Applied Molecular Enzyme Chemistry

Our research focus

Our research group specializes in applied molecular enzyme chemistry, with a particular emphasis on protein-substrate interactions and the impact of non-catalytic substrate-binding modules, like carbohydrate-binding modules (CBMs), on the activity of multimodular enzymes. Our goal is to gain novel insights that enable engineering of enzymes with desired, altered functions.

Research approaches and methods

To achieve our research objectives, we employ a variety of approaches and methods, including:

• Heterologous gene expression: Utilizing E. coli or Pichia pastoris for gene expression
• Enzymatic activity analysis: Conducting reducing-end assays, thin layer chromatography and HPLC
• Functional analysis of specific amino acids and protein domains: Using point mutations or truncations selected using bioinformatics and structure analysis as well as computational design using Rosetta. Furthermore, non-catalytic substrate-binding domains are studied as fusion proteins with green fluorescence protein (GFP)
• Molecular interaction analysis: Employing surface plasmon resonance (SPR), affinity gel electrophoresis, pull-down assays, and holdup assays (in a high-throughput setup)
• Protein stability analysis: Utilizing techniques such as nano differential scanning calorimetry
• Protein structure determination: Applying X-ray crystallography

EDFuSE: Exploiting Domain Fusion Strategies to make better Enzymes

Funded by an Emerging Investigator Grant from the Novo Nordisk Foundation (April 2025 to March 2030) (https://researchleaderprogramme.com/recipients/marie-sofie-moller/)
To tackle the climate and environmental impact of chemical industrial processes, enzymes have emerged as a promising solution capable of radically minimizing the use of harsh chemicals and lowering consumption. Unfortunately, the enzymes available today are rarely directly suitable for these processes. However, fast-growing advanced techniques, such as computational protein design, enable us to improve existing enzymes or even create new ones. What sets EDFuSE apart is its holistic approach considering the enzyme multi-domain structure. Our focus is on the mutual organization and interplay of different domains with polymeric substrates, for optimal performance under industrial conditions. We will delve into the intricate details of enzyme structure and function to convert particularly complex materials and develop a versatile toolbox dedicated to enzyme engineering. EDFuSE represents a significant step toward more sustainable and eco-friendly industrial processes.

Carbohydrate-binding module regulation of enzymatic transglycosylation (CBM4TG)
Funded by a project grant from the Novo Nordisk Foundation (April 2024 to March 2026)
Oligosaccharides and related compounds have many applications, e.g. as prebiotics used by the good bacteria helping our gut stay healthy, in therapeutics, or in cosmetics. These compounds are made in two ways: either by breaking down large sugars or by chemical synthesis from smaller ones. Yet, creating them efficiently and sustainably is tricky. Biotechnology could help as enzymes can replace harsh chemicals and make unique products. One type of enzymes, glycoside hydrolases (GHs) are promising candidates, as some can synthesis new bonds between sugars, in a process called transglycosylation (TG), besides breaking down sugars. But GHs need optimization to promote the TG route. We hypothesize that certain parts of these enzymes, called carbohydrate-binding modules (CBMs), have a say in this and want to find out how CBMs in GHs influence the balance between synthesis and breakdown. This can help us engineer better industrial enzymes able to produce important molecules in a better way.

Substrate-binding domains as facilitators for enzymatic waste management of man-made polymeric materials

Funded by a Project 1 grant from the Independent Research Fund Denmark, Technology and Production Sciences (July 2021 to April 2025) and a grant from the U.S. Department of Energy Joint Genome Institute ( https://doi.org/10.46936/10.25585/60008756) for synthesis of genes
Man-made polymer materials, such as plastics, are causing severe environmental issues. While enzymes capable of degrading plastic have been discovered, their efficiency in industrial settings remains low. This inefficiency is partly due to the difficulty enzymes face in interacting with the plastic surface in a productive manner.
One potential solution is to utilize protein modules known as carbohydrate-binding modules (CBMs), which naturally facilitate interactions between enzymes and resistant polysaccharides like cellulose, starch, and chitin. Although some CBMs have demonstrated the ability to bind to plastics, this binding promiscuity has not been systematically studied. To enhance their binding efficiency to various plastic types, CBMs need to be engineered.
In this project, bioinformatics methods will be employed to select at least 1000 protein modules. These modules will undergo screening for polymer binding ability (to both natural and man-made polymers. Furthermore, selected CBMs will be used for engineering plastic degrading enzymes through module fusion. By fusing optimized protein modules to relevant polymer-degrading enzymes, we aim to develop more efficient enzymes. This advancement could significantly improve the sustainable waste management of man-made polymers.

α-glucan debranching enzymes as tools for producing an array of valuable bioproducts

In nature and industry starch is degraded by a cascade of enzymes. Starch is composed of two types of polysaccharides made from glucose: the essentially linear amylose, and the branched amylopectin having maltooligosaccharide chains interconnected through branch points. Debranching enzymes (DBEs) remove the branches from amylopectin by hydrolysis. Remarkably, some DBEs can catalyse the reverse hydrolysis reaction and create branch points in so-called transglycosylation reactions. The present project is going to utilize such transglycosylation activity of DBEs to obtain starch-derived mostly small to medium sized products with novel structures and functions, e.g. slowly digested carbohydrates with health promoting effects and glycoengineered natural compounds (secondary metabolites) for bioplastic incorporation acting as natural pH indicators. A range of DBEs will be engineered to optimize transglycosylation specificity and yield, and to reduce (ideally abolishing) the hydrolytic reaction.