Journal of Proteomics & Bioinformatics
Open Access
ISSN: 0974-276X
ISSN: 0974-276X
Michael Kotik, Gisela Weiz, Kate Brodsky, Laura S Mazzaferro, Javier D Breccia and Vladimir Kren
Institute of Microbiology, Czech Academy of Sciences, Prague
Universidad Nacional de La Pampa, Argentina
Posters & Accepted Abstracts: J Proteomics Bioinform
α-Rhamnosyl-β-glucosidases are rather rare enzymes and occur in some bacteria, fungi and plants. They hydrolyze the heterosidic linkage of diglycoconjugates such as rutin and hesperidin, generating rutinose (a disaccharide) and the corresponding aglycon as products. Some enzymes exhibit transglycosylation activities, which provide access to various novel diglycoconjugates. The objective of the first part of the project was to over express several fungal α-rhamnosyl-β- glucosidases in a heterologous host and to characterize them. We report on the cloning of several α-rhamnosyl-β-glucosidaseencoding genes from four fungal strains: Aspergillus niger, Mucor circinelloides, Penicillium chrysogenum and Acremonium sp., we succeeded in heterologously expressing these enzymes in active form using Pichia pastoris as an expression host. The enzymes were secreted to the cultivation medium, which greatly simplified the purification of the enzymes. It followed a basic biochemical characterization of the purified enzymes, including determination of the pH optimum and optimal temperatures, thermostabilities and substrate specificities and transglycosylation activities. Catalytically important active site residues were assessed by site directed mutagenesis. The final goal of the project is an x-ray structure of at least one α-rhamnosyl-β- glucosidase. Ideally the structure of the substrate bound to a catalytically inactive enzyme variant will be determined as well. Recent Publications: 1. Šimčíková D, Kotik M, Weignerová L, Halada P, Pelantová H, Adamcová K and K?en V (2015) α-L-Rhamnosyl-β-Dglucosidase (rutinosidase) from Aspergillus niger: characterization and synthetic potential of a novel diglycosidase. Advanced Synthesis & Catalysis 357:107??117. 2. Neher B, Mazzaferro LS, Kotik M, Oyhenart J, Halada P, K?en V and Breccia J D (2016) Bacteria as source of diglycosidase activity: Actinoplanes missouriensis produces 6-O-α-L-rhamnosyl-β-D-glucosidase active on flavonoids. Applied Microbiology and Biotechnology 100:3061??3070. 3. Bassanini I, Krejzová J, Panzeri W, Monti D, K?en V and Riva S (2017) A sustainable one-pot two-enzymes synthesis of naturally occurring arylalkyl glucosides. ChemSusChem 10:2040??2045.