Abstract: In this study, based on the codon preference of Escherichia coli BL21 (DE3), base optimization was carried out on the?tannase?encoding?gene CsTanA of Camellia sinensis. In the optimized gene, the GC content was 51.9%, the codon adaption index (CAI) value was 0.8, and the consistency of the gene sequence before and after optimization was 77.8%. Recombinant expression and enzymatic characterization of the optimized gene using?expression?vector?pET30a were investigated. The recombinant strain, with an A600 nm value of approximately 0.6, was cultured at 30 ℃ in the presence of the inducer isopropyl-β-D-thiogalactoside (IPTG). In the supernatant from the broken cells, the recombinant tannase rCsTanA had a specific activity of 0.35 U/mg, and it was purified 4.4 folds by Ni-NTA affinity chromatography with a specific activity of 1.53 U/mg. The apparent molecular mass of the purified rCsTanA was about 39 kDa, and its optimal reaction temperature and pH were 40 ℃ and 7.0, respectively. The effects of different metal ion concentrations on the enzyme activity were different. At low concentration (1 mmol/L), K+ enhanced the enzyme activity of rCsTanA by 37.28%, while Ag+, Cu2+ and Fe3+ reduced the enzyme activity by more than 80%. At high concentration (5 mmol/L), Cu2+ completely inhibited the activity of rCsTanA. The surfactants sodium dodecyl sulfate (SDS), Tween-80 and cetyltrimethyl ammonium bromide (CTAB), and the polar solvents methanol, ethanol, glycerin, isopropanol and acetone had strong inhibitory effects on rCsTanA activity. The results of this study can provide theoretical support for the further application of plant tannase.

Key words: Camellia sinensis; tannase; heterologous expression; codon optimization; enzymatic properties