Project description (2014-2016)

(For Danish description see below)

Design of proteinaceous inhibitors of industrially important starch degrading enzymes and proteases

Proteinaceous enzyme inhibitors have been shown to not only regulate the activity, but also stabilise their target enzymes. The project focuses on design of inhibitors of two groups of industrial enzymes, starch hydrolases and serine-proteases. Prolonging shelf-life and regulation of enzymes are important goals. Today chemicals are used, e.g. for reducing autoproteolysis of proteases. Inhibitors may also be used for recycling enzymes from industrial processes. A biochemically and structurally characterised limit dextrinase inhibitor (LDI) will serve as template for computational protein design. LDI is from a family of α-amylase and serine-protease inhibitors, and we have evidence for interaction hotspots in the complex with its target enzyme. The combination of advanced computational methods and experimental characterisation of inhibitor variants offers a strong approach to achieve useful potent enzyme inhibitors.

Purpose of project: The purpose of the project is to design proteinaceous inhibitors against industrial serine proteases, α-amylases and pullulanases, which can stabilize and/or regulate these enzymes. The design deploys the high-affinity, very stable limit dextrinase inhibitor (LDI) as template. LDI is a 13.4 kDa protein containing four α-helices connected by loops and four disulphide bonds. The first task is to redesign LDI loop regions and evaluate also amino acid residues in α-helices, as hotspot mutational analysis of LDI binding to limit dextrinase (LD) identified an intermolecular hydrophobic cluster as key to tight binding. Redesign of LDI for recognition of new target enzymes requires computational methods. The project is a timely contribution to advancing the discipline of rational protein design.

Dansk projektbeskrivelse:

Design af proteiner der kan hæmme vigtige industrielle enzymer, som nedbryder stivelse og proteiner

Proteinhæmmere har vist sig at have ikke alene en regulerende effekt, men også en stabiliserende effekt på de enzymer, som de er rettet imod. Projektet vil fokusere på udviklingen af hæmmere af to industrielt relevante grupper af enzymer; stivelsesnedbrydende enzymer og proteaser. I enzymindustrien arbejdes der meget på at opnå opbevaringsstabile enzymer. I dag bruges der f.eks. kemikalier til at reducere proteasers selvnedbrydning under opbevaring. Formålet med projektet er at designe nye proteinbaserede hæmmere, som kan fungere som stabilisatorer under opbevaring og som regulatorer under industrielle processer. Endvidere vil immobiliserede hæmmere kunne anvendes til at genanvende enzymerne. Udgangspunktet er en eksisterende hæmmer fra byg, som selv er et protein. Den kommer fra en familie af α-amylase- og serinprotease-hæmmere, men har dog selv kun hæmmende effekt over for et enzym fra α-amylase-familien, limit dextrinase. Computerbaserede proteindesign-metoder vil blive brugt til at redesigne den eksisterende hæmmer. Brugen af computerbaserede metoder til design af proteiner med nye funktioner vinder større og større indpas og der er adskillige succesrige eksempler på design af nye proteinkomplekser. Dog er hver ”case” en udfordring, hvor erfaringer fra lignende protein-komplekser og fra biokemiske analyser skal inddrages.