide analysis results showed:the 1～20 amino acids in N-terminal was a classical signal peptide sequence;its molecular weight and isoelectric point were estimated to be 60.8 kDa and 4.34.The enzyme was a kind of hydrophilic protein,its 57～527 amino acids constituted a multi-functional superfamily structure domain through protein structure domain prediction and analysis.Then,this gene was ligated to pPIC9K expression vector and successfully expressed in Pichia pastoris.After 144 h induction by shake flask culture,the optimum tannase activity was 1.55 U/mL;through using 5 L fermentation tanks,the optimum tannase activity could reach to 390.4 U/mL,which has a 252 fold increase.Then,the recombinant tannase was purified by using DEAE-Sepharose fast flow ion exchange chromatography,and explored its enzymatic properties.SDS-PAGE analysis demonstrated that its molecular weight was about 85 kDa.The optimum temperature and pH of this recombinant tannase were 80℃ and 6.0;it has excellent thermal stability,and excellent pH stability in neutral and acidic solution.Cu2+,Al3+,mercaptoethanol,dimethyl sulfoxide,N-butyl alcohol,N-propyl alcohol had significant inhibitory effects on the recombinant enzyme;Fe3+,N-hexane acetate,cyclohexane had contributes to tannase activity.Recombinant tannase could hydrolyze propyl gallate(PG),epigallocatechin gallate(EGCG),tannin acid(TA),epicatechin gallate(ECG)and catechin gallate(CG).The Km,kcat and Vmax values of the recombinant enzyme on propyl gallate were 0.073 mM,1.06×103 s-1 and 0.14 μmol/(mL·min),respectively.In addition,the recombinant tannase was effective to catalyze the transformation of catechin ester into non-galloylated catechins in tea stalks extract.Finally,using pMD-19T,2000 bp homologous base sequence,bleomycin gene,green fluorescent protein as plasmid skeleton,recombination identification area,resistance and marker gene,respectively,to construct deletion plasmids.The plasmid was linearized and then recycled to 10 ng at least.Meanwhile fresh mycelia was enzymatic hydrolyzed,washed and resuspend to obtain protoplast.With PEG-mediated method,the recombinant fragment was transformed into fungal cells to screen positive transformants.