共查询到20条相似文献,搜索用时 46 毫秒
1.
S. S. Deshmukh M. Dutta Choudhury V. Shankar 《Applied biochemistry and biotechnology》1993,42(2-3):95-104
Partially purified glucose isomerase fromStreptomyces thermonitrificans when coupled to glutaraldehyde-activated Indion 48-R, retained 30–40% activity of the soluble enzyme. However, an approximately
twofold increase in the activity could be achieved by binding the enzyme in the presence of glucose. Binding the enzyme to
matrices presaturated with either glucose or fructose and influence of lysine modification on the activity of the soluble
enzyme revealed that the comparatively low activity observed in case of the enzyme bound in the absence of substrate is the
result of the nonspecific binding of either substrate or product to the matrix. Immobilization did not affect the pH and temperature
optima of the enzyme, but it lowered the temperature stability. Immobilization resulted in a marginal increase in theK
m
and a threefold decrease in theV
max
. Substrate concentrations as high as 36% glucose could be converted to 18.5% fructose in 5 h, at pH 7.0 and 70‡C. The bound
enzyme, however, showed inferior stability to repeated use and lost approx 40% of its initial activity after five cycles of
use. Indion 48-R bound glucose isomerase could be stored, in wet state, for 30 d without any apparent loss in its initial
activity. 相似文献
2.
S1 nuclease fromAspergillus 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
3.
Dipali Bagal 《Analytica chimica acta》2006,555(2):316-321
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
4.
Selin Çelebi Esin İbibikcan Senem Kiralp Kayahan Başak Yiĝitsoy Levent Toppare 《高分子科学杂志,A辑:纯化学与应用化学》2013,50(8):739-744
Immobilization of invertase in conducting copolymer matrix of 2,5-di(thiophen-2-yl)-1-p-tolyl-1H-pyrrole with pyrrole (poly(DDTP-co-Py)) was achieved via electrochemical polymerization. Kinetic parameters, Michaelis-Menten constant, Km and the maximum reaction rate, Vmax were investigated. Operational stability and temperature optimization of the enzyme electrodes were also examined. Immobilized invertase reveals maximum activity at 50°C and; pH 8 and pH 4 for two copolymer matrices. Although the same two monomers are utilized for the copolymer synthesis, the way the copolymer is produced results in quite different responses in terms of enzyme activity, optimum pH and kinetic parameters. Excellent operational stability of the enzyme electrodes enables their repetitive use in the determination of invert sugar. 相似文献
5.
Commercial yeast invertase (Bioinvert®) was immobilized by adsorption on anion-exchange resins, collectively named Dowex® (1×8:50–400, 1×4:50–400, and 1×2:100–400). Optimal binding was obtained at pH 5.5 and 32°C. Among different polystyrene beads, the complex Dowex-1×4–200/invertase showed a yield coupling and an immobilization coefficient equal to 100%. The thermodynamic and kinetic parameters for sucrose hydrolysis for both soluble and insoluble enzyme were evaluated. The complex Dowex/invertase was stable without any desorption of enzyme from the support during the reaction, and it had thermodynamic parameters equal to the soluble form. The stability against pH presented by the soluble invertase was between 4.0 and 5.0, whereas for insoluble enzyme it was between 5.0 and 6.0. In both cases, the optimal pH values were found in the range of the stability interval. The K m and V max for the immobilized invertase were 38.2 mM and 0.0489 U/mL, and for the soluble enzyme were 40.3 mM and 0.0320 U/mL. 相似文献
6.
Brígida AI Pinheiro AD Ferreira AL Gonçalves LR 《Applied biochemistry and biotechnology》2008,146(1-3):173-187
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
7.
Cellulose-based carriers Granocel were specially prepared and optimised for covalent immobilization of enzymes. The effects
of carrier characteristics such as pore size, chemistry of anchor groups and their density on invertase immobilization efficiency
were evaluated. It was found that the preferential adsorption/binding of the enzyme to a carrier during coupling and its activity
after immobilization depended on microenvironmental effects created by hydrophilic surface of the carrier, functional groups
and their activators. The best preparations (activity approx. 300 U/mL, high storage stability) were obtained for NH2-Granocel activated with glutaraldehyde. It is probably due to Granocel modification with pentaethylenehexamine that gave
a 19-atom spacer arm. The enzyme concentration in coupling mixture was optimised as well. The kinetic parameters of sucrose
hydrolysis for native and immobilized invertase were evaluated. Compared to the native invertase, K
m
value of immobilized enzyme was only twice higher with about three times lower substrate inhibition. Reaction runs in a well
mixed batch reactors with native and immobilized invertase showed slightly slower reaction rate in the case of the enzyme
covalently bound to Granocel. Very good stability of cellulose-based carrier was proved experimentally by 20 successive reaction
runs in a batch reactor. 相似文献
8.
Zehra Olcer Mehmet Murat Ozmen Zeynep M. Sahin Faruk Yilmaz Aziz Tanriseven 《Applied biochemistry and biotechnology》2013,171(8):2142-2152
A novel method was developed for the immobilization of Saccharomyces cerevisiae invertase within supermacroporous polyacrylamide cryogel and was used to produce invert sugar. First, the cross-linking of invertase with soluble polyglutaraldehyde (PGA) was carried out prior to immobilization in order to increase the bulkiness of invertase and thus preventing the leakage of the cross-linked enzyme after immobilization by entrapment. And then, in situ immobilization of PGA cross-linked invertase within cryogel synthesis was achieved by free radical polymerization in semi-frozen state. The method resulted in 100 % immobilization and 74 % activity yields. The immobilized invertase retained all the initial activity for 30 days and 30 batch reactions. Immobilization had no effect on optimum temperature and it was 60 °C for both free and immobilized enzyme. However, optimum pH was affected upon immobilization. Optimum pH values for free and immobilized enzyme were 4.5 and 5.0, respectively. The immobilized enzyme was more stable than the free enzyme at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined. The newly developed method is simple yet effective and could be used for the immobilization of some other enzymes and microorganisms. 相似文献
9.
Sari MM 《Applied biochemistry and biotechnology》2011,163(8):1020-1037
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 m2/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
10.
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
11.
Partially purified RNase T2 (EC 2.7.7.17) from Aspergillus oryzae was bound through its carbohydrate moiety to Concanavalin A-Sepharose. The retention of activity was high, ranging from 70% at low enzyme load to approximately 9% at high enzyme load. Though there was no change in the pH and temperature optima, the pH stability and the Km decreased after immobilization. Compared to the soluble enzyme, the immobilized RNase T2 showed enhanced temperature stability and more resistance to metal ions. Both soluble and immobilized enzymes were stable to 8 M urea. On repeated use, the bound enzyme retained more than 60% of its initial activity after six cycles. 相似文献
12.
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
13.
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
14.
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
15.
J. Stano K. Micieta M. Korenova V. Blanarikova H. Tintemann P. Nemec M. Valsikova 《Chemistry of Natural Compounds》2008,44(6):755-761
Cell suspensions of lemon balm (Melissa officinalis L.) were permeabilized by Tween 20, Tween 80, ethanol, hexadecyltrimethylammonium bromide, and hexadecylpyridinium chloride,
and immobilized by glutaraldehyde. The invertase pH optimum was 4.5 at temperature 50°C. The hydrolysis of substrate was linear
for 4 h, reaching 60% conversion. The cells had high invertase activity and good stability, and in longterm storage they showed
good physicomechanical properties. The culture medium (without cells) was used for the identification and determination of
extracellular enzyme activity. Intracellular activity was estimated from the cell suspension. For the lemon balm cell suspension,
the intracellular activity accounted for 83.7% of the total activity, and the extracellular one for 12.7%. The intracellular
specific activity is 4.2 times higher. Our method permits the rapid, simple, and specific identification and determination
of plant invertase.
Published in Khimiya Prirodnykh Soedinenii, No. 6, pp. 611-615, November-December, 2008. 相似文献
16.
Fatemeh Moradian Khosro Khajeh Hossein Naderi-Manesh Majid Sadeghizadeh 《Applied biochemistry and biotechnology》2009,159(1):33-45
Bacillus sp. HR-08 screened from soil samples of Iran, is capable of producing proteolytic enzymes. 16S rDNA analysis showed that
this strain is closely related to Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus mojavensis, and Bacillus atrophaeus. The zymogram analysis of the crude extract revealed the presence of five extracellular proteases. One of the proteases was
purified in three steps procedure involving ammonium sulfate precipitation, DEAE-Sepharose ionic exchange and Sephacryl S-200
gel filtration chromatography. The molecular mass of the enzyme on SDS-PAGE was estimated to be 29 kDa. The protease exhibited
maximum activity at pH 10.0 and 60 °C and was inhibited by PMSF but it was not affected by cysteine inhibitors, suggesting
that the enzyme is a serine alkaline protease. Irreversible thermoinactivation of enzyme was examined at 50, 60, and 70 °C
in the presence of 10 mM CaCl2. Results showed that the protease activity retains more than 80% and 50% of its initial activity after incubation for 30 min
at 60 and 70 °C, respectively. This enzyme had good stability in the presence of H2O2, nonionic surfactant, and local detergents and its activity was enhanced in the presence of 20% of dimethyl sulfoxide (DMSO),
dimethyl formamide (DMF) and isopropanol. The enzyme retained more than 90% of its initial activity after pre-incubation 1 h
at room temperature in the presence of 20% of these solvents. Also, activation can be seen for the enzyme at high concentration
(50%, v/v) of DMF and DMSO. 相似文献
17.
Deise Juliana Kolling Willian Alexandre Suguino Fábio Cristiano Angonesi Brod Ana Carolina Maisonnave Arisi 《Applied biochemistry and biotechnology》2011,163(2):304-312
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.
Sevda Aydar A. Elif Böyükbayram Merve Şendur Levent Toppare 《高分子科学杂志,A辑:纯化学与应用化学》2013,50(11):855-861
Conductive polymers with donor-acceptor-donor (DAD) type units; benzothiadiazole acceptor unit and 3,4-ethylenedioxythiophene (EDOT) and thiophene (Th) donor units, were investigated for immobilization of invertase. The polymers were prepared potentiodynamically from their monomers, M1 (Th-benzothiadiazole-Th), M2 (EDOT-benzothiadiazole-EDOT), M3 (Th-benzothiadiazole-EDOT) and a copolymer, which is a homolog to homopolymer of M3, was prepared from M1 and M2. Invertase was trapped between a two layer-composite; DAD polymer and polypyrrole. The characterization of immobilized invertase was performed using a spectrophotometric method at 540 nm. Kinetic parameters, V max, maximum reaction rate and Km , Michaelis-Menten constant values of immobilized invertase in DAD type homopolymer (P3), 0.95 μmol min?1 and 22.7 mM, respectively, are found between those of homopolymers P1 and P2 and are better than the copolymer. Polymers and copolymers exhibited a broad optimal temperature profile between 30 and 50°C compared to the free enzyme. Optimum pH (5.0) is the same as for free invertase. The electrodes were found to be stable with 100% activity during one day for 40 consecutive measurements. As to the self-life, 25% of the initial activity was lost in the first ten days, then the electrodes were stable with 75% activity for a 40 day storage at 4°C. 相似文献
19.
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
20.
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 mM) compared to that of the soluble urease (3.0 mM). 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