As a type of polyphenol compound generated to resist a bad growth environment, tannins are extensively present in higher plants (Chung et al., 1998). The formation of stable complexes with various biological macromolecules, such as polysaccharides and proteins, provides lower nutritive value to food, thus making them nutritionally undesirable (Pan et al., 2020). According to some reports, tannins are one of the primary causes of low food intake, slow growth, low level of fodder utilization rate, and low protein breakdown in laboratory animals (Becker and Makkar, 1999; Min et al., 2005; Sengil and Oezacar, 2009). Therefore, the removal of tannins becomes an integral process during food and feed processing. Fortunately, there still exist some special microorganisms which can grow and reproduce stably in an environment containing tannins, making the full utmost of the secreted enzymes to decompose tannins into small-molecule phenolic compounds (Chhokar et al., 2013).

Tannase (EC 3.1.1.20), also known as tannin acylhydrolase, is a hydrolase that can effectively hydrolyze ester bonds and carboxyphenol bonds in hydrolyzable tannins, including ellagitannins (ETs), gallotannins (GTs), and other gallic acid esters to produce polyphenol compounds such as gallic acid (Aguilar and Gutierrez-Sanchez, 2001; Zhang et al., 2015; Govindarajan et al., 2016). The enzymatic degradation of tannins is considered a safe and environmentally friendly method that can effectively overcome the shortcomings of traditional hydrolysis methods and significantly improve the yield and purity of products (Raghuwanshi et al., 2011; Jana et al., 2013; Ghosh and Mandal, 2015; Ebrahimzade et al., 2018). Therefore, tannases have played a significant role in industrial production, especially in the fields of feed, food, brewing, and pharmaceuticals (Chhokar et al., 2013; Wang et al., 2022). Rapeseed meal is considered to be one of the sources of high-quality plant protein feed due to its high protein content and balanced amino acid composition. However, rapeseed meal contains a variety of anti-nutritional factors, like tannins, that seriously affect its palatability as well as nutritive value, severely limiting the application of rapeseed meal in animal feeding (Shim et al., 2003). Interestingly, Lorusso et al. (1996) reported that the tannase produced by Trametes versicolor could degrade more than 80% of tannins in rapeseed meal within 30 min, strengthening the application of tannase in the processing of rapeseed meal feed. Furthermore, the transformation of ester catechins (also belonging to tannins) in green tea to non-ester catechins for improving the taste of tea drinks is another important use for tannases (Ozturk et al., 2016). Therefore, these properties of tannases are of great interest among researchers.

Currently, owing to the characteristics of biochemical diversity and liable genetic manipulations, microbial fermentation is the major approach for the continuous production of tannases (Beniwal et al., 2013). Numerous studies have been conducted for screening tannase-producing microorganisms, of which bacteria and fungi have shown enhanced enzyme production ability (Banerjee et al., 2012; Kanpiengjai et al., 2019). However, tannases produced by bacteria demonstrated limitations in low activity and poor thermostability, imposing major concerns of large-scale application (Rivas et al., 2019). Thus, there are relatively scarce studies of bacterial tannases. On the other hand, the complex heredity of fungi makes genetic manipulations difficult (Beniwal et al., 2010). These collectively present challenges in identifying tannases with better properties for industrial application—for example, tannases play a pivotal role in green tea deep processing to improve the appearance, aroma, and flavor of tea and the extraction efficiency of polyphenols (Cao et al., 2019). During the extraction of green tea, the extraction yield increased with increasing temperature within a certain range (Shao et al., 2020). However, since the enzyme catalysis reaction is usually temperature-limited, tea processing and extraction consume a prolonged time (over 2 h) at a lower range of temperature from 30 to 40°C, thereby reducing product quality and production efficiency (Hong et al., 2013). Therefore, it becomes necessary to screen genetically stable microorganisms with the capability to produce tannases of high vigor and thermal stability. Jana et al. (2013) reported that the high-temperature and solvent-resistant tannase produced from Bacillus subtilis PAB2 had a half-life of up to 4.5 h in an environment of 60°C. Shao et al. (2020) screened Aspergillus niger FJ0118 that could generate tannase rAntan1 with strong temperature tolerance, and its half-life at 60°C was persisting for about 5.4 h. Nevertheless, the majority of the reported thermostable tannases cannot reach food grade due to safety concerns.

Aureobasidium melanogenum T9, with the capability to degrade tannins, has been successfully isolated from the starter of red wine (Zhang et al., 2019). In this study, the gene of encoding tannin-degrading enzyme, tanA, was identified and heterologously expressed in the Yarrowia lipolytica yeast of food grade. The detailed characterization of tannase TanA displayed superior thermal stability and unique robustness, thus laying a solid foundation for its application in the industry.

The previous study of the genes related to tannin degradation in A. melanogenum T9 has suggested that there existed several tannases (including TanA, TanB, and TanC) with different molecular masses, amino acid sequences, gene expression patterns, and diverse properties, which are together responsible for the catalyzed hydrolysis of tannins in the strain T9 (Zhang et al., 2019). The tannases from different categories are often produced in the same fungal species due to species-specific accumulative effects and the impacts of exterior circumstances. Tannase subtypes are distinguished depending on their physical and chemical properties, such as molecular size, family, presence or absence of a signal peptide, and more, which guarantee that microorganisms are able to degrade both intra- and extracellular tannins under various physiological conditions, namely, temperature, pH, ion strength, etc. Therefore, it is of great significance to figure out the metabolic mechanism of tannins in A. melanogenum in future work.

TanA exhibited a typical conserved domain, Gly-X-Ser-X-Gly, almost present in all tannase sequences of fungi and bacteria (boxed in red in Figure 1). This unique sequence of serine hydrolase effectively binds to the tannic substrates, thereby promoting catalytic activity (Zhang et al., 2019). Moreover, a mutation analysis explained that the disulfide bond between Cys202 and Cys458 played a vital part in regulating the activity of AoFaeB (Suzuki et al., 2014). Interestingly, TanA also showed several cysteine residues probably involved in disulfide bond formation at similar sites as that of AoFaeB. For the sake of surveying this, the homology model of TanA was built. Meanwhile, four disulfide bonds with a 3D structure have been predicted (Supplementary Figure S2), including Cys161–Cys415, Cys230–Cys247, Cys256–Cys265, and Cys487–Cys508, among which the first disulfide bond was similar to the one in AoFaeB as described above, indicating that the disruption of disulfide bonds when subjected to ME affected TanA activity and stability to a great extent (Figure 6).

It is shown in Figure 4A that TanA displayed an optimal response at 60°C, with over 80% relative activity from 40 to 70°C, illustrating its thermophilic property and making it suitable for most of the hydrolysis processes assisted by tannases which were carried out at increased temperatures (Abdulhameed et al., 2005). However, a lot of studies demonstrated that many tannases from bacteria (Jana et al., 2014), mold (Iibuchi et al., 1968; Lekha and Lonsane, 1997; Madzak, 2015; Sivashanmugam and Jayaraman, 2011), and even yeast (Wang et al., 2020) showed optimal activity at approximately 40°C. Considering thermal stability, as exhibited in the diagram of Figure 4B, TanA depicted superior heat resistance property at a temperature below 55°C, maintaining up to 61.3% activity even after 12 h of incubation. Studies showed thermostability in the range of 30–50°C for most fungal tannases (Table 3). TanA possessed better temperature stability compared to other tannases from fungi. Although the tannase from P. notatum NCIM923 (Gayen and Ghosh, 2013) and the tannase from K. marxianus NRRL Y-8281 (Mahmoud et al., 2018) could maintain their stability up to 60 and 70°C, respectively, according to their researches (Table 3), their thermostability at high temperatures was, in fact, worse than TanA. The purified tannase from P. notatum retained 60% of its thermostability at 60°C within only 1 h (Gayen and Ghosh, 2013). Meanwhile, the tannase from K. marxianus could also remain stable at 70°C for only 60 min (Mahmoud et al., 2018). Also belonging to thermophilic tannases, the extracellular tannase derived from A. phoenicis, whose optimal temperature was the same as TanA, was stable for 1 h at 40–50°C, with a half-life of only 20 min at 60°C (Riul et al., 2013). In industrial production such as green tea deep processing, the degradation of tannins often takes a longer time at a relatively high temperature, and the tannase TanA retained most of its activity within at least 12 h under 55°C. This concludes that TanA is a thermophilic tannase with sturdy thermostability, which quite better satisfy the industrial application. Contrastingly, the optimal temperature for the tannase rAntan1, with strong thermal stability from A. niger FJ0118, was 80°C. It could persist to be stable at 60°C for about 5.4 h (Shao et al., 2020). Although owning favorable activity and stability at high temperature, rAntan1 expressed by Pichia pastoris cannot reach food grade because of the use of methanol when inducing tannase expression. The thermophilic and thermostable properties of TanA and rAntan1 may be partially attributed to glycosylated formation during expression in yeast (Zhou et al., 2020), corresponding to the predicted N-glycosylation recognition sites (Figure 1). Yang et al. (2007) indicated that the tea steeped in water at 70°C contained the highest contents of active components. Thus, the thermophilic and thermostable tannases, such as TanA and rAntan1, are rare in nature but necessary in industrial production. Some pieces of literature have reviewed the wide application of thermostable tannases in tea extracts to decrease the formation of tea cream (on account of the presence of tannins), thereby improving the taste of tea beverages (Chávez-González et al., 2012). In addition, thermo-tolerant tannases were also applied in the production of instant green tea powder to enhance the color and taste (Ozturk et al., 2016; Cao et al., 2019). In the process of catalytic degradation of tannins, thermostable tannases can enhance high-temperature extraction efficiency, simplify the production process, reduce the costs, and improve the product quality, which are reasons for the application of TanA in industrial production, such as in green tea deep processing.

TanA maintained over 60% activity within the broad pH scope of 3.0–7.0, with maximum activity at pH 6.0 (Figure 5A). For pH stability, TanA still retained over 70% of its activity after incubation for 12 h within the wide pH from 3.5 to 7.5, indicating advisable stability under acidic to weakly alkaline conditions (Figure 5B). It was consistent with the reported fungal tannases—for instance, the tannase derived from A. niger possessed an optimal pH of 6.0 and excellent stability at pH 3.0–8.0 (Shao et al., 2020). The tannase from A. oryzae had an optimum pH of 5.5 along with stability between pH 4.5 and 7.5 and over 80% activity retention (Abdel-Naby et al., 2016). On the other hand, the tannase from E. nidulans (pH 4.0–5.0) and the tannase from Aspergillus thorny (pH 5.0–6.0) displayed limited pH stability (Goncalves et al., 2011; Tanash et al., 2011). Therefore, remarkable stability at high temperatures and a wide pH range become integral properties of TanA, which reduce the risk of pollution during the fermentation process and thereby establish the foundation for its industrial application in tannin biodegradation and gallic acid preparation.

TanA has been incubated with several gallic acid esters to study the substrate specificity. As shown in Table 2, TanA revealed outstanding specific activities to decompose various substrates, illustrating its multifunctional property. The activity of TanA on synthetic substrate PG was significantly superior, whereas the specific activities on some natural substrates (e.g., 703.1 U/mg against GCG, 756.2 U/mg against ECG, and 623.2 U/mg against EGCG) were also quite considerable (Table 2). Many reported tannases exhibited quite lower activities toward some ester catechins that naturally exist in green tea, such as ECG and EGCG (Bhoite et al., 2013; Ramírez-Coronel et al., 2003; Sharma et al., 2008). Although Mahmoud et al. (2018) stated a high activity toward TA (1,026.12 U/mg), the tannase from K. marxianus NRRL Y-8281 was most active on TA (100%), followed by MG (74.3%) and PG (64.5%) and expressing minimum enzymatic activity against EGCG (only 10.5%). Obviously, these enzymes cannot satisfy the application in industrial processing when against natural ester catechins. In addition, the HPLC analysis results of transformation products by TanA further confirmed the almost complete degradation of varying kinds of tannins (especially the natural ester catechins in many plants) at a high temperature within 2 h (Supplementary Figure S3). Previous research reported that immobilized tannase could carry out 98% conversion of TA within 6 h (Mahendran et al., 2006). However, tannase from Bacillus sphaericus converted 90.8% TA after as long as 24 h (Raghuwanshi et al., 2011). By contrast, the higher efficiency of TanA, along with high bioconversion, undoubtedly benefited from its favorable activities toward different gallic acid esters. Moreover, plants in nature, such as oilseed rape and many fruits, usually contain various hydrolyzable tannins (GTs and ETs) (Lorusso et al., 1996). Thus, the multifunction property gives the tannase TanA the capability to decompose diverse gallic acid esters, which is fully conducive when against different kinds of tannins in the plants. The prime cause of the bitter taste of tea is the presence of ester catechins (mainly EGCG and ECG) (Shao et al., 2020). The reported tannase rAntan1 displayed similar substrate specificity as TanA. Enzymatic extraction reduced the proportion of easter catechins in tea polyphenols, weakened the bitter taste, and improved the overall taste of the tea beverage (Shao et al., 2020). Similarly, the TanA-mediated extraction processing can also effectively transform the ester catechins to the non-ester ones (such as EGC and EC) in green tea, thereby improving the taste of tea drinks. Besides this, the green tea catechins possess many biological activities, such as antiviral, antibacterial, antitumor, and antioxidant properties, which are closely related to the presence of multiple hydroxyl groups and galloyl groups in their structures (Xu et al., 2021).

Tannins are ubiquitously present in higher plants, and biodegradation by tannases is essential in industrial processing, such as in feed additives (Lorusso et al., 1996), myrobalan juice (Srivastava and Kar, 2009), and green tea (Cao et al., 2019; Shao et al., 2020). In addition, many active substances, such as catechins and gallic acid, employ tannases for their respective extraction (Chhokar et al., 2013). Rapeseed meal processing is a worthy example illustrating the application of tannases. The agro-industrial chain of canola oil production generates a large mass of rapeseed meals but faces challenges during recycling owing to the high content of tannins, resulting in huge wastes (Lorusso et al., 1996; Shim et al., 2003). Fortunately, the efficient decomposition of tannins through enzymatic catalysis aids in the production of rapeseed meal feed with added value (Lorusso et al., 1996). In other words, tannases play significant roles in various fields, such as food, brewing, pharmacy, and feed. However, tannases of food grade with advisable properties are still rather rare but necessary. In this research, a novel thermophilic tannase, TanA, with favorable thermostability was demonstrated, with detailed characterizations suggesting a potent candidate for the food and agricultural industries.