Optimum Substrate Concentration Research Articles

Purification and characterization of the -amylase isolated from Penicillium camemberti PL21

The amylase family of enzymes is a great significance due to its wide area of potential application. α-amylase from Penicillium camemberti PL21 obtained from I. N. A. (The technological laboratory of Agriculture National Institute Paris- France) and using orange waste as substrates was produced under optimum conditions, after 168 h of incubation and subjected for purification and characterization. The enzyme was purified by ammonium sulfate precipitation, dialysis, Sephadex G-100 and DEAE-Sepharose CL-6B column chromatography. A trial for the purification of α-amylase resulted in an enzyme with specific activity of (154.2 units/ml/mg protein) with (38.5 folds) purification .The α-amylase activity increased by enzyme concentration rise. The optimum substrate concentration for soluble starch was 1 % (w/v) while the optimum incubation temperature was 30oC. The purified α-amylase enzyme had a maximum activity at pH 6 and the Km value for solublestarch was 0.92 mg/ml. Analyses of this enzyme for molecular mass was carried out by SDS-PAGE electrophoresis, which revealed one band 60. 5 Kda. Key words: Penicillium camemberti PL21, α-amylase, purification, SDS-PAGE electrophoresis.

Biotransformation of dl-lactate to pyruvate by a newly isolated Serratia marcescens ZJB-07166

The production of pyruvate using biotransformation from dl-lactate has been recently drawn more and more attentions due to the wide applications of pyruvate in chemicals, drugs, and agrochemicals industries. In the current study, a strain ZJB-07166, which was capable of converting dl-lactate to pyruvate, was newly isolated and characterized and later identified as Serratia marcescens based on the morphology, physiological tests, ATB system and its 16S rDNA sequence. The strain S. marcescens ZJB-07166 was applied in biotransformation of dl-lactate to pyruvate and the detailed time courses for cultivation and biotransformation were investigated. The optimum nitrogen source and carbon source in the microorganism culture for production of lactate dehydrogenase were NH 4Cl and dl-lactate, respectively. The optimum substrate concentration for biotransformation was around 40 mM and EDTA had an obvious stabilizing effect on pyruvate in biotransformation process. The pyruvate production concentration of 210 mM was achieved under the optimum conditions. These results demonstrated that the newly isolated S. marcescens ZJB-07166 was a promising strain for pyruvate production in industrial scale.

Production and characterization of protease enzyme from Bacillus laterosporus

Production and partial purification of protease enzyme by Bacillus laterosporus was the aim of this study. Bacillus laterosporus was allowed to grow in shake flask broth culture for purpose of inducing protease enzyme. The protease enzyme was purified by ammonium sulfate precipitation followed by dialysis and further concentrated by Amicon tubes. After concentration, the protein was subjected to 12 % Zymogram gel with gelatin and the molecular weight of the protease enzyme was 15 kDa. The protease activity increased as the increase in enzyme concentration; optimum substrate concentration (starch) was 1.0% (w/v); an optimum incubation temperature was 40°C. Purified protease enzyme had a maximum activity at pH 7.0 of phosphate buffer and the optimum incubation time was 24 h. The protease isolated from Bacillus latrosporus is a mesophilic protease. It is stable at pH 7, at 40°C temperatures and this enzyme can be exploited commercially.

The Substrate Concentration Effects on Biohydrogen Production in Continuous Stirred Tank Reactor

Substrate concentration effects on H2 production performance in a continuous stirred-tank reactor (CSTR) were investigated. Using molasses as the feeding, the CSTR system was operated at chemical oxygen demand (COD) of 2000-8000 mg/L to identify the optimal working substrate concentration. Increasing substrate concentration (2000~6000 mg/L) gave better biomass content and hydrogen production, signifying that the average cellular activity for H2 production may be enhanced as the substrate concentration increased. The overall maximal biogas and hydrogen production yield were 18.69 L and 6.01 L, respectively, both of them occurred at 6000 mg/L. The gas phase H2 content did not vary considerably regardless of changes in substrate concentration. This reflects that the CSTR was a relatively stable H2-producing system. The major soluble products from hydrogen fermentation were ethanol and acetic acid, accounting for 59% and 23% of total liquid fermentation products, respectively. Thus, the dominant H2 producers in the mixed culture belonged to acidogenic bacteria that underwent ethanol-type fermentation. However, the biomass content and hydrogen production yield tended to decrease as the substrate concentration increased to 8000 mg/L, suggesting that granular sludge formation and cellular activity for H2 production may be inhibited at high substrate concentration. Ethanol, acetic, butyric and propionic were the main liquid fermentation products with the percentages of 31%, 24%, 20% and 18%, which formed the mixed-type fermentation.

Electrochemical detection of transketolase activity using a tyrosinase biosensor

This paper proposes a new concept of transketolase (TK) activity profiling. A tyrosinase (PPO) biosensor, based on the immobilization of this enzyme in a Mg 2Al–Cl layered double hydroxide, was developed for the amperometric detection of N-acetyl- l-tyrosine ethyl ester monohydrate (N-Ac-Tyr-OEt) at −0.2 V. This compound was released during an enzymatic reaction catalyzed by TK with N-acetyl-O-(2 R, 3 S, 5-trihydroxy-4-oxopentyl)- l-tyrosine ethyl ester used as donor substrate. This tyrosinase biosensor was optimized for the detection of TK activity, including PPO optimum substrate concentration, electrolyte nature, pH, and influence of bovine serum albumin (BSA). It was found that N-Ac-Tyr-OEt release is dependent on TK concentration (U/mL) in the electrolyte medium. These results demonstrate the sensitivity and specificity of the tyrosinase biosensor designed for in vitro detection of TK activity, which is known to be involved in several diseases.

Effect of aldicarb insecticide formulation on its detection in postmortem human specimen

A growing body of evidence indicates that young animals exhibit an increased susceptibility to the lethal effects of cholinesterase (ChE)-inhibiting insecticides. Our laboratory is engaged in defining factors which may explain this age-related sensitivity. This report includes results from experiments designed to compare the developmental profiles, kinetic parameters and intrinsic (i.e. in vitro) sensitivity of developing male rat brain acetylcholinesterase (AChE) activity to carbamate and organophosphorus anticholinesterases. Total ChE activity in whole brain for each age was composed of about 90% AChE and 10% butyrylcholinesterase (BuChE) activity for the six ages examined. Brain AChE activity showed an age-related increase in Vmax until postnatal day 17 with no change in Km (average of all six ages ∼72 μM). Optimal substrate (acetylthiocholine) concentration for each age was 1 mM, and there was substantial (≈10%) substrate inhibition at 2.5 mM. IC50s (the concentration of compound that inhibits 50% of the AChE activity in 30 min at 26°C) defined concomitantly for postnatal day 4 and adult brain AChE using either aldicarb, carbaryl, chlorpyrifos-oxon or malaoxon were virtually identical at both ages with average IC50 values being: aldicarb=2.4 μM, carbaryl=1.7 μM, chlorpyrifos-oxon=4.9 nM and malaoxon=140 nM. In summary, AChE in young and adult brain differs mostly in specific activity while the Kms, substrate profiles, and in vitro sensitivity to selected anticholinesterase insecticides are not different. Therefore, these data support and extend the hypothesis that the greater sensitivity of the young animals to anticholinesterase pesticides is not due to the greater sensitivity of the target molecule AChE to these inhibitors.

Statistical optimization of biohydrogen production from sucrose by a co-culture of Clostridium acidisoli and Rhodobacter sphaeroides

Statistically based experimental designs were applied to optimize the fermentation process parameters for hydrogen (H 2) production by co-culture of Clostridium acidisoli and Rhodobacter sphaeroides with sucrose as substrate. An initial screening using the Plackett–Burman design identified three factors that significantly influenced H 2 yield: sucrose concentration, initial pH, and inoculum ratio. These factors were considered to have simultaneous and interdependent effects. A central composite design and response surface analysis were adopted to further investigate the mutual interactions among the factors and to identify the values that maximized H 2 production. The optimal substrate concentration, initial pH, and inoculum ratio of C. acidisoli to R. sphaeroides were 11.43 g/L sucrose, 7.13, and 0.83, respectively. Using these optimal culture conditions, substrate conversion efficiency was determined as 10.16 mol H 2/mol sucrose (5.08 mol H 2/mol hexose), which was near the expected value of 10.70 mol H 2/mol sucrose (5.35 mol H 2/mol hexose).

Hydroxynitrile lyase from Passiflora edulis: Purification, characteristics and application in asymmetric synthesis of (R)-mandelonitrile

A hydroxynitrile lyase from leaves of Passiflora edulis (PeHNL) was purified and characterized for the first time. The enzyme is a monomer of 15kDa and 18kDa by SDS-PAGE, and gel filtration, respectively. Asymmetric synthesis of (R)-mandelonitrile from benzaldehyde and acetone cyanohydrin in a biphasic system employing the PeHNL from rinds of P. edulis was carried out. Several parameters influenced the enantiomeric purity of the product and initial velocity of the reaction. Both pH and temperature were important parameters controlling the enantiomeric purity of the product. The optimum pH and temperature were pH 4 and 10°C, respectively. At the optimum pH and temperature, the spontaneous non-enzymatic reaction yielding the racemic mandelonitrile was almost completely suppressed. The PeHNL was stable (more than 80% residual activity after incubation for 12h) in the system of methyl-t-butyl ether (MTBE), dibutyl ether (DBE), hexane (HEX), and diisopropyl ether (DIPE) while diethyl ether (DEE) and ethyl acetate (EA) were not suitable solvents. The initial velocity was markedly affected by the type of organic solvent in the biphasic system, while high enantiomeric purity was obtained when organic solvents having logP lower than 3.5 were used. The highest initial velocity of reaction and enantiomeric purity of (R)-mandelonitrile were obtained in the biphasic system of DBE with the aqueous phase content of 30% (v/v). The optimum substrate concentrations were 250mM for benzaldehyde and 900mM for acetone cyanohydrin, and the optimum enzyme concentration was 26.7units/ml. The highest enantiomeric purity of (R)-mandelonitrile was successfully obtained with conversion and enantiomeric excess of 31.6% and 98.6%, respectively. The enzyme showed considerable reusability in batch reaction with high enantiomeric purity of product.Herein, we reported the characteristics of a unique (R)-PeHNL from leaves of P. edulis. The PeHNL from rinds had been isolated for the first time and the enzyme showed great ability in transcyanation of (R)-mandelonitrile with high e.e. in DBE as the co-organic solvent in a biphasic system.

Optimization of luminescent assay for screening of cyclin-dependent kinase 2 inhibitors

Cyclin-dependent kinases are most extensively studied targets for cancer chemotherapy since the tumor cells exhibit false checkpoints and can proliferate even if the genome is compromised. Cyclin-dependent kinases ensure the tight regulation of the cell cycle execution by mediating phosphorylation of cellular proteins. Deregulation of the cyclin-dependent kinase 2 activity by cellular and external factors leads to many diseases like cancers. Different methods like radiolabeled, fluorescence and luminescence are available for screening of library of compounds against kinases. However, bioluminescent methods offer several advantages like low background and no effect of fluorescent compound interference. Present study is focused on development, optimization and validation of cyclin-dependent kinase 2 assay which is suitable for identification potent and selective, ATP competitive and non-competitive inhibitors of cyclin-dependent kinase 2. The aim of present investigation was to optimize the assay for cyclin-dependent kinase 2/cylin A and cyclin-dependent kinase 2/cyclin E with use of bioluminescence based biochemical reaction. Both cyclin-dependent kinase 2 which are cyclin-dependent kinase 2/cyclin A and cyclin-dependent kinase 2/cyclin E complexes, have different affinity for ATP. Therefore, both isoform analogs of cyclin-dependent kinase 2 were optimized separately. Optimum cyclin-dependent kinase 2/cyclin A and cyclin-dependent kinase 2/cyclin E concentration were found to be 250 ng/well and 200 ng/well, respectively. Optimum substrate (histone H1) concentration was found to be 2.5 mg/ml for both cyclin-dependent kinase 2 analogs. Optimum reaction time was found to be 20 min for both cyclin-dependent kinase 2/cyclin complexes.

Ionic liquids for enhancing the enantioselectivity of isolated BVMO-catalysed oxidations

The present study describes the first-time usage of an isolated thermostable Baeyer–Villiger monooxygenase (phenylacetone monooxygenase, PAMO) in the presence of ionic liquids. The stability, activity and selectivity of PAMO as an oxidative enzyme in the presence of different ionic liquids were studied. This revealed that the addition of some specific ionic liquids, such Ammoeng™ 102 and [bmim]MeSO4, can significantly enhance the E-value in the oxidation of racemic benzylketones. Moreover, the use of ionic liquids increases the optimal substrate concentration for performing Baeyer–Villiger oxidation, thereby extending the biocatalytic repertoire of PAMO for synthetic applications.

Measurement of plasma renin concentration in cats by use of a fluorescence resonance energy transfer peptide substrate of renin

To evaluate the use of a commercially available 5-carboxyfluorescein-based, intramolecularly quenched, fluorescence resonance energy transfer (FRET) peptide substrate of renin for measurement of plasma renin concentration in cats. Plasma samples obtained during a previous study of renal autograft ischemia-reperfusion injury in 10 cats and samples of fetal bovine serum containing recombinant human renin (rh-renin). Experiments involving samples of fetal bovine serum containing rh-renin were conducted to identify a suitable control vehicle, optimal substrate concentration, and appropriate duration of incubation. With the use of the identified assay conditions, a standard curve was constructed to allow conversion of relative fluorescent units into values of renin concentration (ng/mL). Subsequently, plasma samples obtained from cats before and after renal autograft ischemia-reperfusion injury were assayed to determine endogenous renin concentration. Under conditions of a 1:50 substrate dilution and 4-hour incubation period, the assay detected small amounts of rh-renin in fetal bovine serum. A linear relationship (R(2) = 0.996) between the relative fluorescent units generated and exogenous rh-renin concentration was evident. The assay detected renin in plasma samples obtained from cats after renal autograft ischemia-reperfusion, and renin concentrations on days 1 and 2 after transplant differed significantly. The study data indicated that the assay involving the FRET peptide substrate of renin is potentially a rapid and specific method for measurement of plasma renin concentration in cats.

Optimization of synergistic parameters for thermostable cellulase activity of Aspergillus heteromorphus using response surface methodology

This study was undertaken to find out the optimum reaction conditions, i.e. pH, temperature, enzyme and substrate concentration for the thermostable cellulase enzyme activity of Aspergillus heteromorphus using response surface methodology (RSM). Estimated optimum conditions for cellulase enzyme were found as: pH 4.8, temperature 60 °C, crude enzyme/substrate concentration ratio was 4:6. The maximum enzymatic activity calculated was observed 13.05 U/ml against the predicted value 11.26 U/ml. Cellulase system of A. heteromorphus is thermostable retaining 60.0% of residual activity after 1 h of pre-incubation at 90 °C when assay is carried out at same temperature. Value of correlation coefficient ( R 2 0.9977) and significant value for model p < 0.0001 indicated validity of model fitness and adequate for optimization.

Production of bio-ethanol from corncobs using Aspergillus niger and Saccharomyces cerevisae in simultaneous saccharification and fermentation.

Maize is the most abundant cereal grown in Ghana and is accompanied by enormous amount of agrowastes of which corncobs form 30%. This agrowaste which is currently under utilized was used to produce bio-ethanol. Aspergillus niger isolated from soil sampled from Ejura farms was used to hydrolyze the corncobs into simple sugars. Filtrate obtained from corncobs broth fermented by A. niger was used as crude enzyme in optimization tests on corncobs powder suspended in 50 mM citrate buffer pH 5.0. Optimum temperature, pH and substrate concentration for saccharification were 40oC, 4.0 and 6% respectively. Saccharomyces cerevisae was added to A. niger filtrate to cause fermentation of the corncobs. The highest ethanol concentration of 0.64 g/l was recorded over the 24 h fermentation period.

A study on the improved programming characteristics of flash memory with Si 3N 4/SiO 2 stacked tunneling dielectric

The programming characteristics of memories with different tunneling-layer structures (Si 3N 4, SiO 2 and Si 3N 4/SiO 2 stack) dielectrics are investigated using 2-D device simulator of MEDICI. It is theoretically confirmed that the memory with the SiO 2/Si 3N 4 stacked tunneling layer exhibits better programming characteristics than ones with single tunneling layer of SiO 2 or Si 3N 4 for programming by channel hot electron (CHE) injection. A 10-μs programming time with a threshold-voltage shift of 5 V can be obtained for the memory with SiO 2/Si 3N 4 stacked tunneling layer at V cg = 10 V and V ds = 3.3 V. This is attributed to the fact that the floating-gate voltage is close to drain voltage for the stacked tunneling dielectric (TD), and thus the CHE injection current is the largest. Furthermore, optimal substrate concentration is determined to be 5 × 10 16–2 × 10 17 cm −3, by considering a trade-off between the programming characteristics and power dissipation/lifetime of the devices. Lastly, the effects of interface states on the programming characteristics are investigated. Low interface-state density gives short programming time and small post-programming control-gate current.

Production of hydrogen from marine macro-algae biomass using anaerobic sewage sludge microflora

Hydrogen was produced from various marine macro-algae (seaweeds) through anaerobic fermentation using an undefined bacterial consortium. In this study, anaerobic fermentation from various marine macro-algae for Ulva lactuca, Porphyra tenera, Undaria pinnatifida, and Laminaria japonica was studied. From this analysis Laminaria japorica was determined to be the optimum substrate for hydrogen production. When L. japornica was used as the carbon source for enhanced hydrogen production, the optimum fermentation temperature, substrate concentration, initial pH, and pretreatment condition were determined to be 35°C, 5%, 7.5, and BT120 (Ball mill and thermal treatments at 120°C for 30 min), respectively. In addition, hydrogen production was improved when the sludge was heat-treated at 65°C for 20 min. Under these conditions, about 4,164 mL of hydrogen was produced from 50 g/L of dry algae (L. japonica) for 50 h, with a hydrogen concentration around 34.4%. And the maximum hydrogen production rate and yield were found to be 70 mL/L·h and 28 mL/g dry algae, respectively.

Development and implementation of standardized respiratory chain spectrophotometric assays for clinical diagnosis

Diversity of respiratory chain spectrophotometric assays may lead to difficult comparison of results between centers. The French network of mitochondrial diseases diagnostic centers undertook comparison of the results obtained with different protocols in the French diagnostic centers. The diversity of protocols was shown to have striking consequences, which prompted the network to undertake standardization and optimization of the protocols with respect to clinical diagnosis, i.e. high velocity while maintaining linear kinetics relative to time and enzyme concentration. Assays were set up on animal tissues and verified on control human muscle and fibroblasts. Influence of homogenization buffer and narrow range of optimal concentration of phosphate, substrate and tissue were shown. Experimental data and proposed protocols have been posted on a free access website. Their subsequent use in several diagnostic centers has improved consistency for all assays.

Biodegradation of dimethyl phthalate, diethyl phthalate and di- n-butyl phthalate by Rhodococcus sp. L4 isolated from activated sludge

In this study, an aerobic bacterial strain capable of utilizing dimethyl phthalate (DMP), diethyl phthalate (DEP) and di- n-butyl phthalate (DBP) as sole carbon source and energy was isolated from activated sludge collected from a dyeing plant. According to its morphology, physiochemical characteristics and 16S rDNA sequence, the strain was identified as Rhodococcus ruber. The biodegradation batch tests of DMP, DEP and DBP by the Rhodococcus sp. L4 showed the optimal pH value, temperature and substrate concentration: pH 7.0–8.0, 30–37 °C and PAEs concentration ≤450 mg/L. Kinetics of degradation have also been performed at different initial concentrations. The results show that the degradation can be described with exponential model. The half-life of degradation was about 1.30 days when the concentration of PAEs mixture was lower than 300 mg/L. PAEs contaminated water samples (300 mg/L) with non-emulsification and completed emulsification were prepared to investigate the effect on PAEs degradation rate. Little difference between the above two sample preparations was observed in terms of ultimate degradation rate. Rhodococcus sp. L4 can also grow on phenol, sodium benzoate or naphthalene solution as sole carbon source and energy which suggests its ability in resisting environmental toxicants. This work provides some new evidence for the possibility of applying Rhodococcus for contaminated water remediation in the area of industry.

Specificity of action of piperidylcholine derivatives as substrates of cholinesterases of various origin

The review deals with study of enzymologic properties of a novel highly specific acetylcholinesterase substrate iodomethylate N-beta-(acetoxyethyl)piperidinium ("piperidylcholine") and its 30 derivatives that were tested as effectors of cholinesterases of mammals and various species of Pacific squids. It was proven for the first time that responsible for specificity of action was structure of cyclic ammonium grouping of the alcohol part of molecule of the ester substrate. Analysis of specificity is performed based on enzymatic analysis parameters - activity of catalytic center of cholinesterases and bimolecular constant of the reaction rate that are determined at optimal and low substrate concentrations. Among the specially synthesized group of thioester compounds there is revealed one more highly specific acetylcholine esterase substrate - N-beta-(acetoxyethyl)piperidinium.

Application of organic mono-phase and organic–aqueous two-liquid-phase systems in microalgal conversion of androst-4-en-3,17-dione by Nostoc muscorum

Organic mono-phase and organic–aqueous two-phase systems were applied for 17-carbonyl reduction of androst-4-en-3,17-dione to testosterone by whole cells of the microalga Nostoc muscorum (Nostocaceae). To investigate the correlation between solvent hydrophobicity and biotransformation yield in mono- and biphasic systems, a range of 16 organic solvents with log Poctanol values (logarithm of the solvent partition coefficient in the n-octanol/water system) between − 1.1 and 8.8 were examined. Organic solvents with log Poctanol values greater than 7, such as hexadecane and tetradecane, provided the best biocompatibility with the bioconversion by algal cells. The data also indicated that the highest yields were obtained using organic–aqueous (1:1, v/v) biphasic systems. The optimum volumetric phase ratio, reaction temperature and substrate concentration were 1:1, 30°C and 0.5 mg mL−1, respectively. Under the mentioned conditions a fourfold increase in biotransformation yield (from 7.8±2.3 to 33.4±1.8%) was observed.

Optimal substrate concentration in fermentative process producing hydrogen using activated carbon as solid carriers

Optimal substrate concentration in fermentative process producing hydrogen using activated carbon as solid carriers