Enhancing the Thermostability of a New Tannase Through Rational Design and Site-Directed Mutagenesis: A Quality Improvement Strategy for Green Tea Infusion

Tea has become one of the most popular drinks worldwide thanks to its pleasant sensory attributes and diverse health benefits. However, tannin-rich compositions have several negative effects and significantly impact the quality of tea beverages. Among various detannification methods, tannase treatment appears to be the most secure and environmentally friendly strategy. Although numerous microbial tannases have been identified and used in food processing, they are predominantly mesophilic with compromised heat tolerance, which limit their application in high-temperature tea extraction processing. Computer-assisted rational design and site-directed mutagenesis has emerged as a promising strategy in enzyme engineering to improve the thermostability of industrial enzymes. Nevertheless, relevant studies for tannase thermostability improvement remain lacking. In the present study, a novel thermophilic tannase called TanPL1 from marine fungus Penicillium longicatenatum strain SM102 was expressed in the food-grade host Yarrowia lipolytica. After purification and characterization, the thermostability of this enzyme was improved through site-directed mutagenesis guided by computer-aided rational design and molecular dynamics simulations. Then the thermostable mutant MuTanPL1 was applied in green tea processing for both polyphenol extraction and ester catechin hydrolysis. The tannase yield and specific activity values of 166.4 U/mL and 1059.3 U/mg, respectively, were achieved. The optimum pH and temperature of recombinant TanPL1 were determined to be 5.5 and 55 °C, respectively, and the enzyme exhibited high activity toward various gallic acid ester substrates. The site-directed mutagenesis method successfully generated a single-point mutant, MuTanPL1, with significantly enhanced thermostability and a higher optimum temperature of 60 °C. After 2 h of detannification by MuTanPL1, nearly all gallated catechins in green tea infusion were biotransformed. This resulted in a 202.4% and 12.1-fold increase in non-ester catechins and gallic acid levels, respectively. Meanwhile, the quality of the tea infusion was also markedly improved. Sensory evaluation and antioxidant activity assays revealed notable enhancements in these properties, while turbidity was reduced considerably. Additionally, the α-amylase inhibition activity of the tannase-treated tea infusion declined from 50.49% to 8.56%, revealing a significantly lower anti-nutritional effect. These findings suggest that the thermostable tannase MuTanPL1 holds strong application prospects in tea beverage processing.