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1.
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
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.
Partially purified S1 nuclease was bound through its carbohydrate moiety to Con A-Sepharose containing increasing amounts of lectin. The retention of activity was high, varying essentially from 75% on the "low lectin" matrix (1 mg Con A/mL of Sepharose), to no detectable activity on the "high lectin" matrix (8 mg Con A/mL of Sepharose). However, approximately 50% activity could be restored in "high lectin" matrix when the coupling was carried out in the presence of glucose, suggesting that the loss of activity on the "high lectin" matrix is caused by conformational changes brought about by the multiple attachment of the enzyme to the matrix. Interaction of Con A with S1 nuclease was used to predict the nature of carbohydrate moiety and its location with respect to the active site of the enzyme. Immobilization resulted in an increase in the optimum temperature, pH, and temperature stabilities, but it did not affect the pH optimum. A marginal increase in the apparent Km was observed. The bound enzyme also showed enhanced stability toward 8 M urea. On repeated use, the bound enzyme retained more than 80% of its initial activity after 6 cycles. These results are discussed taking into consideration the factors affecting immobilized enzymes. In addition, the potential use of immobilized S1 nuclease as an analytical tool is discussed. 相似文献
4.
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
5.
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
6.
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
7.
以表面固定Cu2+的改性大尺寸SiO2大孔材料作为载体,考察了时间、pH和给酶量对漆酶固定化效果的影响,并对固定化漆酶的活性和稳定性进行了研究。结果表明:5 h时吸附达到平衡,pH为4.5、漆酶与载体比例为5 mg·g-1时固定化效果最好,酶活回收率可达到100.4%;固定化漆酶的最适pH和最适温度较游离漆酶的均有升高且范围变宽,固定化后,漆酶的pH稳定性和热稳定性都得到显著提高;固定化漆酶的K m值略高于游离漆酶的;固定化漆酶具有良好的操作稳定性,与底物反应反复操作10批次后剩余酶活为72.7%。 相似文献
8.
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
9.
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
10.
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 × 103 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
11.
以自制的多孔、具柔性亲水手臂的壳聚糖–精氨酸树脂为载体,戊二醛为交联剂固定胰凝乳蛋白酶,确定了酶与载体的最佳比例为20 mg酶/g湿树脂,交联剂的最佳用量为10 mL 1.0%戊二醛/1.5 g湿树脂,交联时间为60 min,所得固定化酶的活力回收率达68.95%。固定化胰凝乳蛋白酶的Km为8.36 mg/mL,比游离酶增大1.52倍,其酶促反应10 min达到最大速率,具有接近游离酶的催化时间进程曲线;其最适温度为70 ℃,比游离酶升高10 ℃;其最适pH值为5.92,比游离酶酸性偏移2个pH值。此外,固定化胰凝乳蛋白酶具有良好的热稳定性和贮存稳定性,75 ℃时的半衰期为8 h,4 ℃时的半衰期为46天。 相似文献
12.
Glucoamylase was immobilized onto novel porous polymer supports. The properties of immobilized glucoamylase and the relationship
between the activity of immobilized enzyme and the properties of porous polymer supports were investigated. Compared with
the native enzyme, the temperature profile of immobilized glucoamylase was widened, and the optimum pH was also changed. The
optimum substrate concentration of immobilized glucoamylase was higher than that of native enzyme. After storage for 23 d,
the immobilized glucoamylase still maintained about 84% of its initial activity, whereas the native enzyme only maintained
about 58% of the initial activity. Moreover, after using repeatedly seven times, the immobilized enzyme maintained about 85%
of its initial activity. Furthermore, the properties of porous polymer supports had an effect on the activity of the immobilized
glucoamylase. 相似文献
13.
Madhavan Nampoothiri K Roopesh K Chacko S Pandey A 《Applied biochemistry and biotechnology》2005,120(2):97-108
Escherichia coli NCIM 2569 was evaluated for its potential for amidase production under submerged fermentation. Among the various amide compounds
screened, maximum substrate specificity and enzyme yield (8.1 U/mL) were obtained by using 1% acetamide. Fermentation was
carried out at 30°C in shake-flask culture under optimized process conditions. A maximum of 0.52 U/mL of intracellular amidase
activity was also obtained from cells incubated for 24 h. Studies were also performed to elucidate the optimal conditions
(gel concentration, initial biomass, curing period of beads, and calcium ion concentration in the production medium) for immobilization
of whole cells. By using E. coli cells entrapped in alginate, a maximum of 6.2 U/mL of enzyme activity was obtained after 12 h of incubation under optimized
conditions. Using the immobilized cells, three repeated batches were carried out successfully, and 85% of the initial enzyme
activity was retained in the second and third batches. The study indicated that the immobilized E. coli cells offered certain advantages such as less time for maximum enzyme production, more stability in the enzyme production
rate, and repeated use of the biocatalyst. 相似文献
14.
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
15.
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 higherK
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
16.
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
17.
Hao-Jing Wang Ji-Hong Bai De-Shan Liu Tong Zhang Hai-Meng Zhou 《Applied biochemistry and biotechnology》1999,76(3):183-191
Aminoacylase (EC 3.5.1.14) was immobilized into DEAE-Sephadex A-25 by ion-exchange absorption for optical resolution of N-acyl-dl-alanine. The effects of pH, temperature, and Co2+ concentration on the activity of free and immobilized enzymes were in vestigated along with the operational and the thermal
stability of the immobilized enzyme. The immobilized enzyme retained high catalytic activity. The optimum pH and temperature
for the hydrolysis of N-acyl-l-alanine in the dl-isomer mixture were 8.0 and 65°C, respectively. Co2+ was an activator for the immobilized enzyme in a similarroleas for the free enzyme. Nosignificant loss of activity was observed
for at least 300 h of continuous operation. The yield of l-alanine was about 70% of the theoretical yield. The immobilized aminoacylase column decayed over a very long period of operation,
but could be completely reactivated by regeneration. 相似文献
18.
Surface-anchored poly(2-vinyl-4,4-dimethyl azlactone) brushes as templates for enzyme immobilization
Cullen SP Mandel IC Gopalan P 《Langmuir : the ACS journal of surfaces and colloids》2008,24(23):13701-13709
We explored surface-anchored poly(2-vinyl-4,4-dimethyl azlactone) (PVDMA) brushes as potential templates for protein immobilization. The brushes were grown using atom transfer radical polymerization from surface-anchored initiators and characterized by a combination of ellipsometry, atomic force microscopy, and X-ray photoelectron spectroscopy. RNase A was immobilized as a model enzyme through the nucleophilic attack of azlactone by the amine groups in the lysines located in the protein. The surface density of RNase A increased linearly from 5 to 50 nm. For 50 nm thick poly(2-vinyl-4,4-dimethyl azlactone) brushes, 7.5 microg/cm2 of RNase A was bound. The kinetics and thermodynamics of RNase A immobilization, the activity relative to surface density, and the pH and temperature dependence were examined. A Langmuir-like model for binding kinetics indicates that the kinetics are controlled by the rate of adsorption of RNase A and has an adsorption rate constant, k(ads), of 2.8 x 10(-8) microg(-1) s(-1) cm3. A maximum relative activity of approximately 0.95, which is near the activity of free RNase A, was reached at 1.2 microg/cm2 (approximately 3.0 monolayers) of immobilized RNase A. The immobilized RNase A had a similar temperature and pH dependence as free RNase A, indicating no significant change in conformation. The PVDMA template was extended to other biotechnologically relevant enzymes, such as deoxyribonuclease I, glucose oxidase, glucoamylase, and trypsin, with relative activities higher than or comparable to those of enzymes immobilized by other means. PVDMA brushes offer an efficient route to immobilize proteins via the ring opening of azlactone without the need for activation or pretreatment while retaining high relative activities of the bound enzymes. 相似文献
19.
Concanavalin A (Con A) immobilized poly(2-hydroxyethyl methacrylate) (PHEMA) beads were investigated for specific adsorption of yeast invertase from aqueous solutions. PHEMA beads were prepared by a suspension polymerization technique with an average size of 150-200 microm, and activated by epichlorohydrin. Con A was then immobilized by covalent binding onto these beads. The maximum Con A immobilization was found to be 10 mg/g. The invertase-loading capability of the PHEMA/Con A beads was 107 mg/g. The maximum invertase adsorption capacity on the PHEMA/Con A adsorbents was observed at pH 5.0. The values of the Michaelis constant K(m) of invertase were significantly larger upon adsorption, indicating decreased affinity by the enzyme for its substrate, whereas V(max) was smaller for the adsorbed invertase. Adsorption improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with adsorption. The adsorbed enzyme activity was found to be quite stable in repeated experiments. Storage stability of adsorbed invertase. 相似文献
20.
Sucrose phosphorylase fromLeuconostoc mesenteroides was immobilbilized by covalent linkage to several supports, and the specific activity recovery was 2-11%. The enzyme adsorbed
onto DEAE-cellulose re tained about 18% specific activity and was stable over eight months. The optimum pH (7.0) and temperature
(30°C) did not change after immobilization. Also there was no improvement of thermal stability, and Km for sucrose and phosphate
was lower compared to the soluble enzyme. 相似文献