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dc.contributor.advisorCampopiano, Dominic
dc.contributor.advisorMowat, Christopher
dc.contributor.authorSerpico, Annabel
dc.date.accessioned2019-03-05T10:35:04Z
dc.date.available2019-03-05T10:35:04Z
dc.date.issued2019-07-01
dc.identifier.urihttp://hdl.handle.net/1842/35518
dc.description.abstractOptically active amines and amino acids are ubiquitously distributed in nature where they play many crucial roles. Moreover, it was recently estimated that around 40% of blockbuster drugs and 20% of agrochemicals contain chiral amines in their structure, thus there has been considerable effort in developing efficient, low cost and widely-applicable methods for their production. Compared to classical chemical synthesis, aminotransferase (AT) enzymes have been widely explored as a more efficient and sustainable method for the preparation of optically pure amines from the corresponding ketones. In several cases these biocatalysts have replaced the existing chemical catalysts, which are limited by their high cost and poor regio- and stereoselectivity. Two very interesting, distantly-related class III bacterial ATs have been investigated in this study: the D-phenylglycine (D-Phg) aminotransferase (D-PhgAT) and the amino-pentol AT (FumI). The D-PhgAT from Pseudomonas stutzeri ST-201 catalyses the reversible transamination from L-glutamic acid to benzoylformate, yielding α-ketoglutarate and D-Phg with high (>99%) enantiomeric excess (% ee). The D-PhgAT possesses the unique feature that the amino acid donor and amino acid product display an inverted absolute configuration. Thus, D-PhgAT is a very promising biocatalyst as it yields high value D-amino acids from inexpensive L-amino acid donors. By carrying out a detailed kinetic analysis of recombinant D-PhgAT the best substrates were identified and an optimized method, delivering a range of enantiopure aromatic D-amino acids at 1 g scale, has been developed. Moreover, the x-ray crystal structure of D-PhgAT at 2.25 Å resolution has been determined with its pyridoxal 5’- phosphate (PLP) cofactor bound as an internal aldimine. The active site architecture highlights various residues potentially involved in catalysis and illuminates the basis of the exquisite enantioselectivity of this unique member of the AT superfamily. These studies promote D-PhgAT as a useful tool for the sustainable production of high value, aromatic D-amino acids. Fatty amines (C8-C20) are high value products with a wide variety of applications in fabric softeners, detergents and cosmetic formulations. The unusual Sphingopyxis FumI detoxifies the C20 mycotoxin natural product fumonisin B1 (FB1). FumI catalyses transamination at the C2 position of a hydrolyzed fumonisin B1 (HFB1) derivative with pyruvate as the amino acceptor. A screen of prochiral C2 ketones of varying chain length (C3-C17 2K) with L-alanine as amino donor has revealed that the recombinant FumI is capable of producing alkyl-amines of various chain lengths (C3-C17) by working in the reverse direction. Chiral HPLC analysis has revealed that the enzyme generates (S)-amines with high ee (>99%). The regioselectivity of FumI was also determined using prochiral keto-substrates with substitutions along the chain (such as 3-decanone (C10-3K) and 5-decanone (C10-5K)). Interestingly, C10-3K, but not C10-5K, was converted to the corresponding amine. A range of long chain aldehydes (C10-C20) were also converted by FumI to the corresponding amines with L-Ala as the amino donor. The x-ray crystal structure of the PLP-bound, internal aldimine form of FumI was solved to a resolution of 1.6 Å by molecular replacement using a Bacillus subtilis AT as a model. The structure suggested residues potentially involved in catalysis and revealed a potential hydrophobic binding site for the long alkyl chain substrates. The combination of aldehyde and ketone alkyl chain substrate promiscuity, the high enantioselectivity and the 3D structures of this rare AT suggests that FumI is a very useful addition to the biocatalytic toolbox for the synthesis of high value fatty amines.en
dc.language.isoenen
dc.publisherThe University of Edinburghen
dc.subjectgreen chemistryen
dc.subjectbiocatalysisen
dc.subjectenzymesen
dc.subjectchiral aminesen
dc.subjectaminotransferase enzymesen
dc.subjectD-Phenylgylcine aminotransferaseen
dc.subjectamino-pentol aminotransferase FumIen
dc.subjectD-Phgen
dc.subjectD-PhgATen
dc.subjectFumIen
dc.titleBiocatalytic application of rare PLP-dependent aminotransferases for the synthesis of high value amino acids and aminesen
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen


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