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1.
A commercial inulinase could convert inulin into fructose, which was optimized to be entrapped in the calcium alginate-gelatin beads with the immobilization yield of 86% for free inulinase activities. The optimum pH values and temperatures were 4.5 and 40 °C for the free enzyme and 5.0–5.5 and 45–50 °C for the immobilized enzyme. The kinetic parameters of V max and K m were 5.24 μmol/min and 57.6 mg/mL for the free inulinase and 4.32 μmol/min and 65.8 mg/mL for the immobilized inulinase, respectively. The immobilized enzyme retained 80% of its initial activities at 45 °C for 4 days, which could exhibit better thermal stability. The reuse of immobilized inulinase throughout the continuous batch operations was explored, which had better reusability of the immobilized biocatalyst. At the same time, the stability of immobilized enzyme in the continuous packed-bed bioreactor was estimated, which showed the better results and had its potential scale-up fructose production for inulin conversion. 相似文献
2.
The extracellular inulinase in the supernatant of the cell culture of the marine yeast Cryptococcus aureus G7a was purified to homogeneity with a 7.2-fold increase in specific inulinase activity compared to that in the supernatant
by ultrafiltration, concentration, gel filtration chromatography (Sephadex™ G-75), and anion exchange chromatography (DEAE
sepharose fast flow anion exchange). The molecular mass of the purified enzyme was estimated to be 60.0 kDa. The optimal pH
and temperature of the purified enzyme were 5.0 and 50 °C, respectively. The enzyme was activated by Ca 2+, K +, Na +, Fe 2+, and Zn 2+. However, Mg 2+, Hg 2+, and Ag + acted as inhibitors in decreasing the activity of the purified inulinase. The enzyme was strongly inhibited by phenylmethanesulphonyl
fluoride (PMSF), iodoacetic acid, EDTA, and 1,10-phenanthroline. The K
m and V
max values of the purified enzyme for inulin were 20.06 mg/ml and 0.0085 mg/min, respectively. A large amount of monosaccharides
were detected after the hydrolysis of inulin with the purified inulinase, indicating the purified inulinase had a high exoinulinase
activity. 相似文献
3.
The potential of the modified magnetic nanoparticles for covalent immobilization of porcine pancreatic α-amylase has been investigated. The synthesis and immobilization processes were simple and fast. The co-precipitation method was used for synthesis of magnetic iron oxide (Fe 3O 4) nanoparticles (NPs) which were subsequently coated with silica through sol–gel reaction. The amino-functionalized NPs were prepared by treating silica-coated NPs with 3-aminopropyltriethoxysilane followed by covalent immobilization of α-amylase by glutaraldehyde. The optimum enzyme concentration and incubation time for immobilization reaction were 150 mg and 4 h, respectively. Upon this immobilization, the α-amylase retained more than 50 % of its initial specific activity. The optimum pH for maximal catalytic activity of the immobilized enzyme was 6.5 at 45 °C. The kinetic studies on the immobilized enzyme and its free counterpart revealed an acceptable change of K m and V max. The Km values were found as 4 and 2.5 mM for free and immobilized enzymes, respectively. The V max values for the free and immobilized enzymes were calculated as 1.75 and 1.03 μmol mg ?1 min ?1, in order, when starch was used as the substrate. A quick separation of immobilized amylase from reaction mixture was achieved when a magnetically active support was applied. In comparison to the free enzyme, the immobilized enzyme was thermally stable and was reusable for 9 cycles while retaining 68 % of its initial activity. 相似文献
4.
Based on the characteristics of polycations of chitosan and glucoamylase, which are oppositely charged, they were successfully alternatingly deposited onto the surface of aldehyde‐modified Fe 3O 4 nanoparticles by using a layer‐by‐layer ion exchange method to form magnetic carriers to construct multilayer films (designated as Fe 3O 4@(CS/GA) n). The (CS/GA) n film systems were endowed with the pH‐dependent properties of chitosan as well as the catalytic activity of glucoamylase. The changes in weight loss and surface chemistry, morphology, and magnetic sensitivity were monitored and verified by UV/Vis spectroscopy, zeta potential, TEM, and a vibrating sample magnetometer. Subsequently, the influence of the number of bilayers, storage stability, pH, temperature, and reusability of Fe 3O 4@(CS/GA) 5 biocatalysts on catalytic activity were investigated. The results from characterization and determination remarkably indicate that Fe 3O 4@(CS/GA) 5 presents excellent catalytic activity, storage stability, pH stability, and reusability in comparison with free enzyme. Fe 3O 4@(CS/GA) 5 retained >60 % of its initial activity at 65 °C over 6 h; the optimum temperature and pH also increased to the ranges of 45–65 °C and 2.5–3.5, respectively, and only 27 % activity was lost after 10 cycles. This new strategy simplifies the reaction protocol and improves encapsulation efficiency and catalytic activity for new potential applications in biotechnology. 相似文献
5.
Cyclodextrin glycosyltransferase (CGTase) isolated and purified from Paenibacillus sp . A11 was immobilized on various carriers by covalent linkage using bifunctional agent glutaraldehyde. Among tested carriers,
alumina proved to be the best carrier for immobilization. The effects of several parameters on the activation of the support
and on the immobilization of enzyme were optimized. The best preparation of immobilized CGTase retained 31.2% of its original
activity. After immobilization, the enzymatic properties were investigated and compared with those of the free enzyme. The
optimum pH of the immobilized CGTase was shifted from 6.0 to 7.0 whereas optimum temperature remained unaltered (60°C). Free
and immobilized CGTase showed similar pH stability profile but the thermal stability of the immobilized CGTase was 20% higher.
Kinetic data ( K
M and V
max) for the free and immobilized enzymes were determined from the rate of β-CD formation and it was found that the immobilized
form had higher K
M and lower V
max. The immobilized CGTase also exhibited higher stability when stored at both 4°C and 25°C for 2 months. The enzyme immobilized
on alumina was further used in a batch production of 2- O-α-glucopyranosyl- l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin. The yield of AA-2G was 2.92% and the immobilized CGTase retained
its activity up to 74.4% of the initial catalytic activity after being used for 3 cycles. The immobilized CGTase would have
a promising application in the production of various transglycosylated compounds and in the production of cyclodextrin by
the hydrolysis of starch. 相似文献
6.
A novel hybrid epoxy/nano CaCO 3 composite matrix for catalase immobilization was prepared by polymerizing epoxy resin in the presence of CaCO 3 nanoparticles. The hybrid support was characterized using scanning electron microscopy and Fourier transform infrared spectroscopy. Catalase was successfully immobilized onto epoxy/nano CaCO 3 support with a conjugation yield of 0.67?±?0.01 mg/cm 2 and 92.63?±?0.80 % retention of activity. Optimum pH and optimum temperature of free and immobilized catalases were found to be 7.0 and 35 °C. The value of K m for H 2O 2 was higher for immobilized enzyme (31.42 mM) than native enzyme (27.73 mM). A decrease in V max value from 1,500 to 421.10 μmol (min mg protein) ?1 was observed after immobilization. Thermal and storage stabilities of catalase improved immensely after immobilization. Immobilized enzyme retained three times than the activity of free enzyme when kept at 75 °C for 1 h and the half-life of enzyme increased five times when stored in phosphate buffer (0.01 M, pH 7.0) at 5 °C. The enzyme could be reused 30 times without any significant loss of its initial activity. Desorption of catalase from the hybrid support was minimum at pH 7.0. 相似文献
7.
A new thermophilic inulinase-producing strain, which grows optimally at 60 °C, was isolated from soil samples with medium
containing inulin as a sole carbon source. It was identified as a Bacillus smithii by analysis of 16s rDNA. Maximum inulinase yield of 135.2 IU/ml was achieved with medium pH7.0, containing inulin 2.0%, (NH 4)H 2PO 4 0.5%, yeast extract 0.5%, at 50 °C 200 rpm shaker for 72-h incubation. The purified inulinase from the extracellular extract
of B. smithii T7 shows endoinulinolytic activity. The optimum pH for this endoinulinase is 4.5 and stable at pH range of 4.0–8.0. The optimum
temperature for enzyme activity was 70 °C, the half life of the endoinulinase is 9 h and 2.5 h at 70 °C and 80 °C respectively.
Comparatively lower Michaelis–Menten constant (4.17 mM) and higher maximum reaction velocity (833 IU/mg protein) demonstrate
the endoinulinase’s greater affinity for inulin substrate. These findings are significant for its potential industrial application. 相似文献
8.
The aim of this investigation was to synthesize the adipic acid-modified magnetic nanoparticles for the efficient immobilization
of C-terminally lysine-tagged α-amylase (BACΔNC-Lys 7) from thermophilic Bacillus sp. strain TS-23. The carboxylated magnetic nanoparticles were prepared by the simple co-precipitation of Fe 3+/Fe 2+ in aqueous medium and then subsequently modified with adipic acid. Transmission electron microscopy micrographs showed that
the carboxylated magnetic nanoparticles remained discrete and had no significant change in size after the binding of BACΔNC-Lys 7. Free enzyme was active in the temperature range of 45–70 °C and had an optimum of 60 °C, whereas the thermal stability of
BACΔNC-Lys 7 was improved as a result of immobilization. The immobilized BACΔNC-Lys 7 could be recycled 20 times without a significant loss of the amylase activity and had a better stability during storage with
respect to free enzyme. Taken together, the magnetic nanoparticles coated with this functional moiety can be a versatile platform
for the effective manipulation of various kinds of engineered proteins. 相似文献
9.
The use of nanobiocatalysts, with the combination of nanotechnology and biotechnology, is considered as an exciting and rapidly emerging area. The use of iron oxide magnetic nanoparticles, as enzyme immobilization carriers, has drawn great attention because of their unique properties, such as controllable particle size, large surface area, modifiable surface, and easy recovery. In this study, various γ‐Fe 2O 3/Fe 3O 4 magnetic nanoparticles with immobilized proteases were successfully prepared by three different immobilization strategies including A) direct binding, B) with thiophene as a linker, and C) with triazole as a linker. The oligopeptides syntheses catalyzed by these magnetic nanoparticles (MNPs) with immobilized proteases were systematically studied. Our results show that i) for magnetic nanoparticles immobilized α‐chymotrypsin, both immobilization strategies A and B furnished good reusability for the Z‐Tyr‐Gly‐Gly‐OEt synthesis, the MNPs enzymes can be readily used at least five times without significant loss of its catalytic performance: ii) In the case of Z‐Asp‐Phe‐OMe synthesis catalyzed by magnetic nanoparticles immobilized thermolysin, immobilization Strategy B provided the best recyclability: iii) For the immobilized papain, although Strategy A or B afforded an immobilized enzyme for the first cycle of Z‐Ala‐Leu‐NHNHPh synthesis in good yield, their subsequent catalytic activity decreased rapidly. In general, the γ‐Fe 2O 3 MNPs were better for use as an immobilization matrix, rather than the Fe 3O 4 MNPs, owing to their smaller particle size and higher surface area. 相似文献
10.
New methods of obtaining products containing enzymes reduce the costs associated with obtaining them, increase the efficiency of processes and stabilize the created biocatalytic systems. In the study a catalytic system containing the enzyme α-amylase immobilized on ZnO nanoparticle and Fe3O4 nanoparticles was created. The efficiency of the processes was obtained with variables: concentrations of enzymes, temperatures and times, to define the best conditions for running the process, for which were determined equilibrium and kinetics of adsorption. The most effective parameters of α-amylase immobilization on metal oxides were determined, obtaining 100.8 mg/g sorption capacity for ZnO and 102.9 mg/g for Fe3O4 nanoparticles. Base on the best parameters, ZnO-α-amylase was investigated as an antimicrobial agent and Fe3O4-α-amylase was tested as a catalyst in the process of starch hydrolysis. As a result of the conducted experiments, it was found that α-amylase immobilized on Fe3O4 nanoparticles maintained high catalytic activity (the reaction rate constant KM?=?0.7799 [g/dm3] and the maximum reaction rate Vmax?=?8.660 [g/(dm3min)]). 相似文献
11.
A new epoxidation catalyst has been prepared by grafting a molybdenum(VI)–oxodiperoxo complex containing an oxazine ligand, [MoO(O 2) 2(phox)], on chloro‐functionalized Fe 3O 4 nanoparticles. The synthesized heterogeneous catalyst (MoO(O 2) 2(phox)/Fe 3O 4 was characterized using powder X‐ray diffraction, scanning and transmission electron microscopies, vibrating sample magnetometry, energy‐dispersive X‐ray analysis, Fourier transform infrared spectroscopy and inductively coupled plasma atomic emission spectroscopy. The immobilized complex gave high product yields and high selectivity for epoxide compared to the corresponding homogeneous one in the epoxidation of various olefins in the presence of tert ‐butyl hydroperoxide at 95°C without any co‐solvent. Also, the heterogeneous catalyst can be recycled without a noticeable change in activity and selectivity. 相似文献
12.
Xylanase from Bacillus pumilus strain MK001 was immobilized on different matrices following varied immobilization methods. Entrapment using gelatin (GE)
(40.0%), physical adsorption on chitin (CH) (35.0%), ionic binding with Q-sepharose (Q-S) (45.0%), and covalent binding with
HP-20 beads (42.0%) showed the maximum xylanase immobilization efficiency. The optimum pH of immobilized xylanase shifted
up to 1.0 unit (pH 7.0) as compared to free enzyme (pH 6.0). The immobilized xylanase exhibited higher pH stability (up to
28.0%) in the alkaline pH range (7.0–10.0) as compared to free enzyme. Optimum temperature of immobilized xylanase was observed
to be 8 °C higher (68.0 °C) than free enzyme (60.0 °C). The free xylanase retained 50.0% activity, whereas xylanase immobilized
on HP-20, Q-S, CH, and GE retained 68.0, 64.0, 58.0, and 57.0% residual activity, respectively, after 3 h of incubation at
80.0 °C. The immobilized xylanase registered marginal increase and decrease in K
m and V
max values, respectively, as compared to free enzyme. The immobilized xylanase retained up to 70.0% of its initial hydrolysis
activity after seven enzyme reaction cycles. The immobilized xylanase was found to produce higher levels of high-quality xylo-oligosaccharides
from birchwood xylan, indicating its potential in the nutraceutical industry. 相似文献
13.
This paper describes a new support that permits to efficient immobilization of L-asparaginase (L-ASNase). For this purpose, Fe3O4 magnetic nanoparticles were synthesized and coated by MCM-41. 3-chloropropyltrimethoxysilane (CPTMS) was used as a surface modifying agent for covalent immobilization of L-ASNase on the magnetic nanoparticles. The chemical structure; thermal, morphological, and magnetic properties; chemical composition; and zeta potential value of Fe3O4@MCM-41-Cl were characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential thermal analysis (DTA), differential scanning calorimetry (DSC), vibrating sample magnetometer (VSM), scanning electron microscope (SEM), energy dispersive X-ray (EDX), X-ray diffraction patterns (XRD), and zeta-potential measurement. The immobilization efficiency onto Fe3O4@MCM-41-Cl was detected as 63%. The reusability, storage, pH, and thermal stabilities of the immobilized L-ASNase were investigated and compared to that of soluble one. The immobilized enzyme maintained 42.2% of its original activity after 18 cycles of reuse. Furthermore, it was more stable towards pH and temperature compared with soluble enzyme. The Michaelis–Menten kinetic properties of immobilized L-ASNase showed a lower Vmax and a similar Km compared to soluble L-ASNase. Immobilized enzyme had around 47 and 32.5% residual activity upon storage a period of 28 days at 4 and 25 °C, respectively. In conclusion, the Fe3O4@MCM-41-Cl@L-ASNase core–shell nanoparticles could successfully be used in industrial and medical applications. 相似文献
14.
Candida rugosa lipase was entrapped in silica sol-gel particles prepared by hydrolysis of methyltrimethoxysilane and assayed by p-nitrophenyl palmitate hydrolysis, as a function of pH and temperature, giving pH optima of 7.8 (free enzyme) and 5.0–8.0
(immobilized enzyme). The optimum temperature for the immobilized enzyme (50–55°C) was 19°C higher than for the free enzyme.
Thermal, operational, and storage stability were determined with n-butanol and bytyric acid, giving at 45°C a half-life 2.7 times greater for the immobilized enzyme; storage time was 21 d
at room temperature. For ester synthesis, the optimum temperature was 47°C, and high esterification conversions were obtained
under repeated batch cycles (half-life of 138 h). 相似文献
15.
Urease from pigeonpea ( Cajanus cajan L.) was covalently linked to crab shell chitosan beads using glutaraldehyde. The optimum immobilization (64% activity) was
observed at 4°C, with a protein concentration of 0.24 mg/bead and 3% glutaraldehyde. The immobilized enzyme stored in 0.05
M Trisacetate buffer, pH 7.3, at 4°C had a t
1/2 of 110 d. There was practically no leaching of enzyme (<3%) from the immobilized beads in 30 d. The immobilized urease was
used 10 times at an interval of 24 h between each use with 80% residual activity at the end of the period. The chitosan-immobilized
urease showed a significantly higher Michaelis constant (8.3 m M) compared to that of the soluble urease (3.0 m M). Its apparent optimum pH also shifted from 7.3 to 8.5. Immobilized urease showed an optimal temperature of 77°C, compared
with 47°C for the soluble urease. Time-dependent kinetics of the thermal denaturation of immobilized urease was studied and
found to be monophasic in nature compared to biphasic in nature for soluble enzyme. This immobilized urease was used to analyze
blood urea of some of the clinical samples from the clinical pathology laboratories. The results compared favorably with those
obtained by the various chemical/biochemical methods employed in the clinical pathology laboratories. A column packed with
immobilized urease beads was also prepared in a syringe for the regular and continuous monitoring of serum urea concentrations. 相似文献
16.
The search for an in expensive support has motivated our group to undertake this work dealing with the use of chitosan as
matrix for immobilizing lipase. In addition to its low cost, chitosan has several advantages for use as a support, including
its lack of toxicity and chemical reactivity, allowing easy fixation of enzymes. In this article, we describe the immobilization
of Canada rugosa lipase onto porous chitosan beads for the enzymatic hydrolysis of oliveoil. The binding of the lipase onto the support was
performed by physicalad sorption using hexane as the dispersion medium. A comparativestudy between free and immobilized lipase
was conducted in terms of pH, temperature, and thermal stability. A slightly lower value for optimum pH (6.0) was found for
the immobilized form in comparison with that attained for the soluble lipase (7.0). The optimum reaction temperature shifted
from 37°C for the free lipase to 50°C for the chitosan lipase. The patterns of heat stability indicated that the immobilization
process tends to stabilize the enzyme. The half-life of the soluble free lipase at 55°C was equal to 0.71 h ( K
d=0.98 h −1), whereas for the immobilized lipase it was 1.10 h ( K
d=0.63 h −1). Kinetics was tested at 37°C following the hydrolysis of olive oil and obeys the Michaelis-Menten type of rate equation.
The K
m was 0.15 m M and the V
max was 51 μmol/(min·mg), which were lower than for free lipase, suggesting that the apparent affinity toward the substrate changes
and that the activity of the immobilized lipase decreases during the course of immobilization. 相似文献
17.
A recombinant esterase from Lactobacillus plantarum was immobilized on hydrophobic support polypropylene Accurel MP1000 by adsorption. Adsorption efficiency was 83%, and the
immobilized protein was 12.4 mg/g of support. Esterase activity was determined using p-nitrophenyl butyrate as substrate, and highest activities were observed at 50 °C for immobilized enzyme and 30 °C for free
enzyme extract. Concerning thermal stability, after enzyme incubation at 80 °C for 30 min, immobilized and free enzyme retained
91% and 56% of initial activity, respectively. Immobilized enzyme presented lower V
max and higher K
m than free enzyme. Protein was not released from the support, and esterase activity increased after 3 cycles of reuse. 相似文献
18.
Stable magnetic nanocomposite of gold nanoparticles (Au‐NPs) decorating Fe 3O 4 core was successfully synthesized by the linker of Boc‐L‐cysteine. Transmission electron microscope (TEM), energy dispersive X‐ray spectroscopy (EDX) and cyclic voltammograms (CV) were performed to characterize the as‐prepared Fe 3O 4@Au‐Nps. The results indicated that Au‐Nps dispersed homogeneously around Fe 3O 4 with the ratio of Au to Fe 3O 4 nanoparticles as 5–10/1 and the apparent electrochemical area as 0.121 cm 2. After self‐assembly of hemoglobin (Hb) on Fe 3O 4@Au‐Nps by electrostatic interaction, a hydrogen peroxide biosensor was developed. The Fe 3O 4@Au‐Nps/Hb modified GCE exhibited fast direct electron transfer between heme center and electrode surface with the heterogeneous electron transfer rate constant ( Ks ) of 3.35 s −1. Importantly, it showed excellent electrocatalytic activity towards hydrogen peroxide reduction with low detection limit of 0.133 μM ( S / D =3) and high sensitivity of 0.163 μA μM −1, respectively. At the concentration evaluated, the interfering species of glucose, dopamine, uric acid and ascorbic acid did not affect the determination of hydrogen peroxide. These results demonstrated that the introduction of Au‐Nps on Fe 3O 4 not only stabilized the immobilized enzyme but also provided large surface area, fast electron transfer and excellent biocompatibility. This facile nanoassembly protocol can be extended to immobilize various enzymes, proteins and biomolecules to develop robust biosensors. 相似文献
19.
To address the obstacles facing the use of palladium‐based homogeneous and heterogeneous catalysts in C─C cross‐coupling reactions, a novel semi‐heterogeneous support was developed based on hyperbranched poly(ethylene glycol)‐ block ‐poly(citric acid)‐functionalized Fe 3O 4 magnetic nanoparticles (Fe 3O 4@PCA‐ b ‐PEG). Because of the surface modification of the Fe 3O 4 nanoparticles with amphiphilic and hyperbranched polymers (PCA‐ b ‐PEG), these hybrid materials are not only soluble in a wide range of solvents (e.g. water, ethanol and dimethylformamide) but also are able to trap Pd 2+ ions via complex formation of free carboxyl groups of the PCA dendrimer with metal ions. The reduction of trapped palladium ions in the dendritic shell of Fe 3O 4@PCA‐ b ‐PEG leads to immobilized palladium nanoparticles. The morphology and structural features of the catalyst were characterized using various microscopic and spectroscopic techniques. The catalyst was effectively used in the palladium‐catalysed Mizoroki–Heck coupling reaction in water as a green solvent. In addition, the catalyst can be easily recovered from the reaction mixture by applying an external magnetic field and reused for more than ten consecutive cycles without much loss in activity, exhibiting an example of a sustainable and green methodology. 相似文献
20.
A reversible addition‐fragmentation chain transfer (RAFT) agent was directly anchored onto superparamagnetic Fe 3O 4 nanoparticles (SPNPs) in a simple procedure using a ligand exchange reaction of 2‐[(dodecylsulfanylcarbonylthiolsulfanyl) propionic acid] (DCPA) with oleic acid initially present on the surface of Fe 3O 4 nanoparticles. The DCPA‐modified SPNPs were then used for the surface‐mediated RAFT polymerization of di(ethylene glycol) ethyl ether acrylate and (oligoethylene glycol) methyl ether acrylate to fabricate structurally well‐defined hybrid SPNPs with temperature‐responsive poly[di(ethylene glycol) ethyl ether acrylate‐ co‐(oligoethylene glycol) methyl ether acrylate] shell and magnetic Fe 3O 4 core. Evidence of a well‐controlled surface‐mediated RAFT polymerization was gained from a linear increase of number‐average molecular weight with overall monomer conversions and relatively narrow polydispersity indices of the copolymers grown from the SPNPs. The resultant hybrid nanoparticles exhibited superparamagnetic property with a saturation magnetization of 55.1–19.4 emu/g and showed a temperature‐responsive phenomenon as the temperature changed between 25 and 40 °C. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013 , 51, 3420–3428 相似文献
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