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1.
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
2.
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
3.
An agroindustrial residue, green coconut fiber, was evaluated as support for immobilization of Candida antarctica type B (CALB) lipase by physical adsorption. The influence of several parameters, such as contact time, amount of enzyme
offered to immobilization, and pH of lipase solution was analyzed to select a suitable immobilization protocol. Kinetic constants
of soluble and immobilized lipases were assayed. Thermal and operational stability of the immobilized enzyme, obtained after
2 h of contact between coconut fiber and enzyme solution, containing 40 U/ml in 25 mM sodium phosphate buffer pH 7, were determined.
CALB immobilization by adsorption on coconut fiber promoted an increase in thermal stability at 50 and 60 °C, as half-lives
( t
1/2) of the immobilized enzyme were, respectively, 2- and 92-fold higher than the ones for soluble enzyme. Furthermore, operational
stabilities of methyl butyrate hydrolysis and butyl butyrate synthesis were evaluated. After the third cycle of methyl butyrate
hydrolysis, it retained less than 50% of the initial activity, while Novozyme 435 retained more than 70% after the tenth cycle.
However, in the synthesis of butyl butyrate, CALB immobilized on coconut fiber showed a good operational stability when compared
to Novozyme 435, retaining 80% of its initial activity after the sixth cycle of reaction. 相似文献
4.
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
5.
β-galactosidase from Penicillium canescens was immobilized on chitosan, sepharose-4B, foamable polyurethane and some other carriers. The highest yield of immobilization
(up to 98%) was obtained by using chitosan as a carrier. The optimum pH and temperature were not significantly altered by
immobilization. High stability of immobilized β-galactosidase during storage was demonstrated. Efficient lactose saccharification
(over 90%) in whey was achieved by using immobilized β-galactosidase. 相似文献
6.
Urease was chosen as a model multimeric protein to investigate the utility of reversible denaturation for immobilization to
a hydrophobic support. Of the various procedures investigated, acidic denaturation provided the highest degree of immobilization
and enzymatic activity with lowering of K
m
(apparent). Exposure of hydrophobic clusters in the protein molecule induced by the acidic pH environment was confirmed by
fluorescence studies using 8-anilino-1-naphtalene-sulfonate as a hydrophobic-reporter probe. The catalytic potential of the
enzyme at low pH values was dramatically improved with significant heat and pH stability enhancement on immobilization. Furthermore,
the immobilized preparation was used successfully in continuous catalytic transformations. Based on the results presented
in this article and a recent report involving a relatively more simple monomeric protein, it is suggested that reversible
denaturation may be of general utility for immobilization of proteins, which are not normally adsorbed on hydrophobic supports. 相似文献
7.
Purification and reversible immobilization of d-amino acid oxidase from Trigonopsis variabilis could be simultaneously accomplished by hydrophobic interaction on Phenyl Sepharose CL-4B in the presence of 50 m M pyrophosphate buffer (pH 8.5). The presence of a high salt concentration of 2 M, which is generally required for the hydrophobic interactions, was not essential for the hydrophobic immobilization. The
enzyme in free as well as immobilized form was optimally active between pH 7.0 and 9.0. The immobilized preparation could
be reused in a batch process for the conversion of d-amino acids to α-keto acids. When the activity of the preparation dropped below practical limits, the gel could be regenerated
by water wash and recharged with fresh crude extract from yeast. 相似文献
8.
Mesoporous titania‐Nafion composite doped with carbon nanotube (CNT) has been used for the immobilization of tris(2,2′‐bipyridyl)ruthenium(II) (Ru(bpy) 32+) and alcohol dehydrogenase on an electrode surface to yield a highly sensitive and stable electrogenerated chemiluminescence (ECL) ethanol biosensor. The presence of CNT in the composite film increases not only the sensitivity of the ECL biosensor but also the long‐term stability of the biosensor. The present biosensor responds linearly to ethanol in the wide concentration ranges from 1.0×10 ?5 M to 1.0×10 ?1 M with a detection limit of 5.0×10 ?6 M ( S/ N=3). The present ECL ethanol biosensor exhibited higher ECL response compared to that obtained with the ECL biosensor based on the corresponding composite without CNT. The present CNT‐based ECL biosensor showed good long‐term stability with 75% of its initial activity retained after 2 weeks of storage in 50 mM phosphate buffer at pH 7.0. 相似文献
9.
A novel biosensor was fabricated based on the immobilization of tyrosinase and N ‐acetyl‐ L ‐cysteine‐capped gold nanoparticles onto the surface of the glassy carbon electrode via the film forming by chitosan. The NAC‐AuNPs ( N ‐acetyl‐ L ‐cysteine‐capped gold nanoparticles) with the average size of 3.4 nm had much higher specific surface area and good biocompatibility, which were favorable for increasing the immobilization amount of enzyme, retaining the catalytic activity of enzyme and facilitating the fast electron transfer. The prepared biosensor exhibited suitable amperometric responses at −0.2 V for phenolic compounds vs. saturated calomel electrode. The parameters of influencing on the working electrode such as pH , temperature, working potential were investigated. Under optimum conditions, the biosensor was applied to detect catechol with a linear range of 1.0 × 10 −7 to 6.0 × 10 −5 mol•L −1 , and the detection limit of 5.0 × 10 −8 mol•L −1 ( S / N =3). The stability and selectivity of the proposed biosensor were also evaluated. 相似文献
10.
A nonlabeling electrochemical detection method for analyzing the polymerase-chain-reaction-amplified sequence-specific p16
INK4A
gene, in which the basis for the covalent immobilization of deoxyribonucleic acid (DNA) probe is described, has been developed.
The self-assembly process was based on the covalent coupling of glutaraldehyde (GA) as an arm molecule onto an amino-functional
surface. The p16
INK4A
gene was used as the model target for the methylation detection of early cancer diagnosis. An amino-modified DNA probe was
successfully assembled on the GA-coupling surface through the formation of Schiff base under potential control. The hybridization
of amino-modified DNA probes with the target was investigated by means of electrochemical measurements, including cyclic voltammetry
and square wave voltammetry. Furthermore, the functions of GA coupling for sequence-specific detection were compared with
those obtained based on mercaptopropionic acid. Hybridization experiments indicated that the covalent coupling of GA was suitable
for the immobilization of DNA probe and was sensitive to the electrochemical detection of single-base mismatches of label-free
DNA targets in hybridization. Moreover, reported probe-modified surfaces exhibited excellent stability, and the hybridization
reactions were found to be completely reversible and highly specific for recognition in subsequent hybridization processes.
The strategy provided the potential for taking full advantage of existing modified electrode technologies and was verified
in microarray technology, which could be applied as a useful and powerful tool in electrochemical biosensor and microarray
technology. 相似文献
11.
Nanopolystyrene was used as a solid support for the covalent immobilization of Candida antarctica lipase B (CalB) using the photoreactive reagent 1-fluoro-2-nitro-4-azido benzene (FNAB) as a coupling reagent. The obtained derivative was then used as a biocatalyst in a microwave assisted esterification experiment. Factors such as contact time, pH, and enzyme concentration were investigated during immobilization. The hydrolytic activity, thermal, and operational stability of immobilized-CalB were determined. The maximum immobilized yield (218 μg/mg support) obtained at pH 6.8 exhibited optimum hydrolytic activity (4.42 × 10 3 mU p-nitrophenol/min). The thermal stability of CalB improved significantly when it was immobilized at pH 10, however, the immobilized yield was very low (93.6 μg/mg support). The immobilized-CalB prepared at pH 6.8 and pH 10 retained 50% of its initial activity after incubation periods of 14 and 16 h, respectively, at 60 ℃. The operational stability was investigated for the microwave assisted esterification of oleic acid with methanol. Immobilized-CalB retained 50% of its initial activity after 15 batch cycles in the microwave-assisted esterification. The esterification time was notably reduced under microwave irradiation. The combined use of a biocatalyst and microwave heating is thus an alternative total green synthesis process. 相似文献
12.
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. The V
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 (K i = 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
13.
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 intact E. 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
14.
Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, predictable decay rates
and improved economics. This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase (YLL) by physical adsorption and covalent attachment. The enzyme was adsorbed on octyl–agarose and octadecyl–sepabeads
supports by hydrophobic adsorption at low ionic strength and on MANAE–agarose support by ionic adsorption. CNBr–agarose was
used as support for the covalent attachment immobilization. Immobilization yields of 71, 90 and 97% were obtained when Y. lipolytica lipase was immobilized into octyl–agarose, octadecyl–sepabeads and MANAE–agarose, respectively. However, the activity retention
was lower (34% for octyl–agarose, 50% for octadecyl–sepabeads and 61% for MANAE–agarose), indicating that the immobilized
lipase lost activity during immobilization procedures. Furthermore, immobilization by covalent attachment led to complete
enzyme inactivation. Thermal deactivation was studied at a temperature range from 25 to 45°C and pH varying from 5.0 to 9.0
and revealed that the hydrophobic adsorption on octadecyl–sepabeads produced an appreciable stabilization of the biocatalyst.
The octadecyl–sepabeads biocatalyst was almost tenfold more stable than free lipase, and its thermal deactivation profile
was also modified. On the other hand, the Y. lipolytica lipase immobilized on octyl–agarose and MANAE–agarose supports presented low stability, even less than the free enzyme. 相似文献
15.
S1 nuclease from Aspergillus oryzae (EC 3.1.30.1) was coupled to gelatin-alginate composite matrix using the residual free aldehyde groups on the surface of
glutaraldehyde crosslinked matrix. The immobilized enzyme retained approximately 10% activity of the soluble enzyme. When
partially purified enzyme was bound to the matrix, the immobilized preparation did not show any detectable enzyme activity.
However, the activity could be restored when the coupling was carried out in the presence of a coprotein or substrate. The
optimum pH of the immobilized S1 nuclease shifted to 3.8 from 4.3 for the soluble enzyme. Also, optimum temperature increased
to 65°C after immobilization. Bound S1 nuclease showed increased pH and temperature stabilities. Immobilization brought about
a twofold decrease in the Michaelis-Menton constant ( K
m). 相似文献
16.
Lactococcus lactis CM1, an isolate from homemade “Dahi,” a traditional fermented milk from India, used maltose as carbon source to produce a
high level of bacteriocin. The bacterial cell mass and the bacteriocin production correlated with the initial pH of the medium
and were highest when the initial pH was 11.0. The level of bacteriocin reached its peak at the late log phase with concomitant
reduction of culture pH to 4.2, regardless of the initial pH of the medium. A combination of maltose and an initial medium
pH of 11 resulted in the highest bacteriocin production. The antibacterial spectrum of the bacteriocin was closely similar
to that of nisin and it inhibited a number of food spoilage and pathogenic bacteria. Upon sodium dodecyl sulfate polyacrylamide
gel electrophoresis, the compound migrated close to the position of nisin (3.5 kDa). However, it had higher stability than
nisin at a wide range of pH and temperature. PCR amplification using nisin gene-specific primers and sequencing of the amplified
DNA revealed the structural gene for the bacteriocin to be identical to that of nisZ. 相似文献
17.
A novel potent protease, Urechis unicinctus fibrinolytic enzyme (UFE), was first discovered by our laboratory. In this study, we further investigated the enzymatic properties
and dynamic parameters of UFE. As a low molecular weight protein, UFE appeared to be very stable to heat and pH. When the
temperature was <50°C, the remnant enzyme activity remained almost unchanged, but when the temperature was raised to 60#x00B0;C
the remnant enzyme activity began to decrease rapidly. UFE was quite stable in a pH range of 3.0–12.0, especially at slightly
alkaline pH values. Mn 2+, Cu 2+, and Fe 2+ ions were activators of UFE, whereas Fe 3+ and Ag + ions were inhibitors. Fe 2+ ion along with Fe 3+ ion might regulate UFE activity in vivo. The optimum pH and temperature of UFE were about 8.0 and 50°C, respectively. When
using casein as substrate and a substrate concentration <0.1% casein (w/v), the reaction velocity was increased with substrate
concentration. Also when using casein as substrate, the determined K
m and V
max of UFE were 0.5298 mg/mL and 3.0845 mol of l-tyrosine equivalent, respectively. Our systematic research results are significant
when UFE is applied for medical and industrial purposes. 相似文献
18.
Gold catalysts, supported on a solid base of Mg xAlO hydrotalcite, were prepared by a modified deposition precipitation method for CO selective oxidation. The preparation parameters and pretreatment of the catalysts were investigated. The pH and the HAuCl 4 concentration in the initial solution, and the Mg/Al molar ratio of Mg xAlO affected the pH in the final solution and determined the actual gold loading of the catalyst. The calcination temperatures of the Mg xAlO support and the Au/Mg xAlO catalyst dominated the Au 3+/Au 0 ratio on the catalyst. The pretreatment of the catalyst as well as the gold loading and the Au 3+/Au 0 ratio, critically determined the activity of the catalyst for CO selective oxidation. Based on XPS and in situ DR-FTIR analyses, a mechanism for CO selective oxidation on 2%Au/Mg 2AlO was proposed. The hydroxyl group on Mg 2AlO also participated in the reaction. 相似文献
19.
Bacillus subtilis strain TrigoCor 1448 was grown on wheat middlings in 0.5-l solid-state fermentation (SSF) bioreactors for the production
of an antifungal biological control agent. Total antifungal activity was quantified using a 96-well microplate bioassay against
the plant pathogen Fusarium oxysporum f. sp. melonis. The experimental design for process optimization consisted of a 2 6−1 fractional factorial design followed by a central composite face-centered design. Initial SSF parameters included in the
optimization were aeration, fermentation length, pH buffering, peptone addition, nitrate addition, and incubator temperature.
Central composite face-centered design parameters included incubator temperature, aeration rate, and initial moisture content
(MC). Optimized fermentation conditions were determined with response surface models fitted for both spore concentration and
activity of biological control product extracts. Models showed that activity measurements and spore production were most sensitive
to substrate MC with highest levels of each response variable occurring at maximum moisture levels. Whereas maximum antifungal
activity was seen in a limited area of the design space, spore production was fairly robust with near maximum levels occurring
over a wider range of fermentation conditions. Optimization resulted in a 55% increase in inhibition and a 40% increase in
spore production over nonoptimized conditions. 相似文献
20.
Alkaline Ca-bentonite, obtained upon acid activation and base load of natural bentonite, has a good anion exchange capability. Glu-modified alkaline Ca-bentonites were further prepared by covalent binding with glutamic acid for the immobilization of lipase OF from Candida cylindracea. The obtained immobilized lipase demonstrated a significantly higher catalytic activity than that of unmodified alkaline Ca-bentonite, giving a specific activity of 62.1 U mg−1 protein, twice that of the unmodified carrier, and a total activity of 391.2 U g−1 support, retaining ~ 82.3% of the activity after being reused five times for olive oil emulsion hydrolysis. X-ray diffraction and Fourier transform infrared spectroscopy assays demonstrated the successful immobilization of the lipase on the surface of the bentonite. Upon immobilization, the thermostability of the lipase improved remarkably. At 50 °C, free lipase retained only 6.0% of its initial activity at 6 h, in comparison with 15% for Ca-Bent-lipase and 50% for Glu-Ca-Bent-lipase after 8 h. The Glu-Ca-Bent-lipase is proved as an effective biocatalyst for the biodiesel preparation, improving the transesterification reaction conversion from 52.8% in the condition of free lipase to 99.9% and keeping at 56.2% after being reused five times, while the free lipase was inactive upon two reuses. The above results provide a new route in the use of inexpensive bentonite for the enzyme immobilization. 相似文献
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