Purification, characterization and mass spectroscopic analysis of thermo-alkalotolerant ��-1, 4 endoxylanase from Bacillus sp. and its potential for dye decolorization

A Bacillus sp., isolated from sludge and sediments of pulp and paper mill, was found to produce xylanase in a synthetic culture media containing oat spelt xylan (1% w/v) and 10% black liquor as inducers along with 2.5% (w/v) sucrose as additional carbon source. The purified enzyme was highly thermostable with half-life of 10 min at 90 °C and pH 8. The enzyme was stable over a broad range of pH (pH 6-10) and showed good thermal stability when incubated at 70 °C. Chemicals like EDTA, Hg²⁺, Cu²⁺ and solvents like glycerol and acetonitrile completely inhibited enzyme activity at high concentration. The molecular weights of the purified enzyme, determined by matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF/MS) analysis was analogous to the results obtained from SDS-PAGE, i.e. 55 kDa. Kinetic parameters were determined by using oat spelt xylan as substrate. The K_M and V_max values of the enzyme were 4.4 mg/ml and 287 U/mg respectively. At high xylan concentrations (>70 mg/ml) a substrate inhibition phenomenon of the enzyme was observed. In addition, crude xylanase showed enormous potential for decolorization of various recalcitrant dyes.     

Determination of temozolomide in serum and brain tumor with micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatographic (MEKC) with photodiode-array detection was applied to determine temozolomide (TMZ) in human serum and brain tumor. The limit of quantitation (LOQ) was 0.096 μg/mL using 325 nm as detection wavelength. The method made possible that the TMZ could be detected in in vivo serum samples without sample pretreatment. In order to detect TMZ at lower concentration, an extraction with ethyl acetate was applied to preconcentrate the analyte. Small amount of brain tumor tissues (less than 1g) were lyophilized and pretreated using extraction as a clean up and concentrating step. After removing the organic solvent a final sample volume of only 10 μL was analyzed. The obtained peak concentrations (8.2-10.1 μg/mL) and T(max) (44-65 min) of TMZ in serum were similar to the data reported by others, the in vivo TMZ concentrations found in brain tumor ranged between 0.061 and 0.117 μg/g.     

Group determination of 14-membered macrolide antibiotics and azithromycin using antibodies against common epitopes

Erythromycin (ERY), clarithromycin (CLA), roxithromycin (ROX), and azithromycin (AZI) are macrolide antibiotics widely used in livestock and human medicine. Therefore, they are frequently found as pollutants in environmental water. A method based on indirect competitive enzyme-linked immunosorbent assay (ELISA) for group determination of these macrolides in foodstuffs, human biofluids, and water was developed. Carboxymethyloxime of clarithromycin (CMO–CLA) was synthesized and conjugated to bovine serum albumin (BSA) and gelatin to prepare immunogen and coating antigen with advantageous presentation of target epitopes, l-cladinose and d-desosamine, common for these analytes. Antibodies generated in rabbits were capable of recognizing ERY, CLA, and ROX as a group (100–150%), and AZI (12%) and did not cross-react with ERY degradants, which lack antibiotic activity. Assay displayed sensitivity of determination of 14-membered macrolides (IC₅₀ = 0.13–0.2 ng/ml) and low limit of detection (LOD) that was achieved at 0.02 to 0.03 ng/ml. It allowed performing analysis of milk, muscle, eggs, bovine serum, water, human serum and urine, and avoiding matrix effect without special pretreatment using simple dilution with assay buffer. For 15-membered macrolide AZI, the corresponding characteristics were IC₅₀ = 1.6 ng/ml and LOD = 0.14 ng/ml. The recoveries of veterinary and human medicine macrolides from corresponding matrices were validated and found to be satisfactory.     

Thermostability improvement of maltogenic amylase MAUS149 by error prone PCR

The thermostability of maltogenic amylase from Bacillus sp. US149 (MAUS149) was improved by random mutagenesis using error prone PCR. The library constructed for the mutants obtained was subjected to screening, leading to the selection of a thermostable mutant enzyme named MA-A27. The latter was noted to contain four single mutations, namely D46V, P78L, V145A, and K548E. The half-life times recorded for MA-A27 at 50 °C and 55 °C were 70 min and 25 min, compared to 30 min and 13 min for the wild type, respectively. The results from molecular modeling attributed the increase in thermostability observed for MA-A27 to P78L and K548E substitutions that led to new hydrogen bond and salt bridge formations. Further site-directed mutagenesis studies showed that the P78L and K548E single mutations underwent an increase in thermostability, thus confirming the joint contribution of both substitutions to the increase in thermostability observed for MA-A27.     

A new process of IgG purification by negative chromatography: Adsorption aspects of human serum proteins onto ω-aminodecyl-agarose

The adsorbent ω-aminodecyl-agarose was evaluated as to its feasibility for the adsorption of human serum and plasma proteins, aiming at the purification of immunoglobulin G (IgG). The contribution of electrostatic and hydrophobic interactions (mixed-mode) and the effects of buffer system on the adsorption of serum proteins were also studied. The adsorption isotherm parameters of human serum albumin (HSA) and IgG were evaluated, pointing to the existence of cooperative effects in the process. A positive (n = 2.30 ± 0.38) and negative cooperativity (n = 0.63 ± 0.12) were observed for IgG and HSA binding, respectively. High purity IgG was obtained (based on total protein concentration and nephelometric analysis of HSA, transferrin, and immunoglobulins A, G, and M) with a 75% recovery in Hepes 25 mmol L⁻¹ pH 6.8 feeding human serum. These results indicate that the use of ω-aminodecyl-agarose is a potential technique for purification of IgG from human serum.     

Mono-N-acyl-2,6-diaminopimelic acid derivatives: Analysis by electromigration and spectroscopic methods and examination of enzyme inhibitory activity

Thirteen mono-N-acyl derivatives of 2,6-diaminopimelic acid (DAP)—new potential inhibitors of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18)—were analyzed and characterized by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopies and two capillary electromigration methods: capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Structural features of DAP derivatives were characterized by IR and NMR spectroscopies, whereas CZE and MEKC were applied to evaluate their purity and to investigate their electromigration properties. Effective electrophoretic mobilities of these compounds were determined by CZE in acidic and alkaline background electrolytes (BGEs) and by MEKC in acidic and alkaline BGEs containing a pseudostationary phase of anionic detergent sodium dodecyl sulfate (SDS) or cationic detergent cetyltrimethylammonium bromide (CTAB). The best separation of DAP derivatives, including diastereomers of some of them, was achieved by MEKC in an acidic BGE (500 mM acetic acid [pH 2.54] and 60 mM SDS). All DAP derivatives were examined for their ability to inhibit catalytic activity of DapE from Haemophilus influenzae (HiDapE) and ArgE from Escherichia coli (EcArgE). None of these DAP derivatives worked as an effective inhibitor of HiDapE, but one derivative—N-fumaryl, Me-ester-DAP—was found to be a moderate inhibitor of EcArgE, thereby providing a promising lead structure for further studies on ArgE inhibitors.     

Gene cloning and characterization of a cold-adapted β-glucosidase belonging to glycosyl hydrolase family 1 from a psychrotolerant bacterium Micrococcus antarcticus

The gene bglU encoding a cold-adapted β-glucosidase (BglU) was cloned from Micrococcus antarcticus. Sequence analysis revealed that the bglU contained an open reading frame of 1419 bp and encoded a protein of 472 amino acid residues. Based on its putative catalytic domains, BglU was classified as a member of the glycosyl hydrolase family 1 (GH1). BglU possessed lower arginine content and Arg/(Arg + Lys) ratio than mesophilic GH1 β-glucosidases. Recombinant BglU was purified with Ni2+ affinity chromatography and subjected to enzymatic characterization. SDS-PAGE and native staining showed that it was a monomeric protein with an apparent molecular mass of 48 kDa. BglU was particularly thermolabile since its half-life time was only 30 min at 30 °C and it exhibited maximal activity at 25 °C and pH 6.5. Recombinant BglU could hydrolyze a wide range of aryl-β-glucosides and β-linked oligosaccharides with highest activity towards cellobiose and then p-nitrophenyl-β-d-glucopyranoside (pNPG). Under the optimal conditions with pNPG as substrate, the K_m and k_cat were 7 mmol/L and 7.85 × 103/s, respectively. This is the first report of cloning and characterization of a cold-adapted β-glucosidase belonging to GH1 from a psychrotolerant bacterium.     

Endocrine effects of the GnRH antagonist, acyline, in domestic dogs

Gonadotrophin-releasing hormone (GnRH) antagonists may have a future role in the control of canine reproductive function. In this study, the effects of a single dose of the potent GnRH antagonist, acyline, on serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) were evaluated in male dogs. Blood samples were drawn before (Day −1) and after (30, 60, and 90 min, 3, 6, 9, 12, and 24 h, and 3, 6, 9, 14, 22, and 29 d) treatment with acyline (330 μg/kg, sc); serum concentrations of FSH, LH, and T varied throughout the study period (P < 0.01, <0.05, and <0.01, respectively). Gonadotrophins decreased below pretreatment concentrations 60 min after injection, whereas T took 90 min to decrease below baseline (P > 0.05). Follicle-stimulating hormone, LH and T decreased until Day 9, when they reached their nadir at 2.0 ±1.1 ng/mL (P < 0.01), 1.2 ± 0.2 ng/mL (P > 0.05), and 0.5 ± 0.2 ng/mL (P < 0.05), respectively. Both gonadotrophins and T began increasing on Day 14 after treatment, although FSH and T serum concentrations still remained below baseline on that day (P > 0.05). Follicle-stimulating hormone and T rebounded above baseline on Day 29, whereas LH reached concentrations were similar to baseline at this time (P > 0.05). No local or systemic side effects were detected in any dog following acyline treatment. In conclusion, a single acyline treatment safely and reversibly decreased serum gonadotrophin and T concentrations in dogs for 9 d.     

Strong alkalinization of Chara cell surface in the area of cell wall incision as an early event in mechanoperception

Mechanical wounding of cell walls occurring in plants under the impact of pathogens or herbivores can be mimicked by cell wall incision with a glass micropipette. Measurements of pH at the surface of Chara corallina internodes following microperforation of cell wall revealed a rapid (10–30 s) localized alkalinization of the apoplast after a lag period of 10–20 s. The pH increase induced by incision could be as large as 3 pH units and relaxed slowly, with a halftime up to 20 min. The axial pH profile around the incision zone was bell-shaped and localized to a small area, extending over a distance of about 100 μm. The pH response was suppressed by lowering cell turgor upon the replacement of artificial pond water (APW) with APW containing 50 mM sorbitol. Stretching of the plasma membrane during its impression into the cell wall defect is likely to activate the Ca^2 + channels, as evidenced from sensitivity of the incision-induced alkalinization to the external calcium concentration and to the addition of Ca^2 +-channel blockers, such as La^3 +, Gd^3 +, and Zn^2 +. The maximal pH values attained at the incision site (~ 10.0) were close to pH in light-dependent alkaline zones of Chara cells. The involvement of cytoskeleton in the origin of alkaline patch was documented by observations that the incision-induced pH transients were suppressed by the inhibitors of microtubules (oryzalin and taxol) and, to a lesser extent, by the actin inhibitor (cytochalasin B). The results indicate that the localized increase in apoplastic pH is an early event in mechanoperception and depends on light, cytoskeleton, and intracellular calcium.     

Production of an acidic and thermostable lipase of the mesophilic fungus Penicillium simplicissimum by solid-state fermentation

The production of a lipase by a wild-type Brazilian strain of Penicillium simplicissimum in solid-state fermentation of babassu cake, an abundant residue of the oil industry, was studied. The enzyme production reached about 90 U/g in 72 h, with a specific activity of 4.5 U/mg of total proteins. The crude lipase showed high activities at 35–60 °C and pH 4.0–6.0, with a maximum activity at 50 °C and pH 4.0–5.0. Enzyme stability was enhanced at pH 5.0 and 6.0, with a maximum half-life of 5.02 h at 50 °C and pH 5.0. Thus, this lipase shows a thermophilic and thermostable behavior, what is not common among lipases from mesophilic filamentous fungi. The crude enzyme catalysed the hydrolysis of triglycerides and p-nitrophenyl esters (C4:0–C18:0), preferably acting on substrates with medium-chain fatty acids. This non-purified lipase in addition to interesting properties showed a reduced production cost making feasible its applicability in many fields.     

Definition and characterization of enzymes for maximal biocatalytic solubilization of prebiotic polysaccharides from potato pulp

Potato pulp is a high-volume co-processing product resulting from industrial potato starch manufacturing. Potato pulp is particularly rich in pectin, notably galactan branched rhamnogalacturonan I polysaccharides, which are highly bifidogenic when solubilized. The objective of the present study was to characterize and compare four homogalacturonan degrading enzymes capable of catalyzing the required solubilization of these pectinaceous polysaccharides from potato pulp in a 1 min reaction. An additional purpose was to assess the influence of the pH and the potential buffer chelating effects on the release of these polysaccharides from the potato pulp. The pH and temperature optima of two selected pectin lyases from Emericella nidulans (formerly known as Aspergillus nidulans) and Aspergillus niger were determined to 8.6 and 4.0, respectively, at ≥100 °C within 1 min of reaction. The optima for the two selected polygalacturonases from E. nidulans and Aspergillus aculeatus were determined to pH 4.4 and 46 °C, and pH 3.7 and ≥80 °C, respectively. The polygalacturonase from A. aculeatus was 4-42 times more heat-resistant at 50 °C than the other enzymes. The difference in pH optima of the pectin lyases and the exceptional thermal stabilities of some of the enzymes are proposed to be related to specific amino acid substitutions, stabilizing hydrogen bonding and structural traits of the enzymes. The K_M and V_max values ranged from 0.3-0.6 g/L and 0.5-250.5 U/mg protein, respectively. Phosphate buffer induced release of a higher amount of dry matter than Tris-acetate buffer at pH 6, indicating a chelating effect of the phosphate. Moreover, the phosphate had a higher chelating effect at pH 6 than at pH 4. The optimal conditions for a high yield of polysaccharides from potato pulp were therefore: 1% (w/w) potato pulp treated with 1% (w/w) enzyme/substrate (E/S) pectin lyase from E. nidulans and 1% (w/w) E/S polygalacturonase from A. aculeatus at pH 6.0 and 60 °C for 1 min.     

A thermotolerant and cold-active mannan endo-1,4-β-mannosidase from Aspergillus niger CBS 513.88: Constitutive overexpression and high-density fermentation in Pichia pastoris

The mannan endo-1,4-β-mannosidase gene man26A from Aspergillus niger CBS 513.88 was optimized according to the codon usage bias in Pichia pastoris and synthesized by splicing overlap extension PCR. It was successfully expressed in P. pastoris using constitutive expression vector pGAPzαA. The recombinant endo-beta-1,4-mannanase could work in an extremely board temperature range and over 30% relative activity were retained in the temperature range of 5-60 °C. The optimal pH value and temperature for activity were 5.0 and 45 °C, respectively. It was highly thermotolerant with a half-life time of 15 min at 90 °C. A novel fed-batch strategy was developed successfully for high cell-density fermentation and mannanase activity reached 5069 U/mL after cultivation for 56 h in 50 L fermenter. The broad working temperature range, high thermotolerance and efficient expression made this enzyme possible to be applied in food, animal feed and the production of biofuels.     

Simultaneous determination of clopidogrel and its carboxylic acid metabolite by capillary electrophoresis

A capillary electrophoresis method was developed and validated for the first time for the analysis of clopidogrel and its carboxylic acid metabolite. Prior to method optimization, the pH dependence of effective mobility of both compounds was determined in order to define the initial pH of the running buffer. The optimized method demonstrated to be selective, and linear in the concentration range of 2–100 μM for both compounds. The method limits of detection and quantification were, respectively, 1.2 and 3.7 μM for clopidogrel and 1.1 and 3.2 μM for the carboxylic acid metabolite. Moreover, method validation demonstrated acceptable results for method repeatability (RSD < 7%), intermediate precision (RSD < 7%) and accuracy (85–96%) and is suitable for the quantitative analysis of clopidogrel and its metabolite in serum samples. The validated method was also applied to the determination of the kinetic parameters of the enzymatic hydrolysis of clopidogrel. An apparent K_m of 145 ± 30 μM and V_max of 0.4, 1.5 and 3.4 μM/min, respectively for the enzyme concentrations 1.0, 2.0 and 4.0 U/ml, were obtained.     

A validated stability-indicating HPLC method for the determination of PEGylated puerarin in aqueous solutions

The aim of this study was to develop a validated specific stability-indicating HPLC method for the quantitative determination of PEGylated puerarin (PEG-PUE) in aqueous solutions. The method was validated by subjecting PEG-PUE to forced degradation under stress conditions of acid, alkali, water hydrolysis, and oxidation. Both PEG-PUE and puerarin (PUE) were simultaneously determined and separated on CAPCELL PAK C18 column by gradient elution with 0.2% aqueous phosphoric acid and acetonitrile as the mobile phase. The flow rate was 1.0 mL min⁻¹ and detection wavelength was set at 250 nm. Both calibration curves showed good linear regression (r ≥ 0.9998) within test ranges. The LOD and LOQ of PEG-PUE were determined to be 3 and 9 μg mL⁻¹ respectively. Degradation of PEG-PUE followed pseudo-first-order kinetics with t_1/2 of 59 min at pH 9.0 and 17.79 h at pH 7.4. However, at pH 5.0 and 2.0, there was no significant degradation of PEG-PUE over time. In conclusion, the method was observed to have the necessary specificity, precision, and accuracy, and to be suitable for quantity monitoring the degradation process of PEG-PUE during stability studies. The degradation studies may give insight into useful information for formulation development of PEG-PUE.     

High level expression and characterization of a novel thermostable, organic solvent tolerant, 1,3-regioselective lipase from Geobacillus sp. strain ARM

The mature ARM lipase gene was cloned into the pTrcHis expression vector and over-expressed in Escherichia coli TOP10 host. The optimum lipase expression was obtained after 18 h post induction incubation with 1.0 mM IPTG, where the lipase activity was approximately 1623-fold higher than wild type. A rapid, high efficient, one-step purification of the His-tagged recombinant lipase was achieved using immobilized metal affinity chromatography with 63.2% recovery and purification factor of 14.6. The purified lipase was characterized as a high active (7092 U mg⁻¹), serine-hydrolase, thermostable, organic solvent tolerant, 1,3-specific lipase with a molecular weight of about 44 kDa. The enzyme was a monomer with disulfide bond(s) in its structure, but was not a metalloenzyme. ARM lipase was active in a broad range of temperature and pH with optimum lipolytic activity at pH 8.0 and 65 °C. The enzyme retained 50% residual activity at pH 6.0-7.0, 50 °C for more than 150 min.     

Secreted proprotein convertase subtilisin/kexin type 9 reduces both hepatic and extrahepatic low-density lipoprotein receptors in vivo

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that is known to reduce hepatic low-density lipoprotein receptor (LDLR) levels and increase plasma LDL cholesterol. It is not clear, however, whether secreted PCSK9 degrades extrahepatic LDLRs. We present evidence that recombinant PCSK9, either injected intravenously into or expressed in the liver of C57BL/6 mice, significantly reduced LDLR levels in multiple extrahepatic tissues. During the initial characterization, we found that injected human recombinant PCSK9 at 30 μg/mouse had a half-life of 15 min in serum in mice. Hepatic LDLR levels were reduced within 30 min and the degradation of hepatic LDLR reached the maximum 2 h after the initial protein injection. Endocytosis of PCSK9 in liver occurred within 5 min of protein injection and internalized PCSK9 was only barely detectable within 1 h. When extrahepatic LDLRs were examined by Western blotting analysis, we found significant reductions of LDLRs in multiple extrahepatic tissues including lung, adipose and kidney along with the more dramatic reduction of LDLRs in liver. These studies were further extended using adenoviral expression of human PCSK9 in C57BL/6 mice to demonstrate that PCSK9 produced in liver impacted extrahepatic tissue LDLR levels as well. Taken together, our studies indicate that secreted PCSK9 can potentially impact extrahepatic tissue cholesterol homeostasis by regulating extrahepatic tissue LDLR levels.     

Heat inactivation of Escherichia coli K12 MG1655: Effect of microbial metabolites and acids in spent medium

Aim

The effect of spent medium, obtained after different time-temperature pre-histories, on the heat inactivation of Escherichia coli K12 MG1655 is studied.

Methods and results

Stationary E. coli cells were heated in BHI broth (initial pH 7.5) at different time-temperature scenarios, i.e., (1) 30 °C to 55 °C at 0.14 °C/min, (2) 30 °C to 42 °C at 0.14 °C/min and (3) 30 °C to 42 °C at 0.8 °C/min. After the heat treatment, spent medium was filter-sterilized, non-stressed cells were added and inactivation experiments took place at 54 °C and 58 °C. In all scenarios, increased resistance was observed. The main characteristics of the spent medium - compared to the unmodified BHI broth - are (1) the presence of proteins (proven via SDS-PAGE) and (2) a lower pH of approximately 6. Possibly, the increased resistance is due to these proteins and/or the lower pH. Further experiments revealed that each factor separately may lead to an increased heat resistance.

Conclusions

It can be concluded that this increased heat resistance resulted from both the presence of the heat shock proteins in the spent medium and the lowered pH. Experiments, which separate both effects, showed that mainly the lower pH resulted in the increased thermotolerance.

Significance and impact of study

This study may lead to a better understanding and control of the heat stress adaptation phenomenon as displayed by E. coli at lethal temperatures. Therefore, it contributes to an improved assessment of the effect of temperature during thermal processes in the food industry.     

High-performance liquid chromatography determination of 5-methyl-2'-deoxycytidine, 2'-deoxycytidine, and other deoxynucleosides and nucleosides in DNA digests

This work has undertaken liquid chromatographic separation of nucleosides and deoxynucleosides. Two different columns with three mobile phases (A, deionized water; B, 50 mM phosphate buffer (pH 4.0); C, methanol) and slightly different gradient programs were used. The elution order was as follows: cytidine (C), 2′-deoxycytidine (dC), uridine (U), 5-methyl-2′-cytidine (5mC), 5-methyl-2′-deoxycytidine (5mdC), guanosine (G), deoxyguanosine (dG), 2′-deoxythymidine (dT), adenosine (A), and 2′-deoxyadenine (dA). Using a Luna C18 Phenomenex column (150 × 4.6 mm, 5 μm), the separation was performed at 40 °C with a total flow rate of 1 ml/min and a run time of 10 min. The second column was an Agilent C18 (50 × 3 mm, 1.8 μm), for which the run time was 4.5 min with a flow rate of 0.6 ml/min (25 °C). In application to the DNA digests from human THP-1 cells, the quantification of C, dC, U, 5mC, 5mdC, G, dG, and A was performed. The percentages of global methylation were evaluated based on the 5mdC and dC concentrations (c_5mdC / [c_5mdC + c_dC], where c is concentration in μg/ml) and compared with those calculated from the respective peak areas (A_5mdC / [A_5mdC + A_dC], where A is peak area at 254 nm). For peak area measurements, excellent agreement was obtained with the results reported previously in the same cell line. In the quantitative approach, the results of DNA methylation were higher but consistent with the previous data obtained using mass spectrometric detection. Comparing the analytical features of the two procedures, the use of a smaller column could be recommended because it provides efficient separation (capacity factors in the range of 1.29-10.66), a short run time, and feasibility of nucleoside and deoxynucleoside quantification in real-world samples and because it also minimizes the use of reagents.     

TAMRA- and Cy5-labeled probe for efficient kinetic characterization of caspase-3

Our objective was to create a novel fluorogenic substrate for efficient in vitro kinetic assays on caspase-3. We designed a TAMRA (5′-tetramethylrhodamine-5(6)-carboxamide)- and Cy5 (cyanine 5)-labeled probe that allowed us to evaluate the caspase-3 activity via the changes in fluorescence intensity and wavelength. The prepared probe was found to be an efficient and selective substrate of caspase-3, with V_max of 41.4 ± 3.3 nM/min and K_M of 1.60 ± 0.23 μM. The strategy used in the design of this fluorogenic substrate can be applied in future endeavors to development of substrates for caspase-3 inhibitor screening assays or for real-time detection of apoptosis in living cells.     

Biosensor based on glutamate dehydrogenase immobilized in chitosan for the determination of ammonium in water samples

An optical biosensor based on glutamate dehydrogenase (GLDH) immobilized in a chitosan film for the determination of ammonium in water samples is described. The biosensor film was deposited on a glass slide via a spin-coating method. The ammonium was measured based on β-nicotinamide adenine dinucleotide (NADH) oxidation in the presence of α-ketoglutaric acid at a wavelength of 340 nm. The biosensor showed optimum activity at pH 8. The optimum chitosan concentrations and enzyme loading were found to be at 2% (w/v) and 0.08 mg, respectively. Optimum concentrations of NADH and α-ketoglutaric acid both were obtained at 0.15 mM. A linear response of the biosensor was obtained in the ammonium concentration range of 0.005 to 0.5 mM with a detection limit of 0.005 mM. The reproducibility of the biosensor was good, with an observed relative standard deviation of 5.9% (n = 8). The biosensor was found to be stable for at least 1 month when stored dry at 4 °C.