Degradation of epigallocatechin and epicatechin gallates by a novel tannase Tan_Hcw from Herbaspirillum camelliae

Background: Herbaspirillum camelliae is a gram-negative endophyte isolated from the tea plant. Both strains WT00C and WT00F were found to hydrolyze epigallocatechin-3-gallate (EGCG) and epicatechin-3-gallate (ECG) to release gallic acid (GA) and display tannase activity. However, no tannase gene was annotated in the genome of H. camelliae WT00C.

Results: The 39 kDa protein, annotated as the prolyl oligopeptidase in the NCBI database, was finally identified as a novel tannase. Its gene was cloned, and the enzyme was expressed in E. coli and purified to homogeneity. Moreover, enzymatic characterizations of this novel tannase named Tan_Hcw were studied. Tan_Hcw was a secretary enzyme with a Sec/SPI signal peptide of 48 amino acids at the N-terminus, and it catalyzed the degradation of tannin, methyl gallate (MG), epigallocatechin-3-gallate (EGCG) and epicatechin-3-gallate (ECG). The optimal temperature and pH of Tan_Hcw activities were 30 °C, pH 6.0 for MG and 40 °C, pH 7.0 for both EGCG and ECG. Na⁺, K⁺ Mn²⁺ and Triton-X100, Tween80 increased the enzyme activity of Tan_Hcw, whereas Zn²⁺, Mg²⁺, Hg²⁺, EMSO, EDTA and β-mercaptoethanol inhibited enzyme activity. K_m, k_cat and k_cat /K_m of Tan_Hcw were 0.30 mM, 37.84 s^-1, 130.67 mM^-1 s^-1 for EGCG, 0.33 mM, 34.59 s^-1, 105.01 mM^-1 s^-1 for ECG and 0.82 mM, 14.64 s^-1, 18.17 mM^-1 s^-1 for MG, respectively.

Conclusion: A novel tannase Tan_Hcw from H. camelliae has been identified and characterized. The biological properties of Tan_Hcw suggest that it plays a crucial role in the specific colonization of H. camelliae in tea plants. Discovery of the tannase Tan_Hcw in this study gives us a reasonable explanation for the host specificity of H. camelliae. In addition, studying the characteristics of this enzyme offers the possibility of further defining its potential in industrial application.