Sodium Carbonate Buffer Research Articles

Enhanced Biodiesel Production with Eversa Transform 2.0 Lipase on Magnetic Nanoparticles.

This research investigated the usefulness of magnetic iron oxide nanoparticles (Fe3O4) as a support to immobilize the lipase Eversa Transform 2.0 (ET 2.0) to obtain an active and stable biocatalyst, easily recoverable from the reaction medium for applications in the production of biodiesel. Biodiesel was an alternative fuel composed mainly of fatty acid esters with strong transesterification and esterification capabilities. The study focused on the esterification of oleic acid with ethanol to synthesize ethyl oleate. Magnetic nanoparticles were prepared by coprecipitation, then activated with glutaraldehyde and functionalized with γ-aminopropyltriethoxysilane (APTES). The optimal conditions for immobilizing ET 2.0 were pH 10, 25 mM sodium carbonate buffer, an enzymatic load of 200 U/g, and 1 h of contact time, obtaining 78% yield and enzymatic activity of 205.9 U/g. Postimmobilization evaluation showed that the immobilized enzyme performed better than its free form. Kinetic studies were conducted under these optimized conditions (2-96 h at 150 rpm and 37 °C). The biocatalyst was tested for the synthesis of ethyl oleate using oleic acid as the substrate and ethanol, achieving a conversion of 88.1%. Subsequent recirculation tests maintained approximately 80% conversion until the fourth cycle, confirming the sustainability of ester production. Molecular docking studies revealed that the binding affinity for the enzyme-docked oil composition was estimated at -5.8 kcal/mol, suggesting that the combination of the substrate and lipase was stable and suitable for esterification.

B-151 Development and Validation of Plasma Catecholamines by Mass Spectrometry

Abstract Background Our established method for measuring plasma catecholamines utilized a Shimadzu HPLC coupled to an ESA Coulochem III electrochemical detector. Chromatographic run times were long, resulting in slow turnaround times. Instrument maintenance was labor intensive, and the assay was prone to interferences. To overcome these limitations, we changed the method to liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements. Methods After the addition of a labeled internal standard for each analyte, samples are loaded onto 50 mg of activated alumina at pH 8.6, utilizing a Tris-HCl, sodium carbonate and EDTA buffer. After incubation the alumina is then washed with 50% acetonitrile followed by water. Elution off the alumina is performed with 2% acetic acid. Derivatization is then performed on the eluate utilizing acetaldehyde and a cyanoborohydride buffer. Following derivatization, the samples are analyzed for epinephrine, norepinephrine, and dopamine by LC-MS/MS using a Sciex 7500 coupled to a ThermoFisher Scientific TLX-4 equipped with Vanquish pumps. Chromatographic separation was obtained using a Phenomenex Kinetex 2.6 um BiPhenyl 50x3 mm analytical column. Results Runtimes were significantly shorter than with the previous method, and no interferences were observed. Assay validation consisted of: intra/inter assay precision, accuracy, recovery, linearity, carryover, limit of detection (LOD), and interferences. Three levels were assessed for intra assay precision and each of the analytes monitored yielded %CVs of less than 6%. Four levels were assessed for inter assay precision and each of the analytes monitored yielded %CVs of less than 10%. When using linear regression analysis to compare results with the previous clinical assay, the slope, y-intercept and R2 were the following; dopamine: 1.06, 2.74, 0.978; epinephrine: 0.905, 2.20, 0.989; norepinephrine: 0.919, 9.75, 0.953. Spike and recovery from five samples yielded a mean average recovery of 93.4% for dopamine, 100% for epinephrine and 98.1% for norepinephrine. For linearity 4 samples were diluted and the mean % difference from expected was -1.66% for dopamine, -0.25% for epinephrine and 1.78% for norepinephrine. Blanks were injected following the high standard and carryover was negligible. LOD was determined by running a low sample pool along with a blank over 20 assays. The LOD was determined to be less than the LLOQ. Sixty-two common drugs/vitamins along with varying levels of hemolysis, lipemia, and bilirubin were also tested to ensure no interferences were seen. Conclusions The LC-MS/MS method exceeded the performance of the historical Shimdazu HPLC system with ESA Coluochem III electrochemical detector method. We anticipate that the better analytical performance will improve the testing quality of our patients.

Enhancing biotechnological applications of l-asparaginase: Immobilization on amino-epoxy-agarose for improved catalytic efficiency and stability

l-asparaginase has been shown to be effective in reducing acrylamide formation in food and in treating certain cancers, such as acute lymphoblastic leukemia. This study aimed to improve the application of l-asparaginase in these areas, through its immobilization in two distinct supports: epoxy-agarose and amino-epoxy-agarose. Different immobilization conditions were investigated, including immobilization pH and buffer concentration. The best activity results were obtained employing amino-epoxy-agarose, being the 0.1 M sodium carbonate buffer pH 10 the conditions that offered the highest immobilization yield (76%), immobilization efficiency (89.8%) and relative activity (68.2%). Immobilization of l-asparaginase in amino-epoxy-agarose improved its catalytic efficiency and stability, including at pH 4 and 60 °C. All immobilized biocatalysts preserved more than 70% of their activity after 7 cycles of reuse, and maintained about 82% of their activity after 60 days of storage at 4°C. These results show the efficacy of immobilized biocatalysts in the preservation of enzymatic activity and highlight their potential for biotechnological applications.

Selective H2S Absorption Using the Mixture of NaOH-NaHCO3-Na2CO3 Buffer Solvent Solution

Acid gas enrichment unit (AGEU) involves selective separation of H2S from acid gas mixture, for example using absorption with an NaOH solvent solution. Sodium carbonate (Na2CO3) and sodium bicarbonate (NaHCO3) buffer addition to NaOH solution suppresses CO2 absorption, thereby increasing the selectivity of H2S absorption. This study evaluated the effect of buffer addition to increase H2S absorption selectivity using an NaOH solution. It was shown that both buffer addition and L/G ratio decrease could increase H2S selectivity by limiting CO2 absorption. Based on the simulation results, in the 0.006 to 0.030 L/G ratio range and NaOH solvent concentration greater than 2%-mass, the addition of NaHCO3 with mass ratio greater than 1.5:1 to NaOH and the addition of Na2CO3 at 1.26 times NaHCO3’s mass increased H2S absorption selectivity up to 17.3%. The combination of an L/G ratio of 0.006 and solvent with a composition of 5%-mass NaOH, 15%-mass NaHCO3, and 18.9%-mass Na2CO3 produced the highest H2S selectivity of 23.1 (379.7% H2S selectivity increase).

Precise identification of O-linked β-N-acetylglucosamine peptides based on O-mesitylenesulfonylhydroxylamine elimination reaction

氧连接氮乙酰葡萄糖胺(O-GlcNAc)是一种重要的蛋白质翻译后修饰,它在维持机体正常的生命活动中发挥着重要作用。许多研究证实,O-GlcNAc糖基化修饰稳态的破坏与人类多种疾病的发生相关,大规模富集鉴定O-GlcNAc糖基化修饰蛋白有助于发现新的临床疾病诊断标志物。由于O-GlcNAc糖基化修饰丰度较低,形成的糖苷键不稳定,O-GlcNAc糖基化修饰蛋白/肽段的富集鉴定面临一定挑战。近年来,全乙酰化的非天然糖代谢标记技术被广泛应用于O-GlcNAc糖基化修饰蛋白/肽段的富集鉴定。然而,最新的研究发现,在细胞代谢标记过程中,全乙酰化的非天然单糖会同时标记半胱氨酸的巯基而引入半胱氨酸巯基-叠氮糖人为修饰物。该副反应在一定程度上干扰了O-GlcNAc糖基化修饰蛋白/肽段的富集鉴定。鉴于此,研究发展了一种通过三甲基苯磺酰羟胺(MSH)特异性氧化消除半胱氨酸巯基-叠氮糖人为修饰物的方法,进而显著提高O-GlcNAc糖基化修饰肽段的精准鉴定。该方法建立于温和的磷酸钠缓冲液(50 mmol/L, pH=8)体系,利用过量的MSH,于95 ℃避光振荡反应30 min,可完全消除半胱氨酸巯基-叠氮糖人为修饰物。该方法应用于Hela细胞中,可有效消除叠氮全乙酰化半乳糖胺(Ac4GalNAz)代谢产生的半胱氨酸巯基-叠氮糖人为修饰物,从而成功富集鉴定到157条O-GlcNAc糖基化修饰肽段,归属于130个蛋白质。该方法有效去除了半胱氨酸巯基-叠氮糖人为修饰物对代谢标记结果的干扰,为非天然糖代谢标记技术在糖蛋白组学分析中的应用提供了新的研究策略。

Sodium carbonate versus borate buffer for lactase quenching, laboratory work.

A laboratory exercise for undergraduate advanced students of enzymology and biocatalysis is presented. Since enzyme assays can be quenched or continuous, this experiment compares the performance of two quenching agents for lactase, in a continuous setup. Enzymatic activity of β-galactosidase (Aspergillus oryzae) was determined based on the release of 4-nitrophenol from 4-nitrophenyl β-D-galactopyranoside using a microplate reader. Sodium carbonate and borate buffer were tested as quenching agents, and experimental control was the unstopped assay. Based on released 4-nitrophenol, enzyme activity, and rate constant k, the students could assess the performance of each termination agent. The experiment promotes disciplinary and transversal competencies, including research-based learning, critical thinking, and introduce the students to high-throughput techniques that are common in the research and development environment.

Life-Inspired Endogenous Dynamic Behavior of Lipid Droplet-like Microcompartments in Artificial Adipocyte-like Structures

Chemical systems that can replicate cellular behaviors are gaining increasing attention and are being used to study various biological processes. Here, a protein- or amylose-based assembly at an oi...

Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography

Sertraline is an antidepressant in a group of drugs called selective serotonin reuptake inhibitors. Four stereoisomeric compounds would be produced in its synthetic preparation due to two chiral carbons on its chemical structures. In the present work, stereoselective liquid-liquid extraction of isomeric sertraline with substituted cyclodextrins as stereoselective extractant was investigated. Factors affecting the distribution performance, including organic solvents, types of extractants, pH value, buffer solution of aqueous phase, concentration of extractant and temperature, were investigated. Under optimized conditions, a stereoselectivity of 1.404 was obtained for cis-sertraline and a stereoselectivity of 2.373 was obtained for trans-sertraline when hydroxypropyl-β-cyclodextrin was used as the stereoselective extractant, and a stereoselectivity of 1.685 was achieved for trans-sertraline with methyl-β-cyclodextrin as extractant. An unusual stereoselective combination was observed for trans-sertraline and cis-sertraline when sodium carbonate buffer was used. Successful stereoselective separation of trans-sertraline and cis-sertraline, (1S, 4R) and (1R, 4S)-sertraline by analytical countercurrent chromatography with methyl-β-cyclodextrin as stereoselective selector was achieved, using a biphasic solvent system composed of n-hexane : 0.1 mol L−1 citrate buffer solution with pH7.6 (1:1, v/v).

Racemization without deamidation: Effect of racemization conditions on 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate tagged amino acids

The aim of this research study was to provide a more thorough understanding of the underlying mechanism and to broaden the application field of the recently introduced racemization method employing the amino acid derivatization tag 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC, AccQ) for heat-induced stereoisomerization of common amino acids as well as uniformly isotopically labeled [U-13C15N]-amino acids. The influence of different buffer types such as sodium borate buffer and sodium carbonate buffer as well as their pH and molarity on the racemization and deamidation of amino acids were investigated.It was found that a 0.4 M borate buffer with a pH of 8.0 +/− 0.2 was the most suitable derivatization as well as racemization buffer to ensure degradation free racemization of deamidation prone compounds such as glutamine. Hereby essential was the in-solution pH measurement before and after derivatization with AQC as well as after heat-induced racemization. This strategy provided further insight at which pH an actual racemization event was observed and when an unwanted deamidation of glutamine to glutamic acid occurred. In addition also the influence of the presence of oxygen during racemization was studied. In this context it was possible to determine ideal oxidation and racemization conditions for the production of scalemic mixtures of chiral isotopically labeled methionine AQC-DL-[U-13C15N]-Met as well as its oxidation products, AQC-DL-[U-13C15N]-Met-O and AQC-DL-[U-13C15N]-Met-O2. All stereoselective separations were performed on the zwitterionic Chiralpak ZWIX(+) column combined with HPLC–ESI–QTOF–MS analysis in positive ionization mode.

Dimerization of 9,10-anthraquinone-2,7-Disulfonic acid (AQDS)

The redox flow battery is a leading candidate to supply society with large-scale energy storage. In the category of aqueous organic redox flow batteries, 9,10-anthraquinone-2,7-disulfonic acid (AQDS) is considered a top-performing molecule and shows ideal electrochemical behavior at low concentrations around 1 mM. However, its behavior at higher concentrations was rarely studied. In this work, the correlation between concentration and electrochemical capacity is evaluated for AQDS in 1 M H2SO4 and 1 M alkaline sodium carbonate buffer, employing cyclic voltammetry, rotating disk electrode voltammetry and diffusion NMR. It was found that an electrochemically inactive dimer was formed by the unreduced AQDS molecules, with dimerization equilibrium constants determined to be K = 5 M−1 for the acidic electrolyte and 8 M−1 for the alkaline one, significantly inhibiting full electrochemical utilization of the system. However, upon reduction of the bulk material, the dimerization will shift in favor of monomer or quinhydrone formation, possibly alleviating the impediment to reduction. Finally, it was found that the dimerization could be decreased slightly by increasing the temperature of the system.

Exploring the Long-Term Hydrolytic Behavior of Zwitterionic Polymethacrylates and Polymethacrylamides.

The hydrolytic stability of polymers to be used for coatings in aqueous environments, for example, to confer anti-fouling properties, is crucial. However, long-term exposure studies on such polymers are virtually missing. In this context, we synthesized a set of nine polymers that are typically used for low-fouling coatings, comprising the well-established poly(oligoethylene glycol methylether methacrylate), poly(3-(N-2-methacryloylethyl-N,N-dimethyl) ammoniopropanesulfonate) (“sulfobetaine methacrylate”), and poly(3-(N-3-methacryamidopropyl-N,N-dimethyl)ammoniopropanesulfonate) (“sulfobetaine methacrylamide”) as well as a series of hitherto rarely studied polysulfabetaines, which had been suggested to be particularly hydrolysis-stable. Hydrolysis resistance upon extended storage in aqueous solution is followed by 1H NMR at ambient temperature in various pH regimes. Whereas the monomers suffered slow (in PBS) to very fast hydrolysis (in 1 M NaOH), the polymers, including the polymethacrylates, proved to be highly stable. No degradation of the carboxyl ester or amide was observed after one year in PBS, 1 M HCl, or in sodium carbonate buffer of pH 10. This demonstrates their basic suitability for anti-fouling applications. Poly(sulfobetaine methacrylamide) proved even to be stable for one year in 1 M NaOH without any signs of degradation. The stability is ascribed to a steric shielding effect. The hemisulfate group in the polysulfabetaines, however, was found to be partially labile.

The temporal evolution of magnesium isotope fractionation during hydromagnesite dissolution, precipitation, and at equilibrium

This study provides experimental evidence of the resetting of the magnesium (Mg) isotope signatures of hydromagnesite in the presence of an aqueous fluid during its congruent dissolution, precipitation, and at equilibrium at ambient temperatures over month-long timescales. All experiments were performed in batch reactors in aqueous sodium carbonate buffer solutions having a pH from 7.8 to 9.2. The fluid phase in all experiments attained bulk chemical equilibrium within analytical uncertainty with hydromagnesite within several days, but the experiments were allowed to continue for up to 575 days. During congruent hydromagnesite dissolution, the fluid first became enriched in isotopically light Mg compared to the dissolving hydromagnesite, but this Mg isotope composition became heavier after the fluid attained chemical equilibrium with the mineral. The δ26Mg composition of the fluid was up to ∼0.35‰ heavier than the initial dissolving hydromagnesite at the end of the dissolution experiments. Hydromagnesite precipitation was provoked during one experiment by increasing the reaction temperature from 4 to 50 °C. The δ26Mg composition of the fluid increased as hydromagnesite precipitated and continued to increase after the fluid attained bulk equilibrium with this phase. These observations are consistent with the hypothesis that mineral-fluid equilibrium is dynamic (i.e. dissolution and precipitation occur at equal, non-zero rates at equilibrium). Moreover the results presented in this study confirm (1) that the transfer of material from the solid to the fluid phase may not be conservative during stoichiometric dissolution, and (2) that the isotopic compositions of carbonate minerals can evolve even when the mineral is in bulk chemical equilibrium with its coexisting fluid. This latter observation suggests that the preservation of isotopic signatures of carbonate minerals in the geological record may require a combination of the isolation of fluid-mineral system from external chemical input and/or the existence of a yet to be defined dissolution/precipitation inhibition mechanism.

Bi2O2CO3 Growth at Room Temperature: In Situ X-ray Diffraction Monitoring and Thermal Behavior.

The room-temperature formation of bismuth oxycarbonate (Bi2O2CO3) from Bi2O3 in sodium carbonate buffer was investigated with in situ powder X-ray diffraction (PXRD) in combination with electron microscopy and vibrational spectroscopy. Time-resolved PXRD measurements indicate a pronounced and rather complex pH dependence of the reaction mechanism. Bi2O2CO3 formation proceeds within a narrow window between pH 8 and 10 via different mechanisms. Although a zero-dimensional nucleation model prevails around pH 8, higher pH values induce a change toward a diffusion-controlled model, followed by a transition to regular nucleation kinetics. Ex situ synthetic and spectroscopic studies confirm these trends and demonstrate that in situ monitoring affords vital parameter information for the controlled fabrication of Bi2O2CO3 materials. Furthermore, the β → α bismuth oxide transformation temperatures of Bi2O2CO3 precursors obtained from different synthetic routes differ notably (by min 50 °C) from commercially available bismuth oxide. Parameter studies suggest a stabilizing role of surface carbonate ions in the as-synthesized bismuth oxide sources. Our results reveal the crucial role of multiple preparative history parameters, especially of pH value and source materials, for the controlled access to bismuth oxide-based catalysts and related functional compounds.

Improving the Efficiency of New Automatic Dishwashing Detergent Formulation by Addition of Thermostable Lipase, Protease and Amylase.

The use of T1 lipase in automatic dishwashing detergent (ADD) is well established, but efficiency in hard water is very low. A new enzymatic environmentally-friendly dishwashing was formulated to be efficient in both soft and hard water. Thermostable enzymes such as T1 lipase from Geobacillus strain T1, Rand protease from Bacillus subtilis strain Rand, and Maltogenic amylase from Geobacillus sp. SK70 were produced and evaluated for an automatic dishwashing detergent formulation. The components of the new ADD were optimized for compatibility with these three enzymes. In compatibility tests of the enzymes with different components, several criteria were considered. The enzymes were mostly stable in non-ionic surfactants, especially polyhydric alcohols, Glucopon UP 600, and in a mixture of sodium carbonate and glycine (30:70) buffer at a pH of 9.25. Sodium polyacrylate and sodium citrate were used in the ADD formulation as a dispersing agent and a builder, respectively. Dishwashing performance of the formulated ADDs was evaluated in terms of percent of soil removed using the Leenert‘s Improved Detergency Tester. The results showed that the combination of different hydrolysis enzymes could improve the washing efficiency of formulated ADD compared to the commercial ADD “Finish” at 40 and 50 C.

A novel mass spectrometry-based method for simultaneous determination of asymmetric and symmetric dimethylarginine, l-arginine and l-citrulline optimized for LC-MS-TOF and LC-MS/MS.

Nitric oxide (NO) is a regulatory molecule involved in many biological processes. NO is produced by nitric oxide synthase by conversion of l-arginine to l-citrulline. l-Arginine methylated derivatives, asymmetric and symmetric dimethylarginines (asymmetric dimethylarginine, ADMA, and symmetric dimethylarginine, SDMA), regulate l-arginine availability and the activity of nitric oxide synthase. As such, they have been frequently investigated as potential biomarkers in pathologies associated with dysfunctions in NO synthesis. Here, we present a new multistep analytical methodology based on liquid chromatography combined with mass spectrometry for the accurate identification of l-arginine, l-citrulline, ADMA and SDMA. Compounds are measured as stable 2,3,4,5,6-pentafluorobenzoyl chloride derivatives, which allows for simultaneous analysis of all compounds through chromatographic separation of ADMA and SDMA using a reverse-phase column. Serum aliquots (100 μL) were spiked with isotope-labeled internal standards and sodium carbonate buffer. The derivatization process was carried out at 25°C for 10 minu using pentafluorobenzoyl chloride as derivatization reagent. Calibration demonstrated good linearity (R2 = 0.9966-0.9986) for all derivatized compounds. Good accuracy (94.67-99.91%) and precision (1.92-11.8%) were observed for the quality control samples. The applicability of the method was evaluated in a cohort of angiological patients and healthy volunteers. The method discerned significantly lower l-arginine and l-citrulline in angiologic patients. This robust and fast LC-ESI-MS method may be a useful tool in quantitative analysis of l-arginine, ADMA, SDMA and l-citrulline.

Sodium-Doped Gold-Assisted Laser Desorption Ionization for Enhanced Imaging Mass Spectrometry of Triacylglycerols from Thin Tissue Sections.

The deposition of sodium salts followed by a sputtered layer of gold has been demonstrated to be a power combination for the analysis of triacylglycerols (TAGs) from tissue sections by laser desorption ionization (LDI) imaging mass spectrometry (IMS). Various sodium salts were tested for their capability to ionize TAGs and their ability to produce fast drying, small crystals (≤3 μm). The spray deposition of a sodium acetate and carbonate buffer mixture at pH 10.3 on which a 28 ± 3 nm sputtered layer of gold (Au-CBS) is subsequently deposited was found to provide the most effective combination for TAG analysis by high imaging resolution IMS. Under these conditions, a 30-fold increase in TAG signal intensity was observed when compared to matrix-assisted LDI (MALDI) methods using 2,5-dihydrobenzoic acid as matrix. Furthermore, Au-CBS led to an increase in the number of detected TAG species from ∼7 with DHB to more than 25 with the novel method, while few phospholipid signals were observed. These results were derived from the IMS investigation of fresh frozen mouse liver and rabbit adrenal gland tissue sections with a range of higher spatial resolutions between 35 and 10 μm. Au-CBS-LDI MS presents a highly sensitive and specific alternative to MALDI MS for imaging of TAGs from tissue sections. This novel approach has the potential to provide new biological insights on the role of TAGs in both health and disease.

Detection of Allergens in a Multiple Allergen Matrix and Study of the Impact of Thermal Processing

The objectives of this work were to (a) determine the percentage recovery of three allergens (casein, egg and soy) simultaneously incurred in a flour matrix, (b) investigate the effect of different baking periods on allergen recovery using two different methods of allergen detection, namely enzyme linked immunosorbent assay (ELISA) and flow cytometry, and c) determine the solubility of proteins in the prepared blank dry cookie mix flour, non-cooked dough and baked cookies after baking at different temperatures using different extraction buffers. Sodium carbonate buffer was least effective in extracting protein. For the blank flour and non-cooked samples, PBS buffer was the most effective at extracting protein, whereas the Tris buffer gave the highest protein recoveries among the three buffers for the cooked samples. For all three allergens, thermal processing greatly reduced allergen recovery in the processed food matrix as detected using both the commercial ELISA kits and flow cytometry. Moreover, allergen recovery in the cooked samples decreased with increasing processing temperatures and ranged from 96% to 20% for casein, 26.5% to 3.7% for soy and 12.8% to 0% for egg in an instance where a false negative was detected when a high processing temperature of 450°F was employed.

Adaptation of microcystin thiol derivatization for matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis

Thiol derivatization of microcystins is an emerging technique for simplifying mass spectrometric analysis of microcystins in complex matrices and for distinguishing between Mdha-/Dhb-containing microcystins. The present protocol is not compatible with MALDI-TOF mass spectrometry, but use of sodium carbonate buffer and desalting with ZipTips yielded no loss in sensitivity. Use of ammonium carbonate buffer followed by dilution in MALDI matrix resulted in a small loss of sensitivity but allowed accurate determination of reaction rates.

AB0122 In Vivo Intraarticular Binding of A Biopolymeric Compound in the Course of Experimental Immune Complex Induced Arthritis, Evaluated as A Potential Tool for Targeting Inflamed Synovium

ObjectivesTo evaluate in an in vivo rat experimental model of antigen induced arthritis (AIA) the concentration and persistence of intraarticular (ia) binding of a biocompatible modified polysaccharide, composed of a...

Operation Modes Can Affect the Activity of Immobilized Enzyme onto Silk Fibroin Nanofibrous Membrane

Note ] Int. J. Indust. Entomol. Vol. 27, No. (2), pp. 322-325 (2013) 322 323 Immobilization of CT on SF nanofibrous membrane In order to immobilize CT on the SF, the amine groups of SF were activated with glutaraldehye (GA). To one piece of SF nanofibrous membrane, 10% (v/v) GA in 0.2 M sodium carbonate buffer (pH 9.2) was added to activate the SF. The reaction was continued for 1 h at 25oC. The activated SF was washed 2 times with distilled water and 3 times with 0.1 M sodium phosphate buffers (pH 7.4). The activated SF was mounted on the membrane holder and an ice-cold buffer solution of CT (1 mg/mL) in 0.1 M sodium phosphate buffer (pH 7.4) was circulated overnight at 4oC. The unbound CT was washed out 20 mL of ice-cold 0.5 M of NaCl in 0.1 M sodium phosphate buffer (pH 7.4). Further it was washed 30 mL of the same buffer but without NaCl. Quantification and Activity Test of CT The bound CT was measured by bicinchoninic acid assay methods using bovine serum albumin as standard. The amount of bound CT was calculated by subtracting the amount of remaining CT from the initial amount of CT. N-benzoyl-DLtyrosine-p-nitroanilide hydrochloride (BTPNA) was used as substrate of CT. For general purpose, the substrate solution were prepared by mixing 5 mM BTPNA in DMSO, distilled water and 0.1 M sodium phosphate buffer (pH 7.4) in a ratio of 10:15:3, respectively. In order to measure the activity of the immobilized CT, the increase of absorbance at 400 nm was measured using UV spectrometer (UVICON 923, Kontron Instruments, USA). If the value of absorbance exceeded 2 then it was diluted until it reached below 1.5. The activity of immobilized CT was defined as μmol BTPNA hydrolyzed in 1 min by 1 mg of immobilized CT applying an absorbance coefficient of e400 = 9520 M -1 cm -1 . The Michaelis-Menten parameters was calculated by the Lineweaver– Burk plot after reacting enzyme immobilized SF nanofibrous membrane with the substrate solution having different BTPNA concentration. In this case, the final concentration of BTPNA was 0.0625, 0.125, 0.25, 0.5, 1.0 and 2.0 mM. Operation modes of immobilized CT The substrate solution was reacted with enzyme immobilized SF nanofibrous membrane in two modes. For the batch reactor mode, the enzyme immobilized SF nanofibrous membrane nanofibrous membrane and operated in two different systems. First, it was operated like a batch-type reactor, where the enzyme immobilized SF nanofibrous membrane was immersed into the substrate solution. Secondly, the enzyme immobilized SF nanofibrous membrane was mounted on the membrane holder and the substrate solution was passed through the membrane. We compared the Michaelis-Menten parameters of both operations and examined the effectiveness of the membrane reactor. Materials and Methods