Diglycosidases are endo-β-glucosidases that hydrolyze the heterosidic linkage of diglycoconjugates, thereby releasing in a single reaction the disaccharide and the aglycone. Plant diglycosidases belong to the glycoside hydrolase family 1 and are associated with defense mechanisms. Microbial diglycosidases exhibit higher diversity-they belong to the families 3, 5, and 55-and play a catabolic role. As diglycoconjugates are widespread in the environments, so are the microbial diglycosidases, which allow their utilization as nutritional source and carbon recycling. In the last 10 years, six microbial diglycosidases have been sequenced, and for two of them, the three-dimensional structure has been elucidated. This knowledge allowed the identification of their diverse phylogenetic origin, and gave insights into the understanding of the substrate specificity. Here, the last advances and the applications of microbial diglycosidases are reviewed. Key points• Substrate specificity and phylogenetic relationships of diglycosidases are reviewed.• On-going and potential applications of diglycosidases are discussed.

Actinoplanes missouriensis 431 T (=NRRL B-3342) is a soil isolate, well known for its ability to degrade flavonoids. In this work, we identified an intracellular α-L-rhamnosidase from A. missouriensis 431 T in the genomic vicinity of the previously reported diglycosidase 6-O-α-L-rhamnosyl-β-D-glucosidase (αRβG). The α-L-rhamnosidase (BAL86047.1) was functionally expressed in Escherichia coli. The purified protein (94 kDa) was able to hydrolyze rutinose to the corresponding monosaccharides. The highest activity was found at 40 °C and pH 6 in sodium citrate buffer. Regarding substrate specificity, a preference for rutinose and 7-O-diglycosylated flavanones was observed, and only traces of activity (<5%) were detected with 3-O-rutinosyl flavanols. Minor activity was found with karaya, arabic, and gellan gums. The α-L-rhamnosidase and the αRβG were associated with a pattern of genes possibly involved in the degradation and intake of the glycosidic fraction of 7-O-rutinosylated flavanones. This gene pattern was also found in five related Actinoplanes strains.