Tannins are a type of polyphenolic compounds that are abundant in higher plants, with a molecular weight ranging from 500 Da to 28,000 Da. After cellulose, hemicellulose, and lignin, tannins are the fourth most abundant substance in plants. The presence of phenolic rings and phenol hydroxyl groups in tannins enables them to capture metal ions and form strong interactions with macromolecules such as proteins, cellulose, starch, and other polysaccharides. The resulting complexes can lead to noticeable insolubility and precipitation effects (Ragan, 1986; Schofield et al., 2001; Abebe et al., 2007; Jeong et al., 2013). Chemically, tannins can be classified into three major categories: phlorotannins, condensed tannins, and hydrolyzable tannins (Ragan, 1986). Condensed tannins primarily accumulate in the endothelial layer of rapeseed between the aleurone layer and the outer integument, which contributes to the darker color of the seed coat. Tannins have an unpleasant taste, astringent properties, and are indigestible, and these properties enable them to play a role in the defense against invading pathogenic microorganisms and herbivores by forming stable complexes with biological macromolecules (Buzzini et al., 2008).

Protein digestibility is a critical factor in determining the bioavailability of protein in rapeseed meals (Fuller and Tomé, 2005). Recently, various methods have been employed to degrade tannins and improve protein digestibility in vitro. Heat processing is a practical technique that can enhance protein digestibility, but it often results in nutrient loss (Petitot et al., 2009). Lye soaking solution (Salem et al., 2005; Canbolat et al., 2007) and high-pressure treatment (Xue et al., 2020) have been utilized for tannin degradation and protein digestibility improvement, but their widespread use is limited by high equipment requirements and costs.

Compared to other methods, enzymatic hydrolysis is a mild and effective degradation method that can efficiently degrade anti-nutritional factors in oilseed meals, enabling the protein to be fully utilized (Nair and Duvnjak, 1990; Hao et al., 2020; Wang et al., 2022). However, studies on the microbial degradation of tannins in rapeseed meals are scarce. Some microorganisms are known to be resistant to tannins and produce tannin-degrading enzymes. Tannase (EC 3.1.1.20), also known as tannin acyl hydrolase, directly cleaves the ester bonds in all types of tannins, including gallotannins, ellagitannins, and gallic acid esters. The product of tannase-based degradation is gallic acid and ellagic acid (Ebune et al., 1995; Tripathi and Mishra, 2007; Lomascolo et al., 2012; Mejicanos et al., 2016). Tannase-based degradation of tannins has received significant attention. Li et al. reported that tannase generated through Trametes versicolor was utilized to treat rapeseed meals, and the results indicated that the degradation rate of tannins exceeded 80% within 30 min (Lorusso et al., 1996). Tannase has also been used to prevent chill haze in beer production.

The biotransformation of gallic acid from gallotannins through tannase catalysis has gained increasing importance as a significant application of tannase. This process has been extensively studied by various researchers (Bell, 1984; Lacassagne et al., 1988; Al-Mamary et al., 2001; Şengil and Özacar, 2009; El-Beltagi and Mohamed, 2010; Frazier et al., 2010). Gallic acid, a product of tannin degradation, has been found to possess a range of biological activities, such as anticarcinogenic, antiallergic, antibacterial, anti-inflammatory, and antioxidant properties (Lacassagne et al., 1988; Frazier et al., 2010). Its medicinal properties make it a potential candidate for drug material and food additives. Gallic acid has also been used as a noted medical intermediate in the pharmaceutical industry for synthesizing trimethoprim, a commercial antibacterial drug, and specific gallate esters, which serve as green food preservatives (Lacassagne et al., 1988; Al-Mamary et al., 2001; Abebe et al., 2007; Şengil and Özacar, 2009).

Although the chemical method of producing gallic acid through acid hydrolysis has been widely used, but it has some limitations in terms of its purity and yield. In contrast, the enzymatic method, which utilizes tannase, has demonstrated higher efficiency, purity, and yield with less environmental pollution. However, there is still a dearth of information on the bioconversion of tannic acid to gallic acid via tannase (Lacassagne et al., 1988; Al-Mamary et al., 2001; Abebe et al., 2007; Ozturk et al., 2016). To ensure that tannase can effectively catalyze the hydrolysis of its substrate, tannic acid, it is necessary to maintain a low pH during the reaction. Few characterized tannases thus far exhibit high catalytic activities within a narrow pH range, which is insufficient for gallic acid production (Kanpiengjai et al., 2020). Therefore, tannases working over the broad pH range is demanded in application.

Mangroves, being a typical transitional ecosystem, possess a rich diversity of species, including microorganisms. These microorganisms, due to their proximity to both terrestrial and marine habitats, have been found to secrete degrading enzymes that can utilize plant materials as nutrient substances (Zhang et al., 2019; Wang et al., 2022). In this study, a novel tannase abbreviated as TanF was successfully obtained from Debaryomyces hansenii and expressed effectively in yeast host. This enzyme demonstrated superb pH stability and a unique ability to degrade tannin, making it a potent tool for tannin biodegradation and gallic acid production.

Traditionally, industrial tannases were mostly derived from fungi. Recently, many bacterial tannases have also been well characterized (Ebune et al., 1995; Ebune et al., 1995; Tripathi and Mishra, 2007; Lomascolo et al., 2012). However, tannases from yeast strains were still rare. As shown in Table 1, tannase activity of the yeast strain Y71 was higher than those of A. niger, Cyberlindnera rhodanensis, but lower than those of Sporidiobolus ruineniae and Rhodosporidium diobovatum. Notably, the tannase of strain Y71 was extracellular, which facilitates the purification and application. Meanwhile, potential resources in the form of uncharacterized tannases from yeast need to be developed. By comparing the sequences of TanF and 11 representative tannases, it can be seen directly that the current enzyme sequence contains a conserved domain, namely Gly-X-Ser-X-Gly (shown in the red box of Figure 1), which is a typical as well as common amino acids of serine hydrolase, showing the highest conservation among all tannase sequences of bacteria and fungi (Zhang et al., 2019).

Most tannase hydrolysis processes occur at increased temperatures, which is seen as an advantage for soybean meal biodegradation (Sabu et al., 2005). However, some yeast, bacteria (Jana et al., 2014), and mold tannases (Sabu et al., 2005; Sivashanmugam and Jayaraman, 2011) such as Aspergillus flavus, Penicillium chrysogenum, A. niger, and A. oryzae (Inoue et al., 2016; Wang et al., 2020) have been shown to display the highest activity at about 40°C. Tannase TanF exhibits favorable stability under temperatures below 40°C, maintaining 78.6% activity even after 2 h of incubation at 40°C.

It is reported that the optimal pH of most tannases from bacteria is approximately 7.0–9.0, while most fungal tannases possess superior activity at pH 6.0. For example, TanF from Kluyveromyces marxianus exhibits an optimal pH of 4.5, as shown in Table 2, and displays favorable stability at pH 4.0–4.5. In comparison, tannases from other sources such as Aspergillus spinosus (pH 5.0–6.0) and E. nidulans (pH 4.0–5.0) show limited pH stability (El-Tanash et al., 2011; Gonalves et al., 2011). Tannase from Candida sp. has an optimizing pH of 6.0 (Raghuwanshi et al., 2011; Mahmoud et al., 2018) and maintains high catalytic activities in a narrow pH range of approximately 5.0–7.0 (Ragan, 1986; Schofield et al., 2001; Fuller and Tomé, 2005; Canbolat et al., 2007; Buzzini et al., 2008; Petitot et al., 2009). Efficient and stable tannases at low pH are rare, despite comparisons of tannases from bacteria and fungi (Ebune et al., 1995; Tripathi and Mishra, 2007; Lomascolo et al., 2012; Mejicanos et al., 2016). Yeast tannases have been studied in only a few species, including S. ruineniae, Candida sp., Aureobasidium, B. adeninivorans, and K. marxianus (Lacassagne et al., 1988; Nair and Duvnjak, 1990; Salem et al., 2005; Hao et al., 2020; Xue et al., 2020). Because both the product and substrate of tannase are acidic, TanF tannase characterized in the present paper can be applied as a potential tool for soybean meal biodegradation due to its satisfying pH stability.

With the alternative protein source lacking, some unconventional non-food protein resources have gain great attention. Rapeseed dregs, the solid residue extracted from rapeseed oil, was abundant in vitamins and well-balanced amino acids (Ebune, et al., 1995; Jeong et al., 2013; Pustjens et al., 2013). However, the application of rapeseed protein in animal feeds was severely restricted owing to the existence of tannins, which accounted for 0.6–3% of the dry weight. Tannins affecting the absorption of nutrients and possibly causing toxic effects to poultry, becoming the main anti-nutritional factor in rapeseed meal. Therefore, the removal of tannins using strain processing was one of the focus issues. TanF obtained from mangroves with salt tolerance can be another choice to process the unconventional resources in marine aquatic feed.

TanF demonstrated considerable activity on natural substrates, including CG, MG, and EGCG. The substrate specificity makes TanF suitable for tea leaves treatment. In previous studies, green tea leaves treated with tannase resulted in a higher concentration of theaflavin compared to pure withered leaf fermentation, indicating its potential for improving the tea’s quality. Tannase treatment also leads to the hydrolysis of EGC, GA, ECG, and EGCG, resulting in a significant reduction in tea cream formation and an increase in antioxidant activity (Liang et al., 2022). Furthermore, tannase treatment improves the sensory characteristics of black tea infusions, including the intensity of color and the absence of cream formation. In Hibiscus tea, tannase treatment decreased esterified catechins by 8.91% while increasing non-esterified catechins by 19.76%. Total phenolic compounds in Hibiscus tea also increased significantly by 8.6% due to tannase treatment (Sayed, 2023). Therefore, tannase represents a potential means of producing low-astringency Hibiscus tea while also enhancing its quality. Tea leaves treatment can be new direction of application of TanF.

Gallic acid synthesis using tannase producing microorganisms has been studied extensively. Candida sp. and K. marxianus are two yeast species that have shown potential for the production of extracellular tannase (Bell, 1984; Lorusso et al., 1996; Petitot, et al., 2009). It is important to note that Aspergillus sp. has been widely used for tannase production in previous studies, but their ability to convert tannic acid to gallic acid has not been thoroughly evaluated. Additionally, it has been suggested that the accumulation of gallic acid is not directly associated with the tannase yield of the organism, indicating that Aspergillus sp. may only serve as a tannase producer due to its endogenous metabolism. (Salem et al., 2005; Canbolat et al., 2007). Similar findings were observed in studies involving B. thurangiences and A. Pullulans (Abebe et al., 2007; Xue et al., 2020). The gallic acid yields of these microorganisms were too low to perform commercial production.

Cell wall-associated tannases were better choice for gallic acid synthesis. A. niger Aa-20 and A. aculeatus DBF9 have been reported to produce 7.64 g/L and 6 g/L gallic acid, respectively, from tannic acid (Nair and Duvnjak, 1990; Salem et al., 2005). In a recent study, B. subtilis AM1 and Lactobacillus plantarum CIR1 produced the maximum levels of gallic acid in non-optimized conditions during anaerobic fermentation of 10 g/L tannic acid, with final contents of 2.41 and 2.37 g/L, respectively (Hao et al., 2020). Another cell wall-associated tannase from yeast S. ruineniae can appears to be more efficient for gallic acid production, with a final content of 11.8 g/L (Kanpiengjai et al., 2020).

In this study, concentrated recombinant TanF was used as the catalyst for gallic acid synthesis. The concentration of substrate can be up to 20 g/L. Meanwhile, the yield was improved to above 0.90 g/g, due to the absence of endogenous metabolism in the live cell. Actually, the final gallic acid production achieved 18.32 g/L, much higher than those in studies above mentioned. Therefore, TanF has been demonstrated to be an efficient tool for the preparation of gallic acid. Enzyme modification and process optimization will be carried out to prompt the application of TanF in commercial scale.