Enzyme Kinetics Experiment with the Multienzyme Complex Viscozyme L and Two Substrates for the Accurate Determination of Michaelian Parameters

Abstract

A laboratory experiment in which students study the kinetics of the Viscozyme-L-catalyzed hydrolysis of cellulose and starch comparatively was designed for an upper-division biochemistry laboratory. The main objective of this experiment was to provide an opportunity to perform enhanced enzyme kinetics data analysis using appropriate informatics and statistical tools. By fitting the Michaelis–Menten, Lineweaver–Burk, Eadie–Hofstee, and Hanes equations to the experimental data, students explore the strengths and weaknesses of each plot and determine whether the two Viscozyme-L-catalyzed reactions obey Michaelis–Menten kinetics. Comparing the values of the Michaelian constants (v max and K M) and the efficiencies obtained for each enzyme-catalyzed reaction allows advanced undergraduates to determine if the differences between these parameters are statistically significant.

Optimasi Selulase pada Enzyme Assisted Extraction Lemak dari Caulerpa Lentillifera Menggunakan Response Surface Methodology

“…Larutan sampel diambil sebanyak 1 mL dan ditambah pereaksi DNS sebanyak 3 mL, setelah itu ukur absorbansi larutan pada panjang gelombang 540 nm. (Saropah et al, 2012;Guerra, 2017).…”

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“…The Michaelis-Menten Curve is an indicator that can be used to describe changes in enzyme kinetics and to provide inhibitory characteristics for enzyme activity 19 . Km and Vmax values are two main parameters in the enzyme kinetics study, each of which represents the Michaelis-Menten constanta which can be used to estimate the number of substrates and acceleration reaction 20 . The acceleration reaction will continue to increase until it reaches the limit point indicating that the activity of this enzyme has reached its maximum limit, this point is called Vmax 21 .…”

Enzyme kinetic parameters estimation: A tricky task?

“…Thus, instructors engaged into enzyme kinetics can choose from a wide range of pedagogical strategies and instructional resources, including the use of analogies, 1 simulation and modeling software, 2 suggestions for framing content, [3][4][5][6] guided-inquiry approaches, 7 and laboratory experiments. [8][9][10] Indeed, enzyme kinetics experiments are popular in the undergraduate laboratory, and a wide variety of enzyme experiments has been proposed. Many of these proposed lab experiments revolve around the generation of kinetic data.…”

“…To illustrate this assertion, we will use empirical data generated by our students as well as data previously published by other authors. 8,10 1.1 | Lineweaver-Burk transformation An enzyme is said to be michaelian if the initial velocity of the catalyzed reaction obeys the Michaelis-Menten equation:…”