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1.
《Journal of Molecular Catalysis .B, Enzymatic》2009,56(3-4):169-176
The three new dual-layer matrices (polyacrylonitrile (PAN) membranes coated with physically bound chitosan (CHI)—PANCHI-A and chemically bound chitosan—PANCHI-B and PANCHI-C) for immobilization of acetylcholinesterase (AChE) were obtained. The chemical-modified PAN membrane (PAN-NaOH + ethylenediamine (EDA)) was used as a base for the prepared dual-layer membranes. For chemical chitosan bound membrane, chitosan was tethered onto the membrane surface to form a dual-layer biomimetic membrane in the presence of glutaraldehyde (GA). The basic characteristics (amount of amino groups, hydrophilicity and transport characteristics) of the chitosan-modified membranes were investigated. The SEM analyses were shown essential morphology change in the different chitosan membranes.The relative activities and Vmax of the covalently immobilized enzyme on PANCHI-B and PANCHI-C membranes were higher than that on PANCHI-A membrane and chemical-modified membrane with NaOH + EDA. Km values for the different modified membranes are lower for the chitosan-treated membranes. The pH and temperature optimum of immobilized enzyme were determined. The bound enzymes on PANCHI-B and PANCHI-C have higher thermal and storage stability in comparison with AChE on PANCHI-A membrane and free enzyme. 相似文献
2.
Gabrovska K Marinov I Godjevargova T Portaccio M Lepore M Grano V Diano N Mita DG 《International journal of biological macromolecules》2008,43(4):339-345
Acetylcholinesterase (AChE) was immobilized on two different composite membranes constituted by a chemically modified poly-acrylonitrile (PAN) membrane plus a layer of tethered chitosan of different molecular weight, 10 kDa or 400 kDa. AChE was also directly immobilized on a chemically modified PAN membrane with NaOH and ethylenediamine (EDA) without chitosan. To know how the different supports affected the enzyme activity and the kinetic parameters, the AChE activity was studied in the soluble form and in the insoluble form with all the three types of modified PAN membranes. The best performance was obtained by the modified PAN membrane having the chitosan with the lower molecular weight. The results concerning the AChE inhibition by methyl-paraoxon and the subsequent reactivation by pyridine-2-aldoxime methochloride (2-PAM) are presented and discussed. The composite membrane having chitosan with the lower molecular weight appeared to be potentially useful for applications in the field of biosensors. 相似文献
3.
Gabrovska K Georgieva A Godjevargova T Stoilova O Manolova N 《Journal of biotechnology》2007,129(4):674-680
(Poly)acrylonitrile/chitosan (PANCHI) composite membranes were prepared. The chitosan layer was deposited on the surface as well as on the pore walls of the base membrane. This resulted in the reduction of the pore size of the membrane and in an increase of their hydrophilicity. The pore structure of PAN and PANCHI membranes were determined by TEM and SEM analyses. It was found that the average size of the pore under a selective layer base PAN membrane is 7 microm, while the membrane coated with 0.25% chitosan shows a reduced pore size--small or equal to 5 microm and with 0.35% chitosan--about 4 microm. The amounts of the functional groups, the degree of hydrophilicity and transport characteristics of PAN/Chitosan composite membranes were determined. Urease was covalently immobilized onto all kinds of PAN/chitosan composite membranes using glutaraldehyde. Both the amount of bound protein and relative activity of immobilized urease were measured. The highest activity (94%) was measured for urease bound to PANCHI2 membranes (0.25% chitosan). The basic characteristics (pH(opt), pH(stability), T(opt), T(stability), heat inactivation and storage stability) of immobilized urease were determined. The obtained results show that the poly(acrylonitrile)chitosan composite membranes are suitable for enzyme immobilization. 相似文献
4.
Covalent immobilization of acetylcholinesterase on a novel polyacrylic acid‐based nanofiber membrane
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Bekir Çakıroğlu Aslı Beyler Çiğil Ayşe Ogan M. Vezir Kahraman Serap Demir 《Engineering in Life Science》2018,18(4):254-262
In this study, polyacrylic acid‐based nanofiber (NF) membrane was prepared via electrospinning method. Acetylcholinesterase (AChE) from Electrophorus electricus was covalently immobilized onto polyacrylic acid‐based NF membrane by demonstrating efficient enzyme immobilization, and immobilization capacity of polymer membranes was found to be 0.4 mg/g. The novel NF membrane was synthesized via thermally activated surface reconstruction, and activation with carbonyldiimidazole upon electrospinning. The morphology of the polyacrylic acid‐based membrane was investigated by scanning electron microscopy, Fourier Transform Infrared Spectroscopy, and thermogravimetric analysis. The effect of temperature and pH on enzyme activity was investigated and maxima activities for free and immobilized enzyme were observed at 30 and 35°C, and pH 7.4 and 8.0, respectively. The effect of 1 mM Mn2+, Ni2+, Cu2+, Zn2+, Mg2+, Ca2+ ions on the stability of the immobilized AChE was also investigated. According to the Michaelis–Menten plot, AChE possessed a lower affinity to acetylthiocholine iodide after immobilization, and the Michaelis–Menten constant of immobilized and free AChE were found to be 0.5008 and 0.4733 mM, respectively. The immobilized AChE demonstrated satisfactory reusability, and even after 10 consecutive activity assay runs, AChE maintained ca. 87% of its initial activity. Free enzyme lost its activity completely within 60 days, while the immobilized enzyme retained approximately 70% of the initial activity under the same storage time. The favorable reusability of immobilized AChE enables the support to be employable to develop the AChE‐based biosensors. 相似文献
5.
Heru Susanto A.M. Samsudin Nur Rokhati I.N. Widiasa 《Enzyme and microbial technology》2013,52(6-7):386-392
The glucose oxidase (GOx) enzyme was immobilized on chitosan-based porous composite membranes using a covalent bond between GOx and the chitosan membrane. The chitosan-based porous membranes were prepared by the combination of the evaporation- and non-solvent-induced phase separation methods. To increase the membrane conductivity, carbon nanotubes (CNTs) were added to the chitosan solution. The resulting membranes were characterized in terms of water permeability, surface morphology and surface chemistry. Enzyme immobilization was performed on the chitosan membranes with and without activation using glutaraldehyde (GA). Three different configurations of working electrodes were evaluated to investigate the potential use of the modified membranes in biosensors. The results show that enzyme immobilization capacity was greater for membranes that had been activated than for membranes that had not been activated. In addition, activation increased the stability of the enzyme immobilization. The immobilization of GOx on chitosan-based membranes was influenced by both pH and the concentration of the enzyme solution. The presence of CNTs significantly increased the electrical conductivity of the chitosan membranes. The evaluation of three different configurations of working electrodes suggested that the third configuration, which was composed of an electrode-mediator-(chitosan and carbon nanotube) structure and enzyme, is the best candidate for biosensor applications. 相似文献
6.
Preparation and characterization of epoxy-functionalized magnetic chitosan beads: laccase immobilized for degradation of reactive dyes 总被引:1,自引:0,他引:1
Gulay Bayramoglu Meltem Yilmaz M. Yakup Arica 《Bioprocess and biosystems engineering》2010,33(4):439-448
Cross-linked magnetic chitosan beads were prepared by phase-inversion technique in the presence of epichlorohydrin under alkaline
condition, and used for covalent immobilization of laccase. The activity of the immobilized laccase on the magnetic chitosan
was about 260 U (g/dry beads) with an enzyme loading of about 16.33 ± 0.39 mg [(g/dry beads) mg/g]. Kinetic parameters, V
max and K
m values were determined as 21.7 U/mg protein and 9.4 μM for free enzyme, and 15.6 U/mg protein and 19.7 μM for the immobilized
laccase, respectively. The operational and thermal stabilities of the immobilized laccase were improved compared to free counterpart.
The immobilized laccase was operated in a batch reactor for the decolorization of reactive dyes from aqueous solution. The
laccase immobilized on magnetic chitosan beads was very effective for removal of textile dyes from aqueous solution which
creates an important environmental problem in the discharged textile dying solutions. 相似文献
7.
A new method for immobilization of acetylcholinesterase (AChE) to alginate gel beads by activating the carbonyl groups of
alginate using carbodiimide coupling agent has been successfully developed. Maximum reaction rate (V
max) and Michaelis–Menten constant (K
m) were determined for the free and binary immobilized enzyme. The effects of pH, temperature, storage stability, reuse number
and thermal stability on the free and immobilized AChE were also investigated. For the free and binary immobilized enzyme
on the Ca–alginate gel beads, optimum pH values were found to be 7 and 8, respectively. Optimum temperatures for the free
and immobilized enzyme were observed to be 30 and 35 °C, respectively. Upon 60 days of storage the preserved activity of free
and immobilized enzyme were found as 4 and 68%, respectively. In addition, reuse number, and thermal stability of the free
AChE were increased by as a result of binary immobilization. 相似文献
8.
Xiao-Jun Huang An-Guo Yu Jun Jiang Chao Pan Jing-Wen Qian Zhi-Kang Xu 《Journal of Molecular Catalysis .B, Enzymatic》2009,57(1-4):250-256
In this work, poly(acrylonitrile-co-acrylic acid) (PANCAA) was electrospun into nanofibers with a mean diameter of 180 nm. To create a biofriendly microenvironment for enzyme immobilization, collagen or protein hydrolysate from egg skin (ES) was respectively tethered on the prepared nanofibrous membranes in the presence of 1-ethyl-3-(dimethyl-aminopropyl) carbodiamine (EDC)/N-hydroxyl succinimide (NHS). Confocal laser scanning microscopy (CLSM) was used to verify the surface modification and protein density on the nanofibrous membranes. Lipase from Candida rugosa was then immobilized on the protein-modified nanofibrous membranes by covalent binding using glutaraldehyde (GA) as coupling agent, and on the nascent PANCAA nanofibrous membrane using EDC/NHS as coupling agent, respectively. The properties of the immobilized enzyme were assayed. It was found that different pre-tethered biomacromolecules had distinct effects on the immobilized enzyme. The activity retention of the immobilized lipase on ES hydrolysate-modified nanofibrous membrane increased from 15.0% to 20.4% compared with that on the nascent one, while it was enhanced up to more than quadrupled (activity retention of 61.7%) on the collagen-modified nanofibrous membrane. The kinetic parameter, Km and Vmax, were also determined for the free and immobilized lipases. Furthermore, the stabilities of the immobilized lipases were obviously improved compared with the free one. 相似文献
9.
10.
G��lay Bayramo?lu Beg��m Alt?nta? M. Yakup Ar?ca 《Bioprocess and biosystems engineering》2011,34(2):127-134
Polyacrylonitrile film (PAN) surfaces were modified with chemical polymerization of conductive polyaniline (PANI) in the presence
of potassium dichromate as an oxidizing agent. The conductive films were used for immobilization of uricase. The surface resistance
of the conductive film in this work was found to be 0.97 kΩ/cm. The maximum amount of immobilized enzyme on conductive film
containing 2.4% PANI was about 216 μg/cm2. The optimum pH for free and immobilized enzymes was observed at 7.0 and 7.5, respectively. The K
m values for free and immobilized uricase were found to be 94 and 138 μM, respectively. V
max values were calculated as 1.87 and 1.63 U/mg protein for the free and immobilized enzymes, respectively. Immobilized uricase
exhibited ~68% of its original activity even after 2 months of storage at 4 °C while the free enzyme lost its initial activity
within 4 weeks. 相似文献
11.
Poly(itaconic acid) grafted and/or Fe(III) ions incorporated chitosan membranes were used for reversible immobilization of catalase (from bovine liver) via adsorption. The influences of pH and initial catalase concentration on the immobilization capacities of the CH-g-poly(IA) and CH-g-poly(IA)-Fe(III) membranes have been investigated in a batch system. Maximum catalase adsorption onto CH-g-poly(IA) and CH-g-poly(IA)-Fe(III) membrane were found to be 6.3 and 37.8 mg/g polymer at pH 5.0 and 6.5, respectively. The CH-g-poly(IA)-Fe(III) membrane with high catalase adsorption capacity was used in the rest of the study. The Km value for immobilized catalase on CH-g-poly(IA)-Fe(III) (25.8 mM) was higher about 1.6-fold than that of free enzyme (13.5 mM). Optimum operational temperature was observed at 40 °C, a 5 °C higher than that of the free enzyme and was significantly broader. The optimum operational pH was same for both free and immobilized catalase (pH 7.0). Thermal stability was found to increase with immobilization. Free catalase lost all its activity within 20 days whereas immobilized catalase lost 23% of its activity during the same incubation period. It was observed that the same support enzyme can be repeatedly used for immobilization of catalase after regeneration without significant loss in adsorption capacity or enzyme activity. In addition, the CH-g-poly(IA)-Fe(III) membrane prepared in this work showed promising potential for various biotechnological applications. 相似文献
12.
W. Somers R. T. M. van den Dool G. A. H. de Jong J. A. Jongejan J. A. Duine J. P. van der Lugt 《Biotechnology Techniques》1994,8(6):407-412
Summary Quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni was immobilized on polypyrrole-coated track-etch and microporous membranes. On the track-etch membrane, 3.4 to 4.8 × 10–3 Units of enzyme/cm2 was immobilized whilst on the microporous membrane 0.05 U/cm2 was immobilized. The track-etch membrane was then used in electrochemical studies using ferricyanide as a redox mediator giving a maximum catalytic current of 0.022 mA/cm2 membrane with 1-pentanol as the substrate. The kinetic parameters (Km and Vmax) of the immobilized enzyme are of the same order of magnitude as those of the free enzyme. 相似文献
13.
Wen-Yi Yang Munusamy Thirumavalavan Madasamy Malini Gurusamy Annadurai Jiunn-Fwu Lee 《The Journal of membrane biology》2014,247(7):549-559
The present work was aimed at developing stability enhanced silica gel-supported macroporous chitosan membrane for immobilization of enzymes. The membrane was surface modified using various cross-linking agents for covalent immobilization of enzyme Bovine serum albumin. The results of FT-IR, UV–vis, and SEM analyses revealed the effect of cross-linking agents and confirmed the formation of modified membranes. The presence of silica gel as a support could provide a large surface area, and therefore, the enzyme could be immobilized only on the surface, and thus minimized the diffusion limitation problem. The resultant enzyme immobilized membranes were also characterized based on their activity retention, immobilization efficiency, and stability aspects. The immobilization process increased the activity of immobilized enzyme even higher than that of total (actual) activity of native enzyme. Thus, the obtained macroporous chitosan membranes in this study could act as a versatile host for various guest molecules. 相似文献
14.
Song-Se Yi Jin-Mi Noh Yoon-Sik Lee 《Journal of Molecular Catalysis .B, Enzymatic》2009,57(1-4):123-129
Amino acid modified chitosan beads (CBs) for immobilization of lipases from Candida rugosa were prepared by activation of a chitosan backbone with epichlorohydrin followed by amino acid coupling. The beads were analyzed by elemental analysis and solid state NMR with coupling yields of the amino acids ranging from 15 to 60%. The immobilized lipase on unmodified chitosan beads showed the highest immobilization yield (92.7%), but its activity was relatively low (10.4%). However, in spite of low immobilization yields (15–50%), the immobilized lipases on the amino acid modified chitosan beads showed activities higher than that of the unmodified chitosan beads, especially on Ala or Leu modified chitosan beads (Ala-CB or Leu-CB) with 49% activity for Ala-CB and 51% for Leu-CB. The immobilized lipases on Ala-CB improved thermal stability at 55 °C, compared to free and immobilized lipases on unmodified chitosan beads and the immobilized lipase on Ala-CB retained 93% of the initial activity when stored at 4 °C for 4 weeks. In addition, the activity of the immobilized lipase on Ala-CB retained 77% of its high initial activity after 10 times of reuse. The kinetic data (kcat/Km) supports that the immobilized lipase on Ala-CB can give better substrate specificity than the unmodified chitosan beads. 相似文献
15.
Gabrovska K Ivanov J Vasileva I Dimova N Godjevargova T 《International journal of biological macromolecules》2011,48(4):620-626
A new matrix for enzyme immobilization of urease was obtained by incorporating rhodium nanoparticles (5% on activated charcoal) and chemical bonding of chitosan with different concentration (0.15%; 0.3%; 0.5%; 1.0%; 1.5%) in previously chemically modified AN copolymer membrane. The basic characteristics of the chitosan modified membranes were investigated. The SEM analyses were shown essential morphology change in the different modified membranes. Both the amount of bound protein and relative activity of immobilized enzyme were measured. A higher activity (about 77.44%) was measured for urease bound to AN copolymer membrane coated with 1.0% chitosan and containing rhodium nanoparticles. The basic characteristics (pH(opt), T(opt), thermal, storage and operation stability) of immobilized enzyme on this optimized modified membrane were also determined. The prepared enzyme membrane was used for the construction of amperometric biosensor for urea detection. Its basic amperometric characteristics were investigated. A calibration plot was obtained for urea concentration ranging from 1.6 to 23 mM. A linear interval was detected along the calibration curve from 1.6 to 8.2mM. The sensitivity of the constructed biosensor was calculated to be 3.1927 μAmM(-1)cm(-2). The correlation coefficient for this concentration range was 0.998. The detection limit with regard to urea was calculated to be 0.5mM at a signal-to-noise ratio of 3. The biosensor was employed for 10 days while the maximum response to urea retained 86.8%. 相似文献
16.
An enzymatic membrane for application in the processes of decomposition and removal of urea from aqueous solutions was prepared: jack bean urease was immobilized on an aminated polysulphone membrane by adsorption. The inhibition of the system by boric acid was studied using procedures based on the MICHAELIS-MENTEN integrated equation (non-linear regression, and the linear transformations of WALKER and SCHMIDT, JENNINGS and NIEMANN, and BOOMAN and NIEMANN). The reaction was carried out in a 100 mM phosphate buffer of pH 7.0, containing 2 mM EDTA, obtained by neutralization of orthophosphoric acid with NaOH, at an initial urea concentration of 10 mM, and a temperature of 25 °C. The reaction was initiated by the addition of the enzyme to the urea solution, and was monitored by removing samples of the reaction mixture for NH3 determinations by the phenol-hypochlorite method until the urea was exhausted. The results were compared with those obtained earlier under the same reaction conditions for free urease and urease covalently immobilized on chitosan. The inhibition was found to be competitive, similar to that of the free enzyme and urease immobilized on chitosan, with inhibition constants Ki equal to 0.36, 0.19 and 0.60 mM. The results show that adsorption of the enzyme on a polysulphone membrane changed the enzyme to a lesser degree than covalent immobilization of the enzyme on a chitosan membrane. 相似文献
17.
Soria F Ellenrieder G Oliveira GB Cabrera M Carvalho LB 《Applied microbiology and biotechnology》2012,93(3):1127-1134
α-l-Rhamnosidase from Aspergillus terreus was covalently immobilized on the following ferromagnetic supports: polyethylene terephthalate (Dacron-hydrazide), polysiloxane/polyvinyl
alcohol (POS/PVA), and chitosan. The powdered supports were magnetized by thermal coprecipitation method using ferric and
ferrous chlorides, and the immobilization was carried out via glutaraldehyde. The activity of the Dacron-hydrazide (0.53 nkat/μg
of protein) and POS/PVA (0.59 nkat/μg of protein) immobilized enzyme was significantly higher than that found for the chitosan
derivative (0.06 nkat/μg of protein). The activity–pH and activity–temperature profiles for all immobilized enzymes did not
show difference compared to the free enzyme, except the chitosan derivative that presented higher maximum temperature at 65 °C.
The Dacron-hydrazide derivative thermal stability showed a similar behavior of the free enzyme in the temperature range of
40–70 °C. The POS/PVA and chitosan derivatives were stable up to 60 °C, but were completely inactivated at 70 °C. The activity
of the preparations did not appreciably decrease after ten successive reuses. Apparent K
m of α-l-rhamnosidase immobilized on magnetized Dacron-hydrazide (1.05 ± 0.22 mM), POS/PVA (0.57 ± 0.09 mM), and chitosan (1.78 ± 0.24 mM)
were higher than that estimated for the soluble enzyme (0.30 ± 0.03 mM). The Dacron-hydrazide enzyme derivative showed better
performance than the free enzyme to hydrolyze 0.3% narigin (91% and 73% after 1 h, respectively) and synthesize rhamnosides
(0.116 and 0.014 mg narirutin after 1 h, respectively). 相似文献
18.
Ern? Zádor 《Molecular & general genetics : MGG》1989,218(3):487-490
Summary Acetylcholinesterase (AChE) is mainly membrane bound in the central nervous system (CNS) of larvae and in the head and thorax of adults of Drosophila melanogaster; it is mostly soluble in the larval carcass, the adult abdomen, similar to that of the embryos (Zador et al. 1986). The enzyme shows the same number of isozymes (four or five) in larvae and adults as in the head of the fly or in embryos (Zador et al. 1986). In the Df(3R)GE26/MKRS stock both the membrane bound and the soluble enzyme are at about half normal levels while in the Df(3R)Ace
HD1/MKRS stock this is true only for the membrane bound AChE. Therefore the effect of the above deficiencies in larvae and adults is consistent with that in embryos (Zador et al. 1986). In heat-sensitive combinations of certain Ace mutant alleles both the membrane bound and the soluble enzyme has reduced activity.Abbreviations AChE
acetylcholinesterase (acetylcholine acetyl hydrolase, EC 3.1.1.7)
- BAP
1,5-bis(allyldimethylammonium-phenyl)-pentan-3-one dibromide
- CNS
central nervous system 相似文献
19.
This study was carried out to define how the overall rate of reaction would be influenced by different degrees of diffusional resistance to cofactor transport within an oxidoreductase membrane matrix. To accomplish this, 0.7–6.6μM yeast alcohol dehydrogenase was immobilized in an albumin matrix crosslinked with 2.5 or 5.0% glutaraldehyde to give 102–1685 μM thick membranes. The enzyme half-life was at least doubled at pH 7.5 or 8.8 on immobilization. Values of the kinetic constants for the soluble and immobilized enzyme were determined at 25°C and pH 8.8 over the range of 0.01–1.0M bulk solution concentration of ethanol as substrate and 140–1000μM bulk solution concentration of nicotinamide adenine dinucleotide (NAD+) as cofactor, to give essentially single substrate kinetics in NAD+. Equilibrium partitioning of ethanol and NAD+ between the solution and membrane was measured and used in the data analysis. The four kinetic constants for the soluble enzyme agreed with literature values; and all increased with immobilization of the enzyme. The Michaelis constants for NAD+ and for ethanol were greater for the immobilized enzyme. The diffusional resistance to NAD+ transport, presented in terms of the Thiele modulus, showed that the overall rate of reaction was decreased by about 50% even at values of the modulus as low as 2.0. 相似文献
20.
《Process Biochemistry》2014,49(10):1682-1690
Double enzymes (alcalase and trypsin) were effectively immobilized in a composite carrier (calcium alginate–chitosan) to produce immobilized enzyme beads referred to as ATCC. The immobilization conditions for ATCC were optimized, and the immobilized enzyme beads were characterized. The optimal immobilization conditions were 2.5% of sodium alginate, 10:4 sodium alginate to the double enzymes, 3:7 chitosan solution to CaCl2 and 2.5 h immobilization time. The ATCC beads had greatly enhanced stability and good usability compared with the free form. The ATCC residual activity was retained at 88.9% of DH (degree of hydrolysis) after 35 days of storage, and 36.0% of residual activity was retained after three cycles of use. The beads showed a higher zein DH (65.8%) compared with a single enzyme immobilized in the calcium alginate beads (45.5%) or free enzyme (49.3%). The ATCC kinetic parameters Vmax and apparent Km were 32.3 mL/min and 456.62 g−1, respectively. Active corn peptides (CPs) with good antioxidant activity were obtained from zein in the ethanol phase. The ATCC might be valuable for preparing CPs and industrial applications. 相似文献