Comments on experimental evaluation of usefulness of equations describing the apparent maximum reaction rate and apparent Michaelis constant of an immobilized enzyme reaction

Catalytic activity of catalase (CAT, EC 1.11.1.6), immobilized on carbon black NORIT and soot PM-100, with respect to decomposition of dibenzoyl peroxide (BPO) in non-aque-ous media (acetonitrile and tetrachloromethane), was investigated with a quantitative UV-spectrophotometrical approach. Progress of the above reaction was controlled by selected kinetic parameters: the apparent Michaelis constant (Kmapp), the specific rate constant (Ksp), the activation energy (Ea), the maximum reaction rate (Vmax), and the Arrhenius’ pre-exponential factor (Z0). Conclusions on the tentative mechanism of the catalytic process observed were drawn from the calculated values of the Gibbs energy of activation (ΔG*), the enthalpy of activation (ΔH*), and entropy of activation (ΔS*)

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Calculation of the Effect of Solanine on the Michaelis Constant and the Maximum Reaction Rate of NAT

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.282-283.213 ◽

2011 ◽

Vol 282-283 ◽

pp. 213-217

Author(s):

Yu Bin Ji ◽

Shi Yong Gao ◽

Chen Feng Ji ◽

Xiang Zou

Keyword(s):

High Performance ◽

Reaction Rate ◽

Accurate Method ◽

Competitive Inhibitor ◽

Negative Control ◽

Michaelis Constant ◽

Intact Cells ◽

Maximum Reaction Rate ◽

Maximum Reaction ◽

Reciprocal Method

To explore how to calculate the effect of solanine on the Michaelis constant and the maximum reaction rate of NAT using the double reciprocal method. High performance liquid chromatography (HPLCP) was used, with 2-AF as substrate and its concentration as substrate concentration, and the rate at which 2-AF is acetylated into 2-AAF in intact HepG2 cells or in the cytoplasm of HepG2 cells as the reaction rate. The double reciprocal plot was made, with 1/S (reciprocal of the concentration of the substrate 2-AF) plotted against 1/V (reciprocal of the reaction rate), to yield a regression equation for calculating Km and Vmax. Studies on enzymetic kinetics have shown that, with intact HepG2 cells, the Km and Vmax for the negative control are 2.37×10-3±8.37×10-5 mM and 9.16×10-4±7.54×10-5nmol/106 cells, respectively, and that the Km and Vmax for the solanine group are 2.22×10-3±9.05×10-5mM and 5.14×10-4±3.72×10-5nmol/106 cells, respectively. For the cytoplasm of HepG2 cells, the Km and Vmax for the negative control are 8.95×10-3±2.61×10-4 mM and 2.55×10-6±1.92×10-8nmol/min·mg protein, respectively, and the Km and Vmax of the solanine group are 9.48×10-3±3.63×10-4mM and 2.43×10-6±1.32×10-8 nmol/min·mg protein, respectively. Statistical analysis showed that, for both intact HepG2 cells and cytoplasm of HepG2 cells, Km does not differ significantly between the negative control and the solanine groups, but Vmax does differ significantly for these groups (p<0.001 for the intact cells, p<0.05 for the cytoplasm). Solanine is a non-competitive inhibitor for the 2-AF substrate of NAT, and the double reciprocal method is a convenient and accurate method for calculating the constants of NAT kinetics.

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Calculation of the Effect of Solanine on the Michaelis Constant and the Maximum Reaction Rate of NAT

2008 2nd International Conference on Bioinformatics and Biomedical Engineering ◽

10.1109/icbbe.2008.643 ◽

2008 ◽

Author(s):

Shi-yong Gao ◽

Yu-bin Ji ◽

Xiang Zou ◽

Chen-feng Ji

Keyword(s):

Reaction Rate ◽

Michaelis Constant ◽

Maximum Reaction Rate ◽

Maximum Reaction

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A Novel Sucrose Isomerase Producing Isomaltulose from Raoultella terrigena

Applied Sciences ◽

10.3390/app11125521 ◽

2021 ◽

Vol 11 (12) ◽

pp. 5521

Author(s):

Li Liu ◽

Shuhuai Yu ◽

Wei Zhao

Keyword(s):

Ph Value ◽

Wild Type ◽

Michaelis Constant ◽

Wild Type Enzyme ◽

Maximum Reaction Rate ◽

E Coli ◽

Maximum Reaction ◽

Conversion Rates ◽

Raoultella Terrigena ◽

Sucrose Isomerase

Isomaltulose is widely used in the food industry as a substitute for sucrose owing to its good processing characteristics and physicochemical properties, which is usually synthesized by sucrose isomerase (SIase) with sucrose as substrate. In this study, a gene pal-2 from Raoultella terrigena was predicted to produce SIase, which was subcloned into pET-28a (+) and transformed to the E. coli system. The purified recombinant SIase Pal-2 was characterized in detail. The enzyme is a monomeric protein with a molecular weight of approximately 70 kDa, showing an optimal temperature of 40 °C and optimal pH value of 5.5. The Michaelis constant (Km) and maximum reaction rate (Vmax) are 62.9 mmol/L and 286.4 U/mg, respectively. The conversion rate of isomaltulose reached the maximum of 81.7% after 6 h with 400 g/L sucrose as the substrate and 25 U/mg sucrose of SIase. Moreover, eight site-directed variants were designed and generated. Compared with the wild-type enzyme, the enzyme activities of two mutants N498P and Q275R were increased by 89.2% and 42.2%, respectively, and the isomaltulose conversion rates of three mutants (Y246L, H287R, and H481P) were improved to 89.1%, 90.7%, and 92.4%, respectively. The work identified a novel SIase from the Raoultella genus and its mutants showed a potential to be used for the production of isomaltulose in the industry.

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