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4 Publications visible to you, out of a total of 4
Kinetic characterization of two neuraminic acid synthases and evaluation of their application potential

Abstract (Expand)

Neuraminic acid synthases are an important yet underexplored group of enzymes. Thus, in this research, we performed a detailed kinetic and stability analysis and a comparison of previously known … neuraminic acid synthase from Neisseria meningitidis, and a novel enzyme, PNH5, obtained from a metagenomic library. A systematic analysis revealed a high level of similarity of PNH5 to other known neuraminic acid synthases, except for its pH optimum, which was found to be at 5.5 for the novel enzyme. This is the first reported enzyme from this family that prefers an acidic pH value. The effect of different metal cofactors on enzyme activity, i.e. Co2+, Mn2+ and Mg2+, was studied systematically. The kinetics of neuraminic acid synthesis was completely elucidated, and an appropriate kinetic model was proposed. Enzyme stability study revealed that the purified enzyme exhibits changes in its structure during time as observed by differential light scattering, which cause a drop in its activity and protein concentration. The operational enzyme stability for the neuraminic acid synthase from N. meningitidis is excellent, where no activity drop was observed during the batch reactor experiments. In the case of PNH5, some activity drop was observed at higher concentration of substrates. The obtained results present a solid platform for the future application of these enzymes in the synthesis of sialic acids.

Authors: Mehmet Mervan Çakar, Nevena Milčić, Theofania Andreadaki, Simon Charnock, Wolf-Dieter Fessner, Zvjezdana Findrik Blažević

Date Published: 21st Aug 2024

Publication Type: Journal

Broadening The Substrate Scope of Aldolases Through Metagenomic Enzyme Discovery

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Bio‐processes based on enzymatic catalysis play a major role in the development of green, sustainable processes, and the discovery of new enzymes is key to this approach. In this work, we analysed ten …e analysed ten metagenomes and retrieved 48 genes coding for deoxyribose‐5‐phosphate aldolases (DERAs, EC 4.1.2.4) using a sequence‐based approach. These sequences were recombinantly expressed in Escherichia coli and screened for activity towards a range of aldol additions. Among these, one enzyme, DERA‐61, proved to be particularly interesting and catalysed the aldol addition of furfural or benzaldehyde with acetone, butanone and cyclobutanone with unprecedented activity. The product of these reactions, aldols, can find applications as building blocks in the synthesis of biologically active compounds. Screening was carried out to identify optimized reaction conditions targeting temperature, pH, and salt concentrations. Lastly, the kinetics and the stereochemistry of the products were investigated, revealing that DERA‐61 and other metagenomic DERAs have superior activity and stereoselectivity when they are provided with non‐natural substrates, compared to well‐known DERAs.

Authors: Andrea Rizzo, Maria Carmen Aranda, James Galman, Annette Alcasabas, Akash Pandya, Amin Bornadel, Bruna Costa, Helen C Hailes, John M Ward, Jack W.E. Jeffries, Beatriz Dominguez

Date Published: 2nd Jul 2024

Publication Type: Journal

Cross-acyloin condensation of aldehydes catalysed by transketolase variants for the synthesis of aliphatic α-hydroxyketones

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We described a strategy for the enzymatic synthesis of 1-deoxy and 1,2-deoxyketoses from the aliphatic α-ketoacids, pyruvate and 2-oxobutyrate, as donors and natural aldoses of variable chain length … as acceptors, catalyzed by thermostable transketolase variants from Geobacillus stearothermophilus (TKgst). Analytical studies have been carried out on a panel of TKgst variants with the appropriate substrates allowing to select the best combinations and to apply it to the preparative scale synthesis of 1-deoxy and 1,2-deoxyketoses obtained with good to excellent isolated yields (61%–86%). To optimize the strategy, and as a proof of principle, the α-ketoacids pyruvate and 2-oxobutyrate were generated in situ from the corresponding d-amino acids d-alanine and d-homoalanine respectively, using a thermostable d-amino acid oxidase dAAO4536 that was selected from a screening of 55 putative DAAOs provided by Prozomix Limited. Hence, a one-pot one step procedure was performed at 50°C by coupling dAAO4536 and the best TKgst variant H102L/L118I/ H474S in the presence of d-alanine or d-homoalanine as α-ketoacids precursors and d-erythrose as acceptor substrate. The corresponding 1-deoxy and 1,2-dideoxyketoses were isolated with good yields (64% and 72% respectively, out of two steps)

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Date Published: 30th May 2024

Publication Type: Journal

Synthesis of Deoxysugars from Aliphatic α-Ketoacids and Aldoses Catalyzed by Thermostable Transketolase Variants from Geobacillus stearothermophilus

Abstract

Not specified

Author: Nazim Ocal, Giuseppe Arbia, Aurélie Lagarde, Muriel Joly, Samantha Gittings, Kirsty M. Graham, Franck Charmantray, Laurence Hecquet

Date Published: 14th Dec 2022

Publication Type: Journal

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