On the donor substrate dependence of group‐transfer reactions by hydrolytic enzymes: Insight from kinetic analysis of sucrose phosphorylase‐catalyzed transglycosylation

Klimacek, Mario; Sigg, Alexander; Nidetzky, Bernd

doi:10.1002/bit.27471

Cited by 13 publications

(27 citation statements)

References 53 publications

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“…Especially in the early reaction phase (~ 5 h), LmSucP produced more glucose than BaSucP. This observation was consistent with a recent kinetic study that showed BaSucP to exhibit higher reaction selectivity (glycosylation of glycerol compared with hydrolysis) than LmSucP [ 63 ]. (3) Both enzymes formed 1-GG as secondary glycosylation product to GG.…”

Section: Resultssupporting

confidence: 89%

Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol

et al. 2021

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Background Glucosylglycerol (2-O-α-d-glucosyl-sn-glycerol; GG) is a natural osmolyte from bacteria and plants. It has promising applications as cosmetic and food-and-feed ingredient. Due to its natural scarcity, GG must be prepared through dedicated synthesis, and an industrial bioprocess for GG production has been implemented. This process uses sucrose phosphorylase (SucP)-catalyzed glycosylation of glycerol from sucrose, applying the isolated enzyme in immobilized form. A whole cell-based enzyme formulation might constitute an advanced catalyst for GG production. Here, recombinant production in Escherichia coli BL21(DE3) was compared systematically for the SucPs from Leuconostoc mesenteroides (LmSucP) and Bifidobacterium adolescentis (BaSucP) with the purpose of whole cell catalyst development. Results Expression from pQE30 and pET21 plasmids in E. coli BL21(DE3) gave recombinant protein at 40–50% share of total intracellular protein, with the monomeric LmSucP mostly soluble (≥ 80%) and the homodimeric BaSucP more prominently insoluble (~ 40%). The cell lysate specific activity of LmSucP was 2.8-fold (pET21; 70 ± 24 U/mg; N = 5) and 1.4-fold (pQE30; 54 ± 9 U/mg, N = 5) higher than that of BaSucP. Synthesis reactions revealed LmSucP was more regio-selective for glycerol glycosylation (~ 88%; position O2 compared to O1) than BaSucP (~ 66%), thus identifying LmSucP as the enzyme of choice for GG production. Fed-batch bioreactor cultivations at controlled low specific growth rate (µ = 0.05 h−1; 28 °C) for LmSucP production (pET21) yielded ~ 40 g cell dry mass (CDM)/L with an activity of 2.0 × 104 U/g CDM, corresponding to 39 U/mg protein. The same production from the pQE30 plasmid gave a lower yield of 6.5 × 103 U/g CDM, equivalent to 13 U/mg. A single freeze–thaw cycle exposed ~ 70% of the intracellular enzyme activity for GG production (~ 65 g/L, ~ 90% yield from sucrose), without releasing it from the cells during the reaction. Conclusions Compared to BaSucP, LmSucP is preferred for regio-selective GG production. Expression from pET21 and pQE30 plasmids enables high-yield bioreactor production of the enzyme as a whole cell catalyst. The freeze–thaw treated cells represent a highly active, solid formulation of the LmSucP for practical synthesis.

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Section: Resultssupporting

confidence: 89%

Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol

et al. 2021

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“…The retaining trehalose phosphorylases (THP), on the other hand, are believed to follow a direct front-side nucleophilic displacement, often referred to a so-called internal return-like mechanism [ 33 ]. In this case, the glycosidic oxygen is protonated by a phosphate hydroxyl group, and the glycosidic bond is destabilized by a nucleophilic attack at C-1 atom with the oxygen atom donating a proton to the glycosidic oxygen [ 34 ]. As Fig.…”

Section: Overview Of Disaccharide Phosphorylasesmentioning

confidence: 99%

Disaccharide phosphorylases: Structure, catalytic mechanisms and directed evolution

Sun

You

2021

Synthetic and Systems Biotechnology

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Disaccharide phosphorylases (DSPs) are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties. They are modular enzymes that form active homo-oligomers. From a mechanistic as well as a structural point of view, they are similar to glycoside hydrolases or glycosyltransferases. As the majority of DSPs show strict stereo- and regiospecificities, these enzymes were used to synthesize specific disaccharides. Currently, protein engineering of DSPs is pursued in different laboratories to broaden the donor and acceptor substrate specificities or improve the industrial particularity of naturally existing enzymes, to eventually generate a toolbox of new catalysts for glycoside synthesis. Herein we review the characteristics and classifications of reported DSPs and the glycoside products that they have been used to synthesize.

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“…In this context, glycoside phosphorylases have attracted increasing attention as biocatalysts for glycosyl transfer [21] . In vivo , they catalyze the degradation of di‐ and oligosaccharides with inorganic phosphate, but the reaction is readily reversible in vitro due to the high energy content of the synthesized glycosyl phosphate [22] .…”

Section: Introductionmentioning

confidence: 99%

Enzymatic Synthesis of Phloretin α‐Glucosides Using a Sucrose Phosphorylase Mutant and its Effect on Solubility, Antioxidant Properties and Skin Absorption

et al. 2021

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Glycosylation of polyphenols may increase their aqueous solubility, stability, bioavailability and pharmacological activity. Herein, we used a mutant of sucrose phosphorylase from Thermoanaerobacterium thermosaccharolyticum engineered to accept large polyphenols (variant TtSPP_R134A) to produce phloretin glucosides. The reaction was performed using 10% (v/v) acetone as cosolvent. The selective formation of a monoglucoside or a diglucoside (53% and 73% maximum conversion percentage, respectively) can be kinetically controlled. MS and 2D‐NMR determined that the monoglucoside was phloretin 4’‐O‐α‐D‐glucopyranoside and the diglucoside phloretin‐4’‐O‐[α‐D‐glucopyranosyl‐(1→3)‐O‐α‐D‐glucopyranoside], a novel compound. The molecular features that determine the specificity of this enzyme for 4’‐OH phenolic group were analysed by induced‐fit docking analysis of each putative derivative, using the crystal structure of TtSPP and changing the mutated residue. The mono‐ and diglucoside were, respectively, 71‐ and 1200‐fold more soluble in water than phloretin at room temperature. The α‐glucosylation decreased the antioxidant capacity of phloretin, measured by DPPH and ABTS assays; however, this loss was moderate and the activity could be recovered upon deglycosylation in vivo. Since phloretin attracts a great interest in dermocosmetic applications, we analyzed the percutaneous absorption of glucosides and the aglycon employing a pig skin model. Although the three compounds were detected in all skin layers (except the fluid receptor), the diglucoside was present mainly on superficial layers.

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On the donor substrate dependence of group‐transfer reactions by hydrolytic enzymes: Insight from kinetic analysis of sucrose phosphorylase‐catalyzed transglycosylation

Cited by 13 publications

References 53 publications

Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol

Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol

Disaccharide phosphorylases: Structure, catalytic mechanisms and directed evolution

Enzymatic Synthesis of Phloretin α‐Glucosides Using a Sucrose Phosphorylase Mutant and its Effect on Solubility, Antioxidant Properties and Skin Absorption

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