There is an important need to immobilise enzymes for use in industry, to do this I have the promising idea that by conjugating the enzyme to a hydrogel network, thus fabricating a stable biocatalytic network would be a potential method for immobilising enzymes for the manufacture of fine chemicals, this has not been done before for octapeptide systems. Hydrogels have been previously shown as a viable way of immobilising and stabilising enzymes. In this thesis the octapeptide VKVKVEVK (V is valine, K is lysine and E is glutamic acid) is used to immobilise enzymes tagged with VKVKVEVK. This peptide sequence is chosen as it forms stable hydrogels at enzyme appropriate conditions (pH 7). The enzymes chosen are; PETNR as it is well understood and is therefore a good starting point, CDH and CHMO were also chosen as they could combine with PETNR to form a cascade reaction. PETNR was both chemically conjugated to VKVKVEVK (SpepPETNR) and also genetically modified to express the peptide tag (CpepPETNR), whilst CDH and CHMO were genetically modified to express the tag (NpepCDH and CpepCHMO respectively). For S/CpepPETNR retention within the hydrogels was superior to retention for untagged PETNR. NpepCDH was found to not precipitate within the hydrogel whilst untagged was found to do so. CpepCHMO functionalised hydrogels were found to be heterogeneous. Characterisation of CpepPETNR functionalised hydrogels was undertaken using micro differential scanning calorimetry (µDSC), rheology, small angle neutron scattering (SANS) and atomic force microscopy (AFM). From the µDSC evidence of 'protective immobilisation' was observed by the increase in denaturation energy (+253 kJ mol-1) in the hydrogel in comparison to in solution (+18 kJ mol-1). The ability of S/CpepPETNR functionalised hydrogels to perform the ketoisophorone to levodione biotransformation reaction was explored with yields of 86%. S/CpepPETNR within VKVKVEVK hydrogels was found to retain ~90% conversion for at least 9 months at room temperature. Incubation overnight at 90°C resulted in a yield of 84% of levodione. These two results added more evidence for 'protective immobilisation'. Hydrogels functionalised with NpepCDH or CpepCHMO were characterised using rheology and atomic force microscopy. The biotransformation ability of NpepCDH was elucidated; the overall yield of carvone was a maximum of 54% from the hydrogel phase. NpepCDH was used alongside CpepPETNR for the cascade reaction producing dihydrocarvone in low yields; however, an improvement from 2% to 13% in yield is presented. The yield of lactone products from CpepCHMO functionalised hydrogel was low at 15%. The CpepPETNR/ CpepPETNR cascade reaction proceeded with a yield of 36%. The initial activities of CpepPETNR, NpepCDH and CpepCHMO were assayed in both solution and in gel phase using a modified method. The activities were assessed with varying conditions; temperature, pH, quantity of ethanol and incubation at high and low temperatures. Generally, it was found that immobilisation within the hydrogel phase resulted in 'protective immobilisation' against non-optimal conditions. This work will be of benefit to those who are interesting immobilising enzymes within hydrogels in the future.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:728116 |
| Date | January 2016 |
| Creators | Hickling, Christopher |
| Contributors | Saiani, Aline |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/fabrication-of-stable-biocatalyst-networks-for-the-manufacture-of-fine-chemicals(40ad3b26-bc12-48b9-836c-323e18c1f241).html |
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