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1.
Enzymes are gradually increasingly preferred over chemical processes, but commercial enzyme applications remain limited due to their low stability and low product recovery, so the application of an immobilization technique is required for repeated use. The aims of this work were to produce stable enzyme complexes of cross-linked xylanase on magnetic chitosan, to describe some characteristics of these complexes, and to evaluate the thermal stability of the immobilized enzyme and its reusability. A xylanase was cross-linked to magnetite particles prepared by in situ co-precipitation of iron salts in a chitosan template. The effect of temperature, pH, kinetic parameters, and reusability on free and immobilized xylanase was evaluated. Magnetization, morphology, size, structural change, and thermal behavior of immobilized enzyme were described. 1.0?±?0.1 μg of xylanase was immobilized per milligram of superparamagnetic chitosan nanoparticles via covalent bonds formed with genipin. Immobilized xylanase showed thermal, pH, and catalytic velocity improvement compared to the free enzyme and can be reused three times. Heterogeneous aggregates of 254 nm were obtained after enzyme immobilization. The immobilization protocol used in this work was successful in retaining enzyme thermal stability and could be important in using natural compounds such as Fe3O4@Chitosan@Xylanase in the harsh temperature condition of relevant industries. 相似文献
2.
β-Glucosidase is part of the cellulases and is responsible for degrading cellobiose into glucose, a compound that can be used to produce biofuels. However, the use of the free enzyme makes the process more expensive. Enzyme immobilization improves catalytic characteristics and supports, such as zeolites, which have physical-chemical characteristics and ion exchange capacity that have a promising application in the biotechnological industry. This research aimed to immobilize by adsorption a recombinant β-glucosidase from Trichoderma reesei, obtained in Escherichia coli BL21 (DE3), in a commercial zeolite. A Box Behnken statistical design was applied to find the optimal immobilization parameters, the stability against pH and temperature was determined, and the immobilized enzyme was characterized by SEM. The highest enzymatic activity was determined with 100 mg of zeolite at 35 °C and 175 min. Compared to the free enzyme, the immobilized recombinant β-glucosidase presented greater activity from pH 2 to 4 and greater thermostability. The kinetic parameters were calculated, and a lower K M value was obtained for the immobilized enzyme compared to the free enzyme. The obtained immobilization parameters by a simple adsorption method and the significant operational stability indicate promising applications in different fields. 相似文献
3.
Core shell magnetite nanoparticle (CSMN) was successfully synthesized with diameter around 125 nm according to the determination
with scanning electronic microscopy. SBA-15 with diameter around 31 nm was synthesized in our previous work as another supporter
for immobilized degradation enzymes. The aim of this study was to investigate the influence of silica-derived nano-supporters
on cellobiase after immobilization. With covalent method, glutaraldehyde was introduced to immobilize cellobiase. The immobilized
enzyme efficiency, specific activity, and its characterization, including optimum pH, pH stability, optimum temperature for
enzyme reaction, and enzyme thermal stability were investigated. Results show that the method of enzyme immobilization on
both nano-supporters could improve cellobiase stability under low pH and high temperature conditions compared with the free
enzyme. In the aspect of immobilization efficiency, SBA had higher amount of bounded protein than that of CSMN, but had lower
specific enzyme activity than CSMN, assumably due to the change in silica surface properties caused by process of supporter
synthesis. 相似文献
4.
The present work aims to provide the basic characterization of sol–gel immobilized inulinase, a biocatalyst configuration
yet unexploited, using as model system the hydrolysis of inulin to fructose. Porous xerogel particles with dimensions in slight
excess of 10 μm were obtained, yielding an immobilization efficiency of roughly 80%. The temperature– and pH–activity profiles
displayed a broader bell-shaped pattern as a result of immobilization. In the latter case, a shift of the optimal pH of 0.5
pH units was observed towards a less acidic environment. The kinetic parameters estimated from the typical Michaelis–Menten
kinetics suggest that immobilization in sol–gel did not tamper with the native enzyme conformation, but on the other hand,
entrapment brought along mass transfer limitations. The sol–gel biocatalyst displayed a promising operational stability, since
it was used in more than 20 consecutive 24-hour batch runs without noticeable decay in product yield. The performance of sol–gel
biocatalyst particles doped with magnetite roughly matched the performance of simple sol–gel particles in a single batch run.
However, the operational stability of the former proved poorer, since activity decay was evident after four consecutive 24-hour
batch runs. 相似文献
5.
The immobilization of horseradish peroxidase (HRP) on composite membrane has been investigated. This membrane was prepared
by coating nonwoven polyester fabric with chitosan glutamate in the presence of glutraldehyde as a crosslinking agent. The
physico-chemical properties of soluble and immobilized HRP were evaluated. The soluble HRP lost 90% of its activity after
4 weeks of storage at 4°C, whereas the immobilized enzyme retained 85% of its original activity at the same time. A reusability
study of immobilized HRP showed that the enzyme retained 54% of its activity after 10 cycles of reuse. Soluble and immobilized
HRP showed the same pH optima at pH 5.5. The immobilized enzyme had significant stability at different pH values, where it
had maximum stability at pH 3.0 and 6.0. The kinetic properties indicated that the immobilized enzyme had more affinity toward
substrates than soluble enzyme. The soluble and immobilized enzymes had temperature optima at 30 and 40°C and were stable
up to 40 and 50°C, respectively. The stability of HRP against metal ion inactivation was improved after immobilization. Immobilized
HRP exhibited high resistance to proteolysis by trypsin. The immobilized HRP was more resistant to inactivation induced by
urea, Triton X-100, and organic solvents compared to its soluble counterpart. The immobilized HRP showed very high yield of
immobilization and markedly high stabilization against several forms of denaturants that offer potential for several applications. 相似文献
6.
Cupric ion-chelated poly(hydroxyethyl methacrylate- n-vinyl imidazole) (poly(HEMA-VIM)) microspheres prepared by suspension polymerization were investigated as a specific adsorbent
for immobilization of yeast invertase in a batch system. They were characterized by scanning electron microscopy, surface
area, and pore size measurements. They have spherical shape and porous structure. The specific surface area of the p(HEMA-VIM)
spheres was found to be 81.2 m 2/g with a size range of 70–120 μm in diameter, and the swelling ratio was 86.9%. Then, Cu(II) ion chelated on the microspheres
(546 μmol Cu(II)/g), and they were used in the invertase adsorption. Maximum invertase adsorption was 51.2 mg/g at pH 4.5.
Cu(II) chelation increases the tendency from Freundlich-type to Langmuir-type adsorption model. The optimum activity for both
free and adsorbed invertase was observed at pH 4.5. The optimum temperature for the poly(HEMA-VIM)/Cu(II)-invertase system
was found to be at 55 °C, 10 °C higher than that of the free enzyme at 45 °C. V
max values were determined as 342 and 304 U/mg enzyme, for free and adsorbed invertase, respectively. K
m values were found to be same for free and adsorbed invertase (20 mM). Thermal and pH stability and reusability of invertase
increased with immobilization. 相似文献
7.
β-Galactosidase from the fungus Talaromyces thermophilus CBS 236.58 was immobilized by covalent attachment onto the insoluble carrier Eupergit C with a high binding efficiency of
95%. Immobilization increased both activity and stability at higher pH values and temperature when compared with the free
enzyme. Especially the effect of immobilization on thermostability is notable. This is expressed by the half-lifetime of the
activity at 50°C, which was determined to be 8 and 27 h for the free and immobilized enzymes, respectively. Although immobilization
did not significantly change kinetic parameters for the substrate lactose, a considerable decrease in the maximum reaction
velocity V
max was observed for the artificial substrate o-nitrophenyl-β- d-galactopyranoside (oNPG). The hydrolysis of both oNPG and lactose is competitively inhibited by the end products glucose
and galactose. However, this inhibition is only very moderate as judged from kinetic analysis with glucose exerting a more
pronounced inhibitory effect. It was evident from bioconversion experiments with 20% lactose as substrate, that the immobilized
enzyme showed a strong transgalactosylation reaction, resulting in the formation of galactooligosaccharides (GalOS). The maximum
yield of GalOS of 34% was obtained when the degree of lactose conversion was roughly 80%. Hence, this immobilized enzyme can
be useful both for the cleavage of lactose at elevated temperatures, and the formation of GalOS, prebiotic sugars that have
a number of interesting properties for food applications. 相似文献
8.
An optical pH sensor was developed based on the fluorophor, fluoresceinamine isomer II (FA), covalently immobilized in a sol–gel matrix. This sol–gel matrix was created by the copolymerization of two precursors, methyltriethoxysilane (MTES) and 3-glycidoxypropyltrimethoxysilane (GPTMS), in an ethanolic solution. Fluoresceinamine was covalently bound to the glycidoxypropyl chain group of GPTMS, thereby preventing it from leaching. Moreover, the immobilization of the fluoresceinamine also extended its linear detection range. The sensor showed good repeatability, a short response time of less than 8 s, high long-term stability and no temperature effect in the biologically relevant range. In the pH range of 4–10, the sensor was very sensitive and its linear range was found to be between pH 6 and 9 ( R2 = 0.995). 相似文献
9.
Papain (EC 3.4.22.2) has been chemically modified using two novel reagents including different anhydrides of 1,2,4-benzenetricarboxylic
and pyromellitic acids. Then, the modified papain was immobilized on the activated cotton fabric by a two-step method. The
number of free amino groups in the modified protein was investigated through the 2,4,6-trinitrobenzenesulfonic acid method.
Energy dispersive spectrometer was used to characterize papain immobilization. Some parameters of both modified and native
papain immobilized on cotton fabric, such as optimum temperature, optimum pH, and the stabilities for reservation in various
detergents were studied and compared. The resultant papain had its optimum pH shifted from 6.0 to 9.0. Compared with immobilized
native papain, the thermal stability and the resistance to alkali and washing detergent of immobilized modified enzyme were
improved considerably. When the concentration of detergent was 20 mg/ml, the activity of the immobilized pyromellitic papain
retained about 40% of its original activity, whereas the native papain was almost inhibited. This work demonstrated that the
cotton fabric immobilized modified papain has potential applications in the functional textiles field. 相似文献
10.
以十六烷基三甲基溴化铵(CATB)-辛烷-己醇反胶束体系对醇脱氢酶(ADH)进行固定化,试验了含水量、酶液pH值、CTAB和己醇浓度对ADH固定化的影响。对游离酶和固定化酶的催化动力学性质研究表明:酶促反应的最适pH值分别为8.2和8.8,最适温度分别是31℃和20℃,对乙醇的米氏常数Km分别为12mmol/L和7.4mmol/L。在30℃时,游离酶存放150min后失活90%,固定化酶失活50%,表明反胶束固定化ADH有较好的热稳定性。 相似文献
11.
Invertase or β-d-Fructofuranosidase (E.C.3.2.1.26) was extracted from Cucumis melo. L. fruit (Family-Cucurbitaceae). Soluble, plant invertase enzyme was immobilized in novel composite of agarose-guar gum biopolymer matrix in the form of hydrophilic, porous membranes. The immobilized invertase was characterized for sucrose hydrolytic activity and leakage from the matrix support. The efficiency of immobilization was found to be 91% with negligible leaching. The kinetic parameters Km and Vmax for free and immobilized invertase were also determined. Immobilized invertase was optimally active in the wide pH range of 4.5-6.5. The immobilization process also enhanced the thermal stability of enzyme up to 65 °C. Immobilized invertase membranes showed excellent storage stability with shelf life of 110 days. Entrapped invertase showed better operational stability and reusability up to 12 cycles. The fluorescence spectra of the composite membranes were studied and compared with that of soluble enzyme. All these characteristics of the immobilized invertase membranes make them suitable for the fabrication of biosensors. 相似文献
12.
Lactose has been hydrolyzed using covalently immobilized β-galactosidase on thermally stable carrageenan coated with chitosan
(hydrogel). The hydrogel’s mode of interaction was proven by Fourier transform infrared spectroscopy, differential scanning
calorimetry (DSC), and Schiff’s base formation. The DSC thermogram proved the formation of a strong polyelectrolyte complex
between carrageenan and chitosan followed by glutaraldehyde as they formed one single peak. The modification of carrageenan
improved the gel’s thermal stability in solutions from 35 °C to 95 °C. The hydrogel has been proven to be efficient for β-galactosidase
immobilization where 11 U/g wet gel was immobilized with 50% enzyme loading capacity. Activity and stability of free and immobilized
β-galactosidase towards pH and temperature showed marked shifts in their optimum pH from 4.5–5 to 5–5.5 and temperature from
50 °C to 45–55 °C after immobilization, which reveals higher catalytic activity and reasonable stability at wider pHs and
temperatures. The apparent K
m of the immobilized enzyme increased from 13.2 to 125 mM, whereas the V
max increased from 3.2 to 6.6 μmol/min compared to the free enzyme, respectively. The free and immobilized enzymes showed lactose
conversion of 87% and 70% at 7 h, respectively. The operational stability showed 97% retention of the enzyme activity after
15 uses, which demonstrates that the covalently immobilized enzyme is unlikely to leach. The new carrier could be suitable
for immobilization of other industrial enzymes. 相似文献
13.
Immobilization of cellulase onto acrylamide grafted acrylonitrile copolymer (PAN) membranes by means of glutaraldehyde has been studied. The bound cellulase was verified by X-ray photoelectron spectroscopy. The activities of free cellulase and immobilized cellulase are determined by measuring the amount of glucose made from carboxymethyl cellulase in the given conditions. Results show that immobilization conditions had some effects on the activity of immobilized cellulase. The immobilized cellulase had a higher Km than free cellulase (0.02 mg/ml) did. The immobilized cellulase had better stability with respect to pH or temperature than free cellulase. 相似文献
14.
以烟用醋酸纤维的生物化学改性为目标,研究了以壳聚糖为载体时,吸附交联固定化过氧化氢酶的条件,并考察了固定化酶的性质。结果表明,固定化的最佳条件为:加酶量(酶活2×104C IU/m l)6m l,3%壳聚糖20m l,乙二醛浓度6%(w/v),交联剂用量100m l,吸附时间0.5 h,交联时间2.5h,酶活收率可达42.9%。过氧化氢酶固定化后,动学参数Km值为61.7mmol/L;对活性氧具有较好清除作用。 相似文献
15.
Egg shell membrane is a novel, robust, microporous, cost effective, easily available organic support material. In recent studies egg shell membranes were utilized as organic support for enzyme immobilization. But low conjugation yield limits its application as good support for biotechnological industries. In present study egg shell membrane was chemically treated to introduce free functional groups for covalent linkage of proteins to increase its conjugation yield and stability of conjugate complex. Many enzymes were tested for immobilization on modified egg shell membrane like oxalate oxidase, glucose oxidase, peroxidase and lipase. A fifteen to sixteen fold increase in conjugation yield was observed when immobilization was performed after chemical treatment in comparison to immobilization on native membrane with slight change in specific activity of immobilized enzyme which ranges from 5% to 15%. Egg shell membrane bound enzymes showed slight changes in their kinetic properties after immobilization. Egg shell membrane bound oxalate oxidase shows detection limit of 1.5 μM when used for urinary oxalate determination. Egg shell membrane support shows no interference to enzyme activity and a good correlation of 0.99 was observed with the values estimated using commercially available Sigma kit. The immobilized oxalate oxidase, glucose oxidase, peroxidase and lipase were stable up to duration of 180 days and there is respective loss of 10%, 13%, 24%, and 33% of initial activity. Overall result strengthens our view of using chemically modified egg shell membrane as solid support for better immobilization of enzymes and can be used in various biotechnological applications. 相似文献
16.
Carboxymethyl cellulose-silver nanoparticle (AgNp)-silica hybrids have been synthesized in a modified Stöber process. The hybrid synthesis was optimized to obtain an efficient immobilization matrix for diastase alpha amylase, a multimeric enzyme of high technological significance. The synthesized hybrids were characterized using FTIR, XRD, SEM, TGA and BET studies. The enzyme immobilization was done by adsorption and using the immobilized enzyme, the hydrolysis of soluble starch has been optimized in comparison to free enzyme. The optimum usable pH for the immobilized enzyme ranged from pH 4 to 5, while pH 5 was optimum pH for the free enzyme activity. The kinetic parameters for the immobilized, ( K M = 3.4610 mg ml ?1; V max = 6.3540 mg ml ?1 min ?1) and free enzyme ( K M = 4.1664 mg ml ?1; V max = 4.291 mg ml ?1 min ?1) hydrolysis indicated that the immobilization at the nanohybrid has significantly improved the catalytic property of the enzyme. In the immobilized state, the enzyme remained usable for many repeated cycles like our previous material, gum acacia-gelatin-AgNp-silica. Storage experiments indicated that the immobilization has increased the stability of the enzyme and also that AgNps play a role in stabilizing the immobilized enzyme. 相似文献
17.
Cellulase was covalently immobilized using a hydrophilic polyurethane foam (Hypol®FHP 2002). Compared to the free enzyme, immobilized cellulase showed a dramatic decrease (7.5-fold) in the Michaelis constant for carboxymethylcellulose. The immobilized enzyme also had a broader and more basic pH optimum (pH 5.5–6.0), a greater stability under heat-denaturing or liquid nitrogen-freezing conditions, and was relatively more efficient in utilizing insoluble cellulose substrates. High molecular weight compounds (Blue Dextran) could move throughout the foam matrix, indicating permeability to insoluble celluloses; activity could be further improved 2.4-fold after powdering, foams under liquid nitrogen. The improved kinetic and stability features of the immobilized cellulase combined with advantageous properties of the polyurethane foam (resistance to enzymatic degradation, plasticity of shape and size) suggest that this mechanism of cellulase immobilization has high potential for application in the industrial degradation of celluloses. 相似文献
18.
Phenylalanine dehydrogenase ( l-PheDH) from Sporosarcina ureae was immobilized on DEAE-cellulose, modified initially with 2-amino-4,6-dichloro-s-triazine followed by hexamethylenediamine
and glutaraldehyde. The highest activity of immobilized PheDH was determined as 95.75 U/g support with 56% retained activity.
The optimum pH value of immobilized l-PheDH was shifted from pH 10.4 to 11.0. The immobilized l-PheDH showed activity variations close to the maximum value in a wider temperature range of 45–55 °C, whereas it was 40 °C
for the native enzyme. The pH and the thermal stability of the immobilized l-PheDH were also better than the native enzyme. At pH 10.4 and 25 °C, K
m values of the native and the immobilized l-PheDH were determined as K
m Phe = 0.118, 0.063 mM and K
m NAD+ = 0.234, 0.128 mM, respectively. Formed NADH at the exit of packed bed reactor column was detected by the flow-injection
analysis system. The conversion efficiency of the reactor was found to be 100% in the range of 5–600 μM Phe at 9 mM NAD + with a total flow rate of 0.1 mL/min. The reactor was used for the analyses of 30 samples each for 3 h per day. The half-life
period of the reactor was 15 days. 相似文献
19.
In the present study, the copolymeric hydrogels based on 2-hydroxyethyl methacrylate (HEMA) and itaconic acid (IA) were synthesized by gamma radiation induced radical polymerization, in order to examine the potential use of these hydrogels in immobilization of Citrullus vulgaris urease. Gelation and Swelling properties of PHEMA and copolymeric P (HEMA/IA) hydrogels with different IA contents (96.5/3.5, 94.4/5.6 and 92.5/7.5 mol) were studied in a wide pH range. Initial studies of so-prepared hydrogels show interesting pH sensitivity in swelling and immobilization. C. vulgaris urease was immobilized on HEMA/IA (92.5/7.5) at 6 kGy with 41.3% retention of activity. The properties of free and immobilized urease were compared. Immobilized urease maintained a higher relative activity than free urease at both lower and higher pH levels, indicating that the immobilized urease was less sensitive to pH changes than the free urease. The K m value of the immobilized urease was approximately 2 times higher than that of the free urease. Temperature stability was improved for immobilized enzyme. The free form exhibited a loss about 80% of activity upon incubation for 15 min at 80°C. The influence of various heavy metal ions at the concentration of l mM was improved after enzyme immobilization. The immobilization of C. vulgaris urease on HEMA/IA (92.5/7.5) at 6 kGy showed a residual activity of 47 % after 4 reuses. 相似文献
20.
Urease was covalently immobilized on acrylamide-grafted poly (ethylene terephthalate) fibers after glutaraldehyde activation.
Ureasecontaining fibers showed a very high operational stability and reusability, with about 85% of the initial activity after
90 d. The thermostability of the bound urease was positively influenced, and a slight change in optimum temperature was observed
after immobilization, when compared with the free enzyme. The pH optimum of both types of urease was found to be the same,
but immobilized urease showed an increased stability in a broader range of pH. The kinetic studies exhibited a slightly higher K
m
value for the bound enzyme, with a value of 4.50 mmol dm -3, when compared with the free enzyme (2.82 mmol dm -3), which demonstrated that the immobilization procedure did not cause an unfavorable conformation for the substrate-product
formation and a hindered diffusion. The graft yield was also found effective on maximum activity of immobilized urease. Twenty-five
percent of the acrylamide-grafted fibers exhibited the highest enzymatic activity together with the highest water uptake.
Higher graft yields were not suitable for the immobilization of the enzyme molecules as a result of crosslinks formed between
the poly(acrylamide) chains and glutaraldehyde. 相似文献
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