Introduction

The major flavonoid glycosides found in plants or fruits are quercetin 3-O-rutinoside (rutin), hesperetin 7-O-rutinoside (hesperidin), kaempferol-3-O-rutinoside, and naringenin 7-O-neohesperidoside (naringin) (Mazzaferro and Breccia 2011a). These compounds are involved in the bitter taste or clouding in plant-based foods or beverages, respectively.

Flavonoid glycoside degradation can proceed through two alternative enzymatic pathways: one that is mediated by monoglycosidases, and the other catalyzed by a diglycosidase. Monoglycosidases (e.g., EC 3.2.1.40: α-L-rhamnosidase), the main catalysts for deglycosylation, firstly cleave the glycosidic bond between the monosaccharide moiety and glucose. Subsequently, a β-glucosidase hydrolyzes the link between glucose and the aglycone. In contrast to this, β-diglycosidase performs the flavonoid deglycosylation in a single reaction. Many β-diglycosidases have been identified and characterized from several plants (Imaseki and Yamamoto 1961; Yasuda and Nakagawa 1994; Ogawa et al. 1997; Wirth et al. 2001; Lizotte and Poulton 1988; Mizutani et al. 2002; Suzuki et al. 2002; Baumgertel et al. 2003; Ahn et al. 2004, 2007; Nakanishi et al. 2005; Chuankhayan et al. 2005). To our knowledge, the crystal structure of β-primeverosidase (EC 3.2.1.149) from plant Camellia sinensis has been the only reported β-diglycosidase crystal structure (Saino et al. 2014). There is only one previous review about these enzymes that summarize the functional and biotechnological insights into diglycosidases (Mazzaferro and Breccia 2011a). The present paper reviews the biochemical characteristics and potential industrial applications of β-diglycosidases from microorganisms that break down plant diglycoconjugated flavonoids.

Potential substrate for β-diglycosidases

Quercetin 3-O-β-rutinoside (rutin), keampferol 3-O-β-rutinoside, hesperetin 7-O-β-rutinoside (hesperidin), diosmetin 7-O-β-rutinoside (diosmin), naringenin 7-O-neohesperidoside (naringin), and (S)-linalyl β-primeveroside are the major diglycoconjugated flavonoids of some plants (Fig. 1), mainly buckwheat and tea leaves, and fruits, such as apple, grape, and citrus (Mazzaferro and Breccia 2011). Hydroxynitriles, naphthoquinones, isoflavonoids, and terpenoids also consist of a diglycoside moiety. For instance, β-rutinosidase (6-O-α-L-rhamnosyl-β-D-glucosidase; EC 3.2.1.168) cleaves β-rutinose from rutin, hesperidin, and other rutinose (6-O-α-L-rhamnopyranosyl-β-D-glucopyranose)-containing glycoconjugates (Fig. 2).

Fig. 1
figure 1

Chemical structures of potential substrates for β-glycosidase activity. a Quercetin-3-O-rutinoside (Rutin). b Kaempferol-3-O-rutinoside. c Hesperetin-7-O-rutinoside (Hesperidin). d Diosmetin-7-O-rutinoside (Diosmin). e Naringenin-7-O-neohesperidoside (Naringin). f (S)-Linalyl β-primeveroside

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Fig. 2
figure 2

Reaction scheme of the rutin hydrolysis catalyzed by β-rutinosidase

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Microbial β-diglycosidases

The β-diglycosidases acting on glycoconjugated flavonoids have been predominantly reported in plants. Recently, β-diglycosidases were also described from microorganisms such as fungi, bacteria, and archae (Narikawa et al. 2000; Yamamoto et al. 2002; Tsuruhami et al. 2006; Nam et al. 2012; Šimčíková et al. 2015; Neher et al. 2016; Ishikawa et al. 2018). These diglycosidases hydrolyze β-rutinoside (6-O-α-L-rhamnopyranosyl-β-D-glucopyranoside) or β-primeveroside (6-O-β-D-xylopyranosyl-β-D-glucopyranoside). It was found that Acremonium sp. SES201, Penicillium rugulosum IFO7242, and Aspergillus niger K2 produced β-rutinosidase when 0.5% hesperidin, 2% rutin, 0.5% rutin, respectively, were used as the sole carbon source (Mazzaferro et al. 2010, 2011b; Narikawa et al. 2000; Šimčíková et al. 2015). It has also been reported that the production of Aspergillus oryzae β-1,3-exoglucanase (ExgA) was highest when A. oryzae was grown with a carbon source containing flavonoids, such as quercetin and rutin (Riou et al. 1998). Moreover, β-primeverosidase from Aspergillus fumigatus AP-20 and Penicillium multicolor IAM7153 are extracellular inducible enzymes that uses β-primeveroside-containing substances as the carbon source and inducer (Yamamoto et al. 2002; Tsuruhami et al. 2006). At first, the β-rutinosidase (6-O-α-rhamnopyranosyl-β-glucosidase)-encoding gene was identified from A. niger K2 (Šimčíková et al. 2015). However, this gene was annotated in GenBank as having a putative exo-β-1,3-glucanase-encoding open reading frame. Proximately, the β-rutinosidase-encoding gene was also reported in A. oryzae RIB40 (Ishikawa et al. 2018). The β-rutinosidase from A. oryzae showed high degree of sequence similarity to the β-rutinosidase from A. niger K2 (70%) (Šimčíková et al. 2015) and the β-primeverosidase of Penicillium multicolor TS-5 (58%) (Tsuruhami et al. 2006). Based on the amino acid sequence similarity, the CAZy database (Lombard et al. 2014) classifies fungal β-diglycosidases into the GH5-subfamily 23 (GH5_23) of the glycoside hydrolases (Aspeborg et al. 2012). Twelve sequences of only fungal origin have been deposited in the CAZy database in GH5_23 section (Fig. 3). Meanwhile, sequences for exo-β-1,3-glucanases, which are fungal cell wall modifying enzymes, have been deposited in the GH5_9. The β-rutinosidases from A. niger (AnRutA) and A. oryzae (AoRut) showed low similarities with the exo-β-1,3-glucanases from A. oryzae (ExgA and Exg1) and Lentinula edodes (Exg1) (Tamano et al. 2007; Sakamoto et al. 2005). A thermostable β-glucosidase/β-rutinosidase from the archaea Pyrococcus furiosus (Nam et al. 2012) involved in the production of quercetin from rutin belongs to GH1, similar to the β-diglycosidases from plants. Moreover, the gene encoding the GH55 family member 6-O-α-L-rhamnopyranosyl-β-D-glucosidase was identified from Actinoplanes missouriensis 431T genome. Biochemical analyses of the corresponding recombinant protein purified from Escherichia coli showed specificity for 7-O-rutinosylated flavonoids (Neher et al. 2016). Exo-β-1,3-glucanases from fungi including the genera Aspergillus and Penicillium have also been deposited in the GH55 section of the database.

Fig. 3
figure 3

Phylogenetic trees among β-diglycosidases and exo-β-1,3-glucanases classified as GH1, GH3, GH5_9, GH5_23, and GH55 in the CAZy database. An amino acid sequence alignment was performed using ClustalW (Thompson et al. 1994), and the phylogenetic tree was constructed using molecular evolutionary genetics analysis software version7.0 (Tamura et al. 2007). The accession numbers are as follows: Acremonium sp. α-rhamnosyl-β-glucosidase (AMD11613.1), Actinoplanes missouriensis α-rhamnosyl-β-glucosidase (BAL86042.1), Aspergillus fumigatus ExgO (CAF32160.1), Aspergillus oryzae ExgA (CAC07551.1), Aspergillus oryzae ExgO (BAB92972.1), Aspergillus oryzae IpeA (BAE62006.1), Aspergillus oryzae β-rutinosidase (BAE61018.1), Aspergillus nidulans AN1332.2 (EAA65515.1), Aspergillus niger β-rutinosidase (CAK39791.1), Aspergillus niger An06g02060 (CAK48049.1), Aspergillus phoenicis ExgS (BAB83607.1), Botrytis cinerea BofuT4P72000002001 (CCD33736.1), Camellia sinensis β-primeverosidase (BAC78656.1), Dalbergia nigrescens β-apiosyl-β-glucosidase (A3RF67.1), Fusarium fujikuroi FFUJ_02196 (CCT65263.1), Fusarium fujikuroi FFUJ_03742 (CCT64988.1), Lentinula edodes Exg1 (BAD97445.1), Oerskovia sp. IPase (BAM08953.1), Penicillium multicolor β-primeverosidase (BAG70961.1), Penicillium rubens Pc13g14840 (CAP925533.1), Penicillium sp. ExgP (BAH69264.1), Pyrococcus furiosus β-glucosidase/β-rutinosidase (AAC25555.1), and Viburnum furcatum furcatin hydrolase (BAD14925.1). The bar represents 0.05 amino acid substitutions per site

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A. oryzae RIB40 and Oerskovia sp. Y1 produce isoprimeverose-producing oligoxyloglucan hydrolase (EC 3.2.1.120), a unique β-diglycosidase, that recognizes isoprimeverose (6-O-α-D-xylopyranosyl-β-D-glucopyranoside) units from the non-reducing ends of oligoxyloglucans (Kato et al. 1985; Yaoi et al. 2007; Matsuzawa et al. 2016). The enzyme-encoding genes have been identified (Yaoi and Miyazaki 2012; Matsuzawa et al. 2016). Based on the amino acid sequence, isoprimeverose-producing oligoxyloglucan hydrolase has been classified as a member of the GH3 family.

Properties of microbial β-diglycosidases

Data from the studies cited in Table 1 show that the pH and temperature optima of β-rutinosidase (α-rhamnosyl-β-glucosidase) and β-primeverosidase from fungi ranged from 2.2–5.0 to 45–70 °C, respectively. The β-rutinosidases from P. rugulosum and A. niger, and β-primeverosidase from A. fumigatus are extreme acidophiles (Narikawa et al. 2000; Šimčíková et al. 2015; Yamamoto et al. 2002). Meanwhile, the optimal pH of α-rhamnosyl-β-glucosidase from A. missouriensis belonging to the GH55 family was 7.0 (Neher et al. 2016). α-Rhamnosyl-β-glucosidase from Acremonium sp. and isoprimeverose-producing oligoxyloglucan hydrolase from A. oryzae are thermophilic enzymes (Mazzaferro et al. 2010, 2011b; Kato et al. 1985). Especially, β-glucosidase/β-rutinosidase from archaea P. furiosus is an extremely thermostable enzyme (Nam et al. 2012). However, the specific activity for rutin of β-glucosidase/β-rutinosidase from P. furiosus was approximately 900- and 15,700-fold lower, respectively, than those for isoquercitrin and p-nitrophenyl-β-D-glucoside.

Table 1 Properties of β-diglycosidases from microorganisms
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Furthermore, β-rutinosidase hydrolyzes several rutinose-containing glycoconjugates including flavonoids, such as hesperidin, rutin, kaempferol-3-O-rutinoside, and hesperidin methylchalcone. However, the enzyme does not hydrolyze neohesperidose (2-O-α-L-rhamnopyranosyl-β-D-glucopyranose)-conjugated flavonoids, such as naringin. The β-rutinosidases from P. rugulosum and Arthrobacter sp. show specificity for 3-O-linked rutinosides such as rutin, a 3-O-rutinosylated flavonol (Narikawa et al. 2000; Song-Joon et al. 1990). The catalytic activity of β-rutinosidase from A. niger was almost ten times higher for rutin hydrolysis than that for hesperidin, a 7-O-rutinosylated flavanone (Šimčíková et al. 2015). Meanwhile, among the three substrates examined, the catalytic activity of β-rutinosidase from A. oryzae was highest for kaempferol-3-O-rutinoside, which is a 3-O-rutinosylated flavonol, moderate for rutin, and lowest for hesperidin (Ishikawa et al. 2018). In contrast to this, α-rhamnosyl-β-glucosidase from Acremonium sp. and A. missouriensis hydrolyzes 7-O-linked rutinosides only, such as hesperidin, but not 3-O-linked rutinosides such as rutin (Mazzaferro et al. 2010, 2011b Neher et al. 2016). However, no activity of β-rutinosidases from A. missouriensis and A. oryzae was determined toward diosmin, a 7-O-rutinosylated flavone. This suggests that the structure of flavonoids also determines enzyme specificity.

The hydrolytic activity of the β-primeverosidase from A. fumigatus on the p-nitrophenyl β-gentiobioside was greater than that from P. multicolor (Yamamoto et al. 2002; Tsuruhami et al. 2006). The substrate specificity of these fungal enzymes also differed appreciably from that of tea β-primeverosidase.

Potential industrial applications

In the citrus processing industry, the deglycosylation of flavonoids plays an important role in improving the product quality, such as in the reduction of bitterness, clarification of juices, and in enhancing the aroma in wine and tea (Günata et al. 1998; Hemingway et al. 1999; Wang et al. 2001; Ma et al. 2001; Genovés et al. 2005). This process is also used for controlling aroma compounds, such as monoterpene alcohols, in sweet potato shochu which is a traditional Japanese distilled spirit (Sato et al. 2018). A simple enzymatic-spectrophotometric method for hesperidin quantification in citrus-based foods was developed by means of Acremonium sp. α-rhamnosyl-β-glucosidase (Mazzaferro and Breccia 2012a).

Rutinose-containing compounds have been demonstrated to have interesting pharmaceutical and medicinal applications (Robinson et al. 2004; Knaup et al. 2007). Rutinose-containing flavonoids have been shown to be absorbed in he intestines only after rhamnose hydrolysis and catalyzed by human gut microflora (Nielsen et al. 2006). The transglycosylation potential of the fungal β-rutinosidase has been explored. The biocatalyst has been shown to have broad acceptor specificity toward aliphatic, aromatic, and arylalkyl alcohols using α-rhamnosyl-β-glucosidases from Acremonium sp. (Minig et al. 2011; Mazzaferro et al. 2012b) and A. niger (Šimčíková et al. 2015; Bassanini et al. 2017). Bassanini et al. (2017) have developed two-step two-enzymatic synthesis of coniferin using the α-rhamnosyl-β-glucosidase and A. terreus α-L-rhamnosidase. It has also been reported that rutinosylation of various phenolic acids can increase their antiviral activity against feline calicivirrus, more than the respective aglycone (Katayama et al. 2013).

Conclusions

In conclusion, we present here the biochemical characteristics and potential industrial applications of β-diglycosidases from archaea, bacteria, and fungi that breakdown plant diglycoconjugated flavonoids. Eukaryotic β-diglycosidases are effective catalysts when food technology for aroma modulation and pharmaceutical and medicinal applications are envisioned. Because of their retaining mechanism, transglycosylation activity is to be expected. The synthetic potential of β-diglycosidases from fungi and plants has also been demonstrated, which can glycosylate alkylic, phenolic, and arylalkyl alcohols and phenolic acids in vitro (Mazzaferro et al. 2012b; Šimčíková et al. 2015; Katayama et al. 2013; Bassanini et al. 2017).