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Bernd Nidetzky - One of the best experts on this subject based on the ideXlab platform.
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continuous process technology for glucoside production from Sucrose using a whole cell derived solid catalyst of Sucrose Phosphorylase
Applied Microbiology and Biotechnology, 2021Co-Authors: Andreas Kruschitz, Linda Peinsipp, Martin Pfeiffer, Bernd NidetzkyAbstract:Advanced biotransformation processes typically involve the upstream processing part performed continuously and interlinked tightly with the product isolation. Key in their development is a catalyst that is highly active, operationally robust, conveniently produced, and recyclable. A promising strategy to obtain such catalyst is to encapsulate enzymes as permeabilized whole cells in porous polymer materials. Here, we show immobilization of the Sucrose Phosphorylase from Bifidobacterium adolescentis (P134Q-variant) by encapsulating the corresponding E. coli cells into polyacrylamide. Applying the solid catalyst, we demonstrate continuous production of the commercial extremolyte 2-α-d-glucosyl-glycerol (2-GG) from Sucrose and glycerol. The solid catalyst exhibited similar activity (≥70%) as the cell-free extract (~800 U g−1 cell wet weight) and showed excellent in-operando stability (40 °C) over 6 weeks in a packed-bed reactor. Systematic study of immobilization parameters related to catalyst activity led to the identification of cell loading and catalyst particle size as important factors of process optimization. Using glycerol in excess (1.8 M), we analyzed Sucrose conversion dependent on space velocity (0.075–0.750 h−1) and revealed conditions for full conversion of up to 900 mM Sucrose. The maximum 2-GG space-time yield reached was 45 g L−1 h−1 for a product concentration of 120 g L−1. Collectively, our study establishes a step-economic route towards a practical whole cell-derived solid catalyst of Sucrose Phosphorylase, enabling continuous production of glucosides from Sucrose. This strengthens the current biomanufacturing of 2-GG, but also has significant replication potential for other Sucrose-derived glucosides, promoting their industrial scale production using Sucrose Phosphorylase. • Cells of Sucrose Phosphorylase fixed in polyacrylamide were highly active and stable. • Solid catalyst was integrated with continuous flow to reach high process efficiency. • Generic process technology to efficiently produce glucosides from Sucrose is shown.
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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
Biotechnology and Bioengineering, 2020Co-Authors: Mario Klimacek, Alexander Sigg, Bernd NidetzkyAbstract:Chemical group-transfer reactions by hydrolytic enzymes have considerable importance in biocatalytic synthesis and are exploited broadly in commercial-scale chemical production. Mechanistically, these reactions have in common the involvement of a covalent enzyme intermediate which is formed upon enzyme reaction with the donor substrate and is subsequently intercepted by a suitable acceptor. Here, we studied the glycosylation of glycerol from Sucrose by Sucrose Phosphorylase (SucP) to clarify a peculiar, yet generally important characteristic of this reaction: partitioning between glycosylation of glycerol and hydrolysis depends on the type and the concentration of the donor substrate used (here: Sucrose, α-d-glucose 1-phosphate (G1P)). We develop a kinetic framework to analyze the effect and provide evidence that, when G1P is used as donor substrate, hydrolysis occurs not only from the β-glucosyl-enzyme intermediate (E-Glc), but additionally from a noncovalent complex of E-Glc and substrate which unlike E-Glc is unreactive to glycerol. Depending on the relative rates of hydrolysis of free and substrate-bound E-Glc, inhibition (Leuconostoc mesenteroides SucP) or apparent activation (Bifidobacterium adolescentis SucP) is observed at high donor substrate concentration. At a G1P concentration that excludes the substrate-bound E-Glc, the transfer/hydrolysis ratio changes to a value consistent with reaction exclusively through E-Glc, independent of the donor substrate used. Collectively, these results give explanation for a kinetic behavior of SucP not previously accounted for, provide essential basis for design and optimization of the synthetic reaction, and establish a theoretical framework for the analysis of kinetically analogous group-transfer reactions by hydrolytic enzymes.
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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
Authorea Preprints, 2020Co-Authors: Mario Klimacek, Alexander Sigg, Bernd NidetzkyAbstract:Chemical group-transfer reactions by hydrolytic enzymes have considerable importance in biocatalytic synthesis and are exploited broadly in commercial-scale chemical production. Mechanistically, these reactions have in common the involvement of a covalent enzyme intermediate which is formed upon enzyme reaction with the donor substrate and is subsequently intercepted by a suitable acceptor. Here, we studied the glycosylation of glycerol from Sucrose by Sucrose Phosphorylase (SucP) to clarify a peculiar, yet generally important characteristic of this reaction: partitioning between glycosylation of glycerol and hydrolysis depends on the type and the concentration of the donor substrate used (here: Sucrose, α-D-glucose 1-phosphate (G1P)). We develop a kinetic framework to analyze the effect and provide evidence that, when G1P is used as donor substrate, hydrolysis occurs not only from the β-glucosyl-enzyme intermediate (E-Glc), but additionally from a noncovalent complex of E-Glc and substrate which unlike E-Glc is unreactive to glycerol. Depending on the relative rates of hydrolysis of free and substrate-bound E-Glc, inhibition (Leuconostoc mesenteroides SucP) or apparent activation (Bifidobacterium adolescentis SucP) is observed at high donor substrate concentration. Using G1P at a concentration excluding the substrate-bound E-Glc, the product ratio changes to a value consistent with reaction exclusively through E-Glc, independent of the donor substrate used. Collectively, these results give explanation for a kinetic behavior of SucP not previously accounted for, provide essential basis for design and optimization of the synthetic reaction, and establish a theoretical framework for the analysis of kinetically analogous group transfer reactions by hydrolytic enzymes.
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production of glucosyl glycerol by immobilized Sucrose Phosphorylase options for enzyme fixation on a solid support and application in microscale flow format
Journal of Biotechnology, 2017Co-Authors: Juan M Bolivar, Thornthan Sawangwan, Christiane Luleygoedl, Ernestine Leitner, Bernd NidetzkyAbstract:2-O-(α-d-Glucopyranosyl)-sn-glycerol (αGG) is a natural osmolyte. αGG is produced industrially for application as an active cosmetic ingredient. The biocatalytic process involves a selective transglucosylation from Sucrose to glycerol catalyzed by Sucrose Phosphorylase (SPase). Here we examined immobilization of SPase (from Leuconostoc mesenteroides) on solid support with the aim of enabling continuous production of αGG. By fusing SPase to the polycationic binding module Zbasic2 we demonstrated single-step noncovalent immobilization of the enzyme chimera to different porous supports offering an anionic surface. We showed that immobilization facilitated by Zbasic2 was similarly efficient as immobilization by multipoint covalent attachment on epoxy-activated supports in terms of production of αGG. Enzyme loadings of up to 90mg enzyme g-1 support were obtained and the immobilized SPase was about half as effective as the enzyme in solution. The high regio- and chemo-selectivity of soluble SPase in αGG synthesis was retained in the immobilized enzyme and product yields of >85% were obtained at titers of ∼800mM. The Zbasic2-SPase immobilizates were fully recyclable: besides reuse of the enzyme activity, easy recovery of the solid support for fresh immobilizations was facilitated by the reversible nature of the enzyme attachment. Application of immobilized Zbasic2-SPase for continuous production of αGG in a microstructured flow reactor was demonstrated. Space-time yields of 500mmol αGG L-1h-1 were obtained at product titers of ∼200mM. The continuous microreactor was operated for 16days and an operational half-life of about 10days was determined.
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multivalency effects on the immobilization of Sucrose Phosphorylase in flow microchannels and their use in the development of a high performance biocatalytic microreactor
Chemcatchem, 2017Co-Authors: Donya Valikhani, Martin Pfeiffer, Juan M Bolivar, Bernd NidetzkyAbstract:Microstructured reactors are emergent engineering tools for development of biocatalytic conversions in flow. A promising layout involves microchannels wall-coated with enzyme. Because protein immobilization within closed microstructures is challenging, we suggested a confluent design of enzyme and microreactor: fusion to the silica-binding module Zbasic2 is used to engineer enzymes for high-affinity oriented attachment to the plain wall surface of glass microchannels. In this study of Sucrose Phosphorylase, we examined effects of multiple Zbasic2 modules in a single enzyme molecule on activity and adsorption stability of the Phosphorylase immobilized in a glass microchannel reactor. Compared to the "monovalent" enzyme, two Zbasic2 modules, present in tandem repeat at the N-terminus, separated at the N- and C-terminus of an enzyme monomer or arranged N-terminally in a protein homodimer, boosted the effectiveness of the immobilized Phosphorylase by up to 2-fold. They attenuated (up to 12-fold) the elution of the wall-coated enzyme during continuous reactor operation. About 70% activity could be retained after 690 reactor cycles. Reaction-diffusion regime analysis revealed the absence of mass transport limitations on conversion rate. Synthesis of α-D-glucose 1-phosphate occurred with a productivity of ~14 mM min-1 at 50% substrate conversion (50 mM). The use of wall-coated enzyme microreactors in high-performance biocatalytic reaction engineering is strongly supported.
Tom Desmet - One of the best experts on this subject based on the ideXlab platform.
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Microbial enzymes for glycoside synthesis : development of Sucrose Phosphorylase as a test case
Biotechnology of Microbial Enzymes, 2017Co-Authors: Tom Verhaeghe, Karel De Winter, Tom DesmetAbstract:Glycosylation is a crucial modification of many secondary metabolites, but is a challenging reaction to perform at a larger scale. In this chapter, an engineering pipeline will be discussed that was used to develop Sucrose Phosphorylase as a practical biocatalyst for glycoside synthesis. By addressing both enzyme and process engineering in a systematic fashion, we were able to establish a technology with proven industrial value. First, various homologs were compared to identify the most suitable starting point for further optimization. The stability of the best enzyme was then increased by semirational mutagenesis, after which its active site was rationally engineered to enable the binding of polyphenolic acceptors. Finally, the enzyme’s glycosylation activity was further improved by developing an appropriate solvent system, which enabled the cost-effective production of 1 kg of phenolic glucosides.
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the quest for a thermostable Sucrose Phosphorylase reveals Sucrose 6 phosphate Phosphorylase as a novel specificity
Applied Microbiology and Biotechnology, 2014Co-Authors: Tom Verhaeghe, Margo Diricks, Dirk Aerts, Wim Soetaert, Tom DesmetAbstract:Sucrose Phosphorylase is a promising biocatalyst for the glycosylation of a wide range of compounds, but its industrial application has been hampered by the low thermostability of known representatives. Hence, in this study, the putative Sucrose Phosphorylase from the thermophile Thermoanaerobacterium thermosaccharolyticum was recombinantly expressed and fully characterised. The enzyme showed significant activity on Sucrose (optimum at 55 °C), and with a melting temperature of 79 °C and a half-life of 60 h at the industrially relevant temperature of 60 °C, it is far more stable than known Sucrose Phosphorylases. Substrate screening and detailed kinetic characterisation revealed however a preference for Sucrose 6′-phosphate over Sucrose. The enzyme can thus be considered as a Sucrose 6′-phosphate Phosphorylase, a specificity not yet reported to date. Homology modelling and mutagenesis pointed out particular residues (Arg134 and His344) accounting for the difference in specificity. Moreover, phylogenetic and sequence analysis suggest that glycoside hydrolase 13 subfamily 18 might harbour even more specificities. In addition, the second gene residing in the same operon as Sucrose 6′-phosphate Phosphorylase was identified as well, and found to be a phosphofructokinase. The concerted action of both these enzymes implies a new pathway for the breakdown of Sucrose, in which the reaction products end up at different stages of the glycolysis.
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biphasic catalysis with disaccharide Phosphorylases chemoenzymatic synthesis of α d glucosides using Sucrose Phosphorylase
Organic Process Research & Development, 2014Co-Authors: Karel De Winter, Tom Verhaeghe, Tom Desmet, Vladimir Křen, Tim Devlamynck, Lisa Van Renterghem, Helena Pelantova, Wim SoetaertAbstract:Thanks to its broad acceptor specificity, Sucrose Phosphorylase (SP) has been exploited for the transfer of glucose to a wide variety of acceptor molecules. Unfortunately, the low affinity (K-m > 1 M) of SP towards these acceptors typically urges the addition of cosolvents, which often either fail to dissolve sufficient substrate or progressively give rise to enzyme inhibition and denaturation. In this work, a buffer/ethyl acetate ratio of 5:3 was identified to be the optimal solvent system, allowing the use of SP in biphasic systems. Careful optimization of the reaction conditions enabled the synthesis of a range of alpha-D-glucosides, such as cinnamyl alpha-D-glucopyranoside, geranyl alpha-D-glucopyranoside, 2-O-alpha-D-glucopyranosyl pyrogallol, and series of alkyl gallyl 4-O-alpha-D-glucopyranosides. The usefulness of biphasic catalysis was further illustrated by comparing the glucosylation of pyrogallol in a cosolvent and biphasic reaction system. The acceptor yield for the former reached only 17.4%, whereas roughly 60% of the initial pyrogallol was converted when using biphasic catalysis.
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Biphasic Catalysis with Disaccharide Phosphorylases: Chemoenzymatic Synthesis of α‑d‑Glucosides Using Sucrose Phosphorylase
2014Co-Authors: Karel De Winter, Tom Verhaeghe, Tom Desmet, Tim Devlamynck, Lisa Van Renterghem, Helena Pelantová, Vladimír Křen, Wim SoetaertAbstract:Thanks to its broad acceptor specificity, Sucrose Phosphorylase (SP) has been exploited for the transfer of glucose to a wide variety of acceptor molecules. Unfortunately, the low affinity (Km > 1 M) of SP towards these acceptors typically urges the addition of cosolvents, which often either fail to dissolve sufficient substrate or progressively give rise to enzyme inhibition and denaturation. In this work, a buffer/ethyl acetate ratio of 5:3 was identified to be the optimal solvent system, allowing the use of SP in biphasic systems. Careful optimization of the reaction conditions enabled the synthesis of a range of α-d-glucosides, such as cinnamyl α-d-glucopyranoside, geranyl α-d-glucopyranoside, 2-O-α-d-glucopyranosyl pyrogallol, and series of alkyl gallyl 4-O-α-d-glucopyranosides. The usefulness of biphasic catalysis was further illustrated by comparing the glucosylation of pyrogallol in a cosolvent and biphasic reaction system. The acceptor yield for the former reached only 17.4%, whereas roughly 60% of the initial pyrogallol was converted when using biphasic catalysis
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consensus engineering of Sucrose Phosphorylase the outcome reflects the sequence input
Biotechnology and Bioengineering, 2013Co-Authors: Dirk Aerts, Tom Verhaeghe, Wim Soetaert, Henkjan Joosten, Gert Vriend, Tom DesmetAbstract:Consensus engineering, which is replacing amino acids by the most frequently occurring one at their positions in a multiple sequence alignment (MSA), is a known strategy to increase the stability of a protein. The application of this concept to the entire sequence of an enzyme, however, has been tried only a few times mainly because of the problems determining the consensus in highly variable regions. We show that this problem can be solved by replacing such problematic regions by the corresponding sequence of the natural homologue closest to the consensus. When one or a few sub-families are overrepresented in the MSA the consensus sequence is a biased representation of the sequence space. We examine the influence of this bias by constructing three consensus sequences using different MSAs of Sucrose Phosphorylase (SP). Each consensus enzyme contained about 70 mutations compared to its closest natural homologue and folded correctly and displayed activity on Sucrose. Correlation analysis revealed that the family's co-evolution network was kept intact, which is one of the main advantages of full-length consensus design. The consensus enzymes displayed an "average" thermostability, that is, one that is higher than some but not all known representatives. We cautiously present practical rules for the design of consensus sequences, but warn that the measure of success depends on which natural enzyme is used as point of comparison.
Wim Soetaert - One of the best experts on this subject based on the ideXlab platform.
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the quest for a thermostable Sucrose Phosphorylase reveals Sucrose 6 phosphate Phosphorylase as a novel specificity
Applied Microbiology and Biotechnology, 2014Co-Authors: Tom Verhaeghe, Margo Diricks, Dirk Aerts, Wim Soetaert, Tom DesmetAbstract:Sucrose Phosphorylase is a promising biocatalyst for the glycosylation of a wide range of compounds, but its industrial application has been hampered by the low thermostability of known representatives. Hence, in this study, the putative Sucrose Phosphorylase from the thermophile Thermoanaerobacterium thermosaccharolyticum was recombinantly expressed and fully characterised. The enzyme showed significant activity on Sucrose (optimum at 55 °C), and with a melting temperature of 79 °C and a half-life of 60 h at the industrially relevant temperature of 60 °C, it is far more stable than known Sucrose Phosphorylases. Substrate screening and detailed kinetic characterisation revealed however a preference for Sucrose 6′-phosphate over Sucrose. The enzyme can thus be considered as a Sucrose 6′-phosphate Phosphorylase, a specificity not yet reported to date. Homology modelling and mutagenesis pointed out particular residues (Arg134 and His344) accounting for the difference in specificity. Moreover, phylogenetic and sequence analysis suggest that glycoside hydrolase 13 subfamily 18 might harbour even more specificities. In addition, the second gene residing in the same operon as Sucrose 6′-phosphate Phosphorylase was identified as well, and found to be a phosphofructokinase. The concerted action of both these enzymes implies a new pathway for the breakdown of Sucrose, in which the reaction products end up at different stages of the glycolysis.
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biphasic catalysis with disaccharide Phosphorylases chemoenzymatic synthesis of α d glucosides using Sucrose Phosphorylase
Organic Process Research & Development, 2014Co-Authors: Karel De Winter, Tom Verhaeghe, Tom Desmet, Vladimir Křen, Tim Devlamynck, Lisa Van Renterghem, Helena Pelantova, Wim SoetaertAbstract:Thanks to its broad acceptor specificity, Sucrose Phosphorylase (SP) has been exploited for the transfer of glucose to a wide variety of acceptor molecules. Unfortunately, the low affinity (K-m > 1 M) of SP towards these acceptors typically urges the addition of cosolvents, which often either fail to dissolve sufficient substrate or progressively give rise to enzyme inhibition and denaturation. In this work, a buffer/ethyl acetate ratio of 5:3 was identified to be the optimal solvent system, allowing the use of SP in biphasic systems. Careful optimization of the reaction conditions enabled the synthesis of a range of alpha-D-glucosides, such as cinnamyl alpha-D-glucopyranoside, geranyl alpha-D-glucopyranoside, 2-O-alpha-D-glucopyranosyl pyrogallol, and series of alkyl gallyl 4-O-alpha-D-glucopyranosides. The usefulness of biphasic catalysis was further illustrated by comparing the glucosylation of pyrogallol in a cosolvent and biphasic reaction system. The acceptor yield for the former reached only 17.4%, whereas roughly 60% of the initial pyrogallol was converted when using biphasic catalysis.
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Biphasic Catalysis with Disaccharide Phosphorylases: Chemoenzymatic Synthesis of α‑d‑Glucosides Using Sucrose Phosphorylase
2014Co-Authors: Karel De Winter, Tom Verhaeghe, Tom Desmet, Tim Devlamynck, Lisa Van Renterghem, Helena Pelantová, Vladimír Křen, Wim SoetaertAbstract:Thanks to its broad acceptor specificity, Sucrose Phosphorylase (SP) has been exploited for the transfer of glucose to a wide variety of acceptor molecules. Unfortunately, the low affinity (Km > 1 M) of SP towards these acceptors typically urges the addition of cosolvents, which often either fail to dissolve sufficient substrate or progressively give rise to enzyme inhibition and denaturation. In this work, a buffer/ethyl acetate ratio of 5:3 was identified to be the optimal solvent system, allowing the use of SP in biphasic systems. Careful optimization of the reaction conditions enabled the synthesis of a range of α-d-glucosides, such as cinnamyl α-d-glucopyranoside, geranyl α-d-glucopyranoside, 2-O-α-d-glucopyranosyl pyrogallol, and series of alkyl gallyl 4-O-α-d-glucopyranosides. The usefulness of biphasic catalysis was further illustrated by comparing the glucosylation of pyrogallol in a cosolvent and biphasic reaction system. The acceptor yield for the former reached only 17.4%, whereas roughly 60% of the initial pyrogallol was converted when using biphasic catalysis
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consensus engineering of Sucrose Phosphorylase the outcome reflects the sequence input
Biotechnology and Bioengineering, 2013Co-Authors: Dirk Aerts, Tom Verhaeghe, Wim Soetaert, Henkjan Joosten, Gert Vriend, Tom DesmetAbstract:Consensus engineering, which is replacing amino acids by the most frequently occurring one at their positions in a multiple sequence alignment (MSA), is a known strategy to increase the stability of a protein. The application of this concept to the entire sequence of an enzyme, however, has been tried only a few times mainly because of the problems determining the consensus in highly variable regions. We show that this problem can be solved by replacing such problematic regions by the corresponding sequence of the natural homologue closest to the consensus. When one or a few sub-families are overrepresented in the MSA the consensus sequence is a biased representation of the sequence space. We examine the influence of this bias by constructing three consensus sequences using different MSAs of Sucrose Phosphorylase (SP). Each consensus enzyme contained about 70 mutations compared to its closest natural homologue and folded correctly and displayed activity on Sucrose. Correlation analysis revealed that the family's co-evolution network was kept intact, which is one of the main advantages of full-length consensus design. The consensus enzymes displayed an "average" thermostability, that is, one that is higher than some but not all known representatives. We cautiously present practical rules for the design of consensus sequences, but warn that the measure of success depends on which natural enzyme is used as point of comparison.
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ionic liquids as cosolvents for glycosylation by Sucrose Phosphorylase balancing acceptor solubility and enzyme stability
Green Chemistry, 2013Co-Authors: Karel De Winter, Wim Soetaert, Kristien Verlinden, Vladimir Křen, Lenka Weignerova, Tom DesmetAbstract:Over the past decade, disaccharide Phosphorylases have received increasing attention as promising biocatalysts for glycoside synthesis. Unfortunately, these enzymes typically have a very low affinity for non-carbohydrate acceptors, which urges the addition of cosolvents to increase the dissolved concentration of these acceptors. However, commonly applied solvents such as methanol and dimethyl sulfoxide (DMSO) are not compatible with many intended applications of carbohydrate-derived products. In this work, the solubility of a wide range of relevant acceptors was assessed in the presence of ionic liquids (ILs) as alternative and ‘green’ solvents. The IL AMMOENG 101 was found to be the most effective cosolvent for compounds as diverse as medium- and long-chain alcohols, flavonoids, alkaloids, phenolics and terpenes. Moreover, this IL was shown to be less deleterious to the stability and activity of Sucrose Phosphorylase than the commonly used dimethyl sulfoxide. To demonstrate the usefulness of this solvent system, a process for the resveratrol glycosylation was established in a buffer containing 20% AMMOENG 101, 1 M Sucrose and saturated amounts of the acceptor. A single regioisomer 3-O-α-D-glucopyranosyl-(E)-resveratrol was obtained as proven by NMR spectroscopy.
Lothar Brecker - One of the best experts on this subject based on the ideXlab platform.
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interplay of catalytic subsite residues in the positioning of α d glucose 1 phosphate in Sucrose Phosphorylase
Biochemistry and biophysics reports, 2015Co-Authors: Patricia Wildberger, Lothar Brecker, Gaia A Aish, David L Jakeman, Bernd NidetzkyAbstract:Kinetic and molecular docking studies were performed to characterize the binding of α-d-glucose 1-phosphate (αGlc 1-P) at the catalytic subsite of a family GH-13 Sucrose Phosphorylase (from L. mesenteroides) in wild-type and mutated form. The best-fit binding mode of αGlc 1-P dianion had the phosphate group placed anti relative to the glucosyl moiety (adopting a relaxed 4C1 chair conformation) and was stabilized mainly by hydrogen bonds from residues of the enzyme׳s catalytic triad (Asp196, Glu237 and Asp295) and from Arg137. Additional feature of the αGlc 1-P docking pose was an intramolecular hydrogen bond (2.7 A) between the glucosyl C2-hydroxyl and the phosphate oxygen. An inactive phosphonate analog of αGlc 1-P did not show binding to Sucrose Phosphorylase in different experimental assays (saturation transfer difference NMR, steady-state reversible inhibition), consistent with evidence from molecular docking study that also suggested a completely different and strongly disfavored binding mode of the analog as compared to αGlc 1-P. Molecular docking results also support kinetic data in showing that mutation of Phe52, a key residue at the catalytic subsite involved in transition state stabilization, had little effect on the ground-state binding of αGlc 1-P by the Phosphorylase. However, when combined with a second mutation involving one of the catalytic triad residues, the mutation of Phe52 by Ala caused complete (F52A_D196A; F52A_E237A) or very large (F52A_D295A) disruption of the proposed productive binding mode of αGlc 1-P with consequent effects on the enzyme activity. Effects of positioning of αGlc 1-P for efficient glucosyl transfer from phosphate to the catalytic nucleophile of the enzyme (Asp196) are suggested. High similarity between the αGlc 1-P conformers bound to Sucrose Phosphorylase (modeled) and the structurally and mechanistically unrelated maltodextrin Phosphorylase (experimental) is revealed.
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chiral resolution through stereoselective transglycosylation by Sucrose Phosphorylase application to the synthesis of a new biomimetic compatible solute r 2 o α d glucopyranosyl glyceric acid amide
ChemInform, 2014Co-Authors: Patricia Wildberger, Lothar Brecker, Bernd NidetzkyAbstract:The Sucrose Phosphorylase catalyzed glycosylation of racemic glyceric acid amide (II) with Sucrose (I) affords the new biomimetic glycoside (III) in high yield with complete regio- and diastereoselectivity.
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Chiral resolution through stereoselective transglycosylation by Sucrose Phosphorylase: application to the synthesis of a new biomimetic compatible solute, (R)-2-O-α-D-glucopyranosyl glyceric acid amide.
Chemical communications (Cambridge England), 2014Co-Authors: Patricia Wildberger, Lothar Brecker, Bernd NidetzkyAbstract:Sucrose Phosphorylase catalysed glycosylation of glyceric acid amide with complete regio- and diastereo-selectivity is studied. (R)-2-O-α-D-Glucopyranosyl glyceric acid amide was obtained in high yield from single-step transformation of racemic glyceric acid amide and Sucrose. Non-productive binding of (S)-glyceric acid amide appeared to underlie strict enantiodiscrimination by the enzyme, thus supporting chiral resolutions based on stereoselective transglycosylation.
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regioselective o glucosylation by Sucrose Phosphorylase a promising route for functional diversification of a range of 1 2 propanediols
Carbohydrate Research, 2010Co-Authors: Christiane Luleygoedl, Lothar Brecker, Patricia Wildberger, Thornthan Sawangwan, Bernd NidetzkyAbstract:Propanediol and 3-aryloxy/alkyloxy derivatives thereof are bulk commodities produced directly from glycerol. Glycosylation is a promising route for their functional diversification into useful fine chemicals. Regioselective glucosylation of the secondary hydroxyl in different 1,2-propanediols was achieved by a Sucrose Phosphorylase-catalyzed transfer reaction where Sucrose is the substrate and 2-O-a-D-glucopyr- anosyl products are exclusively obtained. Systematic investigation for optimization of the biocatalytic synthesis included prevention of Sucrose hydrolysis, which occurs in the process as a side reaction of the Phosphorylase. In addition to 'nonproductive' depletion of donor substrate, the hydrolysis also resulted in formation of maltose and kojibiose (up to 45%) due to secondary enzymatic glucosylation of the glucose thus produced. Using 3-ethoxy-1,2-propanediol as the acceptor substrate (1.0 M), the desired transfer product was obtained in about 65% yield when employing a moderate (1.5-fold) excess of Sucrose donor. Loss of the glucosyl substrate to 'glucobiose' by-products was minimal ( 1,2-propanediol > 3-allyloxy-1,2-propanediol > 3-(o-methoxyphenoxy)-1,2-propanediol > 3-tert- butoxy-1,2-propanediol. Glucosylated 1,2-propanediols were not detectably hydrolyzed by Sucrose Phosphorylase so that their synthesis by transglucosylation occurred simply under quasi-equilibrium reaction conditions. 2010 Published by Elsevier Ltd.
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substitution of the catalytic acid base glu237 by gln suppresses hydrolysis during glucosylation of phenolic acceptors catalyzed by leuconostoc mesenteroides Sucrose Phosphorylase
Journal of Molecular Catalysis B-enzymatic, 2010Co-Authors: Johanna Wiesbauer, Alexandra Schwarz, Christiane Goedl, Lothar Brecker, Bernd NidetzkyAbstract:Abstract Stereoselective glycosylation of a phenolic hydroxyl is a key transformation in the (bio)synthesis of natural products. Biocatalytic transglycosylation usually provides the desired glycosidic product in exquisite anomeric purity. However, loss of substrate and product to hydrolysis often limits application of the method. Kinetic studies and in situ proton NMR analysis of reaction time courses were used here to characterize glucosylation of substituted phenol acceptors by Leuconostoc mesenteroides Sucrose Phosphorylase in the presence of α- d -glucose 1-phosphate (αG1P) as donor substrate. In the wild-type enzyme, hydrolysis of the sugar 1-phosphate strongly prevailed (about 10-fold, ∼1.6 U/mg) over glucosyl transfer to the 2,6-difluorophenol acceptor (∼0.17 U/mg) used. A mutated Phosphorylase in which the catalytic acid–base Glu 237 had been replaced by Gln (E237Q) did not display hydrolase activity under transglucosylation conditions and therefore provided substantial (∼7-fold) enhancement of transfer yield. Utilization of the donor substrate was however slowed down (about 400-fold, ∼0.004 U/mg) in E237Q as compared to wild-type enzyme (∼1.6 U/mg). In a series of mono- and disubstituted phenols differing in hydroxyl p K a between 7.02 and 8.71, the transferase activity of E237Q was found to be dependent on steric rather than electronic properties of the acceptor used. Both wild-type and mutated enzyme employed 4-nitrophenyl-α- d -glucopyranoside (4-NPG) as a slow artificial substrate for phosphorolysis and hydrolysis (native: ∼0.12 U/mg; E237Q: ∼0.02 U/mg).
Luciano Caseli - One of the best experts on this subject based on the ideXlab platform.
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acylated carrageenan changes the physicochemical properties of mixed enzyme lipid ultrathin films and enhances the catalytic properties of Sucrose Phosphorylase nanostructured as smart surfaces
Journal of Physical Chemistry B, 2016Co-Authors: Jefferson Muniz Rocha, Adriana Pavinatto, Thatyane M Nobre, Luciano CaseliAbstract:Control over the catalytic activity of enzymes is important to construct biosensors with a wide range of detectability and higher stability. For this, immobilization of enzymes on solid supports as nanostructured films is a current approach that permits easy control of the molecular architecture as well as tuning of the properties. In this article, we employed acylated carrageenan (AC) mixed with phospholipids at the air–water interface to facilitate the adsorption of the enzyme Sucrose Phosphorylase (SP). AC stabilized the adsorption of SP at the phospholipid monolayer, as detected by tensiometry, by which thermodynamic parameters could be inferred from the surface pressure–area isotherm. Also, infrared spectroscopy applied in situ over the monolayer showed that the AC–phospholipid system not only permitted the enzyme to be adsorbed but also helped conserve its secondary structure. The mixed monolayers were then transferred onto solid supports as Langmuir–Blodgett (LB) films and investigated with transfe...
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Acylated Carrageenan Changes the Physicochemical Properties of Mixed Enzyme–Lipid Ultrathin Films and Enhances the Catalytic Properties of Sucrose Phosphorylase Nanostructured as Smart Surfaces
2016Co-Authors: Jefferson M. Rocha, Thatyane M Nobre, Adriana Pavinatto, Luciano CaseliAbstract:Control over the catalytic activity of enzymes is important to construct biosensors with a wide range of detectability and higher stability. For this, immobilization of enzymes on solid supports as nanostructured films is a current approach that permits easy control of the molecular architecture as well as tuning of the properties. In this article, we employed acylated carrageenan (AC) mixed with phospholipids at the air–water interface to facilitate the adsorption of the enzyme Sucrose Phosphorylase (SP). AC stabilized the adsorption of SP at the phospholipid monolayer, as detected by tensiometry, by which thermodynamic parameters could be inferred from the surface pressure–area isotherm. Also, infrared spectroscopy applied in situ over the monolayer showed that the AC–phospholipid system not only permitted the enzyme to be adsorbed but also helped conserve its secondary structure. The mixed monolayers were then transferred onto solid supports as Langmuir–Blodgett (LB) films and investigated with transfer ratio, quartz crystal microbalance, fluorescence spectroscopy, and atomic force microscopy. The enzyme activity of the LB film was then determined, revealing that although there was an expected reduction in activity in relation to the homogeneous environment the activity could be better preserved after 1 month, revealing enhanced stability
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adsorption and enzyme activity of Sucrose Phosphorylase on lipid langmuir and langmuir blodgett films
Colloids and Surfaces B: Biointerfaces, 2014Co-Authors: Jefferson Muniz Rocha, Luciano CaseliAbstract:Abstract The production of bioelectronic devices, including biosensors, can be conducted using enzymes immobilized in ultrathin solid films, for which preserving the enzymatic catalytic activity is crucial for optimal performance. In this sense, nanostructured films that allow for control over molecular architectures are of interest. In this paper, we investigate the adsorption of Sucrose Phosphorylase onto Langmuir monolayers of the phospholipid dimyristoylphosphatidic acid, which caused the surface pressure isotherms to expand. With polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), the amide bands from the enzyme could be identified, with the C–N and C=O dipole moments lying parallel to the air–water interface. Structuring of the enzyme into an α-helix was noted, and this structure was preserved when the mixed enzyme-phospholipid monolayer was transferred in the form of a Langmuir–Blodgett (LB) film. The latter was demonstrated with measurements of the catalytic activity of Sucrose Phosphorylase, which presented the highest enzyme activity for multilayer LB film. The approach presented in this study not only allows for optimized catalytic activity toward Sucrose but also permits to explain why certain film architectures exhibit superior performance.
