Self-Assembled Surface Layer Proteins from Corynebacterium glutamicum as a Matrix for Cutinase Immobilization
Author
Fuh, Derick Shu
Term
4. term
Education
Publication year
2014
Submitted on
2014-06-02
Pages
58
Abstract
Nanovidenskab forsøger at udnytte selv-samling—hvor molekyler spontant organiserer sig i forudsigelige strukturer. I dette projekt bruger vi bakteriers overfladeproteiner, såkaldte S-lag-proteiner, som byggeklodser, fordi de naturligt gendanner ordnede todimensionelle mønstre på mange materialer. Vi isolerede S-lag-proteiner fra den Gram-positive bakterie Corynebacterium glutamicum ved at behandle hele celler med et detergent (SDS) og et chaotropt middel (guanidine hydrochloride), som løsner cellekuverten, så proteinerne kan høstes. Vi bestemte derefter deres størrelse med SDS-PAGE (en gelmetode, der adskiller proteiner efter molekylvægt) og kontrollerede, at de samledes igen på en glimmeroverflade ved hjælp af atomkraftmikroskopi (AFM), som kan skanne overflader på nanometerskala. Til sidst fastgjorde vi et hydrolytisk enzym, cutinase, til det selv-samlede S-lag og målte dets esteraseaktivitet for at bekræfte bindingen. Vi sammenlignede aktiviteten af immobiliseret cutinase med enzymet frit i opløsning. Fordi S-lag har regelmæssigt fordelte bindingssteder, kan de placere enzymer som cutinase i ordnede mønstre—en idé, der er relevant for udvikling af analytiske biosensorer og kan hjælpe med at reducere enzymomkostninger.
Nanoscience seeks to harness self-assembly—molecules organizing themselves into predictable structures. In this project, we use bacterial surface (S-layer) proteins as building blocks because they naturally re-form ordered, two-dimensional patterns on many materials. We isolated S-layer proteins from the Gram-positive bacterium Corynebacterium glutamicum by treating whole cells with a detergent (SDS) and a chaotropic agent (guanidine hydrochloride), which disrupt the cell envelope so the proteins can be collected. We then measured their size by SDS-PAGE (a gel method that separates proteins by molecular weight) and confirmed that they reassembled on a mica surface using atomic force microscopy (AFM), a technique that scans surfaces at the nanometer scale. Finally, we attached a hydrolytic enzyme, cutinase, to the self-assembled S-layer and tested its esterase activity to confirm binding. We compared the activity of immobilized cutinase with the enzyme free in solution. Because S-layers present regularly spaced binding sites, they can position enzymes like cutinase in defined patterns, an approach relevant to analytical biosensors and potentially lowering enzyme costs.
[This abstract was generated with the help of AI]
Keywords
Documents