Determination of the Activity of Immobilized Biocatalysts in the Case of Poorly Soluble Substrates: The Example of D-5Phenylhydantoinase

The present communication describes the determination of activity of immobilized biocatalysts, from progress curves, in the case of a poorly soluble substrate. Computer simulation is used for the comparison of five well known methods for the determination of initial velocity of the reaction with the modified method of smoothing experimental data by cubic spline. The computer simulation data show that, in the case of poorly soluble substrate, it is expedient to use the method of data linearization in the co-ordinate system p/t versus p and method of splines.     

Modeling and simulation of a pressure-swing reactor for the conversion of poorly soluble substrate by immobilized enzyme: the case of d-hydantoinase reaction

An immobilized enzyme reactor system for converting poorly soluble substrate is proposed. In this stirred batch reactor, the solid substrate and immobilized enzyme suspensions are separated by a microporous filter. The advantage of separating the solid substrate from immobilized enzyme is that the fouling and breakage of the immobilized enzyme usually encountered in the stirred tank reactor can be prevented. Pressure swing can be applied to enhance the mass transfer between the two compartments. The hydrolytic reaction converting the poorly soluble substrate p-hydroxyphenylhydantoin (pHPH) into soluble N-carbamoyl-p-d-hydroxyphenylglycine (CpHPG) by immobilized d-hydantoinase is carried out in this reactor. The performance of this pressure-swing reactor is studied by simulation using a simple kinetic model. The pressure-swing operation increases the overall production rate significantly. The pressure swing also makes the reactor perform better for converting the solid substrate at higher concentration.     

Fast determination of operational stability of the soluble acetylacetone-cleaving enzyme Dke1 in an enzyme membrane reactor

The main aim of this study was the determination of the operational stability of soluble Dke1 (EC 1.13.11.50) in an enzyme membrane reactor. In order to calculate the half-life of soluble Dke1, the K _M of oxygen must be known. The determination of this constant was done using progress curve analysis (K _M=260 μmol l⁻¹). In a next step, the reactor system was studied by building a mathematical model for calculation of the reactor system, using Berkeley Madonna ver. 8.0.1 software. After that, the determination of the half-life of Dke1 under operational conditions at different temperatures (5, 10, 15, 25, 30, 35°C) was performed. The quantitative criterion for stability was the value of the first-order rate constant of monomolecular inactivation. The experiments showed that soluble Dke1 is poorly stable. The half-life ranged from 308 min at 5°C to 9 min at 35°C. This method for determining the half-life is quite applicable for enzymes which are poorly stable. In addition, both the storage stability and the operational stability can be determined.     

A colorimetric method for the determination of lipase activity in soil

A colorimetric method for the determination of lipase activity in soil has been developed. Using p-nitrophenyl butyrate as substrate, soil samples are incubated at 30°C and pH 7.25 for 10 min. After cooling on ice and centrifugation, the released p-nitrophenol is determined at 400 nm. To allow for the adsorption of p-nitrophenol onto soil, a calibration curve is prepared in the presence of soil.     

Isolation of phospholipase d producing microorganisms with high transphosphatidylation activity

The transphosphatidylation and hydrolytic activities of phospholipase d in culture supernatants of soil isolates were evaluated by a specific spectrophotometric method for quantitative determination using an artifical substrate, phosphatidyl-p-nitrophenol. Phospholipase d from strain TH-2 showed the highest specific activity and ratio of transphosphatidylation activity to hydrolytic activity among those from the eight soil isolates and commercial Actinomycetes phospholipase d.     

Characterization of recombinant human renin: Kinetics,pH-stability, and peptidomimetic inhibitor binding

The kinetic behavior andpH-stability of recombinant human renin was analyzed using a new fluorogenic substrate based on the normal P₆-P₃ renin cleavage sequence in human angiotensinogen. The design of this fluorogenic substrate makes possible, for the first time, direct monitoring of the kinetics of proteolytic conversion of prorenin to renin. ThepH-stability profile for renin, measured with the substrate at 25°C, indicated a broad plateau of stability betweenpH 6.0 and 10.0. Analysis of thepH-activity profile of renin for the substrate indicated a minimumK _m (1.8 µM) atpH 7.4 and a maximumV _m betweenpH 7.4 and 8.0. The thermodynamics of the binding of a novel, soluble, peptidomimetic inhibitor to renin indicated it is possible to retain the tight-binding characteristics and enthalpy contributions to binding of larger peptide-derived inhibitors, while reducing inhibitor size and entropic contributions to binding. A novel derivative of the fluorogenic substrate, containing a 3-methyl histidine substitution at the P₂ site, was used to test the recent hypothesis that renin functions by virtue of substrate-directed catalysis.     

Temperature and pH dependence of mold α-galactosidase

The effect of temperature and pH on kinetic behavior of α-galactosidase of Mortierella vinacea was investigated on the hydrolysis of p-nitrophenyl-α-D -galactopyranoside (PNPG). A very unusual kinetic behavior was observed for the soluble α-galactosidase i.e., substrate inhibition diminished gradually with increasing temperature or near the neutral pH range, and the kinetics approached the ordinary Michaelis-Menten (MM) type. On the other hand, with decreasing temperature or in acidic pH range, substrate inhibition was accelerated. Therefore, Arrhenius plots based on the initial reaction rate did not give straight lines. Furthermore, the slope in the Arrhenius plot changed with substrate concentration, which would make the determination of a characteristic value using conventional methods meaningless. However, the Arrhenius plots of individual kinetic parameters in the rate equation resulted in straight lines in the temperature range 15 to 50°C. From this, the drastic change in kinetic behavior could be explained in connection with the temperature and pH dependence of kinetic parameters in the model. For mold pellets (whole-cell enzyme), however, the influence of temperature and pH was less apparent than that of soluble enzyme because of the limitation in intraparticle diffusion. By using the rate equation that was determined for soluble enzyme and the theoretically derived effectiveness factor, the overall reaction rate for mold pellets at various temperature and pH could be predicted to some extent.     

Radiochemical high-performance liquid chromatographic assay for the determination of catechol O-methyltransferase activity towards various substrates

A new chromatographic catechol O-methyltransferase (COMT) assay based on S-adenosyl- -[methyl-¹⁴C]methionine and on-line radioactivity detection was developed. With minor modifications in the mobile phase composition the methylation velocities for 30 structurally diverse compounds including simple catechols, neurotransmitters, catecholestrogens and catecholic drugs could be measured using human and rat recombinant soluble COMT. The enzymes showed very similar substrate selectivities. The radiochemical method was validated using 3,4-dihydroxybenzoic acid as a model substrate and it was shown that accurate and reproducible methylation velocity values could be achieved for both of the catecholic hydroxyls. The method proved to be suited for determining the enzyme kinetic parameters and can probably be further used for gathering enzyme kinetic data on differentially substituted catechols in order to construct proper structure-activity relationships for COMT.     

Formation of Inclusion Complexes of Cycldextrin with Ethanol under Anhydrous Conditions

Complex formation of poorly water soluble organic compounds with cyclodextrin (CD) is quite difficult in an aqueous cyclodextrin system. Formation of the inclusion complex of d-limonene, phenyl ethanol, acetophenone, or menthol was investigated in a slurry form of α-, β-, or γ-CD in organic solvents or alcohol under anhydrous conditions. Ethanol and methanol were found to be good solvents for this method. The use of ethanol as the solvent was investigated in greater detail. There existed an optimal amount of ethanol for the maximum inclusion of d-limonene as the guest compound. However, an excess of ethanol inhibited the inclusion. An adsorption model of alcohol on CD, analogous to the substrate inhibition model of enzyme kinetics, could correlate the inclusion ratio with the amount of alcohol added to CD.     

Characterization of soluble thrombomodulin levels in patients with stage 3–5 chronic kidney disease

Abstract

Objective: This study aims to test the serum levels of soluble thrombomodulin (TM) in patients with chronic kidney disease (CKD)3–5 and to assess their connection with the different stages and severity of disease.

Methods: Sixty-seven patients with CKD are included, disease severity was evaluated accordingly to CKD staging and clinical data is collected. Nineteen healthy volunteers served as healthy controls. Serum soluble TM is analyzed by ELISA.

Results: The levels of soluble TM in all patients with CKD were significantly higher than those of healthy controls (p?<?0.001). CKD5 patients showed higher serum levels of soluble TM, in comparison to CKD4 patients (p?=?0.001), CKD3 patients (p?<?0.001), and healthy controls (p?<?0.001). The correlation analysis revealed significant correlation between serum soluble TM and disease severity (r?=?0.714, p?<?0.001). Serum soluble TM was found to be correlated with eGFR (r?=??0.766; p?<?0.001) and serum creatinine (r?=?0.778, p?<?0.001).

Conclusion: Soluble TM concentrations significantly increase in the CKD patients and are associated with the severity of the disease. Soluble TM may play critical roles in the development of CKD, as a biomarker of endothelial cells damage, anticoagulation and anti-inflammation.     

Esterase activity assay by flow injection analysis (FIA)

An automatic flow injection analysis (FIA) system for on-line determination of esterase activity has been developed. It is based on a colorimetric method using p-nitrophenyl propionate as substrate. The system permits a linear range analysis up to 0.18 U ml^–1, although the range can be extended up to 1 U ml^–1 without external dilution of the sample. The sampling frequency is of 4 samples per h with a relative standard deviation of 0.9%.     

Measurement of feruloyl/p-coumaroyl esterase by capillary zone electrophoresis

Ferulic andp-coumaric acid can be separated from their corresponding aliphatic methyl esters by capillary zone electrophoresis, which allows the convenient determination of feruloyl andp-coumaroyl esterase activities using synthetic esters as substrates. A feruloyl-containing sugar ester from wheat bran was also efficiently separated and used as substrate for the enzyme assays.Penicillium expansum was shown to produce feruloyl/p-coumaroyl esterase activity when grown on wheat bran in solid-state culture.The authors are with the Food Microbiology Research Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5PX, UK; A.M. McKay is also affiliated with the Department of Food Science (Microbiology), The Queen's University of Belfast, Newforge Lane, Belfast BT9 5PX, UK.     

Microrecirculation reactor system for characterization of immobilized enzymes

A differential microrecirculation reactor was developed for kinetic analysis of both soluble and immobilized enzymes. The reactor system was easliy fabricated with in the laboratory from readily available materials. The disposable, small reactors allowed for in situ weight determination of the enzyme beads. Routinely, only a 1 ml liquid volume of substrate was used for each kinetic assay. The reactor system was also used for determination of partition coefficients. Both uses of the reactor system required only 5–10 min for completion of a given determination.     

The direct determination of the enantiomers of ketorolac and parahydroxyketorolac in plasma and urine using enantioselective liquid chromatography on a human serum albumin-based chiral stationary phase

An enantioselective assay has been developed for the determination of the enantiomers of ketorolac and its metabolite p-hydroxyketorolac in plasma and urine. The analytical method utilizes a coupled achiral–chiral HPLC system where the initial separation of ketorolac from p-hydroxyketorolac and matrix interferences was achieved on a C₁₈-stationary phase and the enantioselective separations of the two target solutes were accomplished on a human serum albumin-based chiral stationary phase. The two columns were attached in sequence and the assay was carried out without the necessity of column-switching techniques. The method has been validated for use in pharmacokinetic and metabolic studies and represents the initial report of the determination of ketorolac and p-hydroxyketorolac enantiomers in urine. The results of the study indicate that after the administration of racemic ketorolac there was an enantioselective distribution of ketorolac enantiomers in plasma [(R)-ketorolac: (S)-ketorolac = 3.89 ± 0.93 (n = 6) and urine (R)-ketorolac: (S)-ketorolac = 1.26 ± 0.09 (n = 7)]. The mean ratio of the p-hydroxyketorolac enantiomers was 1.77 ± 0.46 (n = 7). Both ketorolac and p-hydroxyketorolac are glucuronized in the acyl carboxyl moiety and the results of this study indicate that this process is not enantiospecific. © 1994 Wiley-Liss, Inc.     

Determination of hammerhead ribozyme kinetic constants at high molar ratio ribozyme-substrate

 Hammerhead ribozymes provide valuable tools in the field of gene therapy due to their cleavage specificity and the broad range of RNA targets. A major prerequisite for the selection of suitable ribozymes for in vivo application is represented by in vitro determination of ribozyme cleavage kinetic constants. From the experimental cleavage data, kinetic constants are usually calculated under the assumption of rapid conversion of the substrate into the ribozyme-substrate complex. However, this condition is often not satisfied for ribozymes carrying additional RNA stretches, due to cloning strategies or necessary for ribozyme expression in the cell. To overcome this problem, we propose a mathematical model which is able to calculate ribozyme kinetic constants in the case of non-rapid conversion of substrate into ribozyme-substrate complex. In addition, our system gives the opportunity to evaluate the nature of the S conversion into ES through the determination of a model parameter. The validity of the proposed model is restricted to the hypothesis of a ribozyme excess over the substrate at the beginning of the cleavage reaction and to the absence of any mass exchange with the external environment. Received: 1 February 2001 / Revised version: 1 September 2001 / Published online: 23 August 2002     

Determination of the Ks values during the growth of Alcaligenes eutrophus on phenol, 2,4-dichlorophenoxyacetic acid and fructose

The determination of the K_S values presented here is based on the estimation of the stationary substrate concentrations in continuous cultivation experiments. The separation of biomass from the suspension was performed by an ultrafiltration step which succeeded within one second. The decay of substrate concentration during sampling was calculated to amount to less than 6% of the stationary substrate concentration at relevant growth rates. The K_S values derived from these reduced substrate concentrations deviated by only 10% from the theoretical values at a biomass concentration of about 1 g/1. Thus relevant kinetic parameters can be calculated from the data obtained by this procedure. Values of 11, 59 and 14 μM were obtained with 2,4-dichlorophenoxyacetic acid (2,4-D), phenol and fructose, respectively. Similar K_S values were derived with 2,4-D and fructose by using a respirationbased determination for reasons of comparison. With phenol this value was only 7 μM which is as cribed to a physiological background.     

Non-Involvement of the Human Monocarboxylic Acid Transporter 1 (MCT1) in the Transport of Phenolic Acid

Phenolic acids such asp-coumaric acid and microbial metabolites of poorly absorbed polyphenols are absorbed by the monocarboxylic acid transporter (MCT)-mediated transport system which is identical to the fluorescein/H⁺ cotransport system. We focus here on the physiological impact of MCT-mediated absorption and distribution. We examined whether MCT1, the best-characterized isoform found in almost all tissues, is involved in this MCT-mediated transport system. The induction of MCT1 expression in Caco-2 cells by a treatment with sodium butyrate (NaBut) did not increase the fluorescein permeability. Moreover, the transfection of Caco-2 cells with an expression vector encoding MCT1 caused no increase in either the permeability or uptake of fluorescein. Furthermore, in the MCT1-expressing oocytes, no increase ofp-coumaric acid uptake was apparent, whereas the uptake of salicylic acid, a substrate of MCT1, nearly doubled. Our data therefore establish that MCT1 was not involved in the MCT-mediated transport of phenolic acids.     

The microbial degradation of chlorophenolic preservatives in spent,pressure-treated timber

The reduction of pentachlorophenol in treated timber, after inoculation with pentachlorophenol-degrading bacterial species,Rhodococcus chlorophenolicus andFlavobacterium sp., and the white-rot fungusPhanerochaete chrysosporium, was monitored in solid substrate systems and in liquid culture suspensions. In solid substrate systems there was no significant pentachlorophenol degradation by the bacterial species under a variety of conditions. Under similar conditions,Phanerochaete chrysosporium transformed over 80% of the starting concentration of 500 ppm to pentachloroanisole. In liquid culture suspensions however, mid-exponential phaseFlavobacterium sp. cells were able to degrade over 99% of the pentachlorophenol in sawdust and wood chips due to the extraction of PCP from the timber as a water soluble salt. There were however no significant changes in the chlorinated dioxin components during this treatment.Abbreviations ATTC American type culture collection - AWPA American Wood Preservers' Association - DSM Deutsche Sammlung für Mikroorganismen - GC/MS gas chromatograph/mass spectrometer - HpCDD heptachlorodibenzo-p-dioxin - HpCDF heptachlorodibenzofuran - HxCDD hexachlorodibenzo-p-dioxin - HxCDF hexachlorodibenzofuran - ¹³C-OCDD carbon 13-labelled octachlorodibenzo-p-dioxin - OCDD octachlorodibenzo-p-dioxin - OCDF octachlorodibenzofuran - PCDDs polychlorinated dibenzo-p-dioxins - PCDFs polychlorinated dibenzofurans - PCP pentachlorophenol - PnCDD pentachlorodibenzo-p-dioxin - PnCDF pentachlorodibenzofuran - TCDD tetrachlorodibenzo-p-dioxin - TCDF terachlorodibenzofuran - TeCP tetrachlorophenol - WHC water holding capacity - w/v weight for volume ratio     

A nonspecific nucleoside hydrolase from Leishmania donovani: implications for purine salvage by the parasite

In contrast to their mammalian hosts, protozoan parasites do not synthesize purines de novo, but depend on preformed nucleotides that they purportedly obtain by salvage pathways. Nucleoside hydrolases may play a crucial role in that salvage process. By screening Leishmania donovani libraries with polyclonal antibodies against promastigote soluble exo-antigens, we have identified a cDNA encoding a protein with significant homology to nonspecific and uridine–inosine-preferring nucleoside hydrolases. Sequence comparison demonstrated that all the residues involved in Ca²⁺-binding and substrate recognition in the active site are conserved among the characterized protozoan nucleoside hydrolases. Genomic analysis suggests that it is a single copy gene in L. donovani, and its homologues are present in members representing other Leishmania species complexes. Both Northern blot and immunoblot analyses indicate that it is constitutively expressed in L. donovani promastigotes. The recombinant enzyme overexpressed in and purified from bacteria showed significant activity with all naturally occurring purine and pyrimidine nucleosides, and efficient utilization of p-nitrophenyl-β- -ribofuranoside as a substrate. Altogether, the sequence comparison and substrate specificity data identify this L. donovani nucleoside hydrolase as a nonspecific nucleoside hydrolase. Further, the nucleoside hydrolase was localized to specific foci in L. donovani promastigotes by immunofluorescent assays. Although the conservation of the nucleoside hydrolases among protozoan parasites offers promise for the design of broad-spectrum anti-parasitic drugs, the existence of multiple and distinct nucleoside hydrolases in a single species demands special consideration.     

Effect of cellobiose supplementation and dietary soluble fibre content on in vitro caecal fermentation of carbohydrate-rich substrates in rabbits

The in vitro caecal fermentation of five substrates low in starch and protein content [d-(+)-glucose (GLU), d-cellobiose (CEL), sugar beet pectin (PEC), sugar beet pulp (SBP) and wheat straw (WS)] was investigated using soft faeces from rabbits receiving different levels of cellobiose and soluble fibre as inoculum. A total of 24 rabbits were supplemented 3 levels of cellobiose in the drinking water (0.0, 7.5, 15.0 g/l) and fed two experimental diets containing either low soluble fibre (LSF) or high soluble fibre (HSF) levels (84.0 and 130 g/kg dry matter). All substrates were subjected to a two-step pepsin/pancreatin in vitro pre-digestion, and the whole residue was used as substrate for the in vitro incubations. Gas production was measured until 144 h, and volatile fatty acid (VFA) production was determined at 24 h incubation. Experimental treatments did not affect SBP fermentation and had only a subtle influence on fermentation of WS and GLU. In contrast, cellobiose supplementation × donors’ diet interactions were detected for most gas production parameters for CEL. Both the fractional gas production (k) and maximal gas production rates were linearly increased (p ≤ 0.042) and the initial delay in the onset of gas production (Lag) linearly decreased (p < 0.001) by cellobiose supplementation with the HSF inoculum, with no differences between the 7.5 and 15.0 doses. In contrast, with the LSF inoculum cellobiose supplementation only affected k values, which were quadratically increased (p = 0.043) and had maximal values for the 7.5 dose. A quadratic effect (p ≤ 0.018) of cellobiose supplementation was observed for total VFA production at 24 h when CEL and PEC were fermented, obtaining the maximal VFA production for the 7.5 dose of cellobiose. Total VFA production for CEL was greater with LSF than with HSF inoculum (20.7 vs. 12.9 mmol/l; p = 0.014), but the opposite was found for WS (3.97 vs. 6.21 mmol/l; p = 0.005). The use of LSF inoculum for CEL fermentation sharply reduced acetate (p = 0.001) and increased butyrate proportions (p ≤ 0.001) compared with the HSF inoculum. A positive relationship between total VFA caecal concentrations in rabbits receiving the same experimental treatments and in vitro values was only observed when WS was used as substrate (r = 0.90; p = 0.015; n = 6). The results suggest that experimental factors influenced the fermentative activity of caecal digesta, but the observed response differed with the incubated substrate, being the CEL the most affected.