Exploring the Hydrolysis of Sucrose by Invertase Using Nuclear Magnetic Resonance Spectroscopy: A Flexible Package of Kinetic Experiments

Kehlbeck, Joanne D.; Slack, Clancy C.; Turnbull, Marilyn T.; Kohler, Susan J.

doi:10.1021/ed300889s

Cited by 24 publications

(23 citation statements)

References 17 publications

(20 reference statements)

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“…Furthermore, the sucrose peak at 5.41 ppm from the 1 Hγ proton is well resolved even at proton resonance frequencies as low as 60 MHz. 12 The quality and reproducibility of the NMR based approach critically depends on how reliably the peak areas can be converted into concentrations. As NMR spectroscopic methods take a central role in many applications, such as metabolomics, quantitative NMR (qNMR) spectroscopy are becoming a routine.…”

Section: ■ Resultsmentioning

confidence: 99%

Real-Time Enzyme Kinetics by Quantitative NMR Spectroscopy and Determination of the Michaelis–Menten Constant Using the Lambert-W Function

et al. 2015

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Enzyme kinetics is an essential part of a chemistry curriculum, especially for students interested in biomedical research or in health care fields. Though the concept is routinely performed in undergraduate chemistry/biochemistry classrooms using other spectroscopic methods, we provide an optimized approach that uses a real-time monitoring of the kinetics by quantitative NMR (qNMR) spectroscopy and a direct analysis of the time course data using Lambert-W function. The century old Michaelis−Menten equation, one of the fundamental concepts in biochemistry, relates the time derivative of the substrate to two kinetic parameters (the Michaelis constant K M and the maximum rate V max ) and to the concentration of the substrate. The exact solution to the Michaelis− Menten equation, in terms of the Lambert-W function, is not available in standard curve-fitting tools.The high-quality of the real-time qNMR data on the enzyme kinetics enables a revisit of the concept of applying the progress curve analysis. This is particularly made feasible with the advent of analytical approximations of the Lambert-W function. Thus, the combination of NMR experimental timecourse data with progress curve analysis is demonstrated in the case of enzyme (invertase) catalyzed hydrolysis reaction (conversion of sucrose to fructose and glucose) to provide students with direct and simple estimations of kinetic parameters of Michaelis−Menten. Complete details on how to implement the experiment and perform data analysis are provided in the Supporting Information.

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Section: ■ Resultsmentioning

confidence: 99%

Real-Time Enzyme Kinetics by Quantitative NMR Spectroscopy and Determination of the Michaelis–Menten Constant Using the Lambert-W Function

et al. 2015

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“…4 In the present study, invertase from whole yeast cells (a lowcost and accessible enzyme source) is used to introduce students to Michaelis−Menten kinetics, study the effects of enzyme immobilization on the kinetic parameters, illustrate the use of immobilized cells in bioprocesses, and to model the behavior of three types of classical reactors.…”

Section: ■ Experimentsmentioning

confidence: 99%

Student Collaboration in a Series of Integrated Experiments To Study Enzyme Reactor Modeling with Immobilized Cell-Based Invertase

et al. 2015

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An integrative laboratory study addressing fundamentals of enzyme catalysis and their application to reactors operation and modeling is presented. Invertase, a β-fructofuranosidase that catalyses the hydrolysis of sucrose, is used as the model enzyme at optimal conditions (pH 4.5 and 45°C). The experimental work involves 3 h of laboratory time for each student per week over four weeks. Students, organized in laboratory sessions of three groups with 3 or 4 students per group, work in a collaborative manner to obtain a set of replicates of initial reaction rate for different substrate concentrations, using both free and calcium alginate-immobilized Saccharomyces bayanus cells containing biologically active invertase. The results are shared by all of the groups for statistical data treatment, calculation of Michaelis−Menten kinetic parameters, and modeling of three types of classical reactors, a Continuous Stirred Tank Reactor (CSTR), a Plug Flow Reactor (PFR), and a Fluidized Bed Reactor (FBR), operating in continuous mode with immobilized invertase. For each reactor, the experimental conversion profile as a function of the feed flow rate is compared to predicted profiles based upon the kinetic parameters obtained.

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“…As a fundamental tool for the monitoring of hydrolysis process, a kinetic measurement technology is significant for the studies involving sucrose hydrolysis. Rapid analytical methods, such as nuclear magnetic resonance (NMR) spectroscopy [ 5 , 6 ] and in situ FTIR spectroscopy [ 7 ], have been reported for the kinetic and mechanistic study of sucrose hydrolysis. Besides, the kinetics of sucrose hydrolysis was also monitored with chemical reactors [ 8 , 9 ].…”

Section: Introductionmentioning

confidence: 99%

An Optical Chiral Sensor Based on Weak Measurement for the Real-Time Monitoring of Sucrose Hydrolysis

Weng

Ruan

et al. 2021

Sensors

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A chiral sensor with optical rotation detection based on weak measurement for the kinetic study of sucrose hydrolysis is presented. Based on the polarization modulation to the pre-selection state, the optical rotation of chiral sample was accurately determined through the central wavelength shift of the output spectrum. With this approach, the concentration response curves of sucrose and its hydrolysis products, i.e., fructose and glucose, were experimentally obtained for the hydrolysis analysis. By collecting the output spectrum with a frequency of 100 Hz and fitting the central wavelength shift synchronously during the measurement, the sucrose hydrolysis process was monitored in real time. Different hydrolysis conditions with varied concentration of invertase enzyme and citrate were implemented in this work. As a consequence, the real-time hydrolysis curves of the hydrolysis process with distinct velocities was achieved and analyzed. Such a kinetic monitoring about sucrose hydrolysis with optical rotation detection technology played a critical role in the researches involving sucrose, and also revealed the great potential of weak measurement in intersections, such as food safety inspection and chemical analysis.

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Exploring the Hydrolysis of Sucrose by Invertase Using Nuclear Magnetic Resonance Spectroscopy: A Flexible Package of Kinetic Experiments

Cited by 24 publications

References 17 publications

Real-Time Enzyme Kinetics by Quantitative NMR Spectroscopy and Determination of the Michaelis–Menten Constant Using the Lambert-W Function

Real-Time Enzyme Kinetics by Quantitative NMR Spectroscopy and Determination of the Michaelis–Menten Constant Using the Lambert-W Function

Student Collaboration in a Series of Integrated Experiments To Study Enzyme Reactor Modeling with Immobilized Cell-Based Invertase

An Optical Chiral Sensor Based on Weak Measurement for the Real-Time Monitoring of Sucrose Hydrolysis

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