scholarly journals Selective and predicable amine conjugation sites by kinetic characterization under excess reagents

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Wei-Chun Huang ◽  
Li-Juan Huang ◽  
Liang-Sheng Hsu ◽  
Shih-Ting Huang ◽  
Wen-Ting Lo ◽  
...  
Keyword(s):  
Hot Spots ◽  
Drug Efficacy ◽  
High Yield ◽  
Protein Ratio ◽  
Maximum Reaction Rate ◽  
Tunable Range ◽  
Maximum Reaction ◽  
Site Selectivity ◽  
Amine Groups ◽  
Kinetics Of

AbstractThe site selectivity for lysine conjugation on a native protein is difficult to control and characterize. Here, we applied mass spectrometry to examine the conjugation kinetics of Trastuzumab-IgG (Her-IgG) and α-lactalbumin under excess linker concentration ([L]0) based on the modified Michaelis–Menten equation, in which the initial rate constant per amine (kNH2 = Vmax/NH2/KM) was determined by the maximum reaction rate (Vmax/NH2) under saturated accessible sites and initial amine–linker affinity (1/KM). Reductive amination (RA) displayed 3–4 times greater Vmax/NH2 and a different panel of conjugation sites than that observed for N-hydroxysuccinimide ester (NHS) chemistry using the same length of polyethylene glycol (PEG) linkers. Moreover, faster conversion power rendered RA site selectivity among accessible amine groups and a greater tunable range of linker/protein ratio for aldehyde-linkers compared to those of the same length of NHS-linkers. Single conjugation with high yield or poly-conjugations with site homogeneity was demonstrated by controlling [L]0 or gradual addition to minimize the [L]0/KM ratio. Formaldehyde, the shortest aldehyde-linker with the greatest 1/KM, exhibited the highest selectivity and was shown to be a suitable probe to predict conjugation profile of aldehyde-linkers. Four linkers on the few probe-predicted hot spots were elucidated by kinetically controlled RA with conserved drug efficacy when conjugated with the payload. This study provides insights into controlling factors for homogenous and predictable amine bioconjugation.

scholarly journals Inhibition of phospholipase A2 by Virasole derivation

2021 ◽  
Vol 65 (3) ◽  
pp. 309-319
Author(s):  
N. M. Litvinko
Keyword(s):  
Phospholipase A2 ◽  
Competitive Inhibition ◽  
Antiviral Drug ◽  
Maximum Reaction Rate ◽  
Pancreatic Phospholipase ◽  
Maximum Reaction ◽  
Lipophilic Derivative ◽  
Kinetics Of ◽  
Ks Values

Kinetics of phosphatidylcholine (PC) hydrolysis under the action of pancreatic phospholipase A2 IB, (EC 3.1.1.4, PLA2) in the presence of a lipophilic derivative of the antiviral drug Virazole 1-(3-((tert-butyldimethylsilyl)oxy)-4-hydroxy-5- (((4-methoxyphenyl)diphenylmethoxy)methyl)tetrahydrofuran-2-yl)-1H-1,2,4-triazole-3-carboxamide (Virazole2ЗГ) was studied. The both steps of phospholipolysis were quantitatively characterized: the binding of the enzyme to the lipid-water interface (Ks) and directly the catalytic act (Km) with the determination of the maximum reaction rate (Vmax). It was found that Virazole2ЗГ at a concentration of 0.5 μmol/ml does not affect the Ks value; on the contrary, the Michaelis constant, Km, increases by a factor of 1.8 along with the constancy of the parameter Vmax. Based on the constancy of the Ks values, it seems to be assumed that there is no inhibition of the disintegration of the enzyme-micelle complex in the presence of the effector under the studied reaction conditions. The kinetic parameters of the reaction (the increase in Km and the constancy of Vmax in the presence of Virazole2ЗГ) testify in favor of a moderate competitive inhibition of pancreatic PLA2, Ki = 65 mM, which indicates the possibility of searching for the biological activity of the anti-pancreatitis action in the series of pro-drugs of nucleoside nature.

scholarly journals Derivation of volatile fatty acids concentration-independent optimum trace elements configuration and elucidation of optimization kinetics of biomethanization processes

2021 ◽  
Vol 38 (1) ◽  
pp. 194-208
Author(s):  
N.C. Ezebuiro
Keyword(s):  
Fatty Acids ◽  
Trace Elements ◽  
Volatile Fatty Acids ◽  
Desirability Function ◽  
Surface Model ◽  
Maximum Reaction Rate ◽  
Maximum Reaction ◽  
Two Parameters ◽  
Effective Use ◽  
Kinetics Of

Trace elements (TEs) requirements for improved volatile fatty acids (VFA) degradation during biomethanization depend on VFA  concentration of a reactor and the temperature of the process. While temperature remains relatively constant, VFA concentrations change in the course of biomethanization and this implies that for efficient VFA degradation, different trace elements configurations (TEC) should be supplemented. While this is the most efficient approach, it is impractical and constitutes a challenge for the effective use of TEs in the optimization of biomethanization processes. To alleviate this challenge, we modelled the biomethanization efficiency of various VFA  concentration-dependent (VCD) TEs configuration as scenarios and derived a TEs configuration that produced optimum biomethanization across a wider range of VFA concentrations. The study was carried out at 37oC using different concentrations of fixed VFA composition and TEs configurations as scenarios. Response surface model and desirability function were used to determine and compare the  biomethanization efficiency of the scenarios, and to derive a VFA concentration-independent (VCI) TEs configuration. Michaelis-Menten kinetics for two parameters was used to ascertain that the mechanism by which TEs supplementation enhanced mesophilic biomethanization was through an increase in maximum reaction rate (MRR). However, the enhancement was accompanied by an  insignificant decline in inverse affinity (IA).

scholarly journals Modelling and Multi-Objective Optimization of the Sulphur Dioxide Oxidation Process

Processes ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 1072
Author(s):  
Mohammad Reza Zaker ◽  
Clémence Fauteux-Lefebvre ◽  
Jules Thibault
Keyword(s):  
Sulphuric Acid ◽  
Sulphur Dioxide ◽  
Variable Number ◽  
Inlet Temperature ◽  
Multi Objective Optimization ◽  
Absorption Column ◽  
Maximum Reaction Rate ◽  
Multi Objective ◽  
Maximum Reaction

Sulphuric acid (H2SO4) is one of the most produced chemicals in the world. The critical step of the sulphuric acid production is the oxidation of sulphur dioxide (SO2) to sulphur trioxide (SO3) which takes place in a multi catalytic bed reactor. In this study, a representative kinetic rate equation was rigorously selected to develop a mathematical model to perform the multi-objective optimization (MOO) of the reactor. The objectives of the MOO were the SO2 conversion, SO3 productivity, and catalyst weight, whereas the decisions variables were the inlet temperature and the length of each catalytic bed. MOO studies were performed for various design scenarios involving a variable number of catalytic beds and different reactor configurations. The MOO process was mainly comprised of two steps: (1) the determination of Pareto domain via the determination a large number of non-dominated solutions, and (2) the ranking of the Pareto-optimal solutions based on preferences of a decision maker. Results show that a reactor comprised of four catalytic beds with an intermediate absorption column provides higher SO2 conversion, marginally superior to four catalytic beds without an intermediate SO3 absorption column. Both scenarios are close to the ideal optimum, where the reactor temperature would be adjusted to always be at the maximum reaction rate. Results clearly highlight the compromise existing between conversion, productivity and catalyst weight.

scholarly journals A Novel Sucrose Isomerase Producing Isomaltulose from Raoultella terrigena

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.

scholarly journals Synthesis of DHA/EPA Ethyl Esters via Lipase-Catalyzed Acidolysis Using Novozym® 435: A Kinetic Study

Catalysts ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 565 ◽  
Author(s):  
Chia-Hung Kuo ◽  
Chun-Yung Huang ◽  
Chien-Liang Lee ◽  
Wen-Cheng Kuo ◽  
Shu-Ling Hsieh ◽  
...  
Keyword(s):  
Ethyl Ester ◽  
Ethyl Esters ◽  
Raw Material ◽  
Maximum Reaction Rate ◽  
Mechanism Model ◽  
Maximum Reaction ◽  
Substrate Ratio ◽  
Free Fatty Acid Form ◽  
The Relationship ◽  
Predictive Relationship

DHA/EPA ethyl ester is mainly used in the treatment of arteriosclerosis and hyperlipidemia. In this study, DHA+EPA ethyl ester was synthesized via lipase-catalyzed acidolysis of ethyl acetate (EA) with DHA+EPA concentrate in n-hexane using Novozym® 435. The DHA+EPA concentrate (in free fatty acid form), contained 54.4% DHA and 16.8% EPA, was used as raw material. A central composite design combined with response surface methodology (RSM) was used to evaluate the relationship between substrate concentrations and initial rate of DHA+EPA ethyl ester production. The results indicated that the reaction followed the ordered mechanism and as such, the ordered mechanism model was used to estimate the maximum reaction rate (Vmax) and kinetic constants. The ordered mechanism model was also combined with the batch reaction equation to simulate and predict the conversion of DHA+EPA ethyl ester in lipase-catalyzed acidolysis. The integral equation showed a good predictive relationship between the simulated and experimental results. 88–94% conversion yields were obtained from 100–400 mM DHA+EPA concentrate at a constant enzyme activity of 200 U, substrate ratio of 1:1 (DHA+EPA: EA), and reaction time of 300 min.

Quantifying Target Occupancy of Small Molecules Within Living Cells

2020 ◽  
Vol 89 (1) ◽  
pp. 557-581 ◽  
Author(s):  
M.B. Robers ◽  
R. Friedman-Ohana ◽  
K.V.M. Huber ◽  
L. Kilpatrick ◽  
J.D. Vasta ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Drug Efficacy ◽  
Living Cells ◽  
Drug Binding ◽  
Therapeutic Window ◽  
Target Engagement ◽  
Binding Properties ◽  
Structure Activity ◽  
Sar Analysis ◽  
Kinetics Of

The binding affinity and kinetics of target engagement are fundamental to establishing structure–activity relationships (SARs) for prospective therapeutic agents. Enhancing these binding parameters for operative targets, while minimizing binding to off-target sites, can translate to improved drug efficacy and a widened therapeutic window. Compound activity is typically assessed through modulation of an observed phenotype in cultured cells. Quantifying the corresponding binding properties under common cellular conditions can provide more meaningful interpretation of the cellular SAR analysis. Consequently, methods for assessing drug binding in living cells have advanced and are now integral to medicinal chemistry workflows. In this review, we survey key technological advancements that support quantitative assessments of target occupancy in cultured cells, emphasizing generalizable methodologies able to deliver analytical precision that heretofore required reductionist biochemical approaches.

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