State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, 430062, China.
Xianning Central Hospital, Tongji Xianning Hospital, Xianning, Hubei Province, China.
Microb Cell Fact. 2021 Oct 12;20(1):197. doi: 10.1186/s12934-021-01685-1.
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.
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 were studied. Tan 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 activities were 30 °C, pH 6.0 for MG and 40 °C, pH 7.0 for both EGCG and ECG. Na, K Mn and Triton-X100, Tween80 increased the enzyme activity of Tan, whereas Zn, Mg, Hg, EMSO, EDTA and β-mercaptoethanol inhibited enzyme activity. K, k and k /K of Tan were 0.30 mM, 37.84 s, 130.67 mM s for EGCG, 0.33 mM, 34.59 s, 105.01 mM s for ECG and 0.82 mM, 14.64 s, 18.17 mM s for MG, respectively.
A novel tannase Tan from H. camelliae has been identified and characterized. The biological properties of Tan suggest that it plays a crucial role in the specific colonization of H. camelliae in tea plants. Discovery of the tannase Tan 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.
从茶树中分离出的革兰氏阴性内生菌 Herbaspirillum camelliae 可以水解表没食子儿茶素没食子酸酯（EGCG）和表儿茶素没食子酸酯（ECG），释放出没食子酸（GA）并显示单宁酶活性。然而，在 H. camelliae WT00C 的基因组中没有注释到单宁酶基因。
最终鉴定出在 NCBI 数据库中被注释为脯氨酰寡肽酶的 39 kDa 蛋白为一种新型单宁酶。其基因被克隆，并在大肠杆菌中表达并纯化至均一性。此外，还研究了这种新型单宁酶 Tan 的酶学特性。Tan 是一种分泌型酶，其 N 端具有 48 个氨基酸的 Sec/SPI 信号肽，它可以催化单宁、甲基没食子酸（MG）、表没食子儿茶素没食子酸酯（EGCG）和表儿茶素没食子酸酯（ECG）的降解。Tan 的最适温度和 pH 值分别为 30°C（pH 6.0）和 40°C（pH 7.0）。Na+、K+、Mn2+和 Triton-X100、Tween80 均能提高 Tan 的酶活性，而 Zn2+、Mg2+、Hg2+、EMSO、EDTA 和β-巯基乙醇则抑制酶活性。Tan 对 EGCG 的 K、k 和 k/K 分别为 0.30 mM、37.84 s 和 130.67 mM s，对 ECG 的 K、k 和 k/K 分别为 0.33 mM、34.59 s 和 105.01 mM s，对 MG 的 K、k 和 k/K 分别为 0.82 mM、14.64 s 和 18.17 mM s。
从 Herbaspirillum camelliae 中鉴定和表征了一种新型单宁酶 Tan。Tan 的生物学特性表明它在 Herbaspirillum camelliae 对茶树的特定定殖中起着关键作用。本研究中发现的单宁酶 Tan 为 Herbaspirillum camelliae 的宿主特异性提供了合理的解释。此外，研究该酶的特性为进一步确定其在工业应用中的潜力提供了可能。
