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Almulhim, Nourah (2021) Engineering novel S-glycosidase activity into extremo-adapted β-glucosidase by rational design. PhD thesis, University of Nottingham.

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Abstract

Biocatalysts have proven their efficiency and superiority in industry for the last few decades. Enzymes are made by nature to carry out specific functions required by the host organisms, therefore the practical applications of enzymes can be limited to their natural functions and this has encouraged efforts in the development of these natural catalysts to work in different conditions and with a significantly broader range of substrates.

Enzymes from extremophile organisms have significantly higher tolerance than mesophilic counterparts to temperature and/or pH, making them attractive for industrial applications. In industrial biotechnology, hydrolases, which are one of the six classes of enzymes, are the most commonly used biocatalysis.

Engineering extremo-adapted glycoside hydrolases to broaden their substrate scope towards β-thioglycosidase activity could significantly increase their potential applications. The breakdown of sulphur glycosidic bonds by β-thioglycosidases can produce isothiocyanate, a chemoprotective agent linked to the prevention of cancers, however only a handful of enzymes have been identified that are known to catalyse this reaction. Structural studies of the myrosinase enzyme, which is the only example among natural catalysts capable of hydrolysing the thioglycosidic bond, has identified residues that may play important roles in sulphate group recognition.

Protein engineering techniques can be used to introduce new amino acid residues into enzymes to improve their properties. By using rational design, two extremo-adapted - glycosidases from the species Thermus nonproteolyticus (TnoGH1) and Halothermothrix orenii (HorGH1) were engineered in this study towards thioglycosidic substrates. Twelve variants, six for TnoGH1and six for HorGH1, were assayed for activity. Remarkable enhancement of the specificity (kcat/KM) of TnoGH1 and HorGH1 towards β-thioglycoside was observed in the single mutants TnoGH1-V287R (2500 M-1 s -1 ) and HorGH1-M229R, (13480 M-1 s -1 ) which showed a 3-fold increase with no loss in turnover rate when compared to the WT enzymes. Thus, the role of arginine is key to induce β-thioglycosidase activity. Thorough kinetic investigation of the different mutants has shed light on the mechanism of -glycosidases when acting on the native substrate.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Paradisi, Francesca
Keywords:	Novel S-glycosidase, Extremo-adapted β-glucosidase, Rational design
Subjects:	Q Science > QD Chemistry
Faculties/Schools:	UK Campuses > Faculty of Science > School of Chemistry
Item ID:	64702
Depositing User:	Almulhim, Nourah
Date Deposited:	31 Jul 2021 04:40
Last Modified:	31 Jul 2023 04:31
URI:	https://eprints.nottingham.ac.uk/id/eprint/64702

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