mM Of Substrate Research Articles

Characterization of a vacuolar importer of secologanin in Catharanthus roseus

Monoterpenoid indole alkaloid (MIA) biosynthesis in Catharanthus roseus is a paragon of the spatiotemporal complexity achievable by plant specialized metabolism. Spanning a range of tissues, four cell types, and five cellular organelles, MIA metabolism is intricately regulated and organized. This high degree of metabolic differentiation requires inter-cellular and organellar transport, which remains understudied. Here, we have characterized a vacuolar importer of secologanin belonging to the multidrug and toxic compound extrusion (MATE) family, named CrMATE1. Phylogenetic analyses of MATEs suggested a role in alkaloid transport for CrMATE1, and in planta silencing in two varieties of C. roseus resulted in a shift in the secoiridoid and MIA profiles. Subcellular localization of CrMATE1 confirmed tonoplast localization. Biochemical characterization was conducted using the Xenopus laevis oocyte expression system to determine substrate range, directionality, and rate. We can confirm that CrMATE1 is a vacuolar importer of secologanin, translocating 1 mM of substrate within 25 min. The transporter displayed strict directionality and specificity for secologanin and did not accept other secoiridoid substrates. The unique substrate-specific activity of CrMATE1 showcases the utility of transporters as gatekeepers of pathway flux, mediating the balance between a defense arsenal and cellular homeostasis.

(S)-Thienyl and (R)-Pirydyl phosphonate Derivatives Synthesized by Stereoselective Resolution of Their Racemic Mixtures With Rhodotorula mucilaginosa (DSM 70403) - Scaling Approaches.

Rhodotorula mucilaginosa was successfully applied as a biocatalyst for the enantioselective resolution of the racemic mixtures of heteroatom phosphonates derivatives, resulting in receiving the following enantiomers: (S)-1-amino-1(2-thienyl)methylphosphonic acid (Product 1) and (R)-1-amino-1-(3′pirydyl) methylphosphonic acid (Product 2). Biological synthesis of both products is reported for the first time. Pure (S)-1-amino-1-(2-thienyl)methylphosphonic acid (Product 1) was isolated with a conversion degree of 50% after 24 h of biotransformation was conducted on a laboratory scale under moderate conditions (1.55 mM of substrate 1, 100 mL of distilled water, 135 rpm, 25°C; Method A). The scale was enlarged to semi-preparative one, using a simplified flow-reactor (Method C; 3.10 mM of substrate 1) and immobilized biocatalyst. The product was isolated with a conversion degree of 50% just after 4 h of biotransformation. Amino-1-(3′pirydyl)methylphosphonic acid (Substrate 2) was converted according to novel procedure, by the immobilized biocatalyst - Rhodotorula mucilaginosa. The process was carried out under moderate conditions (3.19 mM – substrate 2 solution; Method C1) with the application of a simplified flow reactor system, packed with the yeasts biomass entrapped in 4% agar-agar solution. Pure (R)-amino-1-(3′pirydyl)methylphosphonic (50% of conversion degree) was received within only 48 h.

Process properties of an l-amino acid oxidase from Hebeloma cylindrosporum for the synthesis of phenylpyruvic acid from l-phenylalanine

The biocatalytic oxidation of amino acids represents an attractive approach towards the synthesis of α-keto acids, which are interest for various industrial applications. As l-amino acids are readily available from fermentation processes, these natural amino acids can serve as substrates in combination with an l-amino acid oxidase. Besides an aqueous phase as reaction medium, a further advantage of such a process is the utilization of air as oxidation agent. In this study, we studied the organic-synthetic properties of a literature-known recombinant l-amino acid oxidase from the fungus Hebeloma cylindrosporum with respect to its suitability to catalyze the formation of α-keto acids exemplified for the synthesis of phenylpyruvic acid starting from l-phenylalanine as a substrate. In our study the enzyme displayed a reasonable operational stability in the reaction system and as well as promising applicability data with respect to substrate and product inhibition. In a biotransformation, 20 mM of substrate were converted after 4 h reaction. The formation of undesired by-products was suppressed using a commercially available catalase enzyme.

Immobilization of Cholesterol Oxidase from Streptomyces Sp. on Magnetite Silicon Dioxide by Crosslinking Method for Cholesterol Oxidation.

Enzymatic biosensor has been paid much attention to the research fields due to its advantage in medical application. As one of the application, we determined the optimum value of cholesterol oxidase against cholesterol. In this work, we studied the behavior of cholesterol oxidation by enzymatic reaction to get the optimum condition for cholesterol oxidation. The enzyme that used were in two form, free cholesterol oxidase, and immobilized cholesterol oxidase. Cholesterol oxidase was produced from Streptomyces sp. by using solid state fermentation method and identified had high enzyme activity to be 5.12U/mL. Cholesterol oxidase was simultaneously crosslinked immobilized onto magnetite coated by silicon dioxide (M-SiO2). The support was characterized by Fourier transform infrared (FTIR) to determine the functional group of modified particle and scanning electron microscope (SEM) to observe the morphological or our prepared particle. Cholesterol oxidase sensitivity to substrate was analyzed by using HPLC with different interval time measurements. The oxidation of cholesterol by free enzyme and immobilized enzyme was also investigated. The best sensitivity of cholesterol oxidase was estimated to oxidize Cso (concentration of substrate) 1.46mM of substrate with Ce (concentration of enzyme) 20mg/mL for 180min. Final oxidation value of cholesterol by immobilized enzyme was greater than 60%. The results of this study revealed that immobilized enzyme for cholesterol oxidation was stable, reproducible, and sensitive.

Sound insulation of lightweight extensive green roofs

This paper examines factors affecting the sound insulation of lightweight extensive green roofs. The research had three objectives: (1) To examine the extent to which the sound insulation of lightweight extensive green roofs is affected by the variable conditions of their substrate and vegetation layer (soil's distribution, water content and compaction level); (2) To quantify how sound insulation is affected by the main elements making up the roof (substrate and vegetation layer, drainage layer and added cavity); (3) To identify applications for such roofs in relation to their sound insulation performance. For objective 1, it was found that the variable conditions of the substrate and vegetation layer do not significantly affect the sound insulation properties of lightweight extensive green roofs (variations in Rw of no more than 1 dB). For objective 2, results indicated that the addition of a cavity represents the most effective solution for improving sound insulation (+13 dB in Rw for a 50 mm deep cavity with mineral wool), whilst increasing the amount of substrate or using heavier drainage layers tend to provide more limited improvements in Rw (+3 dB for 25 mm of substrate and +2 dB for gravel vs. lightweight drainage membrane). Finally, regarding objective 3, the systems tested were found to provide appropriate sound insulation for both commercial and residential types of constructions. All the results were obtained from tests carried out on small 1 m2 samples, so their accuracy and validity will need to be identified through comparisons with large scale tests.

A chronoamperometric screen printed carbon biosensor based on alkaline phosphatase inhibition for W(IV) determination in water, using 2-phospho-L-ascorbic acid trisodium salt as a substrate.

This paper presents a chronoamperometric method to determine tungsten in water using screen-printed carbon electrodes modified with gold nanoparticles and cross linked alkaline phosphatase immobilized in the working electrode. Enzymatic activity over 2-phospho-l-ascorbic acid trisodium salt, used as substrate, was affected by tungsten ions, which resulted in a decrease of chronoamperometric current, when a potential of 200 mV was applied on 10 mM of substrate in a Tris HCl buffer pH 8.00 and 0.36 M of KCl. Calibration curves for the electrochemical method validation, give a reproducibility of 5.2% (n = 3), a repeatability of 9.4% (n = 3) and a detection limit of 0.29 ± 0.01 μM. Enriched tap water, purified laboratory water and bottled drinking water, with a certified tungsten reference solution traceable to NIST, gave a recovery of 97.1%, 99.1% and 99.1% respectively (n = 4 in each case) and a dynamic range from 0.6 to 30 μM. This study was performed by means of a Lineweaver–Burk plot, showing a mixed kinetic inhibition.

Lipase from Carica papaya latex presents high enantioselectivity toward the resolution of prodrug (R,S)-2-bromophenylacetic acid octyl ester

Abstract Besides the well-known papain, lipolytic activity is another interesting enzymatic activity present in latex from Carica papaya . This lipolytic activity is strongly attached to the latex solid phase, resulting in a naturally immobilized biocatalyst. In this work we describe the kinetic resolution of ( R , S )-2-bromophenylacetic acid octyl ester by Carica papaya crude latex and two partially purified latex fractions. Several parameters, such as substrate concentration and solvent effects were studied. The best results were obtained using decane as solvent with 50 mM of substrate and 50 mg/mL enzyme/reaction medium; under these conditions, a high enantioselectivity ( E >200) was obtained with crude latex. A twofold increase of the initial rate maintaining E >200 was obtained using purified fractions without protease and without esterase. Lipase from Carica papaya latex is the most enantioselective wild-type enzyme ever described for the studied reaction.

Asymmetric reduction of (4R)-(?)-carvone catalyzed by Baker's yeast in aqueous mono- and biphasic systems

Abstract (1 R ,4 R )-dihydrocarvone ( 2 ), an important renewable building block, was prepared with good conversions and excellent diastereoisomeric excess through the reduction of the α,β-unsatured carbon–carbon double bond of (4 R )-(−)-carvone ( 1 ) mediated by Baker's yeast (BY) in aqueous mono- and biphasic systems. Some parameters that may alter this bioreduction reaction, such as the concentrations of yeast and substrate, temperature, and pH, were evaluated. The effect of the addition of different additives on the course of 1 biotransformation was also investigated. The results showed that the conversion and diastereoisomeric excesses were strongly dependent on these variables. The optimum reaction conditions were 100 g L −1 of BY, 16.6 mM of substrate, and pH 7.5 at 26 °C in the presence of DMSO, trehalose, or sucrose as additives. Under the optimum conditions, the (1 R ,4 R )-dihydrocarvone was recovered with diastereoisomeric excesses of 92–99% and with conversions of 70–74%.

Microfluidic device to investigate factors affecting performance in biosensors designed for transdermal applications

In this work we demonstrate a novel microfluidic based platform to investigate the performance of 3D out-of-plane microspike array based glucose and lactate biosensors. The microspike array was bonded with a glass slide and modified with glucose oxidase or lactate oxidase using covalent coupling chemistry. An epoxy-polyurethane based membrane was used to extend the linear working range (from 0 to 25 mM of substrate) of these biosensors. Both lactate and glucose sensors performed well in the clinically relevant substrate concentration range. Glucose microspikes were further investigated with respect to the effects of substrate transfer by incorporation into a microfluidic system. Data from the microfluidic system revealed that the sensor response is mainly dependent on enzyme kinetics rather than membrane permeability to glucose. The robustness of the sensors was demonstrated by its consistency in performance extending over 48 h.

Green Roof Plant Responses to Different Substrate Types and Depths under Various Drought Conditions

Plants suitable for extensive green roofs must tolerate extreme rooftop conditions, and the substrates in which they grow must fulfill horticultural and structural requirements. Deeper substrates may retain more water for plants during dry periods, but will also weigh more, especially when near saturation. A study in central Pennsylvania was conducted to evaluate the influence of substrate type and depth on establishment of five green roof plants. Two stonecrops [white stonecrop (Sedum album) and tasteless stonecrop (Sedum sexangulare)], one ice plant (Delosperma nubigenum), and two herbaceous perennials [maiden pink (Dianthus deltoides) and saxifrage pink (Petrorhagia saxifraga)] were planted in three depths (30, 60, and 120 mm) of two commercially available green roof substrates (expanded shale and expanded clay). Study flats inside a plasticulture tunnel received three drought treatments (no drought, 2 weeks early drought, and 2 weeks late drought). The two stonecrops performed well under most conditions, although tasteless stonecrop was stunted by early drought. Ice plant only grew well when provided with water. When subjected to any drought, the herbaceous perennials had the fewest survivors in the expanded shale. Saxifrage pink flowered profusely wherever it survived. The study plants were most affected by substrate depth, except for maiden pink, which responded solely to drought. When subjected to early drought conditions, the herbaceous perennials did not survive in 30 mm of either substrate, or in 60 mm of expanded shale. Although the stonecrops performed well in 60 mm of substrate when subjected to drought, their performance was superior in the expanded clay compared with shale.

Peroxisomal and Mitochondrial Oxidation of Fatty Acids in the Heart, Assessed from the 13C Labeling of Malonyl-CoA and the Acetyl Moiety of Citrate

We previously showed that a fraction of the acetyls used to synthesize malonyl-CoA in rat heart derives from partial peroxisomal oxidation of very long and long-chain fatty acids. The 13C labeling ratio (malonyl-CoA)/(acetyl moiety of citrate) was >1.0 with 13C-fatty acids, which yields [13C]acetyl-CoA in both mitochondria and peroxisomes and < 1.0 with substrates, which yields [13C]acetyl-CoA only in mitochondria. In this study, we tested the influence of 13C-fatty acid concentration and chain length on the labeling of acetyl-CoA formed in mitochondria and/or peroxisomes. Hearts were perfused with increasing concentrations of labeled docosanoate, oleate, octanoate, hexanoate, butyrate, acetate, or dodecanedioate. In contrast to the liver, peroxisomal oxidation of 1-13C-fatty acids in heart does not form [1-13C]acetate. With [1-13C]docosanoate and [1,12-13C2]dodecanedioate, malonyl-CoA enrichment plateaued at 11 and 9%, respectively, with no detectable labeling of the acetyl moiety of citrate. Thus, in the intact rat heart, docosanoate and dodecanedioate appear to be oxidized only in peroxisomes. With [1-13C]oleate or [1-13C]octanoate, the labeling ratio >1 indicates the partial peroxisomal oxidation of oleate and octanoate. In contrast, with [3-13C]octanoate, [1-13C]hexanoate, [1-13C]butyrate, or [1,2-13C2]acetate, the labeling ratio was <0.7 at all concentrations. Therefore, in rat heart, (i) n-fatty acids shorter than 8 carbons do not undergo peroxisomal oxidation, (ii) octanoate undergoes only one cycle of peroxisomal beta-oxidation, (iii) there is no detectable transfer to the mitochondria of acetyl-CoA from the cytosol or the peroxisomes, and (iv) the capacity of C2-C18 fatty acids to generate mitochondrial acetyl-CoA decreases with chain length.

THE EFFECT OF SUBSTRATE DEPTH AND SUPPLEMENTARY WATERING ON THE GROWTH OF NINE HERBACEOUS PERENNIALS IN A SEMI-EXTENSIVE GREEN ROOF

THE EFFECT OF SUBSTRATE DEPTH AND SUPPLEMENTARY WATERING ON THE GROWTH OF NINE HERBACEOUS PERENNIALS IN A SEMI-EXTENSIVE GREEN ROOF

Simultaneous synthesis of enantiomerically pure ( S)-amino acids and ( R)-amines using α/ω-aminotransferase coupling reactions with two-liquid phase reaction system

An efficient simultaneous synthesis of enantiopure ( S)-amino acids and chiral ( R)-amines was achieved using α/ω-aminotransferase (α/ω-AT) coupling reaction with two-liquid phase system. As, among the enzyme components in the α/ω-AT coupling reaction systems, only ω-AT is severely hampered by product inhibition by ketone product, the coupled reaction cannot be carried out above 60 mM substrates. To overcome this problem, a two-liquid phase reaction was chosen, where dioctylphthalate was selected as the solvent based upon biocompatibility, partition coefficient and effect on enzyme activity. Using 100 mM of substrates, the AroAT/ω-AT and the AlaAT/ω-AT coupling reactions asymmetrically synthesized ( S)-phenylalanine and ( S)-2-aminobutyrate with 93% (>99% ee S ) and 95% (>99% ee S ) of conversion yield, and resolved the racemic α-methylbenzylamine with 56% (95% ee R ) and 54% (96% ee R ) of conversion yield, respectively. Moreover, using 300 mM of 2-oxobutyrate and 300 mM of racemic α-methylbenzylamine as substrates, the coupling reactions yielded 276 mM of ( S)-2-aminobutyrate (>99% ee) and 144 mM of ( R)-α-methylbenzylamine (>96% ee) in 9 h. Here, most of the reactions take place in the aqueous phase, and acetophenone mainly moved to the organic phase according to its partition coefficient.

Modification of analytical procedures for determining vitamin C enzyme (L-gulonolactone oxidase) activity in swine liver

Modifications of the analytical method to determine L-gulono-γ-lactone oxidase (EC 1.1.8) enzyme activity were conducted in pig liver by evaluating the concentration of added substrate (L-gulono-γ-lactone), glutathione, and various tissue sample-to-buffer ratios in the incubation mixture. Sampling different liver sites (lobes), the effect of different cooling temperatures of the liver immediately after collection, and the effect of tissue storage length on subsequent enzyme activity were evaluated. Our results demonstrated that 10 mM of substrate added to the reaction media maximized L-gulono-γ-lactone oxidase enzyme activity, whereas increasing levels of glutathione did not greatly affect enzyme activity. High sample-to-buffer ratios resulted in higher L-gulono-γ-lactone oxidase activities but sample analytical variations and background interferences were greater. A 1:4 tissue sample to buffer ratio (weight:weight) resulted in repeatable values, but the importance of maintaining the same ratio of the two components seems to be critical within an experiment. Expressing L-gulono-γ-lactone oxidase enzyme activity on a liver protein rather than on a liver weight basis also resulted in more consistent results. No difference in liver L-gulono-γ-lactone oxidase enzyme activities or ascorbic acid concentrations occurred between liver lobes. L-gulono-γ-lactone oxidase enzyme activity from 0 to 90 day of storage was not affected when tissue samples were immediately frozen in liquid nitrogen, or placed on crushed ice. During a 90-day storage the oxidized form of ascorbic acid (dehydroascorbic acid) decreased (P < 0.01), the reduced (ascorbic acid) form increased (P < 0.01), while total ascorbic acid concentration remained constant.

Response surface methodological study of esterification of lactic acid with palmitic acid catalyzed by immobilized lipases from Mucor miehei and porcine pancreas

s), enzyme/substrate (E/S) ratios (X e) and incubation time (X t) on the preparation of palmitoyl lactic acid using Mucor miehei lipase (MML) and porcine pancreas lipase (PPL). Experimental ester yields were found to be in good agreement with predictions. In the case of MML, the ester yield was found to increase with X e and X t and a maximum yield of 27 mM was obtained at X e=128 activity units(AU)/mM of substrate, X s=64–74 mM and X t=72 h. In the case of PPL, the ester yield was found to increase with increases in X e and X s. Optimum conditions for PPL were X e=45 AU/mM, X s=85–90 mM and X t=72 h, at which the maximum ester yield of 15 mM was obtained.

Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase.

The aminopeptidase activity of Phaseolus vulgaris seeds was measured using L-Leu-p-nitroanilide and the L-aminoacyl-ss-naphthylamides of Leu, Ala, Arg and Met. A single peak of aminopeptidase activity on Leu-ss-naphthylamide was eluted at 750 microS after gradient elution chromatography on DEAE-cellulose of the supernatant of a crude seed extract. The effluent containing enzyme activity was applied to a Superdex 200 column and only one peak of aminopeptidase activity was obtained. SDS-polyacrylamide gel electrophoresis (10%) presented only one protein band with molecular mass of 31 kDa under reducing and nonreducing conditions. The aminopeptidase has an optimum pH of 7.0 for activity on all substrates tested and the highest Vmax/K M ratio for L-Leu-ss-naphthylamide. The enzyme activity was increased 40% by 0.15 M NaCl, inhibited 94% by 2.0 mM Zn2+, inhibited 91% by sodium p-hydroxymercuribenzoate and inhibited 45% by 0.7 mM o-phenanthroline and 30 microM EDTA. Mercaptoethanol (3.3 mM), dithioerythritol (1.7 mM), Ala, Arg, Leu and Met (70 microM), p-nitroaniline (0.25 mM) and ss-naphthylamine (0.53 mM) had no effect on enzyme activity when assayed with 0.56 mM of substrate. Bestatin (20 microM) inhibited 18% the enzyme activity. The aminopeptidase activity in the seeds decayed 50% after two months when stored at 4 degrees C and room temperature. The enzyme is leucyl aminopeptidase metal- and thiol group-dependent.

Molecular Mechanisms for the Hepatic Uptake of Magnetic Resonance Imaging Contrast Agents

The mechanisms were investigated for the hepatic transport of 4 different gadolinium complexes used as contrast agents for magnetic resonance imaging (MRI). In basolateral rat hepatocyte plasma membrane vesicles, Gd-DTPA uptake was indistinguishable from non-specific binding to vesicles; Gd-BOPTA and Gd-EOB-DTPA entered plasma membrane vesicles following a linear, concentration-dependent mechanism up to 1.5 mM of substrate. By contrast, Gd-B 20790 uptake followed a saturative kinetic with an apparent Kmof 92 ± 15 μM and a Vmaxof 143 ± 42 pmol/mg prot/15 sec, and it occurred into an osmotic-sensitive space. Sulfobromophthalein ant taurocholate, but not unconjugated bilirubin inhibited the uptake rate of Gd-B 20790 but not that of the other three compounds. Injection intoXenopus laevisoocytes of 5 ng of human OATP cRNA resulted, after 3 days, in a ≥2-fold stimulation (p < 0.001) of transport of Gd-B 20790 but not of Gd-BOPTA or Gd-EOB-DTPA. Collectively, these data indicate that the hepatic uptake of the MRI contrast agent Gd-B 20790 is a carrier-mediated mechanism operated by OATP while MRI compounds with other chemical structures enter the hepatocyte by other mechanisms.

Bone alkaline phosphatase kinetics studied by a new method.

Studies of enzyme kinetics by histochemical methods has been seriously impaired by available methods. This study combines two newer methods, i.e., sectioning undecalcified bone from glycol methacrylate and the use of atomic absorpton spectrophotometry for photographic densitometry. The results indicate that the substrate concentration for zero order kinetics of the alkaline phosphatase reaction in bone is 0.5 mg/ml of substrate; and the Km of the same reaction is estimated to be 0.25 mg/ml or 0.56 mM of substrate.

Histochemical demonstration of N-acetyl-beta-galactosaminidase in the human tissues.

N-acetyl-β-galactosaminidase was first demonstrated by the histochemical method using naphthol AS-BI N-acetyl-β-galactosaminide as a substrate and hexazonium pararosanilin as a diazo reagent. The optimal staining reaction for the human tissues was obtained with 0.5mM of substrate and 3.6mM of hex-azonium pararosanilin at pH 4.4 for 100 minutes at 37°C. The dark red dye was seen mostly as discrete granules at the site of the enzyme activity. Generally, cytological localization of the enzyme in the human tissues was quite similar to that of other lysosomal enzymes.