Enzymatic Characterization and Biological Function Analysis of Tannases JrTA1 and JrTA2 in Walnut (Juglans regia L.)

Tannins are key compounds determining the astringency of walnuts. Elucidating the structural characteristics of tannin cells in walnut inner seed coats and the accumulation patterns of esterified catechins (e.g., EGCG and ECG) is of significant importance for both quality regulation of walnuts and the high-value utilization of tannin resources. However, the enzymatic properties and biological functions of walnut tannases (JrTAs) have not been systematically investigated. Thus, the enzymatic characteristics of walnut tannase and its hydrolytic function on tannin-like substances were analyzed. It showed that tannin accumulation in the inner seed coat of ‘Nonghe 1’ walnut was closely associated with the development of tannin cells. During seed coats development, the total tannin content initially decreased and then increased, while the levels of monomeric phenolics related to tannin synthesis (GC, EGC and EC) continuously increased. Two walnut tannase genes, JrTA1 and JrTA2, were cloned and the recombinant proteins were purified. In vitro enzymatic activity tests confirmed that both enzymes effectively hydrolyzed ester-type catechins ECG and EGCG after 20 min of reaction at 40 °C and pH 7.0. Moreover, the transgenic Arabidopsis systems and green tea infusion study demonstrated that JrTA1 and JrTA2 retained their ability to specifically cleave the ester bonds of ester-type catechins in heterologous systems, achieving efficient tannin degradation. This study systematically elucidates the enzymatic functions of JrTAs, which provides a theoretical foundation for the further development and application of walnut tannases.