Orange debittering by a novel thermostable flavonoid glycosidase
Yaoguang Huang1, Yingjun Liu2, Haijiao Wang1
1Guangdong Provincial Key Laboratory of Applied Botany, Key State Laboratory of Plant Diversity and Specialty Crops, Key Laboratory of National Forestry and Grassland Administration on Plant Conservation and Utilization in Southern China, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Abstract:
Naringin, a bitter flavanone glycoside in citrus with diverse bioactivities, consists of a glycosyl group linked to naringenin. Naringinase-catalyzed hydrolysis cleaves this glycosidic bond, converting bitter naringin into tasteless naringenin. Current naringinases suffer from limited diversity, poor thermostability, and narrow substrate specificity. While the thermostable glycosidase from Pyrococcus furiosus enables broad-spectrum debittering via transglycosylation, its low catalytic efficiency and complex purification hinder practical application. This study employed semi-rational design to engineer PfNG. Structural analysis yielded single mutants (F265A, M328S) and combinatorial mutant V1, exhibiting optimal activity at 95 °C with a 2.48-fold higher Kcat/Km than wild-type. We further developed a streamlined one-step thermal purification protocol, providing an efficient alternative to chromatography. Structural characterization revealed enhanced activity likely originates from: (1) contracted binding pocket volume, (2) widened substrate channel entrance, (3) novel hydrogen bonds, and (4) increased conformational flexibility. V1 effectively reduced naringin content (>91 %) in green citrus extracts and mitigated juice bitterness. Our work resolves key limitations of PfNG, advancing its industrial deployment for citrus debittering and biopharmaceutical applications.
