Engineering novel S-glycosidase activity into extremo-adapted β-glucosidase by rational design

Almulhim, Nouarh; Moody, Nicholas R.; Paradisi, Francesca (2020). Engineering novel S-glycosidase activity into extremo-adapted β-glucosidase by rational design. Applied microbiology and biotechnology, 104(10), pp. 4407-4415. Springer 10.1007/s00253-020-10582-3

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The breakdown of sulphur glycosidic bonds in thioglycosides can produce isothiocyanate, a chemoprotective agent linked to the prevention of cancers; however, only a handful of enzymes have been identified that are k0nown to catalyse this reaction. Structural studies of the myrosinase enzyme, which is capable of hydrolysing the thioglycosidic bond, have identified residues that may play important roles in sulphur bond specific activity. Using rational design, two extremo-adapted β-glycosidases from the species Thermus nonproteolyticus (TnoGH1) and Halothermothrix orenii (HorGH1) were engineered towards thioglycoside substrates. Twelve variants, six for TnoGH1 and 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 (13,260 M−1 s−1) which showed a 3-fold increase with no loss in turnover rate when compared with the wild-type 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 β-glycosidases when acting on the native substrate.

Item Type:

Journal Article (Original Article)

Division/Institute:

08 Faculty of Science > Departement of Chemistry and Biochemistry

UniBE Contributor:

Paradisi, Francesca

Subjects:

500 Science > 570 Life sciences; biology
500 Science > 540 Chemistry

ISSN:

0175-7598

Publisher:

Springer

Funders:

[UNSPECIFIED] EraNet SUSFOOD2
[UNSPECIFIED] King Faisal University in Saudi Arabia

Projects:

[UNSPECIFIED] SUSFOOD2

Language:

English

Submitter:

Francesca Paradisi

Date Deposited:

12 May 2020 14:52

Last Modified:

12 May 2020 14:58

Publisher DOI:

10.1007/s00253-020-10582-3

BORIS DOI:

10.7892/boris.143154

URI:

https://boris.unibe.ch/id/eprint/143154

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