PhD-position | Targeted interaction of enzymes with cell wall polymers

Description of the project:

Protein engineering is a powerful tool to change the activity of enzymes. This project investigates how carbohydrate binding modules (CBMs) can alter the binding and activity of an enzyme towards cell wall polymers like cellulose and hemicelluloses. The enzyme lytic polysaccharide monooxygenase (LPMO) will be engineered to produce variants and fusion enzymes with an altered substrate interaction to study the influence of CBMs on substrate targeting and activity. This project is correlated with Program 3 of Circular Bioengineering, but financed by BIOTOPIA, the biomolecular technology of protein interactions PhD program (https://biotop.boku.ac.at). The selection process will give you the opportunity to enroll in this PhD program without further selection process.

Background:

Many enzymes feature substrate binding domains that are not part of their active site and located in a different domain. Cellulosic and hemicellulosic enzymes often feature carbohydrate binding modules (CBMs). CBMs bind the enzyme to their substrate and establish an equilibrium of bound and soluble enzymes. Only substrate-bound enzymes are catalytically active and their activity is influenced by the binding strength of the CBM. Soluble enzymes can target newly created positions in the cell wall’s polymer network. The affinity of a CBM therefore not only regulates the catalytic activity, but also influences the efficiency of other enzymes by targeting mutually beneficial positions for subsequent catalytic action.

Aims:

After the selection and engineering of CBMs to achieve varying affinities towards cellulose, they will be fused to LPMO. The hypothesis is that a weak binding increases the soluble fraction of LPMO and therefore supports the targeting of new plant cell wall areas for its catalytic action, whereas a CBM with a high affinity restricts the mobility of an enzyme, but increases its activity locally. Th project aims to modulate the affinity of the LPMO-CBM fusion enzyme to optimize binding and mobility for maximum enzymatic activity on cellulose and other carbohydrate polymers.

Methods:

- Bioinformatic (sequence and structure based) selection of CBMs and LPMOs.

- Structure-based protein engineering

- Enzyme production and purification in a yeast expression system

- Biochemical characterization (protein analysis and kinetic measurements)

- Fluorescence microscopy and electrochemical detection methods

- Small-scale application studies in COE Program 1.

Where to apply:

www.circularbioengineering.at