Efficient hydrolysis of glycosides by Pichia kudriavzevii with high-activity β-glucosidase: a critical role in enhancing terpene aroma in beer.

Beer aroma is strongly influenced by terpenes, many of which exist in hops as non-volatile glycosides. β-Glucosidase (BGL) can release these bound terpenes, but its role in brewing remains underexplored. In this study, twelve non-Saccharomyces yeasts were screened, and Pichia kudriavzevii KD4 was identified as the best BGL producer. Co-fermentation with Saccharomyces cerevisiae US05 under optimized conditions enhanced floral and fruity aromas of beer, increasing esters and terpene alcohols, particularly linalool, nerol, and geraniol. The BGL from KD4 (PkBGL) was heterologously expressed in P. pastoris, exhibiting optimal activity at 50 °C and pH 5.5, and showing stability under low-temperature, acidic, ethanol-, and isoα-acid-rich conditions. Enzymatic assays confirmed efficient hydrolysis of hop glycosides, while molecular docking revealed stable binding of linalyl and nerol glycosides via hydrogen bonding and hydrophobic interactions. These results highlight PkBGL as a promising tool for aroma enhancement in beer through selective terpene release.