Enzymatic Reaction System Research Articles

3-Hydroxybutyrate Oligomer Hydrolase and 3-Hydroxybutyrate Dehydrogenase Participate in Intracellular Polyhydroxybutyrate and Polyhydroxyvalerate Degradation in Paracoccus denitrificans

Genes encoding 3-hydroxybutyrate oligomer hydrolase (PhaZc) and 3-hydroxybutyrate dehydrogenase (Hbd) were isolated from Paracoccus denitrificans. PhaZc and Hbd were overproduced as His-tagged proteins in Escherichia coli and purified by affinity and gel filtration chromatography. Purified His-tagged proteins had molecular masses of 31 kDa and 120 kDa (a tetramer of 29-kDa subunits). The His-tagged PhaZc hydrolyzed not only 3-hydroxybutyrate oligomers but also 3-hydroxyvalerate oligomers. The His-tagged Hbd catalyzed the dehydrogenation of 3-hydroxyvalerate as well as 3-hydroxybutyrate. When both enzymes were included in the same enzymatic reaction system with 3-hydroxyvalerate dimer, sequential reactions occurred, suggesting that PhaZc and Hbd play an important role in the intracellular degradation of poly(3-hydroxyvalerate). When the phaZc gene was disrupted in P. denitrificans by insertional inactivation, the mutant strain lost PhaZc activity. When the phaZc-disrupted P. denitrificans was complemented with phaZc, PhaZc activity was restored. These results suggest that P. denitrificans carries a single phaZc gene. Disruption of the phaZc gene in P. denitrificans affected the degradation rate of PHA.

A new mechanism and kinetic model for the enzymatic synthesis of short-chain fructooligosaccharides from sucrose

A kinetic model based on a ping-pong mechanism was developed under the steady-state hypothesis to account for the short-chain fructooligosaccharides (sc-FOS) synthesis using the commercial cellulolytic enzyme preparation, Rohapect CM. This new mechanism takes into account the interactions between the enzyme species and potential substrates (sucrose and sc-FOS) as a single complex reaction, allowing a better understanding of the reaction kinetics.The initial reaction rate laws appropriately describe the kinetic profiles of the examined substrates. Whereas sucrose exhibited Michaelis–Menten behavior with substrate inhibition, 1-kestose and nystose followed Michaelis–Menten and sigmoid enzyme kinetics. In addition, the enzyme was competitively inhibited by glucose and exhibited significant hydrolytic activity in the presence of nystose.The overall model was simultaneously fitted to experimental data from three initial sucrose concentrations (0.5, 1.5 and 2.1M) using a multi-response regression with kinetic parameters that have biochemical relevance and are independent of the enzyme concentration. According to the model, sucrose acts almost exclusively as a fructosyl donor substrate. The mathematical development described herein is expected to be suitable for modeling similar enzymatic reaction systems.

Rapid decolorization of azo dyes by crude manganese peroxidase from Schizophyllum sp. F17 in solid-state fermentation

The production of ligninolytic enzymes by the fungus Schizophyllum sp. F17 using a cost-effective medium comprised of agro-industrial residues in solid-state fermentation (SSF) was optimized. The maximum activities of the enzymes manganese peroxidase (MnP), laccase (Lac), and lignin peroxidases (LiP) were 1,200, 586, and 109 U/L, respectively, on day 5 of SSF. In vitro decolorization of three structurally different azo dyes by the extracellular enzymes was monitored to determine its decolorization capability. The results indicated that crude MnP, but not LiP and Lac, played a crucial role in the decolorization of azo dyes. After optimization of the dye decolorization system with crude MnP, the decolorization rates of Orange IV and Orange G, at an initial dye concentration of 50 mg/L, were enhanced to 76 and 57%, respectively, after 20 min of reaction at pH 4 and 35°C. However, only 8% decolorization of Congo red was observed. This enzymatic reaction system revealed a rapid decolorization of azo dyes with a low MnP activity of 24 U/L. Thus, this study could be the basis for the production and application of MnP on a larger scale using a low-cost substrate.

Regioselective glycosylation of hydroquinone to α-arbutin by cyclodextrin glucanotransferase from Thermoanaerobacter sp.

Hydroquinone glycosides were produced by transglycosylation reactions catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. (Toruzyme® 3.0L). The reactions were carried out in an aqueous system containing hydroquinone (HQ) and maltodextrin as acceptor and donor substrate molecules respectively. The conditions for the synthesis of hydroquinone glucoside (α-arbutin) were 9mM hydroquinone, maltodextrin (5%, w/v) in 20mM citrate phosphate buffer, pH 5.5 and 0.025mg/ml toruzyme at 40°C for 24h. The transfer efficiency of hydroquinone glycosylation was 31.8% and 29.2% respectively, when α-cyclodextrin and maltodextrin were employed as donor substrates. The major glycoside product was identified as hydroquinone-1-O-α-d-glucopyranoside (α-arbutin) on the basis of mass spectrometric, nuclear magnetic resonance analysis and component analysis of its enzymatic hydrolysates. The highest molar yield of α-arbutin (21.2%) was obtained when α-cyclodextrin was used as the donor substrate. A two step enzymatic reaction system comprising of CGTase and amyloglucosidase helped to attain a molar yield of 30% for α-arbutin. At room temperature the solubility of α-arbutin in water was determined to be 12.8g/100ml which is approximately 1.8 fold higher than that of hydroquinone.

Modeling of a control induced system for product formation in enzyme kinetics

Double substrate enzyme kinetics has a leading role for product quantification and optimization in different chemical and biochemical sectors. Mathematical approach for controlling these reactions in different stages by suitable parameters adds a new dimension in this interdisciplinary field of research. Applying control theoretic approach in the reversible backward stages of double substrate enzymatic model, time economization with regard to product formation is significant. In this article, we formulate a double substrate mathematical model of enzymatic dynamical reaction system with control measures with a view to observe the effect of changes of these measures with respect to the concentration of substrates, enzyme, complexes and finally product. Here, Pontryagin Minimum Principle is used for observing the effect of control measures in the system dynamics with the help of Hamiltonian. We compare the relevant numerical solutions for the substrates, enzyme, complexes and product concentration profile for a specified time interval with respect to control factors.

Model Development and Optimal Experimental Design of A Kinetically Controlled Synthesis System

A mathematical model has been developed for an enzymatic process with kinetically controlled synthesis. Model reduction and detailed system analysis have been undertaken to examine the main properties of this enzyme reaction system. Optimal experimental design (OED) is developed to obtain the experimental conditions that will generate the most informative measurement data for parameter estimation. Both single-input and multiple-inputs optimisation strategies have been investigated to determine the best intensity levels of control inputs. The results demonstrate that parameter estimation quality can be improved through proper model-based experimental design.

Analytical expressions for the concentrations of substrate, oxygen and mediator in an amperometric enzyme electrode

The theoretical model of Martens and Hall [N. Martens, E.A.H. Hall, Model for an immobilized oxidase enzyme electrode in the presence of two oxidants, Anal. Chem. 66 (1994) 2763–2770] in an immobilized oxidase enzyme electrodes is discussed. This model contains a non-linear term related to enzyme reaction system. In this paper, we obtain approximate analytical solutions for the non-linear equations under steady-state condition by using the homotopy perturbation method (HPM) and homotopy analysis method (HAM). Simple and approximate polynomial expressions for the concentration of substrate, oxygen, reduced mediator and current were obtained in terms of Thiele moduli and the normalized surface concentrations of species. Furthermore, in this work the numerical simulation of the problem is also reported using Scilab/Matlab program. An agreement between analytical and numerical results is noted.

Optimization and Characterization of Nicosulfuron-Degrading Enzyme from Bacillus subtilis Strain YB1

A strain of Bacillus subtilis strain YB1, isolated and preserved in our lab., showed a high nicosulfuron-degrading activity. Optimization of culture conditions on production of nicosulfuron-degrading enzyme from Bacillus subtilis strain YB1 was carried out through mono-factor experiments. The characterization of degrading enzyme(s) was studied in this paper. The results showed that B. subtilis YB1 can use nicosulfuron as sole carbon source under aerobic condition. The key enzyme(s) involved in the initial biodegradation of nicosulfuron was localized to extracellular proteins and showed to be induced expressed. Enzyme-specific activity was up to 89.34 U mg−1 at pH 8.0 and 30°C, incubation for 96 h, inoculum 4.5×108 CFU mL−1 in Luria-Bertani liquid medium with nicosulfuron of 40 mg L−1. The maximum degradation rate of extracellular crude enzymes on nicosulfuron was 66% at pH 9.0, 35°C in the enzymatic reaction system with nicosulfuron of 5 mg L−1. This degrading enzyme(s) was sensitive to high temperature, but kept high activity under alkaline conditions.

On the precision of quasi steady state assumptions in stochastic dynamics

Many biochemical networks have complex multidimensional dynamics and there is a long history of methods that have been used for dimensionality reduction for such reaction networks. Usually a deterministic mass action approach is used; however, in small volumes, there are significant fluctuations from the mean which the mass action approach cannot capture. In such cases stochastic simulation methods should be used. In this paper, we evaluate the applicability of one such dimensionality reduction method, the quasi-steady state approximation (QSSA) [L. Menten and M. Michaelis, "Die kinetik der invertinwirkung," Biochem. Z 49, 333369 (1913)] for dimensionality reduction in case of stochastic dynamics. First, the applicability of QSSA approach is evaluated for a canonical system of enzyme reactions. Application of QSSA to such a reaction system in a deterministic setting leads to Michaelis-Menten reduced kinetics which can be used to derive the equilibrium concentrations of the reaction species. In the case of stochastic simulations, however, the steady state is characterized by fluctuations around the mean equilibrium concentration. Our analysis shows that a QSSA based approach for dimensionality reduction captures well the mean of the distribution as obtained from a full dimensional simulation but fails to accurately capture the distribution around that mean. Moreover, the QSSA approximation is not unique. We have then extended the analysis to a simple bistable biochemical network model proposed to account for the stability of synaptic efficacies; the substrate of learning and memory [J. E. Lisman, "A mechanism of memory storage insensitive to molecular turnover: A bistable autophosphorylating kinase," Proc. Natl. Acad. Sci. U.S.A. 82, 3055-3057 (1985)]. Our analysis shows that a QSSA based dimensionality reduction method results in errors as big as two orders of magnitude in predicting the residence times in the two stable states.

Enzyme hydrolysis of soybean oil in a slug flow microsystem

In this paper, we report on the development of a continuous microfluidic reaction system for hydrolysis of soybean oil catalyzed with Thermomyces lanuginosus lipase (Lipolase 100L). The microfluidic reaction system generates water–oil emulsions in the form of a hydrodynamically well controlled slug flow and automatically separates the oil phase after the hydrolysis by employing two microfluidic separators. All elements of this reaction system were tested at different hydrodynamic conditions and showed the ability to operate in a wide range of reactant flow rates. 25–30% conversion of triglyceride was reached in setting the residence time of the emulsion mixture to 10min. This conversion increased to almost 50% for the residence time of 1h. These results are comparable with those published for the same enzyme reaction system. This feature along with the benefits stemming from the use of microfluidics make our developed system a useful, easy to control and easy to scale-up technology for fast production of fine chemicals. Moreover, our calculations indicate that our slug flow system allows for significant savings of the mechanical energy.

Systematic Studies of Sulfation and Glucuronidation of 12 Flavonoids in the Mouse Liver S9 Fraction Reveal both Unique and Shared Positional Preferences

Sulfation and glucuronidation are the principal metabolic pathways of flavonoids, and extensive phase II metabolism is the main reason for their poor bioavailabilities. The purpose of this study was to compare the similarities and differences in the positional preference of glucuronidation versus sulfation in the mouse liver S9 fraction. The conjugating rates of seven monohydroxyflavones (HFs) (i.e., 2'-, 3'-, 4'-, 3-, 5-, 6-, and 7-HF), and five dihydroxyflavones (diHFs) (i.e., 6,7-, 4',7-, 3,7-, 5,7-, and 3,4'-diHF) were determined in three separate enzymatic reaction systems: (A) sulfation only, (B) glucuronidation only, or (C) simultaneous sulfation and glucuronidation (i.e., Sult-Ugt coreaction). In general, glucuronidation rates were much faster than sulfation rates. Among the HFs, 7-HF was the best substrate for both conjugation reactions, whereas 3-HF was rapidly glucuronidated but was not sulfated. As a result, the rank order of sulfation was very different from that of glucuronidation. Among the diHFs, regiospecific glucuronidation was limited to 7-OH and 3-OH positions, whereas regiospecific sulfation was limited to 7-OH and 4'-OH positions. Other positions (i.e., 6-OH and 5-OH) in diHFs were not conjugated. The positional preferences were essentially maintained in a Sult-Ugt coreaction system, although sulfation was surprisingly enhanced. Lastly, sulfation and glucuronidation displayed different regiospecific- and substrate-dependent characteristics. In conclusion, glucuronidation and sulfation shared the same preference for 7-OH position (of flavonoids) but displayed unique preference in other positions in that glucuronidation preferred the 3-OH position whereas sulfation preferred the 4'-OH position.

2PT218 分子混み合いが小胞内反応系に与える影響の理論的考察(日本生物物理学会第50回年会(2012年度))

2PT218 分子混み合いが小胞内反応系に与える影響の理論的考察(日本生物物理学会第50回年会(2012年度))

Geranyl modification on the tryptophan residue of ComXRO-E-2 pheromone by a cell-free system

ComX pheromone is an isoprenoidal oligopeptide containing a modified tryptophan residue, which stimulates natural genetic competence in the gram-positive bacterium Bacillus. Since posttranslational prenylation on the tryptophan residue has not been reported except in ComX pheromone, the universality of this modification has not yet been elucidated. In this paper, we established a cell-free system, whereby the tryptophan residue in peptides is modified with a geranyl group by modifying enzyme ComQ. In addition, we investigated enzymatic reaction conditions using an in vitro enzyme reaction system. This is the first report of in vitro geranylation on the tryptophan residue. This system is potentially a useful tool for elucidating the universality of prenylation on the tryptophan residue.

Model Reduction of Chemical Reaction Systems using Elimination

There exist different schemes of model reduction for parametric ordinary differential systems arising from chemical reaction systems. In this paper, we focus on some schemes which rely on quasi-steady states approximations. We show that these schemes can be formulated by means of differential and algebraic elimination. Our formulation is simpler than the classical ones. It permitted us to obtain an approximation of the basic enzymatic reaction system which is different from those of Henri-Michaelis-Menten and Briggs-Haldane.

Spectrophotometric quantification of lactose in solution with a peroxidase-based enzymatic cascade reaction system

A spectrophotometric assay was developed for the quantification of lactose in aqueous solution via a one-pot enzymatic cascade reaction at 25 °C and pH 7.2. Lactose (0.2-1.8 mM), E. coli β-galactosidase (β-Gal), Aspergillus niger glucose oxidase (GOD), horseradish peroxidase (HRP) and o-phenylenediamine (OPD) were incubated, and the increase in absorbance at 417 nm (A (417)) due to the formation of DAP (2,3-diaminophenazine), the dimeric oxidation product of OPD, was followed. The increase in A (417) was found to depend linearly on the initial lactose concentration via three consecutive but simultaneously occurring enzymatic reaction steps catalyzed by β-Gal, GOD, and HRP. No pre-incubation of lactose with β-Gal is needed with this simple lactose assay.

Bioconverion of Glutamic Acid to Gamma-Aminobutyric Acid in Soybean by High Pressure with Precursor Feeding

Soybean (Glycine max L.) is rich in nutrients and bioactive substances such as γ-aminobutyric acid (GABA), which has physiological functions. In general, soybean is often soaked in water prior to cooking. During water sorption into soybean matrix, component in soybean could be changed by internal enzymes responsible for proteolysis, amino acid metabolisms, and other biological synthetic reactions. In this study, we focused on the enzymatic reaction of glutamic acid (Glu) to GABA catalyzed by glutamate decarboxylase (GAD). We applied high pressure (HP) on water-soaked and Glu (0.01, 0.05 g/ml)-soaked soybean cotyledon as a cellular biological material, and investigate the effects of HP on bioconversion of Glu to GABA from the viewpoints of the cell structure and enzymatic reaction system. Damage to cell structure was evaluated by measuring dielectric properties using the Cole-Cole arc, the radius of which decreased as pressure level increased. Results suggested that cell structure was damaged by HP. After preservation, concentrations of most amino acids increased in pressurized soybeans after soaking in water compared with unpressurized ones. HP caused a slight increase in GABA concentration in water-soaked samples, but would be predicted to cause a greater increase in Glu-soaked samples, since Glu is a precursor of GABA. The highest GABA concentration was 4.20μmol/g (12.35μmol/g dry basis, d.b.) in Glu-soaked (0.05 g/ml) pressurized soybean at 2-d, while that in water-soaked at 0-d was 0.90μmol/g. GABA concentration eventually increased 5-fold during preservation. We concluded that HP with precursor feeding could lead to high accumulation of target compound. These events can be considered “high-pressure induced transformation (Hi-Pit)” of soybean.

Novel Chemical Kinetics for a Single Enzyme Reaction: Relationship between Substrate Concentration and the Second Moment of Enzyme Reaction Time

We report a robust quadratic relation between the inverse substrate concentration and the second moment, <t(2)>, of the catalytic turnover time distribution for enzyme reactions. The results hold irrespective of the mechanism and dynamics of the enzyme reaction and suggest a novel single molecule experimental analysis that provides information about reaction processes of the enzyme-substrate complex and ergodicity of the enzyme reaction system, which is beyond the reach of the conventional analysis for the mean reaction time, <t>. It turns out that <t(2)> - 2<t>(2) is linear in inverse substrate concentration for an ergodic homogeneous enzyme system given that the enzyme substrate encounter is a simple rate process, and its value at the high substrate concentration limit provides direct information about if any non-Poisson reaction process of the enzyme-substrate complex. For a nonergodic heterogeneous reaction system, the corresponding quantity becomes a quadratic function of the inverse substrate concentration. This leads us to suggest an ergodicity measure for single enzyme reaction systems. We obtain a simple analytic expression of the randomness parameter for the single catalytic turnover time, which could provide a quantitative explanation about the previously reported randomness data of the beta-galactosidase enzyme. In obtaining the results, we introduce novel chemical kinetics applicable to a non-Poisson reaction network with arbitrary connectivity, as a generalization of the conventional chemical kinetics.

Kinetic Analysis of L‐Carnosine Formation by β‐Aminopeptidases

AbstractThe β,α‐dipeptide L‐carnosine occurs in high concentrations in long‐lived innervated mammalian tissues and is widely sold as a food additive. On a large scale L‐carnosine is produced by chemical synthesis procedures. We have established two aqueous enzymatic reaction systems for the preparation of L‐carnosine using the dissolved bacterial β‐aminopeptidases DmpA from Ochrobactrum anthropi and BapA from Sphingosinicella xenopeptidilytica as catalysts and investigated the kinetics of the enzyme‐catalyzed peptide couplings. DmpA catalyzed the formation of L‐carnosine from C‐terminally activated β‐alanine derivatives (acyl donor) and L‐histidine (acyl acceptor) in an aqueous reaction mixture at pH 10 with high catalytic rates (Vmax=19.2 μmol min−1 per mg of protein, kcat=12.9 s−1), whereas Vmax in the BapA‐catalyzed coupling reaction remained below 1.4 μmol min−1 per mg of protein (kcat=0.87 s−1). Although the equilibrium of this reaction lies on the side of the hydrolysis products, the reaction is under kinetic control and L‐carnosine temporarily accumulated to concentrations that correspond to yields of more than 50% with respect to the employed acyl donor. However, competing nucleophiles caused unwanted hydrolysis and coupling reactions that led to decreased product yield and to formation of various peptidic by‐products. The substitution of L‐histidine for L‐histidine methyl ester as acyl acceptor shifted the pKa of the amino functionality from 9.25 to 6.97, which caused a drastic reduction in the amount of coupling by‐products in an aqueous reaction system at pH 8.

Generation of Free Amino Acids and γ-Aminobutyric Acid in Water-Soaked Soybean by High-Hydrostatic Pressure Processing

The effects of high-hydrostatic pressure processing (HPP) on soybean cotyledon as a cellular biological material were investigated from the viewpoints of the cell structure and enzyme reaction system. Damage to cell structure was evaluated by measuring dielectric properties using the Cole-Cole arc, the radius of which decreased as pressure level increased. Results suggested that cell structure was damaged by HPP. The distribution of free amino acids was measured after HPP (200 MPa) of soybean soaked in water or sodium glutamate (Glu) solution. HPP resulted in high accumulation of free amino acids in water-soaked soybean, due to proteolysis. HPP of soybean in Glu solution caused higher accumulation of gamma-aminobutyric acid, suggesting that both proteolysis and specific Glu metabolism were accelerated by HPP. We concluded that HPP partially degraded cell structure and accelerated biochemical reactions by allowing enzyme activities to remain. These events can be considered "high-pressure induced transformation" of soybean.

A Study of Mutation Breeding of High-Yielding Tryptophanase Escherichia coli by Low-Energy N+ Ion Beam Implantation

Low energy ion beam has been widely applied in microbe breeding, plant breeding, gene transfer and cell modification. In this study, the Escherichia coli (E.coli) strain producing tryptophanase was irradiated by a low energy nitrogen ion beam with an energy of 10 keV at a fluence of 13 × 1014 N+/cm2 when glycerin at a 15% concentration was used as a protector. The effect on the biomass of E. coli after N+ implantation was analyzed in detail by statistic methods. The screening methods used in this study were proven to be effective. After continuous mutagenicity, a high-yield tryptophanase strain was selected and both its biomass and enzymatic activity were higher than those of the parent strain. The results of scale-up production showed that the biomass could reach wet weight 8.2 g/L and 110 g L-tryptophan could be formed in the volume of the 1l enzymatic reaction system.