The hyperthermophilic glycosidase of Pyrococcus furiosus ssp. woesei (PwGly) is a potential enzyme for the sustainable synthesis of industrially relevant glycosides. The aims of the project were the characterization of the enzyme; defining a microbial production system, early optimization of the enzyme production, the in-silico substrate screening, and expanding the scope of further enzyme variants followed by experimental validation. The Elling core group defined a broad substrate spectrum for the PwGly transglycosylation reaction. Among those are products of cellulose and pectin degradation as donor substrates (transfer of glucose or galactose) and a range of alkanols, allyl- and aryl-alcohols as acceptor substrates. Also, hydroxyl-functionalized monomers such as hydroxyethyl methacrylate (HEMA) are accepted giving potential rise to novel glycopolymers. The Jaeger core group developed E. coli Rosetta strains for efficient intracellular enzyme production. The optimized purification protocol resulted in a lyophilized enzyme preparation in good yield facilitating enzyme application.The Büchs core group performed a screening of PwGly producing strains by online monitoring of respiration activities in 96-deep-well MTP for determination of optimal cultivation conditions and classification of their productivity. The E. coli Rosetta and Rosetta2 strains were classified as enzyme producers with high productivity confirming the results of CG Jaeger. The Gohlke core group generated a structural PwGly model with open binding pocket conformation. The model was used to rationalize that PwGly does not accept xylobiose as a donor substrate for transglycosylation but accepts its analogous pNP-glycoside in hydrolysis. Furthermore, CG Gohlke suggested PwGly variants with a different substrate scope and that longer cello-oligo substrates can bind and could principally take part in transglycosylation reactions, and provide support for experimental activity data where the enzyme is more active at higher HEMA concentrations. In summary, the milestones of the GlycoHype project were achieved and build the basis to proceed with the topic in this consortium with an advanced project proposal.
Prof. Dr. Lothar Elling
RWTH Aachen University
phone: +49 241 80 28350
Prof. Dr. Karl-Erich Jaeger, Dr. Andreas Kanpp, Molecular Enzyme Technology, Heinrich Heine University Düsseldorf
Prof. Dr.-Ing. Jochen Büchs, Biochemical Engineering, RWTH Aachen University
Prof. Dr. Holger Gohlke, Computational Pharmaceutical Chemistry and Molecular Bioinformatics, Heinrich Heine University
01.09.2019 - 31.10.2020
The total budget of GlycoHype was 172,531 €. GlycoHype is part of the NRW-Strategieprojekt BioSC and thus funded by the Ministry of Culture and Science of the German State of North Rhine-Westphalia.