In situ proton NMR of glycosidase catalyzed hydrolysis and reverse hydrolysis

Tyl, Catrin; Felsinger, Susanne; Brecker, Lothar

doi:10.1016/j.molcatb.2004.01.003

Cited by 10 publications

(7 citation statements)

References 32 publications

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“…[22 -24] 2D COSY and TOCSY as well as 1D selTOCSY spectra are recorded directly from the reaction mixture for additional structural analysis of not yet described intermediates. [25,26] An in situ recorded 1 H NMR spectrum is given in Fig. 1 compounds during α-D-glucosyl-1-formate (α-2a) generation, formyl group migration, and hydrolysis.…”

Section: Resultsmentioning

confidence: 99%

“…1D and 2D 1 H NMR measurements of these reactions are measured in non-uniform intervals. [25,26] (A) (B) (C) …”

Section: In Situ 1 H Nmr Measurementsmentioning

confidence: 99%

“…[26] The overwhelming HDO signal in 1 H NMR spectra is suppressed by presaturation. For selective excitation in selTOCSY Gaussian pulses with 50 ms duration are used and the mixing times varied between 25 and 100 ms. After zero filling to 65 536 data points the free induction decays are Fourier transformed to spectra of 6000 Hz range.…”

Section: Nmr Spectroscopic Measurementsmentioning

confidence: 99%

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In situ proton NMR study of acetyl and formyl group migration in mono‐O‐acyl D‐glucose

Brecker

Mahut

Schwarz

et al. 2009

Magnetic Reson in Chemistry

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Acetyl and formyl group migration, mutarotation, and hydrolysis of mono-O-acylated glucose are studied by in situ 1D and 2D (1)H NMR spectroscopy. Alpha-D-glucosyl-1-acetate and alpha-D-glucosyl-1-formate serve as sole starting materials. They are generated in situ by configuration retaining glucosyltransfer from alpha-D-glucosyl-1-phosphate to formate and acetate, which is catalyzed by the Glu-237 --> Gln mutant of Leuconostoc mesenteroides sucrose phosphorylase. Temporary accumulated regio-isomeric mono-O-acyl D-glucoses are identified, characterized, and quantified directly from the reaction mixture. Time courses of the transformations give insight into pH dependence of acyl group migration and mutarotation as well as into the stability of various regioisomers.

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Section: Resultsmentioning

confidence: 99%

“…1D and 2D 1 H NMR measurements of these reactions are measured in non-uniform intervals. [25,26] (A) (B) (C) …”

Section: In Situ 1 H Nmr Measurementsmentioning

confidence: 99%

Section: Nmr Spectroscopic Measurementsmentioning

confidence: 99%

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In situ proton NMR study of acetyl and formyl group migration in mono‐O‐acyl D‐glucose

Brecker

Mahut

Schwarz

et al. 2009

Magnetic Reson in Chemistry

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“…Sample preparation involved removal of the enzyme from the reaction mixture by centrifugal ultrafiltration, lyophilization of the filtrate and dissolution into D 2 O. NMR spectra were recorded on a Bruker DRX 600 AVANCE spectrometer with a triple resonance xyz-gradient inverse probe at 600.13 MHz ( 1 H) and 150.86 MHz ( 13 C) as well as on a Bruker DRX 400 AVANCE spectrometer with a double resonance z-gradient inverse probe at 400.13 MHz ( 1 H) and 100.57 MHz ( 13 C). Proton chemical shifts and coupling constants were compared with earlier reported spectroscopic data (Albini et al, 1994;Tyl et al, 2004).…”

Section: Nmrmentioning

confidence: 99%

Enzymatic synthesis of β‐glucosylglycerol using a continuous‐flow microreactor containing thermostable β‐glycoside hydrolase CelB immobilized on coated microchannel walls

Schwarz¹,

Thomsen²,

Nidetzky³

2009

Biotech & Bioengineering

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beta-Glucosylglycerol (betaGG) has potential applications as a moisturizing agent in cosmetic products. A stereochemically selective method of its synthesis is kinetically controlled enzymatic transglucosylation from a suitable donor substrate to glycerol as acceptor. Here, the thermostable beta-glycosidase CelB from Pyrococcus furiosus was used to develop a microstructured immobilized enzyme reactor for production of betaGG under conditions of continuous flow at 70 degrees C. Using CelB covalently attached onto coated microchannel walls to give an effective enzyme activity of 30 U per total reactor working volume of 25 microL, substrate conversion and formation of transglucosylation product was monitored in dependence of glucosyl donor (2-nitrophenyl-beta-D-glucoside (oNPGlc), 3.0 or 15 mM; cellobiose, 250 mM), the concentration of glycerol (0.25-1.0 M), and the average residence time (0.2-90 s). Glycerol caused a concentration-dependent decrease in the conversion of the glucosyl donor via hydrolysis and strongly suppressed participation of the substrate in the reaction as glucosyl acceptor. The yields of betaGG were > or =80% and approximately 60% based on oNPGlc and cellobiose converted, respectively, and maintained up to near exhaustion of substrate (> or =80%), giving about 120 mM (30 g/L) of betaGG from the reaction of cellobiose and 1 M glycerol. The structure of the transglucosylation products, 1-O-beta-D-glucopyranosyl-rac-glycerol (79%) and 2-O-beta-D-glucopyranosyl-sn-glycerol (21%), was derived from NMR analysis of the product mixture of cellobiose conversion. The microstructured reactor showed conversion characteristics similar to those for a batchwise operated stirred reactor employing soluble CelB. The advantage of miniaturization to the microfluidic format lies in the fast characterization of full reaction time courses for a range of process conditions using only a minimum amount of enzyme.

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“…NMR also allows reaction products to be characterised in situ, and is particularly useful for enzymatic reactions that yield multiple products. For example, NMR‐based kinetic assays have been applied to characterise complex enzyme catalytic systems including (but not limited to) carbohydrate‐processing enzymes, enzymes that are related to antibiotic resistance,, and oxygenases ,. On this basis, NMR spectroscopy is a useful technique to consider for monitoring PPO kinetics and inhibition.…”

Section: Introductionmentioning

confidence: 99%

Development and Application of an NMR‐Based Assay for Polyphenol Oxidases

et al. 2017

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Polyphenol oxidases (PPOs) are enzymes that catalyse the oxidation of phenolic compounds. We report a NMR‐based assay that can be used as a screening and validation tool for PPO activity modulators, which was demonstrated using a series of PPO inhibitors and antioxidants. This method is exemplified by work in tandem with virtual screening and molecular modelling, which led to the discovery of several novel PPO inhibitors.

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In situ proton NMR of glycosidase catalyzed hydrolysis and reverse hydrolysis

Cited by 10 publications

References 32 publications

In situ proton NMR study of acetyl and formyl group migration in mono‐O‐acyl D‐glucose

In situ proton NMR study of acetyl and formyl group migration in mono‐O‐acyl D‐glucose

Enzymatic synthesis of β‐glucosylglycerol using a continuous‐flow microreactor containing thermostable β‐glycoside hydrolase CelB immobilized on coated microchannel walls

Development and Application of an NMR‐Based Assay for Polyphenol Oxidases

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