ABSTRACT
Glucosamine-chitosan synthesized by the Maillard reaction was combined with montmorillonite to obtain a nanohybrid composite to immobilize horseradish peroxidase. The material combines the advantageous properties of clay with those of the chitosan derivative; has improved water solubility and reduced molecular weight and viscosity; involves an eco-friendly synthesis; and exhibits ion exchange capacity, good adhesiveness, and a large specific surface area for enzyme adsorption. The physicochemical characteristics of the composite were analyzed by infrared spectroscopy and X-ray diffraction to determine clay-polycation interactions. The electrochemical response of the different polyphenols to glassy carbon electrodes modified with the composite was evaluated by cyclic voltammetry. The sensitivity and detection limit values obtained with the biosensor toward hydroquinone, chlorogenic acid, catechol, and resorcinol are (1.6 ± 0.2) × 102 µA mM-1 and (74 ± 8) nM; (1.2 ± 0.1) × 102 µA mM-1 and (26 ± 3) nM; (16 ± 2) µA mM-1 and (0.74 ± 0.09) µM; and (3.7± 0.3) µA mM-1 and (3.3 ± 0.2) µM, respectively. The biosensor was applied to quantify polyphenols in pennyroyal and lemon verbena extracts.
Subject(s)
Bentonite , Biosensing Techniques , Chitosan , Electrochemical Techniques , Enzymes, Immobilized , Glucosamine , Horseradish Peroxidase , Polyphenols , Bentonite/chemistry , Polyphenols/analysis , Chitosan/chemistry , Horseradish Peroxidase/chemistry , Enzymes, Immobilized/chemistry , Glucosamine/analysis , ElectrodesABSTRACT
As chitin is gaining an increased attention as feedstock for industry, quantification thereof is becoming increasingly important. While gravimetric procedures are long, not specific and highly labour-intensive, acidic hydrolysis of chitin into glucosamine followed by quantification of the latter is more performant. Even though several quantification procedures for the determination of chitin can be found in the literature, they give inconsistent results and their accuracy was not assessed due to the lack of certified analytical standards. Therefore, in the present study, commercially available chitin from practical grade was characterised in detail, allowing the assessment of method accuracy. The procedure for the hydrolysis of chitin into glucosamine and subsequent quantification via UPLC-MS was investigated in detail as well. Using 9-fluorenylmethyl chloroformate (FMOC-Cl) as derivatisation reagent, glucosamine was quantified using reversed-phase chromatography. For the chitin hydrolysis, the highest glucosamine recovery was obtained with 8.0 M HCl for 2 h at 100 °C. The entire procedure for chitin quantification, including the hydrolysis, was characterised by high interday and intraday precision and accuracy. The specificity of the procedure was assessed as well by analysing different mixtures of cellulose and chitin. Chitin recoveries from these analyses ranged from 98.8 to 105.8% while no signal was observed for 100% cellulose, indicating the high specificity of the procedure. It was also concluded that the procedure is much faster and less labour-intensive compared to the gravimetric procedure.
Subject(s)
Chitin/analysis , Chromatography, Liquid/methods , Glucosamine/analysis , Mass Spectrometry/methods , Calibration , Chitin/standards , Hydrolysis , Limit of Detection , Reference Standards , Reproducibility of ResultsABSTRACT
Several species of fungus from the genus Aspergillus are implicated in pulmonary infections in immunocompromised patients. Broad screening methods for fungal infections are desirable, as cultures require a considerable amount of time to provide results. Herein, we developed degradation and detection methods to produce and detect D-glucosamine (GlcN) from Aspergillus niger, a species of filamentous fungus. Ultimately, these techniques hold the potential to contribute to the diagnosis of pulmonary fungal infections in immunocompromised patients. In the following studies, we produced GlcN from fungal-derived chitin to serve as a marker for Aspergillus niger. To accomplish this, A. niger cells were lysed and subjected to a hydrochloric acid degradation protocol. Products were isolated, reconstituted in aqueous solutions, and analyzed using hydrophilic interaction liquid chromatography (HILIC) in tandem with electrospray ionization time-of-flight mass spectrometry. Our results indicated that GlcN was produced from A. niger. To validate these results, products obtained via fungal degradation were compared to products obtained from the degradation of two chitin polymers. The observed retention times and mass spectral extractions provided a two-step validation confirming that GlcN was produced from fungal-derived chitin. Our studies qualitatively illustrate that GlcN can be produced from A. niger; applying these methods to a more diverse range of fungi offers the potential to render a broad screening method for fungal detection pertinent to diagnosis of fungal infections.
Subject(s)
Aspergillosis/diagnosis , Aspergillus niger/isolation & purification , Glucosamine/analysis , Lung Diseases, Fungal/diagnosis , Aspergillosis/microbiology , Biomarkers/analysis , Chromatography, Liquid/methods , Glucosamine/standards , Humans , Lung Diseases, Fungal/microbiology , Reference Standards , Tandem Mass Spectrometry/methodsABSTRACT
Next to cellulose, chitin is the most abundant biopolymer on Earth and participates, in particular, in the assembly of the fungal cell walls. Easy monitoring of the amount of chitinous polymers could be advantageous during biotechnological cultivation and mushroom farming in order to control and optimize the production process. In this study, a modified colorimetric glucosamine (GlcN) assay was validated using high-performance anion exchange chromatography (HPAE) coupled with pulsed amperometric detection (PAD) following acid hydrolysis of the Fomes fomentarius cell walls. The average data obtained from both methods were well comparable. The HPAE-PAD analysis was suggested to be preferable because it had less data scattering, but it could be replaced with the simplified colorimetric GlcN screening in some cases such as low budgetary resources or an absence of suitable equipment, materials, or highly skilled staff. The robust, cost-effective colorimetric GlcN assay presented here can be readily requested, considering the rapidly growing market as well as the scientific and industrial potential of gourmet and medicinal mushrooms.
Subject(s)
Agaricales , Coriolaceae , Colorimetry , Coriolaceae/chemistry , Glucosamine/analysis , HumansABSTRACT
Glucosamine (GlcN) was recently proposed as an agent with an excellent safety profile to detect cancer with the chemical exchange saturation transfer (CEST) MRI technique. Translation of the GlcN CEST method to the clinical application requires evaluation of its sensitivity to the different frequency regions of irradiation. Hence, imaging of the GlcN signal was established for the full Z spectra recorded following GlcN administration to mice bearing implanted 4T1 breast tumors. Significant CEST effects were observed at around 1.5, 3.6 and -3.4 ppm, corresponding to the hydroxyl, amine/amide exchangeable protons and for the Nuclear Overhauser Enhancement (NOE), respectively. The sources of the observed CEST effects were investigated by identifying the GlcN metabolic products as observed by 13 C NMR spectroscopy studies of extracts from the same tumor model following treatment with [UL-13 C] -GlcN·HCl. The CEST contribution can be attributed to several phosphorylated products of GlcN, including uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), which is a substrate for the O-linked and N-linked glycosylated proteins that may be associated with the increase of the NOE signal. The observation of a significant amount of lactate among the metabolic products hints at acidification as one of the sources of the enhanced CEST effect of GlcN. The proposed method may offer a new approach for clinical molecular imaging that enables the detection of metabolically active tumors and may play a role in other diseases.
Subject(s)
Glucosamine/analysis , Magnetic Resonance Spectroscopy/methods , Mammary Neoplasms, Experimental/diagnostic imaging , Animals , Cell Line, Tumor/transplantation , Female , Hydrogen-Ion Concentration , Lactates/analysis , Magnetic Resonance Imaging/methods , Mice , Mice, Inbred BALB C , Nuclear Magnetic Resonance, Biomolecular/methods , Tissue Extracts/chemistryABSTRACT
A precise quantification of insect chitin is needed in order to avoid overestimation of crude protein due to chitin-bound nitrogen. An UPLC/FLR method was optimized and validated for the determination of glucosamine (GlcN) hydrolyzed from chitin in insect materials. The method was applied for quantifying the chitin content in mealworms (Tenebrio molitor) and crickets (Acheta domesticus). A baseline separation was obtained using an Acquity HSS T3 C18 column, with an external calibration curve of excellent linearity, and a low limit of detection and quantification of GlcN. Even though the recovery of GlcN from spiked cricket material was slightly lower compared to that using spectrophotometric method, the UPLC/FLR method proved a sensitive and specific method of quantification of insect chitin. Chitin contents in T. molitor and A. domesticus were 4.6 ± 0.1% and 4.5 ± 0.0% on dry matter basis, respectively. Less than 0.01% of chitin was present in insect protein-enriched fractions extracted with 0.1 N NaCl at pH 10.
Subject(s)
Chitin/analysis , Chromatography, High Pressure Liquid/methods , Insecta/chemistry , Animals , Chitin/metabolism , Glucosamine/analysis , Glucosamine/metabolism , Gryllidae/chemistry , Hydrolysis , Insecta/metabolism , Tenebrio/chemistryABSTRACT
Solid-state fermentation with food-grade fungal strains can be applied to enhance the bioactive parameters of agro-industrial by-products. Tempe-type fermentation can be adapted to various substrates, but the key factor is the appropriate strain selection. The aim of this study was to compare the potential of Rhizopus strains for obtaining products of improved antioxidant activity from pumpkin oil cake. For this purpose, substances reacting with the Folin-Ciocalteu reagent, with free radical scavenging potential, as well as reducing power were assessed. The effect of the fermentation on the phytate level and inositol phosphate profile in the material was also monitored. The fermentation resulted in the significant enhancement of the antioxidant potential of pumpkin oil cake in the case of all the strains tested, but the most efficient one was R. oligosporus ATCC 64063. During the course of fermentation, the level of phytate in the material decreased (the highest reduction rate was observed in the oil cake fermented with R. oryzae CBS 372.63), while peptides and fungal glucosamine were accumulated. Tempe-type fermentation can be considered as an alternative way of improving the bioactive parameters of pumpkin oil cake and, thanks to the various activities of different Rhizopus strains, it is possible to obtain products of desired parameters.
Subject(s)
Cucurbita/chemistry , Fermentation , Food Handling , Food Microbiology , Plant Oils/metabolism , Rhizopus/metabolism , Antioxidants/chemistry , Antioxidants/pharmacology , Food Analysis , Glucosamine/analysis , Inositol Phosphates/metabolism , Peptides/analysis , Proteins/analysis , Species SpecificityABSTRACT
BACKGROUND: There are several methods for the quantification of biomass in SSF, such as glucosamine measurement, ergosterol content, protein concentration, change in dry weight or evolution of CO2 production. However, all have drawbacks when obtaining accurate data on the progress of the SSF due to the dispersion in cell growth on the solid substrate, and the difficulty encountered in separating the biomass. Studying the disadvantages associated with the process of biomass quantification in SSF, the monitoring of the growth of biomass by a technique known as digital image processing (DIP), consists of obtaining information on the production of different compounds during fermentation, using colorimetric methods based on the pixels that are obtained from photographs. OBJECTIVE: The purpose of this study was to know about the state of the technology and the advantages of DIP. METHODS: The methodology employed four phases; the first describes the search equations for the SSF and the DIP. A search for patents related to SSF and DIP carried out in the Free Patents Online and Patent inspiration databases. Then there is the selection of the most relevant articles in each of the technologies. As a third step, modifications for obtaining the best adjustments were also carried out. Finally, the analysis of the results was done and the inflection years were determined by means of six mathematical models widely studied. RESULTS: For these models, the inflection years were 2018 and 2019 for both the SSF and the DIP. Additionally, the main methods for the measurement of biomass in SSF were found, and are also indicated in the review, as DIP measurement processes have already been carried out using the same technology. CONCLUSION: In addition, the DIP has shown satisfactory results and could be an interesting alternative for biomass measurement in SSF, due to its ease and versatility.
Subject(s)
Colorimetry/methods , Fermentation/physiology , Fungi/metabolism , Image Processing, Computer-Assisted/methods , Models, Statistical , Biomass , Biotechnology/methods , Carbon Dioxide/analysis , Carbon Dioxide/metabolism , Databases, Bibliographic , Ergosterol/analysis , Ergosterol/metabolism , Food Technology/methods , Glucosamine/analysis , Glucosamine/metabolism , Humans , Image Processing, Computer-Assisted/statistics & numerical data , Patents as Topic , Proteins/analysis , Proteins/metabolismABSTRACT
Reducing sugars can react with 1-phenyl-3-methyl-5-pyrazolone (PMP) to form sugar-PMP derivatives, which can be detected by HPLC-UV or HPLC-DAD due to their high UV absorbance at 248â¯nm. Six different sugars were synthesized with PMP with aid of response surface methodology (RSM), by which the parameters of the synthesis were designed within temperature ranged between 60⯰C and 90⯰C, and time from 60 to 180â¯min, respectively. Consequently, optimal conditions of the glucose (Glu)-, glucosamine (GluN)-, galactose (Gal)-, glucuronic acid (GluA), galacturonic acid (GalA) and glucose-6-phosphate (G6P-PMP) reactions were determined at 71⯰C for 129â¯min, 73⯰C for 96â¯min, 70⯰C for 117â¯min, 75⯰C for 151â¯min, 76⯰C for 144â¯min, and 70⯰C for 154â¯min, respectively. Experiments demonstrated that unique functional groups and delicate differences of carbohydrates' inner pH environment could significantly influence the sugar-PMP reactions. However, sugar stereoisomers did not have remarkable impacts on the reactions.
Subject(s)
Carbohydrates/analysis , Carbohydrates/chemistry , Edaravone/chemistry , Chromatography, High Pressure Liquid , Galactose/analysis , Galactose/chemistry , Glucosamine/analysis , Glucosamine/chemistry , Glucose/analysis , Glucose/chemistry , Glucose-6-Phosphate/analysis , Glucose-6-Phosphate/chemistry , Glucuronic Acid/analysis , Glucuronic Acid/chemistry , Hexuronic Acids/analysis , Hexuronic Acids/chemistry , StereoisomerismABSTRACT
Chondroitin sulfate and glucosamine, commercialized as anti-osteoarthritis food supplements, do not undergo the strict quality controls of pharmaceuticals. In this paper a systematic multi-analytical approach was designed to analyse 25 food supplements from 8 European countries compared to 2 pharmaceuticals by using high performance anion-exchange chromatography with pulsed amperometric detection, size exclusion chromatography with triple detector array, capillary electrophoresis, mono and bi-dimensional NMR. Furthermore the biological activity was assessed on in vitro human synoviocyte and chondrocyte primary cell models. Most of the samples (over 19 out of 25) showed lower condroitin sulfate and glucosamine contents than the declared ones (up to -60.3%) while all of them showed a KS contamination (up to 47.1%). Mixed animal origin chondroitin sulfate and multiple molecular weight species were determined in more than 32% of the samples. Only 1 on 5 biologically screened samples had an effective action in vitro almost comparable to the pharmaceuticals.
Subject(s)
Chondroitin Sulfates/analysis , Dietary Supplements/analysis , Glucosamine/analysis , Keratan Sulfate/chemistry , Osteoarthritis/drug therapy , Cells, Cultured , Chondrocytes/drug effects , Drug Contamination , Europe , Humans , Synoviocytes/drug effectsABSTRACT
To detect and characterise compounds in complex matrices, it is often necessary to separate the compound of interest from the matrix before analysis. In our previous work, we have developed the coupling of supercritical fluid chromatography (SFC) with nuclear magnetic resonance (NMR) spectroscopy for the analysis of nonpolar samples [Van Zelst et al., Anal. Chem., 2018, 90, 10457]. In this work, the SFC-NMR setup was successfully adapted to analyse polar samples in complex matrices. In-line SFC-NMR analysis of two N-acetylhexosamine stereoisomers was demonstrated, namely N-acetyl-mannosamine (ManNAc) and N-acetyl-glucosamine (GlcNAc). ManNAc is a metabolite that is present at elevated concentrations in patients suffering from NANS-mediated disease. With our SFC-NMR setup it was possible to distinguish between the polar stereoisomers. Until now, this was not possible with the standard mass-based analysis techniques. The concentrations that are needed in the SFC-NMR setup are currently too high to be able to detect ManNAc in patient samples (1.7 mM vs. 0.7 mM). However, several adaptations to the current setup will make this possible in the future.
Subject(s)
Chromatography, Supercritical Fluid , Glucosamine/analysis , Hexosamines/analysis , Chromatography, Supercritical Fluid/instrumentation , Glucosamine/analogs & derivatives , Magnetic Resonance Spectroscopy/instrumentation , Molecular ConformationABSTRACT
INTRODUCTION: Glucosamine dietary supplements are commonly used for the management of osteoarthritis (OA). However, clinical trials have reported varying outcomes with regard to joint function and disease progression. One of the possible reasons for variability in observed effects of glucosamine could be that, unlike prescription drugs, the quality of manufactured dietary supplements is not closely monitored in many countries. Therefore, there is the possibility that the actual amount of glucosamine present in a dietary supplement is different from that claimed on the label. The quality control of glucosamine supplements is further complicated by the unavailability of a simple and effective analytical method for the analysis of glucosamine. Therefore, the aim of this study was to develop a simple analytical method that could be easily adapted by the pharmaceutical industry for routine analysis of glucosamine. AIMS: To develop a novel high-performance liquid chromatography (HPLC) method for the quantification of glucosamine, and determine the amount of glucosamine present in a sample of dietary supplements commercially available in Australia and India. METHODS: Chromatographic separation of glucosamine was achieved using a zwitter-ionic hydrophilic interaction liquid chromatography column with a mobile phase consisting of 60% acetonitrile and 40% of 85 mM ammonium acetate, at a flow rate of 0.3 mL/min and column temperature 40°C. The developed method was validated for intra- and inter-day linearity, accuracy, precision, and reproducibility. The newly-developed method was subsequently used to analyse 12 glucosamine supplements. RESULTS: The developed method was selective for glucosamine, which had a retention time of 5.9 min. The standard curve was linear with a correlation coefficient (r2) exceeding 0.99, over the range of 10-200 µg/mL for glucosamine. The relative standard deviations for intra- and inter-day accuracy, precision and reproducibility were all less than 4%. The amount of glucosamine determined in six Australian and six Indian glucosamine supplements ranged between 98.7-101.7% and 85.9-101.8% of the labelled values, respectively. DISCUSSION: Unlike previous HPLC methods, this newly-developed HPLC technique does not require pre-derivatisation and can separate glucosamine from both hydrochloride and sulphate salts, and from other amino sugars, such as chondroitin sulphate present in dietary supplements. This simple and effective technique can be employed by analytical laboratories for the quality control of glucosamine dietary supplements. CONCLUSION: The current study has developed a new analytical technique using HPLC-Corona CAD, which can analyse underivatised glucosamine hydrochloride and sulphate within 6 minutes. Using the novel assay, we confirmed that unlike the tested Australian dietary supplements, only half of the tested Indian products had a glucosamine content within ±10% of what was claimed on the label.
Subject(s)
Chromatography, High Pressure Liquid/methods , Dietary Supplements/analysis , Glucosamine/analysis , Aerosols , Australia , India , Reproducibility of ResultsABSTRACT
Amino sugars (AS) are routinely used as microbial biomarkers to investigate the dynamics of soil carbon (C) and nitrogen (N) under different environments. However, the effect of any AS on soil C and N, or other AS, is not well-defined. In this study, acid soils from Dongbei (D) and Fujian (F) and alkaline soil from Henan (H) were selected to perform an incubation experiment under glucosamine addition for 36 days. In the present study, the dynamics of soil soluble organic C (SOC), NH4+-N, NO3--N, soluble organic N (SON), and four AS: glucosamine (GluN), mannosamine (ManN), galactosamine (GalN), and muramic acid (MurN), were investigated. The results showed that AS was different among the three soils, but had similar dynamics in the same soil. The higher total C and inorganic N in the D and F relative to the H soil were related to the greater AS in two soils. With incubation, AS decreased in D soil and increased in F soil before 1 week, while after 1 week, the inverse dynamics were observed, which suggest that SOC or SOC combined with inorganic N may be a mechanism to adjust the dynamics of C from AS. Overall, glucosamine addition did not significantly affect AS in D, while the reverse was true for F and H soils. Glucosamine addition decreased AS at day 0 for D soil and at day 3 for F and H soils, and increased SOC. The lowered NH4+-N and AS in D soil, but the higher values of these, were observed in F soil after 1 week of incubation. The increase of SON in D soil with glucosamine addition might be due to the depolymerization of soil organic matter (SOM) into SON. However, the decrease of SON in F soil could be attributed to the mineralization of SON.
Subject(s)
Amino Sugars/analysis , Environmental Biomarkers , Forests , Nitrogen/analysis , Soil/chemistry , Carbon/analysis , Glucosamine/analysis , Hydrogen-Ion Concentration , Soil MicrobiologyABSTRACT
BACKGROUND: Morus alba and Morus nigra leaves which have been widely used as herbal teas in Anatolian region of Turkey, were extracted twice by 50â¯mM HCI solution, derivatized with 9-fluorenylmethyl chloroformate and analyzed by reversed phase HPLC equipped with a fluorescence detector. HYPOTHESIS/PURPOSE: This study was performed to determine the main antidiabetic active compounds 1-deoxynojirimycin by HPLC method and evaluate the in-vitro antioxidant and antidiabetic activity of ethanol extracts prepared from Morus alba L. and Morus nigra leaves. STUDY DESIGN: A reliable simple, and rapid high-performance liquid chromatographic (HPLC) method for the determination of 1-deoxynojirimycin in M. alba L. and M. nigra leaves with fluorimetric detection after pre-column derivatization with 9-fluorenylmethyl chloroformate was developed. In addition, the chemical composition of ethanol extract of mulberry leaves was analyzed with GC-MS. METHODS: Separation and quantitation were performed on C18, 250â¯×â¯4.6â¯mm, 5⯵m analytical column. Mobile phase consisted of acetonitrile and 0.1% acetic acid solution (1:1, v/v) was performed applied to the column 1.0â¯ml/min flow rate at 26 °C. Potential antioxidant activity of ethanol extract of different mulberry varieties were evaluated by DPPH, and ABTS radical scavenging assay as well as total phenol and flavonoid content were determined. In addition, α-amylase and α-glucosidase activity was determined by 96-well plate method to evaluate the probable antidiabetic potential use of Turkish mulberry leaves. RESULTS: The isocratic HPLC method showed excellent correlation coefficient (r2â¯=â¯0.9985) between 0.3 and 30⯵g/ml calibration points. The method was specific and sensitive with detection and quantification limits of 1.07 and 3.27â¯ng/ml, respectively. Intraday and interday method precision (nâ¯=â¯5) wereâ¯<â¯7.3 (RSD%). Intraday and interday method accuracy (nâ¯=â¯5) were between 3.77 and (-8.35) (RE%). The average method recovery (nâ¯=â¯3) was 102.5%. The results showed that the content of 1-deoxynojirimycin in leaves of Morus alba L. was 0.103% (nâ¯=â¯3), and in leaves of M. nigra L. was 0.102%. 2-hexadecen-1-ol, oleamide, 2-propenoic acid, and cyclododecane were identified as the major compounds by GC-MS in the ethanol extract of mulberry leaves. CONCLUSION: The obtained robustness values from emission and excitation detection, mobile phase ingredients and flow rates changes showed that method was very strong. This work contributes to the knowledge of antioxidant and antidiabetic properties of Morus species, thus may be provide useful data in evaluation of food products and pharmaceutical preparations produced from Morus species.
Subject(s)
1-Deoxynojirimycin/analysis , Antioxidants/pharmacology , Chromatography, High Pressure Liquid/methods , Glycoside Hydrolase Inhibitors/pharmacology , Morus/chemistry , alpha-Amylases/antagonists & inhibitors , Chromatography, Reverse-Phase , Fluorenes/chemistry , Gas Chromatography-Mass Spectrometry , Glucosamine/analogs & derivatives , Glucosamine/analysis , Glycoside Hydrolase Inhibitors/chemistry , Limit of Detection , Plant Leaves/chemistry , Reproducibility of Results , TurkeyABSTRACT
Saccharides and their derivatives are typical polar analytes without a suitable UV-chromophore that are nowadays analyzed by HPLC (high-performance liquid chromatography) under HILIC (hydrophilic interaction liquid chromatography) mode. Usually an evaporative light scattering detector (ELSD) is utilized which, however, gives a nonlinear response. A procedure to overcome the problem of mutarotating (time-varying) analytes recorded with such a nonlinear response detector is described. The procedure was applied for determination of glucosamine in two commercially available pharmaceutical formulations containing the common inorganic ions that the detector gives a response to. Under optimized conditions, both the anomers of glucosamine were separated and could be determined separately. Owing to the short retention time of the analyte (a run time <4 min) and relatively slow kinetics of the anomeric conversion (equilibration time 2.5 h), mutarotation could be monitored and corresponding rate constants calculated.
Subject(s)
Chromatography, Liquid/methods , Glucosamine/analysis , Glucosamine/chemistry , Scattering, Radiation , Hydrophobic and Hydrophilic Interactions , Isomerism , Light , Linear Models , Reproducibility of ResultsABSTRACT
Glucosamine (GlcN) is widely used in the nutraceutical and pharmaceutical industries. Currently, GlcN is mainly produced by traditional multistep chemical synthesis and acid hydrolysis, which can cause severe environmental pollution, require a long prodution period but a lower yield. The aim of this work was to develop a whole-cell biocatalytic process for the environment-friendly synthesis of glucosamine (GlcN) from N-acetylglucosamine (GlcNAc). We constructed a recombinant Escherichia coli and Bacillus subtilis strains as efficient whole-cell biocatalysts via expression of diacetylchitobiose deacetylase (Dacph) from Pyrococcus furiosus. Although both strains were biocatalytically active, the performance of B. subtilis was better. To enhance GlcN production, optimal reaction conditions were found: B. subtilis whole-cell biocatalyst 18.6 g/l, temperature 40°C, pH 7.5, GlcNAc concentration 50 g/l and reaction time 3 h. Under the above conditions, the maximal titer of GlcN was 35.3 g/l, the molar conversion ratio was 86.8% in 3-L bioreactor. This paper shows an efficient biotransformation process for the biotechnological production of GlcN in B. subtilis that is more environmentally friendly than the traditional multistep chemical synthesis approach. The biocatalytic process described here has the advantage of less environmental pollution and thus has great potential for large-scale production of GlcN in an environment-friendly manner.
Subject(s)
Acetylglucosamine/metabolism , Bacterial Proteins/metabolism , Glucosamine/biosynthesis , Hydrolases/metabolism , Metabolic Engineering , Pyrococcus furiosus/enzymology , Acetylglucosamine/analysis , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Biocatalysis , Escherichia coli/genetics , Escherichia coli/metabolism , Glucosamine/analysis , Hydrogen-Ion Concentration , Hydrolases/genetics , Pyrococcus furiosus/genetics , Reaction Time , TemperatureABSTRACT
N-acetyl-d-glucosamine (GlcNAc) is a microbial fermentation product, and NIR spectroscopy is an effective process analytical technology (PAT) tool in detecting the key quality attribute: the GlcNAc content. Meanwhile, the design of NIR spectrometers is under the trend of miniaturization, portability and low-cost nowadays. The aim of this study was to explore a portable micro NIR spectrometer with the fermentation process. First, FT-NIR spectrometer and Micro-NIR 1700 spectrometer were compared with simulated fermentation process solutions. The Rc2, Rp2, RMSECV and RMSEP of the optimal FT-NIR and Micro-NIR 1700 models were 0.999, 0.999, 3.226â¯g/L, 1.388â¯g/L and 0.999, 0.999, 1.821â¯g/L, 0.967â¯g/L. Passing-Bablok regression method and paired t-test results showed there were no significant differences between the two instruments. Then the Micro-NIR 1700 was selected for the practical fermentation process, 135 samples from 10 batches were collected. Spectral pretreatment methods and variables selection methods (BiPLS, FiPLS, MWPLS and CARS-PLS) for PLS modeling were discussed. The Rc2, Rp2, RMSECV and RMSEP of the optimal GlcNAc content PLS model of the practical fermentation process were 0.994, 0.995, 2.792â¯g/L and 1.946â¯g/L. The results have a positive reference for application of the Micro-NIR spectrometer. To some extent, it could provide theoretical supports in guiding the microbial fermentation or the further assessment of bioprocess.
Subject(s)
Fermentation/physiology , Glucosamine/analysis , Spectroscopy, Near-Infrared/methods , Bioreactors , Feasibility Studies , Glucosamine/metabolism , Microtechnology , Regression AnalysisABSTRACT
Since the determination of the fermentation kinetics is one of the main challenges in solid state fermentation, the quantitative measurement of biomass growth during microbial pretreatment by FTIR spectroscopy in Attenuated Total Reflectance mode was evaluated. Peaks at wave numbers of 1651â¯cm-1 and 1593â¯cm-1 showed to be affected during pretreatment of poplar wood particles by Phanerochaete chrysosporium MUCL 19343. Samples with different microbial biomass fractions were obtained from two different experiments, i.e., shake flask and fixed-bed reactor experiments. The glucosamine concentration was compared to the normalized absorbance ratio of the 1651â¯cm-1 to 1593â¯cm-1 peak, measured by FTIR-ATR, and resulted in a linear relationship. The application of a normalized absorbance ratio in function of time provided a graph that was similar to the microbial growth curve. Application of FTIR in ATR mode to follow-up kinetics during solid state fermentation seems to be a fast and easy alternative to laborious measurement techniques, such as glucosamine determination.
Subject(s)
Phanerochaete/growth & development , Populus/microbiology , Spectroscopy, Fourier Transform Infrared/methods , Batch Cell Culture Techniques , Bioreactors , Cell Wall/drug effects , Chitin/analysis , Chitin/metabolism , Glucosamine/analysis , Glucosamine/metabolism , Kinetics , Lignin/analysis , Lignin/metabolism , Pentanones/pharmacology , Phanerochaete/drug effects , Sulfuric Acids/pharmacologyABSTRACT
Ion mobility spectrometry (IMS) is an analytical technique that separates gas-phase ions drifting under an electric field according to their size to charge ratio. We used electrospray ionization-drift tube IMS coupled to quadrupole mass spectrometry to measure the mobilities of glucosamine (GH+ ) and caffeine (CH+ ) ions in pure nitrogen or when the shift reagent (SR) 2-butanol was introduced in the drift gas at 6.9 mmol m-3 . Binding energies of 2-butanol-ion adducts were calculated using Gaussian 09 at the CAMB3LYP/6-311++G(d,p) level of theory. The mobility shifts with the introduction of 2-butanol in the drift gas were -2.4% (GH+ ) and -1.7% (CH+ ) and were due to clustering of GH+ and CH+ with 2-butanol. The formation of GBH+ was favored over that of CBH+ because GBH+ formed more stable hydrogen bonds (83.3 kJ/mol) than CBH+ (81.7 kJ/mol) for the reason that the positive charge on CH+ is less sterically available than on GH+ and the charge is stabilized by resonance in CH+ . These results are a confirmation of the arguments used to explain the drift behavior of these ions when ethyl lactate SR was used (Bull Kor Chem Soc 2014, 1023-1028). This study is a step forward to predict IMS separations of overlapping peaks in IMS spectra, simplifying a procedure that is trial and error by now.
Subject(s)
Butanols/chemistry , Caffeine/analysis , Glucosamine/analysis , Ion Mobility Spectrometry/methods , Gases/chemistry , Ions/chemistry , Models, Molecular , Nitrogen/chemistry , ThermodynamicsABSTRACT
The development and application of polyelectrolytic gel electrodes (PGEs) for a microfluidic photothermal absorbance detection system is described. The PGEs are used to measure changes in conductivity based on heat generation by analytes absorbing light and changing the solution viscosity. The PGEs are suitable for direct contact conductivity measurements since they do not degrade with exposure to high electric fields. Both a 2-electrode system with DC voltages and a 3-electrode system with AC voltages were investigated. Experimental factors including excitation voltage, excitation frequency, laser modulation frequency, laser power, and path length were tested. The limits of detection for the 3-electrode and 2-electrode systems are 500nM and 0.55nM for DABSYL-tagged glucosamine, respectively. In addition, an electrokinetic separation of a potassium, DABSYL-tagged glucosamine, Rhodamine 6G, and Rhodamine B mixture was demonstrated.