Engineering a Paraburkholderia pallida Tannase for Improved Catalytic Efficiency, Thermal Stability, and Green Tea Clarification

Abstract Tannases can reduce haze and astringency in tea beverages, but their application is often limited by poor thermal stability and catalytic efficiency. A tannase from Paraburkholderia pallida (Tanpp) was engineered using active-site mutagenesis and computational hotspot prediction. The double mutant F384T/N307A showed the best performance, with a 2.7-fold increase in catalytic efficiency and an 11-fold longer half-life at 50 °C than wild-type Tanpp. Structural modeling and molecular dynamics simulations suggested that N307A widened the substrate-entry region, whereas F384T compacted the catalytic pocket. The double mutant remained more stable at 323.15 K. In green tea clarification assays, F384T/N307A produced the lowest turbidity and promoted conversion of galloylated catechins into non-galloylated products and gallic acid. It retained superior performance across the tested enzyme dosages and treatment times. These findings show that coordinated active-site remodeling can improve both catalytic performance and thermal durability, supporting F384T/N307A as a promising tannase for tea clarification.