This project aims to investigate the effect of post-translational modifications (PTMs) – particularly glycosylation on protein structure, function, and stability. Eukaryotic and prokaryotic proteins often undergo chemical changes after synthesis, known as PTMs, which include functional group additions that significantly influence their activity, stability, localization, and interactions within cellular processes.
Project Focus: This study examines glycosylation which is a key post-translational modification and its impact on enzyme stability and activity. We will investigate two industrially relevant classes: glycosyl hydrolases and metalloproteases. Emphasis will be on glycosylation patterns to identify strategies that enhance structural integrity, protease resistance, and performance in home-care formulations.
Glycosylation is one of the most critical PTMs, involving the attachment of sugar moieties (glycans) to proteins. The two most common types are:
• N-linked glycosylation: Glycans are attached to the nitrogen atom of asparagine residues.
• O-linked glycosylation: Glycans are attached to the oxygen atom of serine or threonine residues.
Industrial Relevance: Enzymes are widely used across industries from pulp and paper to biofuels. In home-care applications, enzymes play a key role in sustainable cleaning, reducing reliance on fossil fuels and lowering the carbon footprint. However, many wild-type enzymes are unstable in home-care formulations due to surfactants and other cleaning ingredients like proteases, significantly lowering enzyme efficacy; therefore, methods to enhance stability are pertinent.
Scientific Background: A broad body of literature confirms that glycosylation enhances both structural and proteolytic stability of enzymes. This offers a promising strategy to improve enzyme performance without extensively altering the amino acid sequence. However, glycosylation can also negatively impact activity depending on the type and extent of modification. For example, hyper N-linked glycosylation in the catalytic domain of Cel7a from T. reesei reduces activity, and the size of N-glycans can influence function, with larger glycans sometimes impairing performance. Conversely, glycosylation can have beneficial effects when appropriately positioned. For instance, O-linked glycosylation in linker regions of Cel7a and Cel45a helps maintain an extended conformation crucial for activity. These findings highlight the need for deeper understanding of how glycosylation type, size, and extent influence enzyme stability and activity.
Hypotheses:
1. Glycosylation enhances structural and proteolytic stability of enzymes.
2. The type, size, and extent of glycosylation significantly influence enzyme activity and stability. 3. Specific glycosylation patterns can optimize both stability and activity.
Methodology: To test these hypotheses, we will use glycoproteomics, biochemical assays, structural bioinformatics, and enzyme activity measurements.
• Expression and Purification: Komagataella phaffii (Pichia pastoris) will be the primary host; other microbial systems may be explored to manipulate glycosylation.
• Glycosylation Mapping: Mass spectrometry-based glycoproteomics to identify and map glycosylation sites.
• Stability Analysis: Biochemical assays and differential scanning fluorimetry (DSF) will assess glycosylation’s impact on enzyme stability.
• Activity Assays: Kinetic assays will evaluate correlations between glycosylation patterns and catalytic activity.
• Structural Bioinformatics: In-silico modelling and computational tools will predict glycosylation sites and guide mutagenesis or expression strategies.
• Data Integration: Results from glycoproteomics, structural analysis, and activity assays will be integrated to identify correlations and inform chassis design.
Representative Publications / References
- Greene ER, Himmel ME, Beckham GT, Tan Z. Glycosylation of Cellulases: Engineering Better Enzymes for Biofuels. Adv Carbohydr Chem Biochem. 2015;72:63-112. doi: 10.1016/bs.accb.2015.08.001. Epub 2015 Oct 24. PMID: 26613815.
- Liu Y, Hoppenbrouwers T, Wang Y, Xie Y, Wei X, Zhang H, Du G, Imam KMSU, Wichers H, Li Z, Bastiaan-Net S. Glycosylation Contributes to Thermostability and Proteolytic Resistance of rFIP-nha (Nectria haematococca). Molecules. 2023 Aug 31;28(17):6386. doi: 10.3390/molecules28176386. PMID: 37687215; PMCID: PMC10490071.
- Beckham GT, Dai Z, Matthews JF, Momany M, Payne CM, Adney WS, Baker SE, Himmel ME. Harnessing glycosylation to improve cellulase activity. Curr Opin Biotechnol. 2012 Jun;23(3):338-45. doi: 10.1016/j.copbio.2011.11.030. Epub 2011 Dec 18. PMID: 22186222.