Discovery and engineering of novel cold-active cellobiose 2-epimerases with both epimerase and isomerase activity

• Applicant: Professor Dr. Lutz Fischer
• Subject Area: Biological Process Engineering ; Biochemistry
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 492716110

The aim of our project is the discovery and molecular modification of novel cold-adapted cellobiose 2-epimerases (EC 5.1.3.11, CE), catalyzing the bioconversion of lactose into the prebiotics epilactose and lactulose more efficiently at low temperature. We want to understand the correlation between thermostability and activity underpinning the cold-adaptation of these enzymes.To reach these goals metagenomic libraries from cold environment samples are created and two metagenomic approaches are used to discover novel CE genes. The sequence based (in-silico) selection of putative cellobiose 2-epimerases from the database via the “Basic Local Alignment Search Tool” (BLAST) is done based on their phylogenetic hierarchy and the prior knowledge of reported CEs. For the function based metagenomic approach a sensitive high-throughput plate screening has to be established. Identified CE candidates from the metagenomic library and from the in-silico screening will be cloned for recombinant production and enzyme activity determination at low temperatures. Further, protein engineering via molecular modifications using directed evolution methods and semi-rational and rational design are promising tools to generate CEs with more favorable kinetic properties. The identification of residue substitutions that increase the accessibility of the catalytic center or reduce the stability of the active sites, will hopefully improve the kinetic properties of the wild-type CEs at low temperature (8°C). The novel CEs, which can catalyze bioconversion of lactose to epilactose and lactulose at low temperatures, will be a benefit for the usage in the dairy industry to produce a prebiotic product out of a by-product like whey.

• DFG Programme: Research Grants