Gene Cloning, Heterologous Expression And Characterization Of Fungal Tannases

Tannic acid, i.e. tannin, is a phenolic secondary metabolite in plants. Tannin acylhydrolase （tannase, EC3.1.1.20） is present in animals, plants and microorganisms, andcatalyzes the breakdown of ester and depside bonds of tannin, releasing glucose and gallic acid.Tannase has great application potentials for gallic acid production in medicines, as clarifyingagents in industrial processing of tea, fruit juices and brewry, and to remove anti-nutritionaltannin from animal feed. However, due to its complex structure, difficult purification and lowexpression level, tannase rarely been studied. This study aimed to obtain tannase-coding genesfrom fungi, to achive high-level tannase expression by constructing recombinant plasmids, andto provide excellent tannases for industrial application.The fungal strains Aspergillus fumigates DW8and Thielavia subthermophila XZ7,isolated from snow-lotus rhizospheric soil and dessert soil, respectively, had ability to producetannases. By using degenerate PCR, TAIL-PCR and over-lap PCR, two tannase genes,tanDW8and tanXZ7, were cloned from strains DW8and XZ7, respectively。TanDW8was1713bp in length, and encoded a polypeptide of570amino acid residues. Its molecular weightand isoelectric point were estimated to be62.0kDa and4.82. Deduced TanDW8containedeight potential glycosylation sites, and shared99%amino acid sequence idemtity with thehyoertheoretical tannase from A. fumigates （XP₇46534.1）. TanXZ7contained1722bp thatencoded573amino acid residues. Mature TanXZ7was estimated to have a molecular weightof62.5kDa, an isoelectric point of5.03, and seven potential glycosylation sites. DeducedTanXZ7shared77%identity with the putative protein of Byssochlamys spectabilis No.5。Thesequence identity of deduced TanDW8and TanXZ7was76.1%.Both tanDW8and tanXZ7were successfully expressed in Pichia pastoris. Purifiedrecombinant TanDW8and TanXZ7were determined to be polymers of two subunits withdifferent molecular weights （30/33kDa and28/30kDa）. Their pH optima were5.0and6.0,respectively, and retained stable at pH3.0-8.0. The temperature optima were40C, andTanDW8showed better thermostability than TanXZ7at50C and above. The Km and Vmax ofTanDW8and TanXZ7were2.33/2.02mM and35.92/105.46μmol/min/mg, respectively. Theirenzymatic activities were partially affected by some tested metal ions, surfactants and organissolvents.In summary, two tannase genes were cloned from two fungal strains, and high-levelexpression, purification and characterization of these gene products were achieved. Bothrecombinant enzymes had similar protein structures and enzymatic properties, such aspolymeric structure of two subunits, mesophilic and acid propensity, but varied in otherproperties. TanDW8had better thermostability than TanXZ7, but TanXZ7was more efficientthan TanDW8in catalysis. These good properties make TanDW8and TanXZ7prospective forapplication in the animal feed, food, and medicine industries.