enzyme immobilization
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β-Glucans comprise a group of β-D-glucose polysaccharides (glucans) that occur naturally in the cell walls of bacteria, fungi, and cereals. Its degradation is catalyzed by β-glucanases, enzymes that catalyze the breakdown of β-glucan into cello-oligosaccharides and glucose. These enzymes are classified as endo-glucanases, exo-glucanases, and glucosidases according to their mechanism of action, being the lichenases (β-1,3;1,4-glucanases, EC 3.2.1.73) one of them. Hence, we aimed to enhance lichenase production by Thermothelomyces thermophilus through the application of response surface methodology, using tamarind (Tamarindus indica) and jatoba (Hymenaea courbaril) seeds as carbon sources. The crude extract was immobilized, with a focus on improving lichenase activity, using various ionic supports, including MANAE (monoamine-N-aminoethyl), DEAE (diethylaminoethyl)-cellulose, CM (carboxymethyl)-cellulose, and PEI (polyethyleneimine)-agarose. Regarding lichenase, the optimal conditions yielding the highest activity were determined as 1.5% tamarind seeds, cultivation at 50 °C under static conditions for 72 h. Moreover, transitioning from Erlenmeyer flasks to a bioreactor proved pivotal, resulting in a 2.21-fold increase in activity. Biochemical characterization revealed an optimum temperature of 50 °C and pH of 6.5. However, sustained stability at varying pH and temperature levels was challenging, underscoring the necessity of immobilizing lichenase on ionic supports. Notably, CM-cellulose emerged as the most effective immobilization medium, exhibiting an activity of 1.01 U/g of the derivative (enzyme plus support), marking a substantial enhancement. This study marks the first lichenase immobilization on these chemical supports in existing literature.
- MeSH
- enzymy imobilizované * metabolismus chemie MeSH
- fungální proteiny * metabolismus chemie MeSH
- glykosidhydrolasy * metabolismus chemie biosyntéza MeSH
- koncentrace vodíkových iontů MeSH
- ovoce metabolismus MeSH
- semena rostlinná metabolismus MeSH
- Sordariales MeSH
- stabilita enzymů MeSH
- Tamarindus metabolismus mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
MicroRNAs (miRNAs) are small non-coding RNAs (18-22 nucleotides) that regulate gene expression and are associated with various diseases, including Laryngeal Cancer (LCa), which has a high mortality rate due to late diagnosis. Traditional methods for miRNA detection present several drawbacks (time-consuming steps, high cost and high false positive rate). Early-stage diagnosis and selective detection of miRNAs remain challenging. This study proposes a 3D flexible biosensor that combines nanofibers (NFs), gold nanoparticles (AuNPs), and an inverse molecular sentinel (iMS) for enzyme-free, SERS-based detection of miRNA-223-3p, evaluated as a potential LCa biomarker. The electrospun flexible nanofibers decorated with AuNPs enhance Raman signal. Selective detection of miRNA-223-3p is achieved by immobilizing an iMS-DNA probe labeled with a Raman reporter (Cyanine 3) on the AuNPs. The iMS distinctive stem-and-loop structure undergoes a conformational change upon interaction with the miRNA-223-3p, producing an "on to off" SERS signal. The proposed sensor demonstrated a linear detection range from 10 to 250 fM, with a limit of detection (LOD) of 19.50 ± 0.05 fM. The sensor selectivity was confirmed by analyzing the SERS signal behaviour in the presence of both Non-complementary miRNA and miRNA with three mismatched base pairs. This easily fabricable sensor requires no amplification and offers key advantages, including sensitivity, flexibility, and cost-effectiveness.
- MeSH
- biosenzitivní techniky * metody MeSH
- časná detekce nádoru metody MeSH
- kovové nanočástice * chemie MeSH
- lidé MeSH
- limita detekce MeSH
- mikro RNA * analýza genetika MeSH
- nádory hrtanu * diagnóza genetika MeSH
- nanovlákna * chemie MeSH
- Ramanova spektroskopie * metody MeSH
- zlato * chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Biotransformation of steroids by fungi has been raised as a successful, eco-friendly, and cost-effective biotechnological alternative for chemical derivatization. Endophytic fungi live inside vegetal tissues without causing damage to the host plant, making available unique enzymes that carry out uncommon reactions. Moreover, using nanofibrous membranes as support for immobilizing fungal cells is a powerful strategy to improve their performance by enabling the combined action of adsorption and transformation processes, along with increasing the stability of the fungal cell. In the present study, we report the use of polyacrylonitrile nanofibrous membrane (PAN NFM) produced by electrospinning as supporting material for immobilizing the endophytic fungus Penicillium citrinum H7 aiming the biotransformation of progesterone. The PAN@H7 NFM displayed a high progesterone transformation efficiency (above 90%). The investigation of the biotransformation pathway of progesterone allowed the putative structural characterization of its main fungal metabolite by GC-MS analysis. The oxidative potential of P. citrinum H7 was selective for the C-17 position of the steroidal nucleus.
- MeSH
- biotransformace MeSH
- nanovlákna * chemie MeSH
- progesteron MeSH
- Publikační typ
- časopisecké články MeSH
The purpose of the current study was to evaluate the functional activity and storage viability (at 4 °C and 35 °C) of an immobilized as well as lyophilized multienzyme, viz., pectinase, cellulase, and amylase (PCA) that was produced by Bacillus subtilis NG105 under solid state fermentation (SSF) at 35 °C for 10 days using mosambi peel as a substrate. After SSF, the culture media was divided into two aliquots. From the first aliquot, the produced ME was extracted, precipitated, and further immobilized on calcium alginate beads (MEICA). In order to immobilize on mosambi peel matrix, the second aliquot was mixed with acetone and subsequently lyophilized (MELMP). Thus, ready MEICA and MELMP extracted 87.5 and 91.5% juice from mango pulp, respectively. In the reusability study, after 5 cycles, MEICA exhibited 23.8%, 24.4%, and 36.5% PCA activity, respectively. The PCA activity of MEICA and MELMP was examined after 60 days of storage at 4 °C. The result revealed that the PCA for MEICA declined from 100 to 66%, 58.2%, and 64.5%, respectively, while for MELMP, it dropped from 100 to 84.2%, 82.1%, and 69.7%, respectively. Further, after 60 days of storage, the reduction of total protein content (TPC) in free multienzyme (FME), MEICA, and MELMP was 92.2%, 91.5%, and 36.3% observed, respectively. In the localization study, the maximum levels of multienzyme activity were found in cell exudates. This study demonstrated that immobilizing of multienzyme through lyophilization on waste substrates like mosambi peel boosted its stability and shelf-life along with greatly reducing the cost of products.
BACKGROUND: Ultra-high field magnetic resonance imaging (MRI) has obvious advantages in acquiring high-resolution images. 7 T MRI has been clinically approved and 21.1 T MRI has also been tested on rodents. PURPOSE: To examine the effects of ultra-high field on mice behavior and neuron activity. STUDY TYPE: Prospective, animal model. ANIMAL MODEL: Ninety-eight healthy C57BL/6 mice and 18 depression model mice. FIELD STRENGTH: 11.1-33.0 T SMF (static magnetic field) for 1 hour and 7 T for 8 hours. Gradients were not on and no imaging sequence was used. ASSESSMENT: Open field test, elevated plus maze, three-chambered social test, Morris water maze, tail suspension test, sucrose preference test, blood routine, biochemistry examinations, enzyme-linked immunosorbent assay, immunofluorescent assay. STATISTICAL TESTS: The normality of the data was assessed by Shapiro-Wilk test, followed by Student's t test or the Mann-Whitney U test for statistical significance. The statistical cut-off line is P < 0.05. RESULTS: Compared to the sham group, healthy C57/6 mice spent more time in the center area (35.12 ± 4.034, increased by 47.19%) in open field test and improved novel index (0.6201 ± 0.02522, increased by 16.76%) in three-chambered social test a few weeks after 1 hour 11.1-33.0 T SMF exposure. 7 T SMF exposure for 8 hours alleviated the depression state of depression mice, including less immobile time in tail suspension test (58.32% reduction) and higher sucrose preference (increased by 8.80%). Brain tissue analysis shows that 11.1-33.0 T and 7 T SMFs can increase oxytocin by 164.65% and 36.03%, respectively. Moreover, the c-Fos level in hippocampus region was increased by 14.79%. DATA CONCLUSION: 11.1-33.0 T SMFs exposure for 1 hour or 7 T SMF exposure for 8 hours did not have detrimental effects on healthy or depressed mice. Instead, these ultra-high field SMFs have anti-depressive potentials. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 1.
This article describes the characterization and application of collagenase-based chitosan nanofiber membranes with rat burns. Electrospun chitosan nanofibers were functionalized with clostridial collagenase using carbodiimide chemistry. The immobilized collagenase was characterized by enzyme activity, kinetic constants, and dry storage stability measurements using a Pz-peptide substrate. The apparent kinetic constants KM and Vmax of immobilized collagenase showed a high affinity for the peptide substrate compared to the free enzyme. Drying of chitosan membranes with immobilized collagenase ensured 98 % stability of enzyme activity after rehydration. The effect of collagenase immobilized on chitosan nanofibers on the burn of the rat model was compared with a control treatment with chitosan nanofibers. The healing of the wound with both materials was terminated after 30 days at the same time, although the collagenase wound healed more rapidly during healing. The scar area size after the application of collagenase-containing chitosan nanofiber membranes was 31.6 % smaller than when only chitosan nanofibers were used.
- MeSH
- chitosan terapeutické užití MeSH
- Clostridium histolyticum MeSH
- enzymy imobilizované MeSH
- hojení ran * účinky léků MeSH
- krysa rodu rattus MeSH
- kůže zranění MeSH
- mikrobiální kolagenasa * metabolismus terapeutické užití MeSH
- nanovlákna terapeutické užití MeSH
- pilotní projekty MeSH
- rány a poranění farmakoterapie patologie MeSH
- stabilita enzymů MeSH
- výsledek terapie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
With the increased demand for beef in emerging markets, the development of quality-control diagnostics that are fast, cheap and easy to handle is essential. Especially where beef must be free from pork residues, due to religious, cultural or allergic reasons, the availability of such diagnostic tools is crucial. In this work, we report a label-free impedimetric genosensor for the sensitive detection of pork residues in meat, by leveraging the biosensing capabilities of graphene acid - a densely and selectively functionalized graphene derivative. A single stranded DNA probe, specific for the pork mitochondrial genome, was immobilized onto carbon screen-printed electrodes modified with graphene acid. It was demonstrated that graphene acid improved the charge transport properties of the electrode, following a simple and rapid electrode modification and detection protocol. Using non-faradaic electrochemical impedance spectroscopy, which does not require any electrochemical indicators or redox pairs, the detection of pork residues in beef was achieved in less than 45 min (including sample preparation), with a limit of detection of 9% w/w pork content in beef samples. Importantly, the sample did not need to be purified or amplified, and the biosensor retained its performance properties unchanged for at least 4 weeks. This set of features places the present pork DNA sensor among the most attractive for further development and commercialization. Furthermore, it paves the way for the development of sensitive and selective point-of-need sensing devices for label-free, fast, simple and reliable monitoring of meat purity.
Antibiotics are antimicrobial substances that can be used for preventive and therapeutic purposes in humans and animals. Their overdose usage has led to uncontrolled release to the environment, contributing significantly to the development of antimicrobial resistance phenomena. Here, enzyme-immobilized self-propelled zinc oxide (ZnO) microrobots are proposed to effectively target and degrade the released antibiotics in water bodies. Specifically, the morphology of the microrobots is tailored via the incorporation of Au during the synthetic process to lead the light-controlled motion into having on/off switching abilities. The microrobots are further modified with laccase enzyme by physical adsorption, and the immobilization process is confirmed by enzymatic activity measurements. Oxytetracycline (OTC) is used as a model of veterinary antibiotics to investigate the enzyme-immobilized microrobots for their removal capacities. The results demonstrate that the presence of laccase on the microrobot surfaces can enhance the removal of antibiotics via oxidation. This concept for immobilizing enzymes on self-propelled light-driven microrobots leads to the effective removal of the released antibiotics from water bodies with an environmentally friendly strategy.
- MeSH
- antibakteriální látky MeSH
- chemické látky znečišťující vodu * MeSH
- lakasa metabolismus MeSH
- lidé MeSH
- oxid zinečnatý * MeSH
- oxytetracyklin * MeSH
- voda MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Mikrobiální enzymy jsou používány v širokém spektru průmyslové výroby, ve farmacii, v medicíně, nebo jako součást detekčních metod či biosenzorů, díky nízkým nákladům na produkci za krátkou časovou jednotku. Imobilizace enzymů na pevné povrchy se ukázala jako metoda zlepšující potřebné nároky, jako jsou vyšší efektivita enzymatické reakce v čase, lepší pH a tepelná stabilita, možnost opakovaného použití, snadná separace enzymu a dlouhodobá stabilita. V průmyslu se imobilizace využívá v mnoha procesech výroby potravin, v detergentech a při jejich přípravě, v textilním průmyslu nebo při produkci bio paliv. Ve farmacii jsou imobilizované enzymy součástí výroby léčiv, či jako složka léčiv samotných, v medicíně se imobilizace enzymů využívá k léčbě a diagnostice chorob. Imobilizované enzymy se ukázaly jako vhodná součást detekčních metod, jsou součástí biosenzorů pro stanovení specifických markerů otrav a nemocí nebo se využívají jako detekční zařízení pro stanovení znečistění vod, půd či jako ekologická varianta nahrazující toxické chemikálie. Imobilizace enzymů na pevné povrchy se prosazuje v mnoha oborech a do budoucna v sobě skýtá velký potenciál.
Microbial enzymes are used in a wide range of industrial production, in pharmacy, medicine or as part of detection methods or biosensors, due to low-cost production in short time. Immobilization of enzymes on solid surfaces has been shown to improve essential requirements, such as higher efficiency of the enzymatic reaction per unit time, better pH and thermo stability, repeated use, easy separation of enzyme and long-term stability. In industry, immobilization is used in food production processes, in detergents and their preparation, in the textile industry or in the production of biofuels. In pharmacy, immobilized enzymes are a part of the production of drugs, or as a part of drugs themselves, in medicine, immobilized enzymes are used to treat or diagnosis of diseases. Immobilized enzymes are a suitable part of detection methods, segment of biosensors for the determination of specific markers of poisoning and diseases, also are used for the determination of water and soil pollution or as an ecological variant replacing toxic chemicals. Immobilization of enzymes on solid surfaces is used in many areas and offers great potential for the future.
Biosensors containing cholinesterase are analytical devices suitable for the assay of neurotoxic compounds. In the research on biosensors, a new platform has appeared some years ago. It is the digital photography and scoring of coloration (photogrammetry). In this paper, a colorimetric biosensor is constructed using 3D-printed multiwell pads treated with indoxylacetate as a chromogenic substrate and gold nanoparticles with the immobilized enzyme butyrylcholinesterase. A smartphone camera served for photogrammetry. The biosensor was tested for the assay of carbofuran and paraoxon ethyl as two types of covalently binding inhibitors: irreversible and pseudoirreversible. The biosensor exerted good sensitivity to the inhibitors and was able to detect carbofuran with a limit of detection for carbofuran 7.7 nmol/l and 17.6 nmol/l for paraoxon ethyl. A sample sized 25 μl was suitable for the assay lasting approximately 70 minutes. Up to 121 samples can be measured contemporary using one multiwell pad. The received data fully correlated with the standard spectrophotometry. The colorimetric biosensor exerts promising specifications and appears to be competitive to the other analytical procedures working on the principle of cholinesterase inhibition. Low-cost, simple, and portable design represent an advantage of the assay of the biosensor. Despite the overall simplicity, the biosensor can fully replace the standard spectroscopic methods.
- Publikační typ
- časopisecké články MeSH
