Abstract:
Objective: To develop tannases resources with excellent enzymatic properties. Methods: Primary screening was conducted using the clear zone plate method, followed by a secondary screening using the methanol rhodanine colorimetric assay. A fungal strain with the highest tannase activity was isolated from the Daqu samples. The strain was identified through morphological observation and ITS sequence analysis. The liquid fermentation conditions for tannase production were optimized. The enzyme (
PbTanA) was purified through ammonium sulfate precipitation and DEAE weak anion-exchange chromatography, obtaining electrophoretic-grade pure enzyme. The enzymatic properties of
PbTanA were further investigated, and its application in hydrolyzing tannic acid to produce gallic acid was evaluated. Results: The strain was identified as
Penicillium bilaiae and named
Penicillium bilaiae CAU46-1. After fermentation at 30 ℃ and pH5.5 for 4 days, a tannase activity of 28.7 U/mL was achieved. This was achieved under conditions where 3% tannic acid served as inducer, 4% wheat bran as carbon source, and 0.2% ammonium chloride as nitrogen source, resulting in a tannase activity 3.3 times higher than the initial level. The tannase (
PbTanA) was purified using ammonium sulfate precipitation followed by DEAE weak anion exchange chromatography. It was a molecular weight of 79.0 kDa. The enzyme exhibited good acid tolerance and thermal stability, with an optimal pH of 5.0 and good stability within the pH range of 2.0~7.0. Its optimal temperature was 60 ℃, and it remained stable up to 50 ℃.
PbTanA hydrolyzed 20 g/L tannic acid for 8 h, producing a gallic acid concentration of 18.4 g/L with a yield of 91.8%. Conclusion: Tannase
PbTanA from
Penicillium bilaiae CAU46-1 showed excellent enzymatic properties and efficiently hydrolyzed tannic acid to produce gallic acid, demonstrating its potential for application.