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1.
A method for simultaneous covalent immobilization of glucose oxidase and peroxidase with previously oxidized carbohydrate
residues to urea derivative of regenerated acetylcellulose granules is described. The effect of immobilization on the catalytic
properties of the separately immobilized enzymes are studied. The immobilized enzymes manifested no change in their pH and
temperature optima and slight increase ofK
m
x compared to data for the soluble enzymes. A column packed with simultaneously immobilized enzymes is used for manual glucose
determination in blood sera. The results are in high correlation with those obtained by the Beckman Glucose Analyzer method
(r = 0.976). The method is economic (the enzyme-carrier conjugate may be used more than 300 times), easy to perform, and less
time consuming than the manual methods utilizing soluble enzymes. The established manual method can be proposed for emergency
clinical analysis and smaller clinical laboratories. 相似文献
2.
Properties of pectinesterase and endo-d-polygalacturonase coimmobilized in a porous glass support 总被引:1,自引:0,他引:1
A. Manjón J. L. Iborra C. Romero M. Cánovas 《Applied biochemistry and biotechnology》1992,37(1):19-31
Derivatives of pectinesterase and polygalacturonase, both individually immobilized and coimmobilized, were obtained and characterized.
Homologous soluble systems were also studied to establish differences between the effect of the immobilization process and
the presence of the other enzyme. Immobilization or coimmobilization did not change the optima pH or temperature for the enzymes.
However, optimum ionic strength was displaced toward higher values for immobilized pectinesterase, while for polygalacturonase
immobilization resulted in a wider range for activity.K
m
value remained nearly unchanged for pectinesterase, and decreased for polygalacturonase. TheV
m
value decreased with the immobilization process for the two enzymes, except for polygalacturonase immobilization in presence
of pectinesterase. Soluble pectinesterase activity showed a competitive inhibition by polygalacturonic acid (Ki = 0.44 mg/mL). Either immobilization or presence of polygalacturonase rendered the enzyme insensitive to the inhibitory effect.
Thermal stability of pectinesterase was not improved after immobilization. On the contrary, the thermal stability of endo-D-polygalacturonase
was improved slightly by presence of pectinesterase, and in a greater extent by immobilization. Individually immobilized and
coimmobilized pectinesterase activities kept 90 and 60%, respectively, of their initial values after more than one year stored
at 3-5 °C. The two endo-D-polygalacturonase derivatives showed the same activity decay pattern along 10 mo storage at 3-5
°C. The two immobilized pectinesterase derivatives showed similar operational stabilities during continuous operation. The
presence of pectinesterase remarkably increased the operational stability of the immobilized endo-D-poly galacturonase. 相似文献
3.
Crude extracellular invertase fromSclerotium rolfsii, when coupled to glutaraldehyde activated Indion 48-R, retained 70–80% activity of the soluble enzyme. Immobilization resulted
in a decrease in the pH and temperature optima but it increased the temperature stability. Km and Vmax also increased as a result of immobilization. Both soluble and immobilized invertase showed inhibition at high substrate
concentrations. The bound enzyme showed excellent stability to repeated use and retained approx 90% of its initial activity
after 8 cycles of use. 相似文献
4.
Pereira Ernandes B. De Castro Heizir F. De Moraes Flávio F. Zanin Gisella M. 《Applied biochemistry and biotechnology》2001,91(1-9):739-752
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 mM 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
5.
Khaja Basheeruddin Vicki Rothman Simeon Margolis 《Applied biochemistry and biotechnology》1985,11(2):133-140
We have immobilized E.coli alkaline phosphatase (EC 3.1.3.1) by linking it covalently to sepharose 4B. This preparation has several advantages over
the soluble enzyme. The immobilized enzyme is easily separable from other constituents in incubation mixtures. The immobilized
enzyme can be reused repeatedly and is more stable than the soluble enzyme to heat treatment in the presence of 10 mM Mg2+. The insoluble and soluble phosphatases removed 75 and77%, respectively, of the inorganic phosphorus from casein. The immobilized enzyme inactivated two enzymes believed to be active
in the phosphorylated state, acyl-CoA : cholesterol acyltransferase (ACAT) by 39% and NADPH-cytochrome P-450 reductase by
89%. The utility of immobilized alkaline phosphatase for studying the phosphorylation and dephosphorylation of soluble or
membrane-bound enzymes and proteins is discussed. 相似文献
6.
George G. Guilbault 《Applied biochemistry and biotechnology》1982,7(1-2):85-98
Immobilized enzymes are becoming increasingly popular as analytical reagents because of their reusability, stability, and
sensitivity to many inhibitors that would seriously interfere in assays using soluble enzymes. In this article, some of the
kinetic and catalytic effects of immobilized enzymes in analysis will be discussed. The shift of the activity-pH profile curves
on immobilization, the changes in temperature dependence. the inhibitor constants (K1). Michaelis constants (K
m
), and the maximum velocity (Vmax). plus others, will be discussed. Finally, the use of these immobilized enzymes in fluorometric and electrochemical monitoring
systems will be shown, and the future of these reagents in various areas will be discussed. A survey of enzyme electrodes
will be presented as an example of the use of immobilized enzymes. Application of immobilized enzyme technology to the assay
of BUN, glucose, uric acid, amino acids, ethanol. and other metabolites will be discussed. 相似文献
7.
E. A. Markvicheva N. E. Tkachuk S. V. Kuptsova T. N. Dugina S. M. Strukova Yu. E. Kirssh V. P. Zubov L. D. Rumish 《Applied biochemistry and biotechnology》1996,61(1-2):75-84
A new one-step procedure for entrapping proteases into a polymeric composite calcium alginate-poly(N-vinyl caproladam) hydrogel was developed that provided 75–90% retention of the activity of entrapped enzymes compared to
soluble ones. Properties of entrapped carboxypeptidase B, trypsin, and thrombin were investigated. The immobilized enzymes
were active within a wide pH range. The temperature optima of entrapped trypsin and carboxypeptidase B were approx 25°C higher
than that of the soluble enzymes, and the resistance to heating was also increased. The effects of various polar and nonpolar
organic solvents on the entrapped proteases were investigated. The immobilized enzymes retained their activity within a wide
concentration range (up to 90%) of organic solvents. Gel-entrapped trypsin and carboxypeptidase (CPB) were successfully used
for obtaining human insulin from recombinant proinsulin. The developed stabilization method can be used to catalyze various
reactions proceeding within wide pH and temperature ranges. 相似文献
8.
A novel affinity covalent immobilization technique of glucoamylase enzyme onto ρ-benzoquinone-activated alginate beads was
presented and compared with traditional entrapment one. Factors affecting the immobilization process such as enzyme concentration,
alginate concentration, calcium chloride concentration, cross-linking time, and temperature were studied. No shift in the
optimum temperature and pH of immobilized enzymes was observed. In addition, K
m values of free and entrapped glucoamylase were found to be almost identical, while the covalently immobilized enzyme shows
the lowest affinity for substrate. In accordance, V
m value of covalently immobilized enzyme was found lowest among free and immobilized counter parts. On the other hand, the
retained activity of covalently immobilized glucoamylase has been improved and was found higher than that of entrapped one.
Finally, the industrial applicability of covalently immobilized glucoamylase has been investigated through monitoring both
shelf and operational stability characters. The covalently immobilized enzyme kept its activity over 36 days of shelf storage
and after 30 repeated use runs. Drying the catalytic beads greatly reduced its activity in the beginning but recovered its
lost part during use. In general, the newly developed affinity covalent immobilization technique of glucoamylase onto ρ-benzoquinone-activated
alginate carrier is simple yet effective and could be used for the immobilization of some other enzymes especially amylases. 相似文献
9.
Manchumas Hengsakul Prousoontorn Supranee Pantatan 《Journal of inclusion phenomena and macrocyclic chemistry》2007,57(1-4):39-46
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
10.
Huseyin Bekir Yildiz Dilek Odaci Demirkol Serkan Sayin Mustafa Yilmaz Ozcan Koysuren Musa Kamaci 《高分子科学杂志,A辑:纯化学与应用化学》2013,50(10):1075-1084
Accumulation of cholesterol in human blood can cause several health problems such as heart disease, coronary artery disease, arteriosclerosis, hypertension, cerebral thrombosis, etc. Therefore, simple and fast cholesterol determination in blood is clinically important. In this study, two types of amperometric cholesterol biosensors were designed by physically entrapping cholesterol oxidase in conducting polymers; thiophene capped poly(ethyleneoxide)/polypyrrole (PEO-co-PPy) and 3-methylthienyl methacrylate-co-p-vinyl benzyloxy poly(ethyleneoxide)/polypyrrole (CP-co-PPy). PEO-co-PPy and CP-co-PPy were synthesized electrochemically and cholesterol oxidase was immobilized by entrapment during electropolymerization. The amperometric responses of the enzyme electrodes were measured by monitoring oxidation current of H2O2 at +0.7 V in the absence of a mediator. Kinetic parameters, such as Km and Imax, operational and storage stabilities, effects of pH and temperature were determined for both entrapment supports. Km values were found as 1.47 and 5.16 mM for PEO-co-PPy and CP-co-PPy enzyme electrodes, respectively. By using these Km values, it can be observed that ChOx immobilized in PEO-co-PPy shows higher affinity towards the substrate. 相似文献
11.
We report the first application of hydrophobic interaction between graphene oxide (GO) and negatively charged enzymes to fabricate CE-integrated immobilized enzyme microreactors (IMERs) by a simple and reliable immobilization procedure based on layer by layer assembly. L -lactate dehydrogenase (L -LDH), which is negatively charged during the enzymatic reaction, is selected as the model enzyme. Various spectroscopic techniques, including SEM, FTIR, and UV-vis are used to characterize the fabricated CE-IMERs, demonstrating the successful immobilization of enzymes on the negatively charged GO layer in the capillary surface. The IMER exhibits excellent repeatability with RSDs of inter-day and batch-to-batch less than 3.49 and 6.37%, respectively, and the activity of immobilized enzymes remains about 90% after five-day usage. The measured Km values of pyruvate and NADH of the immobilized L -LDH are in good agreement with those obtained by free enzymes. The results demonstrate that the hydrophobic interactions and/or π-π stacking is significant between the GO backbone and the aromatic residues of L -LDH and favorable to fabrication of CE-integrated IMERs. Finally, the method is successfully applied to the determination of pyruvate in beer samples. 相似文献
12.
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
13.
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
14.
Li Gaoxiang Huang Jiayu Kou Xiufen Zhang Shuzheng 《Applied biochemistry and biotechnology》1982,7(5):325-341
Glucoamylase (EC 3.2.1.3) was immobilized to alkylamine porous glass with glutaraldehyde. The choice and pretreatment of carrier
and conditions for immobilization have been investigated. The immobilized enzyme contained about 4.0–8.0% protein and its
activity was about 1000–1700 U/g. Some characteristics of the immobilized enzyme and the native enzyme have been comparatively
investigated. The optimum temperature and the pH stability of the preparation were almost identical to the native one. However,
the optimum pH of bound glucoamylase shifted 1.3 pH units toward the alkaline side compared to the native one. The Michaelis
constant(K
m
) of bound glucoamylase for soluble starch was about four times higher than that of the native enzyme, whileK
m
values for maltose approached those of the native material. At 45‡C the half-life of IMG was 104 days under operational conditions.
Alkaline protease, α-amylase, asparaginase, and penicillin acylase were also chemically coupled to porous glass by the same
method and high relative activities were obtained. 相似文献
15.
Parra A Casero E Pariente F Vázquez L Lorenzo E 《Analytical and bioanalytical chemistry》2007,388(5-6):1059-1067
A rapid, simple and reproducible two-step method for constructing cholesterol biosensors by covalently bonding cholesterol
oxidase (ChOx) to a 3,3′-dithiodipropionic acid di(N-succinimidyl ester) (DTSP)-modified gold electrode is described. Exhaustive characterizations of both the immobilization
process and the morphological properties of the resulting ChOx monolayer were performed via a quartz crystal microbalance
(QCM) and atomic force microscopy (AFM) operated under liquid conditions, respectively. In addition, scanning electrochemical
microscopy (SECM) measurements were performed in order to check that the immobilized enzyme retains its catalytic activity.
The replacement of the natural electron acceptor (O2) in the enzymatic reaction with an artificial mediator, hydroxymethylferrocene (HMF), was also studied. Finally, cholesterol
was amperometrically determined by measuring the hydrogen peroxide produced during the enzymatic reaction at +0.5 V. The optimized
cholesterol biosensor exhibited a sensitivity of 54 nA mM−1 and a detection limit of 22 μM. 相似文献
16.
《Analytical letters》2012,45(7):525-540
Abstract A sensitive method for the rapid determination of activities of soluble or immobilized enzymes, based on the electrochemical detection of hydrogen peroxide is described. Kinetic studies (Vmax and KM determinations) can be performed for all H2O2 generating enzymes (i.e. most of the oxidases) using an amperometric probe with a platinum anode at a fixed potential. When associated with an immobilized glucose oxidase membrane, this sensor constitutes a glucose electrode and the activity of any hydrolase which releases glucose can be measured. There is no need for other auxiliary enzymes and no preincubation step is required. The possibility to carry out continuous analysis constitutes the main advantage of the described method. 相似文献
17.
Quanpeng Chen Shiyun Ai Hai Fan Jun Cai Qiang Ma Xiangbin Zhu Huanshun Yin 《Journal of Solid State Electrochemistry》2010,14(9):1681-1688
A novel and sensitive biosensor was developed for the determination of nitrite. Firstly, multi-walled carbon nanotubes–poly(amidoamine)–chitosan
(MWNT–PAMAM–Chit) nanocomposite along with the incorporation of DNA was used to modify the glassy carbon electrode. Then the
immobilization of Cyt c was accomplished using electrochemical deposition method by consecutive cyclic voltammetry (CV) scanning in a neutral Cyt
c solution. CV behaviors of the modified electrodes showed that the MWNT–PAMAM–Chit nanocomposite is a good platform for the
immobilization of DNA and Cyt c in order, at the same time, an excellent promoter for the electron transfer between Cyt c and the electrode. At high potential, the immobilized Cyt c could be further oxidized into highly reactive Cyt c π-cation by two-step electrochemical oxidation, which could oxidize NO2
− into NO3
− in the solution. Therefore, a nitrite biosensor based on the biocatalytic oxidation of the immobilized Cyt c was fabricated, which showed a fast response to nitrite (less than 5 s). The linear range of 0.2–80 μM and a detection limit
of 0.03 μM was obtained. Finally, the application in food analysis using sausage as testing samples was also investigated. 相似文献
18.
C. Burstein H. Ounissi M. D. Legoy G. Gellf D. Thomas 《Applied biochemistry and biotechnology》1981,6(4):329-338
The use of immobilized enzymes has opened the possibility of large scale utilization of NAD+-linked dehydrogenases, but the applications of this technique were limited by the necessity of providing the large amounts
of NAD+ required by its stoichiometric consumption in the reaction. After immobilization of alcohol dehydrogenase and intactE. coli by glutaraldehyde in the presence of serum albumin, the respiratory chain was found to be capable of regenerating NAD+ from NADH. This NAD+ can be recycled at least 100 times, and thus the method is far more effective than any other, and, moreover, does not require
NADH oxydase purification. The total NADH oxidase activity recovered was 10–30% of the initial activity.
Although, NADH is unable to cross the cytoplasmic membrane, it was able to reach the active site of NADH dehydrogenase after
immobilization. The best yield of NADH oxidase activity with immobilized bacteria was obtained without prior treatment of
the bacteria to render them more permeable. The denaturation by heat of NADH oxidase in cells that are permeabilized was similar
before and after immobilization. In contrast, the heat denaturation of soluble Β-galactosidase required either a higher temperature
or a longer exposure after immobilization. The sensitivity of immobilized NADH oxidase to denaturation by methanol was decreased
compared to permeabilized cells. As a result, it is clear that the system can function in the presence of methanol, which
is necessary as a solvent for certain water insoluble substrates. 相似文献
19.
The catalase (fromAspergillus niger) has been immobilized by a chemical method on the pous SiO2 modified with γ-aminopropyltrietoxysilane, followed with glutaraldehyde and by a physical method in alginate and γ-carrageenan
gel. Optimum support:enzyme ratios and pH values were determined for modified SiO2 in a series of immobilization reactions of catalase in the presence of the crosslinking agent glutaraldehyde, and for alginate
and γ-carrageenan in the presence of hemoglobin and bovine serum albimine. pH and temperaturedependent activity variations
and the stability properties of immobilized catalase preparations were investigated. Rate constants for H2O2 decomposition and catalase deactivation were determined. The decomposition rate of H2O2 used in the cold pasteurizatioan of milk were investigated in a discontinuous batch type reactor system. Activity half-lives
of immobilized catalase were determined. 相似文献
20.
A simple method based on the principle of Wills has been developed for the isoelectric point (pI) determination of immobilized
enzymes. This approach has been applied in the case of five alkylamine immobilized enzymes, i.e., sweet potato β-amylase,B. subtilis α(-amylase, yeast invertase, bacterial dextranase andTrichoderma cellulase employing both polyanionic inhibitors like suramin, inorganic polyphosphates, dextran sulphate and polyanethol
sulphonate and polycationic inhibitors like poly-L-arginine, poly-L-ornithine, and poly-L-histidine. The overall competence
limitations of the method have been discussed. 相似文献