Protein Engineering of a Pyridoxal-5′-Phosphate-Dependent l-Aspartate-α-Decarboxylase from Tribolium castaneum for β-Alanine Production

In the present study, a pyridoxal-5′-phosphate (PLP)-dependent L-aspartate-α-decarboxylase from Tribolium castaneum (TcPanD) was selected for protein engineering to efficiently produce β-alanine. A mutant TcPanD-R98H/K305S with a 2.45-fold higher activity than the wide type was selected through error-prone PCR, site-saturation mutagenesis, and 96-well plate screening technologies. The characterization of purified enzyme TcPanD-R98H/K305S showed that the optimal cofactor PLP concentration, temperature, and pH were 0.04% (m/v), 50 °C, and 7.0, respectively. The 1mM of Na+, Ni2+, Co2+, K+, and Ca2+ stimulated the activity of TcPanD-R98H/K305S, while only 5 mM of Ni2+ and Na+ could increase its activity. The kinetic analysis indicated that TcPanD-R98H/K305S had a higher substrate affinity and enzymatic reaction rate than the wild enzyme. A total of 267 g/L substrate l-aspartic acid was consumed and 170.5 g/L of β-alanine with a molar conversion of 95.5% was obtained under the optimal condition and 5-L reactor fermentation.

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Computer Simulation of Amperometric Biosensor Response to Mixtures of Compounds

Nonlinear Analysis Modelling and Control ◽

10.15388/na.2002.7.2.15190 ◽

2002 ◽

Vol 7 (2) ◽

pp. 3-14 ◽

Cited By ~ 3

Author(s):

R. Baronas ◽

J. Christensen ◽

F. Ivanauskas ◽

J. Kulys

Keyword(s):

Computer Simulation ◽

Enzymatic Reaction ◽

Diffusion Equations ◽

Response Data ◽

Finite Difference Technique ◽

Stationary Diffusion ◽

Biosensor Array ◽

Menten Kinetic ◽

Biosensor Response

A mathematical model of amperometric biosensors has been developed. The model bases on non-stationary diffusion equations containing a non-linear term related to Michaelis-Menten kinetic of the enzymatic reaction. The model describes the biosensor response to mixtures of multiple compounds in two regimes of analysis: batch and flow injection. Using computer simulation, large amount of biosensor response data were synthesised for calibration of a biosensor array to be used for characterization of wastewater. The computer simulation was carried out using the finite difference technique.

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Characterization of Two NMN Deamidase Mutants as Possible Probes for an NMN Biosensor

International Journal of Molecular Sciences ◽

10.3390/ijms22126334 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6334

Author(s):

Alessandra Camarca ◽

Gabriele Minazzato ◽

Angela Pennacchio ◽

Alessandro Capo ◽

Adolfo Amici ◽

...

Keyword(s):

Enzymatic Reaction ◽

Promising Candidate ◽

Beneficial Effects ◽

Steady State Fluorescence ◽

Nicotinamide Mononucleotide ◽

Recognition Elements ◽

Steady State Fluorescence Spectroscopy ◽

Similar Affinity ◽

Micromolar Range

Nicotinamide mononucleotide (NMN) is a key intermediate in the nicotinamide adenine dinucleotide (NAD+) biosynthesis. Its supplementation has demonstrated beneficial effects on several diseases. The aim of this study was to characterize NMN deamidase (PncC) inactive mutants to use as possible molecular recognition elements (MREs) for an NMN-specific biosensor. Thermal stability assays and steady-state fluorescence spectroscopy measurements were used to study the binding of NMN and related metabolites (NaMN, Na, Nam, NR, NAD, NADP, and NaAD) to the PncC mutated variants. In particular, the S29A PncC and K61Q PncC variant forms were selected since they still preserve the ability to bind NMN in the micromolar range, but they are not able to catalyze the enzymatic reaction. While S29A PncC shows a similar affinity also for NaMN (the product of the PncC catalyzed reaction), K61Q PncC does not interact significantly with it. Thus, PncC K61Q mutant seems to be a promising candidate to use as specific probe for an NMN biosensor.

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Synthesis and Polymerization Kinetics of Rigid Tricyanate Ester

Polymers ◽

10.3390/polym13111686 ◽

2021 ◽

Vol 13 (11) ◽

pp. 1686

Author(s):

Andrey Galukhin ◽

Roman Nosov ◽

Ilya Nikolaev ◽

Elena Melnikova ◽

Daut Islamov ◽

...

Keyword(s):

Kinetic Analysis ◽

Single Step ◽

Polymerization Kinetics ◽

Scanning Calorimetry ◽

Modulated Differential Scanning Calorimetry ◽

Diffusion Controlled ◽

Polymerization Product ◽

The One ◽

Kinetics Of

A new rigid tricyanate ester consisting of seven conjugated aromatic units is synthesized, and its structure is confirmed by X-ray analysis. This ester undergoes thermally stimulated polymerization in a liquid state. Conventional and temperature-modulated differential scanning calorimetry techniques are employed to study the polymerization kinetics. A transition of polymerization from a kinetic- to a diffusion-controlled regime is detected. Kinetic analysis is performed by combining isoconversional and model-based computations. It demonstrates that polymerization in the kinetically controlled regime of the present monomer can be described as a quasi-single-step, auto-catalytic, process. The diffusion contribution is parameterized by the Fournier model. Kinetic analysis is complemented by characterization of thermal properties of the corresponding polymerization product by means of thermogravimetric and thermomechanical analyses. Overall, the obtained experimental results are consistent with our hypothesis about the relation between the rigidity and functionality of the cyanate ester monomer, on the one hand, and its reactivity and glass transition temperature of the corresponding polymer, on the other hand.

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Characterization of Saccharomyces cerevisiae thymidylate kinase, the CDC8 gene product. General properties, kinetic analysis, and subcellular localization.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)42612-3 ◽

1984 ◽

Vol 259 (23) ◽

pp. 14394-14398 ◽

Cited By ~ 5

Author(s):

A Y Jong ◽

J L Campbell

Keyword(s):

Saccharomyces Cerevisiae ◽

Subcellular Localization ◽

Kinetic Analysis ◽

Gene Product ◽

Thymidylate Kinase ◽

General Properties

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Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction

International Journal of Molecular Sciences ◽

10.3390/ijms22020734 ◽

2021 ◽

Vol 22 (2) ◽

pp. 734

Author(s):

Paul K. Varghese ◽

Mones Abu-Asab ◽

Emilios K. Dimitriadis ◽

Monika B. Dolinska ◽

George P. Morcos ◽

...

Keyword(s):

Catalytic Activity ◽

Large Scale ◽

Magnetic Beads ◽

Enzymatic Reaction ◽

Oculocutaneous Albinism ◽

Transmission Electron ◽

Hill Kinetics ◽

Rate Limiting ◽

Human Tyrosinase

Human Tyrosinase (Tyr) is the rate-limiting enzyme of the melanogenesis pathway. Tyr catalyzes the oxidation of the substrate L-DOPA into dopachrome and melanin. Currently, the characterization of dopachrome-related products is difficult due to the absence of a simple way to partition dopachrome from protein fraction. Here, we immobilize catalytically pure recombinant human Tyr domain (residues 19–469) containing 6xHis tag to Ni-loaded magnetic beads (MB). Transmission electron microscopy revealed Tyr-MB were within limits of 168.2 ± 24.4 nm while the dark-brown melanin images showed single and polymerized melanin with a diameter of 121.4 ± 18.1 nm. Using Hill kinetics, we show that Tyr-MB has a catalytic activity similar to that of intact Tyr. The diphenol oxidase reactions of L-DOPA show an increase of dopachrome formation with the number of MB and with temperature. At 50 °C, Tyr-MB shows some residual catalytic activity suggesting that the immobilized Tyr has increased protein stability. In contrast, under 37 °C, the dopachrome product, which is isolated from Tyr-MB particles, shows that dopachrome has an orange-brown color that is different from the color of the mixture of L-DOPA, Tyr, and dopachrome. In the future, Tyr-MB could be used for large-scale productions of dopachrome and melanin-related products and finding a treatment for oculocutaneous albinism-inherited diseases.

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Mechanistic Study on the Reaction of pinB-BMes2 with Alkynes Based on Experimental Investigation and DFT Calculations: Gradual Change of Mechanism Depending on the Substituent

Chemical Science ◽

10.1039/d1sc02863d ◽

2021 ◽

Author(s):

Linlin Wu ◽

Chiemi Kojima ◽

Ka Ho Lee ◽

Shogo Morisako ◽

Zhenyang Lin ◽

...

Keyword(s):

Experimental Investigation ◽

Transition Metal ◽

Dft Calculations ◽

Kinetic Analysis ◽

Reaction Rate ◽

Mechanistic Study ◽

Isomer Ratio ◽

Gradual Change ◽

Isomeric Mixture ◽

Metal Free

Transition-metal free direct and base-catalyzed 1,2-diborations of arylacetylenes using pinB-BMes2 provided syn/anti-isomeric mixture of diborylalkenes. The kinetic analysis showed that the reaction rate and isomer ratio were affected by reaction...

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