Sequential Enzymatic Reactions Research Articles

Divergent Synthesis of Heparan Sulfate Oligosaccharides.

Heparan sulfates are implicated in a wide range of biological processes. A major challenge in deciphering their structure and activity relationship is the synthetic difficulties to access diverse heparan sulfate oligosaccharides with well-defined sulfation patterns. In order to expedite the synthesis, a divergent synthetic strategy was developed. By integrating chemical synthesis and two types of O-sulfo transferases, seven different hexasaccharides were obtained from a single hexasaccharide precursor. This approach combined the flexibility of chemical synthesis with the selectivity of enzyme-catalyzed sulfations, thus simplifying the overall synthetic operations. In an attempt to establish structure activity relationships of heparan sulfate binding with its receptor, the synthesized oligosaccharides were incorporated onto a glycan microarray, and their bindings with a growth factor FGF-2 were examined. The unique combination of chemical and enzymatic approaches expanded the capability of oligosaccharide synthesis. In addition, the well-defined heparan sulfate structures helped shine light on the fine substrate specificities of biosynthetic enzymes and confirm the potential sequence of enzymatic reactions in biosynthesis.

Abstract C118: Choline kinase inhibition with the novel pharmacological inhibitor ICL-CCIC-0019 reprograms cellular metabolism and inhibits cancer cell growth

Abstract The glycerophospholipid phosphatidylcholine (PC) is the most abundant phospholipid species of eukaryotic membranes and essential for structural integrity and signaling function of cell membranes required for cancer cell growth. PC is synthesized via the CDP-choline pathway, whereby choline kinase alpha (CHKA) denotes the first committed step in this sequence of enzymatic reactions. CHKA phosphorylates choline to form phosphocholine and its overexpression in many solid tumors is linked to progression of normal cells to malignancy. We developed the highly selective, choline-competitive small-molecule ICL-CCIC-0019 (IC50 of 0.27±0.06 μM). Across a panel of 131 human kinases, the inhibitor showed minimal off-target effects (only 5 kinases were inhibited more than 20% at a concentration of 10 μM). ICL-CCIC-0019 potently inhibited growth of a panel of 60 cancer cell lines with median GI50 of 1.12 μM. Importantly, proliferation of normal cells was only minimally affected (MCF-10A and ST-T1b: GI50 30-120 μM). ICL-CCIC-0019 decreased phosphocholine levels and the fraction of labeled choline in lipids, and induced G1 arrest, endoplasmic reticulum stress and apoptosis. Changes in phosphocholine cellular levels following treatment could be detected non-invasively in tumor xenografts by 18F-fluoromethyl-[1,2-2H4]-choline positron emission tomography. Pharmacokinetic modeling revealed that the macro parameter Ki denoting the net irreversible uptake rate was significantly decreased in tumor after 48 hours (Ki (1/min): control, 0.0054±0.00060; ICL-CCIC-0019, 0.0032±0.00064), confirming in vivo target inhibition. This resulted in potent antitumor activity in HCT116 xenografts. We further reveal a previously unappreciated effect of choline metabolism on mitochondria function. Comparative metabolomics demonstrated that phosphatidylcholine pathway inhibition leads to a metabolically stressed phenotype analogous to mitochondria toxin treatment but without reactive oxygen species activation. Drug treatment decreased TCA cycle activity, oxygen consumption rate and elevated extracellular acidification rate. This was associated with a reduction of citrate synthase expression and AMP kinase activation. Glucose and acetate uptake were increased in an attempt to overcome the metabolic stress. This study indicates that choline pathway pharmacological inhibition is a valid therapeutic strategy and critically affects the metabolic function of the cell beyond reduced synthesis of phospholipids. This work was supported in part by Cancer Research UK-Engineering and Physical Sciences Research Council Grant C2536/A10337. Citation Format: Sebastian Trousil, Maciej Kalisczcak, Zachary Schug, Quang-De Nguyen, Giampaolo Tomasi, Rosy Favicchio, Diana Brickute, Robin Fortt, Frazer J. Twyman, Laurence Carroll, Andrew Kalusa, Naveenan Navaratnam, Thomas Adejumo, David Carling, Eyal Gottlieb, Eric O. Aboagye. Choline kinase inhibition with the novel pharmacological inhibitor ICL-CCIC-0019 reprograms cellular metabolism and inhibits cancer cell growth. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C118.

Using a new molecular genetic of genotype and liquid culture medium for rapid diagnosis tb

This paper presents the results of molecular genetic test system GenoType multyresistentens MTBDRplus. It was established that the presence of mutations associated with resistance to isoniazid, only 93.1 % of cases of MBT to isoniazid during the test in a liquid medium. Work carried out under the National Programme to combat tuberculosis Materials and methods. We investigated the clinical sputum samples from patients with pulmonary tuberculosis. The applied system GenoType. Principle DNA strip technology GenoType is that the DNA-coated strip specific test that are complementary to the derived PCR amplicon. After the single-stranded amplicon denaturation associated with tests on strip (hybridize), and visualized in a sequential enzymatic reaction with streptavydynom and alkaline phosphatase. Evaluation of hybridization is performed automatically. For culturing sputum liquid culture medium used - Middlebrook broth 7N9 VASTES MGIT system. Results and discussion . The results of molecular genetic studies of samples of sputum-concentrated and concentrated by a system GenoType not differed (P>0.05). Diagnostic value of two methods (molecular and genetic – system GenoType and phenotype – VASTES MGIT 960 system) was very high (100%). Two systems have tested positive in the study 756 (95.5 %) Mycobacterium strains that were identified in the system VASTES MGIT 960, formed Cord Factor and the results were positive identification test ID MTB MGIT they attributed to Mycobacterium tuberculosis complex. 36 (4.5 %) samples from positive MGIT tubes were negative. As a result of molecular-genetic identification of nontuberculous mycobacteria complex it was found that 18 (2.3 %) strains of mycobacteria belonging to the M. avium-intracellulare, 12 (1.5 %) mycobacterial cultures were attributed to M. kansasii, 6 (0, 7 %) cultures were identified as M. fortuitum. The results of the molecular study of MS on Mycobacterium resistance profile INN + RIF coincided in 95.5 % (894 strains) the results of testing by phenotypic proportions. In the presence of mutations associated with resistance to INH, only 93.1 % of cases observed MC M. tuberculosis to INH during TMCH in a liquid medium Middlebrook 7N9. In the presence of mutations in the genes responsible for resistance to the presence of Q, in a liquid medium only 288 (90.6 %) strains of M. tuberculosis have MS to Ofx. The strains of mycobacteria DNA were detected mutations in genes associated with MS to aminoglycosides / cyclic peptides in 299 (94.0%) cases were resistant to Am and 302 (94.9 %) cases were resistant to the results Cm DST system VASTES MGIT 960. In determining the resistance to E major differences were found between the productivity of molecular genetic and phenotypic research methods – only 206 (64, 2 %) strains of M. tuberculosis were resistant to the analysis system VASTES MGIT 960. Conclusions. GenoType system allows you to quickly carry out the identification and differentiation of Mycobacterium strains of nontuberculous mycobacteria. The results of the molecular genetic studies multyrezystents system GenoType MTBDRplus match in 95,5 % of the phenotypic test results by proportions. Using DNA strip technology GenoType to determine mutations in the genes of Mycobacterium responsible for the IPU to TDC, necessarily requires a parallel DST setting in a liquid medium

Photo‐electrochemical Bioanalysis of Guanosine Monophosphate Using Coupled Enzymatic Reactions at a CdS/ZnS Quantum Dot Electrode

A photo-electrochemical sensor for the specific detection of guanosine monophosphate (GMP) is demonstrated, based on three enzymes combined in a coupled reaction assay. The first reaction involves the adenosine triphosphate (ATP)-dependent conversion of GMP to guanosine diphosphate (GDP) by guanylate kinase, which warrants substrate specificity. The reaction products ADP and GDPare co-substrates for the enzymatic conversion of phosphoenolpyruvate to pyruvate in a second reaction mediated by pyruvate kinase. Pyruvate in turn is the co-substrate for lactate dehydrogenase that generates lactate via oxidation of nicotinamide adenine dinucleotide (reduced form) NADH to NAD(+). This third enzymatic reaction is electrochemically detected. For this purpose a CdS/ZnS quantum dot (QD) electrode is illuminated and the photocurrent response under fixed potential conditions is evaluated. The sequential enzyme reactions are first evaluated in solution. Subsequently, a sensor for GMP is constructed using polyelectrolytes for enzyme immobilization.

Layer-by-Layer Assembled Enzyme Cascade Bioanode for Catalyzing Oxidation of Sucrose

We report here a novel layer-by-layer (LbL) assembled enzyme cascade bioanode incorporated with CNT-invertase and CNT-glucose dehydrogenase for sucrose oxidation in a biofuel cell. Methylene green (MG) was used as redox mediator for electrocatalytic oxidation of NADH at reduced overpotentials on screen printed electrode (SPE). The LbL architecture showed advantages for sequential enzymatic reaction that favored the efficient penetration of substrate and products in a cascade system. The maximum current density and power density achieved were 412 ± 36 μA/cm2 and 85±6μW/cm2.The high current density/power density obtained showed LbL assembly is of great advantage and can be employed as a simple and efficient way to construct enzyme cascade bioanode for biofuel cell applications.

Structure-function correlations in tyrosinases.

Tyrosinases are metalloenzymes belonging to the type-3 copper protein family which contain two copper ions in the active site. They are found in various prokaryotes as well as in plants, fungi, arthropods, and mammals and are responsible for pigmentation, wound healing, radiation protection, and primary immune response. Tyrosinases perform two sequential enzymatic reactions: hydroxylation of monophenols and oxidation of diphenols to form quinones which polymerize spontaneously to melanin. Two other members of this family are catechol oxidases, which are prevalent mainly in plants and perform only the second oxidation step, and hemocyanins, which lack enzymatic activity and are oxygen carriers. In the last decade, several structures of plant and bacterial tyrosinases were determined, some with substrates or inhibitors, highlighting features and residues which are important for copper uptake and catalysis. This review summarizes the updated information on structure-function correlations in tyrosinases along with comparison to other type-3 copper proteins.

Layer-By-Layer Assembled Carbon Nanotube Immobilized Enzyme Cascade for Sucrose/O2 Biofuel Cell Utilization

A layer by layer (LbL) assemble technique for the first time was employed to modify glassy carbon electrode (GCE) and screen printed electrode (SPE) utilizing multiwalled carbon nanotube (MWCNTs) based polyelectrolytes and enzyme cascade to facilitate efficient electron transfer in Sucrose/O2 biofuel cell. Previously, our group demonstrated layer-by-layer (LbL) assembly in fabrication of carbon nanotube (CNT) immobilized multi-enzyme biosensors for discriminative detection of multiple analytes. In this study, electrostatically interacted CNT-Invertase (INV), CNT-glucose dehydrogenase (GDH, NAD+-dependent) with cushioning bilayers CNT-polyethyleneimine (PEI) and CNT-DNA were alternatively assembled in LbL fashion on SPE (0.2 cm2) and GCE (0.07065 cm2). [Ni(phendion)(phen)]Cl2 complex and methylene green (MG) were investigated as redox mediator for electrocatalytic oxidation of NADH at reduced overpotentials. The LbL architecture showed advantages for sequential enzymatic reaction that favored the efficient penetration of substrate and products in a cascade system while MWCNT facilitated the direct electron transfer from sucrose oxidation and enhancement of the current density. With GCE as modified bioanode using MG and Ni complex, the biofuel cell produced a higher power density (μW/cm2) with 134.4% and 34.3% enhancement comparing with SPE bioanode, respectively. With Ni complex compared modified on GCE and SPE, the biofuel cell produced a higher power density (μW/cm2) with 16.6% and 101.9 % higher comparing with MG modified on GCE and SPE, respectively. The LbL assembly showed great feasibility as a simple and efficient way to construct controlled CNT-multi-enzyme modified electrodes for biofuel cell applications.

Substrate channeling between the human dihydrofolate reductase and thymidylate synthase.

In vivo, as an advanced catalytic strategy, transient non-covalently bound multi-enzyme complexes can be formed to facilitate the relay of substrates, i. e. substrate channeling, between sequential enzymatic reactions and to enhance the throughput of multi-step enzymatic pathways. The human thymidylate synthase and dihydrofolate reductase catalyze two consecutive reactions in the folate metabolism pathway, and experiments have shown that they are very likely to bind in the same multi-enzyme complex in vivo. While reports on the protozoa thymidylate synthase-dihydrofolate reductase bifunctional enzyme give substantial evidences of substrate channeling along a surface "electrostatic highway," attention has not been paid to whether the human thymidylate synthase and dihydrofolate reductase, if they are in contact with each other in the multi-enzyme complex, are capable of substrate channeling employing surface electrostatics. This work utilizes protein-protein docking, electrostatics calculations, and Brownian dynamics to explore the existence and mechanism of the substrate channeling between the human thymidylate synthase and dihydrofolate reductase. The results show that the bound human thymidylate synthase and dihydrofolate reductase are capable of substrate channeling and the formation of the surface "electrostatic highway." The substrate channeling efficiency between the two can be reasonably high and comparable to that of the protozoa.

Novel process for producing 6-deoxy monosaccharides from l-fucose by coupling and sequential enzymatic method

We biosynthesized 6-deoxy-L-talose, 6-deoxy-L-sorbose, 6-deoxy-L-gulose, and 6-deoxy-L-idose, which rarely exist in nature, from L-fucose by coupling and sequential enzymatic reactions. The first product, 6-deoxy-L-talose, was directly produced from L-fucose by the coupling reactions of immobilized D-arabinose isomerase and immobilized L-rhamnose isomerase. In one-pot reactions, the equilibrium ratio of L-fucose, L-fuculose, and 6-deoxy-L-talose was 80:9:11. In contrast, 6-deoxy-L-sorbose, 6-deoxy-L-gulose, and 6-deoxy-L-idose were produced from L-fucose by sequential enzymatic reactions. D-Arabinose isomerase converted L-fucose into L-fuculose with a ratio of 88:12. Purified L-fuculose was further epimerized into 6-deoxy-L-sorbose by D-allulose 3-epimerase with a ratio of 40:60. Finally, purified 6-deoxy-L-sorbose was isomerized into both 6-deoxy-L-gulose with an equilibrium ratio of 40:60 by L-ribose isomerase, and 6-deoxy-L-idose with an equilibrium ratio of 73:27 by D-glucose isomerase. Based on the amount of L-fucose used, the production yields of 6-deoxy-L-talose, 6-deoxy-L-sorbose, 6-deoxy-L-gulose, and 6-deoxy-L-idose were 7.1%, 14%, 2%, and 2.4%, respectively.

Layer-By-Layer Assembled Enzyme Cascade for Catalyzing Oxidation of Sucrose for Biofuel Cells

Feasibility of layer-by-layer (LbL) assembly via electrostatic interaction in development of multi-enzyme based biosensors was successfully demonstrated in our previous research. In this study, application of LbL technique was extended for the first time in assembly of multi-enzyme, invertase (INV) and NAD+-dependent glucose dehydrogenase (GDH) for cascade catalytic oxidation of sucrose for biofuel cell application. The system was constructed through LbL assembly of electrostatically interacted INV and GDH armored multi-walled carbon nanotubes (MWCNT-INV and MWCNT-AChE) with a set of cushioning bilayers consisting of MWCNT-polyethyleneimine (PEI) and MWCNT-DNA on screen printed electrode (SPE). The LbL architecture shows great advantages for sequential enzymatic reaction that it favors the efficient penetration of substrate and products in a cascade system. The utilization of MWCNT facilitates the direct electron transfer from sucrose oxidation and enhances the current generated. The current density of 104.08±0.85 μA/cm2 using Amperometry was currently achieved with 50 mM sucrose and 1 mM NAD+ on SPE with a surface area of 0.2 cm2 in PB buffer 6.0 at room temperature. The current density of LbL assembled electrode can be further improved by optimization of buffer pH, concentration of NAD+ as well as sucrose concentration. Therefore, the results indicated that the versatile LbL assembly is a simple and efficient way to co-immobilize CNT-multi-enzyme for cascade system, showing great potential in enzymatic biofuel cell development.

Plastid-cytosol partitioning and integration of metabolic pathways for APS/PAPS biosynthesis in Arabidopsis thaliana.

Plants assimilate sulfate from the environment to synthesize biologically active sulfur-containing compounds required for growth and cellular development. The primary steps of sulfur metabolism involve sequential enzymatic reactions synthesizing adenosine 5′-phosphosulfate (APS) and 3′-phosphoadenosine 5’-phosphosulfate (PAPS). Recent finding suggests that an adenosine nucleotide transport system facilitating the exchange of PAPS and 3′-phosphoadenosine 5′-phosphate across the plastid envelope is essential for establishing an intimate connection between the plastidic and cytosolic sulfate assimilation pathways in plants. Subcellular partitioning and integration of metabolic pathways provide focal points for investigating metabolic flux regulations. This perspective article presents an integrative view of sulfur metabolic flux control mechanisms with an emphasis on subcellular partitioning of APS/PAPS biosynthetic pathways in Arabidopsis thaliana.

Single‐Pot Enzymatic Reaction Sequence for the Synthesis of D‐Glyceraldehyde‐3‐Phosphate

Single‐Pot Enzymatic Reaction Sequence for the Synthesis of <i>D</i>‐Glyceraldehyde‐3‐Phosphate

Prozessentwicklung zur enzymatischen Synthese eines biologisch abbaubaren Polymers

A single-pot enzymatic reaction sequence has been designed for the synthesis of a key intermediate metabolite called D-glyceraldehyde-3-phosphate (D-GAP). The reaction sequence consists of three enzymes and uses D-F16BP as a starting material (Fig.). The rabbit muscle aldolase (RAMA)catalyzed reaction step is in favor of D-F16BP formation with an equilibrium constant of 10M [1]. Therefore, the single-pot reaction sequence has been designed to shift the equilibrium and regenerate NADH. The capability of the reaction sequence was demonstrated by the enhanced equilibrium conversion from 1.4% to 96%. The reaction system was optimized in detail with regard to activity and stability of the enzymes and stability of the cofactors (NADH and NAD) and D-GAP. Reaction kinetics models for each of the enzymes were formulated. The effect of co-substances on the activity of the enzymes was evaluated. The results elucidated that D-F16BP and sn-G3P do not influence the activity of FDH and HCOO does not affect the activity of RAMA, whereas D-F16BP and HCOO suppress the activity of sn-G3PDH. From the kinetics perspective and due to the stability of the enzymes, a continuous operation based on an enzyme membrane reactor was preferred.

Structural Insights into the Substrate Specificity of (S)-Ureidoglycolate Amidohydrolase and Its Comparison with Allantoate Amidohydrolase

In plants, the ureide pathway is a metabolic route that converts the ring nitrogen atoms of purine into ammonia via sequential enzymatic reactions, playing an important role in nitrogen recovery. In the final step of the pathway, (S)-ureidoglycolate amidohydrolase (UAH) catalyzes the conversion of (S)-ureidoglycolate into glyoxylate and releases two molecules of ammonia as by-products. UAH is homologous in structure and sequence with allantoate amidohydrolase (AAH), an upstream enzyme in the pathway with a similar function as that of an amidase but with a different substrate. Both enzymes exhibit strict substrate specificity and catalyze reactions in a concerted manner, resulting in purine degradation. Here, we report three crystal structures of Arabidopsis thaliana UAH (bound with substrate, reaction intermediate, and product) and a structure of Escherichia coli AAH complexed with allantoate. Structural analyses of UAH revealed a distinct binding mode for each ligand in a bimetal reaction center with the active site in a closed conformation. The ligand directly participates in the coordination shell of two metal ions and is stabilized by the surrounding residues. In contrast, AAH, which exhibits a substrate-binding site similar to that of UAH, requires a larger active site due to the additional ureido group in allantoate. Structural analyses and mutagenesis revealed that both enzymes undergo an open-to-closed conformational transition in response to ligand binding and that the active-site size and the interaction environment in UAH and AAH are determinants of the substrate specificities of these two structurally homologous enzymes.

Design and construction of a non-natural malate to 1,2,4-butanetriol pathway creates possibility to produce 1,2,4-butanetriol from glucose.

1,2,4-butanetriol (BT) is an important bulk chemical mainly used for producing the superior energetic plasticizer (1,2,4-butanetriol trinitrate) in propellant and explosive formulations. BT is commercially produced by chemical synthesis from petroleum-based feedstocks; until recently a costly biosynthetic route from xylose or arabinose was reported. Here we designed a novel biosynthetic pathway for BT from malate, for the purpose of using glucose as an alternative and cheaper substrate in future. This biosynthetic pathway was achieved through six sequential enzymatic reactions. Following tests of several combinations of enzymes for the pathway, five enzymes including malate thiokinase, succinate-semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyrate CoA-transferase and bifunctional aldehyde/alcohol dehydrogenase were finally chosen. All enzyme genes were expressed on two compatible plasmids in E. coli, and their functions verified separately. Following assembly of two functional modules, BT was detected in the fermentation broth upon addition of malate, proving BT can be biosynthesized from malate. Furthermore, BT was detected in the fermentation using glucose as the sole carbon source, suggesting that such novel BT biosynthetic pathway has created the possibility for the production of BT from the cheaper substrate glucose.

A model study of sequential enzyme reactions and electrostatic channeling

We study models of two sequential enzyme-catalyzed reactions as a basic functional building block for coupled biochemical networks. We investigate the influence of enzyme distributions and long-range molecular interactions on reaction kinetics, which have been exploited in biological systems to maximize metabolic efficiency and signaling effects. Specifically, we examine how the maximal rate of product generation in a series of sequential reactions is dependent on the enzyme distribution and the electrostatic composition of its participant enzymes and substrates. We find that close proximity between enzymes does not guarantee optimal reaction rates, as the benefit of decreasing enzyme separation is countered by the volume excluded by adjacent enzymes. We further quantify the extent to which the electrostatic potential increases the efficiency of transferring substrate between enzymes, which supports the existence of electrostatic channeling in nature. Here, a major finding is that the role of attractive electrostatic interactions in confining intermediate substrates in the vicinity of the enzymes can contribute more to net reactive throughput than the directional properties of the electrostatic fields. These findings shed light on the interplay of long-range interactions and enzyme distributions in coupled enzyme-catalyzed reactions, and their influence on signaling in biological systems.

Efficient and cost-effective production of D-p-hydroxyphenylglycine by whole-cell bioconversion

D-p-hydroxyphenylglycine (D-HPG) is a widely used intermediate for the synthesis of semi-synthetic antibiotics. It can be produced from DL-5-p-hydroxyphenylhydantoin through two sequential enzymatic reactions catalyzed by D-hydantoinase and N-carbamoylase. However, the low productivity and high production cost of the current process significantly limit its industrial application. To set up an efficient and cost-effective whole-cell bioconversion process for D-HPG production, a recombinant E. coli strain was constructed by co-expressing D-hydantoinase and N-carbamoylase from Agrobacterium sp. Then a cheap medium formulation, which uses glycerol and corn steep liquor (CSL) as carbon and nitrogen sources and without addition of any foreign inducer, was developed for high level of enzyme expression. Galactose, melibiose, and raffinose in CSL were found to be capable of inducing T7 promoter. Moreover, this CSL-containing cheap medium exhibited higher expression levels than the traditional LB+IPTG medium for several different enzymes tested, indicating that this medium might be a better alternative to the commonly used LB+IPTG medium for enzyme expression under the control of a T7 promoter. Further optimization revealed that low expression temperature not only increased enzyme expression, but also stabilized the enzyme-expressing cells and the plasmids therein. Wholecell bioconversion was carried out in 55 t water containing 1.8 t substrate and the resting cells expressed in 40 t medium. The specific D-HPG productivity reached 1.68 g/h/g dry cell weight, with a molar yield of 97.8%. To the best of our knowledge, this is the highest productivity reported to date and the first description of this process using large-scale production.

High throughput automated colorimetric method for the screening of l-lactic acid producing microorganisms

Lactic acid is a valuable and fully degradable organic acid with promising applications in poly-lactic acid production (Taskila S and Ojamo, 2013 [1]). Despite their efficiency, the cost of the current lactic acid bio-processes is still an obstacle to this application (Miller et al., 2011 [2]). To ameliorate lactic acid producing strains, researchers are using mutations and metabolic engineering techniques, as well as medium optimization. All these studies necessitate a good and high throughput screening method. Currently, researchers mostly use HPLC methods which often necessitate sample preparation, are not stereospecific and do not allow high throughput. To help optimizing l-lactic acid production, we developed a high throughput colorimetric method inspired by the blood l-lactic acid detection method used for diagnosis (Lin et al., 1999 [3]).•Two sequential enzymatic reactions using l-lactate oxidase, peroxidase and ABTS (2,2′-azino-di-[3-ethylbenzthiazoine-sulfonate]), a chromogenic peroxidase substrate, are used to quantify l-lactate between 13.8 and 90mg/l.•The accuracy of the method was ascertained before automation.•The method was successfully applied for the direct determination of l-lactate content in fungal culture supernatants.

Kinetics of Sequential Enzyme Reactions and Electrostatic Channeling

In cells, many enzyme-catalyzed reactions are coupled as a series of biochemical steps. Thus, the reaction rate is dependent on the kinetics of intermediate reaction steps. In this work, we study the case of two coupled enzyme reactions, where the product of the first reaction serves as the substrate of the second reaction. Using a diffusion-limited reaction model, we demonstrate that the overall kinetics is strongly dependent on the separation distance between two enzymes, although interestingly, we find that in some cases, the maximal rate occurs at a finite, non-smallest separation between enzymes. This is because the second enzyme in proximity can block the access of substrate for the first enzyme reaction, which leads to the decrease of product of the first reaction (substrate for the second reaction). We further demonstrate how this reaction rate is additionally dependent on the nature of electrostatic interactions between reactants and the two enzymes. We demonstrate the interplay of these concepts for the dihydrofolate reductase-thymidylate synthase (DHFR-TS) systems, whereby methylene tetrahydrofolate is converted to tetrahydrofoloate.Our study suggests the role of species-specific electrostatic and geometric factors in optimizing reaction rates of substrate-channeling systems.

Extracellular metabolism-dependent uptake of lysolipids through cultured monolayer of differentiated Caco-2 cells

Glycerophospholipids are known to be hydrolyzed in the intestinal lumen into free fatty acids and lysophospholipids that are then absorbed by the intestinal epithelial cells. A monolayer of enterocyte-differentiated Caco-2 cell is often used to assess the intestinal bioavailability of nutrients. In this study, we examined how differentiated Caco-2 cells process lysoglycerolipids such as lysophosphatidylcholine (LPC). Our findings were twofold. (1) Caco-2 cells secreted both a lysophospholipase A-like enzyme and a glycerophosphocholine-phosphodiesterase enzyme into the apical, but not basolateral, lumen, suggesting that food-derived LPC is converted to a free fatty acid, sn-glycerol-3-phosphate, and choline through two sequential enzymatic reactions in humans. The release of the latter enzyme was differentiation-dependent. (2) Fatty acid-releasing activities toward exogenous fluorescent LPC, lysophosphatidic acid and monoacylglycerol were shown to be higher on the apical membranes of Caco-2 cells than on the basolateral membranes. These results suggest that human intestinal epithelial cells metabolize lysoglycerolipids by two distinct mechanisms involving secreted or apical-selective expression of metabolic enzymes.