共查询到20条相似文献,搜索用时 546 毫秒
1.
Pratap Singh 《Applied biochemistry and biotechnology》1984,9(2):161-172
PstI has been immobilized in agarose. A solution of low melting agarose containing 1,6-hexamethylenediamine and PstI formed
a gel that was effective in the linearization of pBR322 DNA. The gel containing PstI could be treated with 1,5-bis(N-acetylamino-N-succinimidoxy carbonyl)pentane, a crosslinking agent, without affecting the enzyme activity. Polymerization of acrylamide
in presence of PstI led to conisderably reduced enzyme activity, although EcoRI under identical conditions showed high activity.
It was found that acetylation of amino groups in PstI, by reaction with hydroxysuccinimide acetate, led to total inactivation
of the enzyme activity. This reaction showed the presence of reactive amino groups that were essential for the enzyme activity
of PstI. Involvement of these amino groups in binding to activated Sepharose 4B, during covalent immobilization, was responsible
for inactive enzyme preparations. 相似文献
2.
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
3.
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
4.
V. Podzemná M. Slováková L. Kourková L. Svoboda 《Journal of Thermal Analysis and Calorimetry》2010,101(2):715-719
In this article, the monitoring of an enzymatic reaction by means of a miniaturized batch type IC-calorimeter was performed.
The aim of this work was focused on an investigation of enthalpy and rate of enzymatic reaction of trypsin with Nα-benzoyl-l-arginine-p-nitroanilide hydrochloride (BApNA). Both the parameters were determined for reactions in different buffers and for varying
concentration of enzyme at 37 °C. The rate of reaction decreased with the increasing concentration of enzyme caused by trypsin
autolysis. 相似文献
5.
Continuous production of L-aspartic acid 总被引:2,自引:0,他引:2
For the continuous production ofl-aspartic acid from fumaric acid and ammonia by the action of aspartase, the enzyme extracted fromEscherichia coli orE. coli cells having high aspartase activity were immobilized by various methods.
In 1973 we succeeded in the industrial production ofl-aspartic acid usingE. coli cells immobilized with polyacrylamide gel.
For the improvement of this process, we developed a novel technique using κ-carrageenan as the immobilizing matrix forE. coli cells. Further, EAPc-7 strain, having higher aspartase activity, was contracted from the parentE. coli by continuous cultivation with a definite medium. The aspartase activity was about seven times higher than that of the parent
cells. In 1982 we changed from the conventional method to the improved method, using EAPc-7 strain immobilized with κ-carrageenan. 相似文献
6.
Yubin Ge Hui Zhou Wei Kong Yi Tong Shuyan Wang Wei Li 《Applied biochemistry and biotechnology》1998,69(3):203-215
Bilayer glucose isomerase was immobilized in porousp-trimethylamine-polystyrene (TMPS) beads, through a molecular deposition technique. Some of the factors that influence the
activity of immobilized glucose isomerase were optimized, with the enzyme concentration of 308 IU/mL, enzyme:matrix ratio
of 924 IU/g wet carrier, and hexamethylenebis(trimethylammonium iodine) concentration of 15 mg/mL, giving the maximum catalytic activity (2238 IU/g dry gel) of the immobilized
bilayer glucose isomerase, retaining 68.5% of the initially added activity. The half-life of the immobilized bilayer glucose
isomerase was approx 45 d at pH 8.5, 60°C, with 50% (w/v) glucose as substrate. The specific productivity of the immobilized
bilayer glucose isomerase was 223 g dry D-glucose/g dry immobilized enzyme per day. 相似文献
7.
F. Pittner 《Applied biochemistry and biotechnology》1981,6(2):85-90
myo-Inositol-1-phosphate synthase (EC 5.5.1.4) from rat testes, an NAD+-containing enzyme, which convertsd-glucose 6-phosphate to 1l-myo-inositol 1-phosphate, could be immobilized together with its cofactor and bovine serum albumin by crosslinking with glutaraldehyde
at pH 4.5. The enzyme bound to the gel showed a specific activity of 5.6% of that of the native enzyme, but the activity could
be increased to 21% by pretreatment with urea. 相似文献
8.
Qian Zhao Jianzhong Sun Hua Ren Qiyun Zhou Qincai Lin 《Journal of polymer science. Part A, Polymer chemistry》2008,46(6):2222-2232
Thermosensitive hydrogel made up of poly(N‐isopropylacrylamide) (PNIPA)‐chitosan semi‐interpenetrating network (semi‐IPN) with ultrarapid responding rate was synthesized. Horseradish peroxidase (HRP) was then immobilized on this hydrogel that acted as an enzyme‐carrier by glutaraldehyde bridge. Polymerization of acrylamide was initiated by a redox system (hydrogen peroxide/acetylacetone (Acac)) and was catalyzed by the immobilized enzyme at room temperature. The attention was focused on the properties of the carrier‐enzyme systems. The hydrogel was proofed to be macroporous by environmental scanning electron microscope images. Swelling properties of the hydrogel such as swelling ratio and deswelling–reswelling kinetics were measured. The properties of the immobilized enzyme such as enzyme activity, storage stability, and thermostability were also studied. The immobilized enzyme could be used repeatedly. Gel permeation chromatography measurement of the resulted polyacrylamide (PAAm) showed that the molecular weight reduced as the repeated times of the immobilized enzyme catalysis increased. In conclusion, the macroporous hydrogel would be a suitable enzyme carrier for practical applications in future. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 2222–2232, 2008 相似文献
9.
Magda Ibrahim Martine Decolin Anne-Marie Batt Edith Dellacherie Gerard Siest 《Applied biochemistry and biotechnology》1986,12(3):199-213
Microsomes from pig liver were covalently coupled to Sepharose activated by CNBr and to Sephadex activated by 1,1’-carbonyldiimidazole.
Microsomes were also entrapped inside Ca-alginate andk-carrageenan gels. The concentration of immobilized cytochrome P-450 was determined by CO-difference spectra. The activity
of the monooxygenase system was demonstrated by theN-demethylation of aminopyrine, theO-demethylation ofp-nitroanisole, and the hydroxylation of perhexiline maleate. Upon immobilization, a 30–40% and a 60–70% decrease in V
max
app
for theOandN-demethylations were respectively observed. The V
max
app
values for the hydroxylation of perhexiline maleate were essentially the same for the different immobilized forms and for
the freely suspended microsomal cytochrome P-450.
Under storage at 4°C, microsomes entrapped insidek-carrageenan and Ca-alginate were less stable than the free microsomes, whereas immobilization on CNBr-activated Sepharose
improved the stability of the hepatic microsomal monooxygenase system at the same temperature. These types of immobilized
microsomes have the advantage of being easily recovered and reused for other assays. Finally, microsomes entrapped insidek-carrageenan or Ca-alginate can be used to follow up the continuous metabolization ofp-nitroanisole for several hours in a stirred-batch reactor. 相似文献
10.
Yubin Ge Hui Zhou Wei Kong Yi Tong Shuyan Wang Wei Li 《Applied biochemistry and biotechnology》1998,69(1):17-29
Bilayer glucose isomerase was immobilized in porousp-trimethylaminepolystyrene (TMPS) beads through a molecular deposition technique. Some of the factors that influence the activity
of immobilized glucose isomerase were optimized, with the enzyme concentration of 308 IU/mL, enzyme-to-matrix ratio of 924
IU/g wet carrier, and hexamethylene bis(trimethylammonium iodine) concentration of 15 mg/mL giving the maximum catalytic activity
(2238 IU/g dry gel) of the immobilized bilayer glucose isomerase, retaining 68.5% of the initially added activity. The half-life
of the immobilized bilayer glucose isomerase was approx 45 d at pH 8.5, 60°C, with 50% (w/v) glucose as substrate. The specific
productivity of the immobilized bilayer glucose isomerase was 223 g dry D-glucose/g dry immobilized enzyme per d. 相似文献
11.
Jaime G. Mayoral Francisco J. Alarcón Tomás F. Martínez Pablo Barranco F. Noriega 《Applied biochemistry and biotechnology》2010,160(1):1-8
A novel end-point fluorimetric procedure based on the use of rhodamine-110-labeled specific substrate was developed to determine
trypsin activities in biological samples. We evaluated the ability of trichloroacetic acid and acetic acid to stop the enzymatic
reaction without hindering the detection of the fluorescence of rhodamine-110 released into the reaction mixture from the
specific substrate (CBZ-l-alanyl-l-arginine)2-rhodamine-110. Trichloroacetic acid decreased markedly the fluorescence of rhodamine-110, even at low concentrations. On
the other hand, the addition of 50 mmol/l acetic acid inactivated efficiently trypsin activity, causing minor effects on rhodamine-110
fluorescence. The proposed procedure was more sensitive than the spectrophotometric end-point method using N-α-benzoyl-dl-arginine-p-nitroanilide as substrate. The possibility of carrying out end-point fluorimetric assays improves the performance of monocell
fluorimeters by setting specific conditions optimal for each enzyme activity independently of the fluorimeter. This method
also allows replicate assays to be conducted simultaneously, resulting in considerable time saving and in increased performance
of low-cost equipment. 相似文献
12.
D. V. Kapustin A. A. Vikhrov I. V. Gorokhova A. N. Generalova O. V. Kalyazina T. G. Murzabekova V. P. Zubov 《Russian Chemical Bulletin》2005,54(2):452-457
Composite matrices based on macroporous silica modified by N-vinylcaprolactam copolymers with diallyldimethylammonium chloride and with 2-hydroxyethyl methacrylate were obtained. Lipase from Pseudomonas fluorescens was immobilized on the obtained materials. The temperature dependence of the hydrolytic activity of the immobilized lipase preparations in the triacetin hydrolysis was investigated. The hydrolytic activity of lipase immobilized on the matrix modified by the N-vinylcaprolactam copolymer with 2-hydroxyethyl methacrylate can be regulated by varying the temperature of the reaction medium. The temperature dependence of the hydrolytic activity of the immobilized enzyme has a maximum at 40 °C, the activity of the immobilized lipase being ∼3.5 times higher compared to that at 20 °C. After immobilization on these composite materials, lipase retained the activity in the acetylation of 1-(RS)-phenylethanol with vinyl acetate in ButOMe.__________Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 2, pp. 443–448, February, 2005. 相似文献
13.
Whole cells ofBrevibacterium flavum having fumarase activity were immobilized using K-carrageenan. The stabilities of fumarase activity in the immobilized cells
against external factors, including heat, pH, organic solvents, and protein denaturing reagents, were compared with those
of free cells and native enzyme. The stabilities of fumarase activity in immobillized cells against external factors were
highest, and those of native enzyme were lowest. In the “gel-state,” K-carrageenan-immobilized cells showed a much higher
stabilization effect for external factors than “sol-state” immobilized cells. 相似文献
14.
Luis Gerardo Ramírez-Ramírez David Enrique Zazueta-lvarez Hctor Alonso Fileto-Prez Damin Reyes-Jquez Cynthia Manuela Núez-Núez Juan de Dios Galindo-De la Rosa Javier Lpez-Miranda Perla Guadalupe Vzquez-Ortega 《Molecules (Basel, Switzerland)》2022,27(13)
β-Glucosidase is part of the cellulases and is responsible for degrading cellobiose into glucose, a compound that can be used to produce biofuels. However, the use of the free enzyme makes the process more expensive. Enzyme immobilization improves catalytic characteristics and supports, such as zeolites, which have physical-chemical characteristics and ion exchange capacity that have a promising application in the biotechnological industry. This research aimed to immobilize by adsorption a recombinant β-glucosidase from Trichoderma reesei, obtained in Escherichia coli BL21 (DE3), in a commercial zeolite. A Box Behnken statistical design was applied to find the optimal immobilization parameters, the stability against pH and temperature was determined, and the immobilized enzyme was characterized by SEM. The highest enzymatic activity was determined with 100 mg of zeolite at 35 °C and 175 min. Compared to the free enzyme, the immobilized recombinant β-glucosidase presented greater activity from pH 2 to 4 and greater thermostability. The kinetic parameters were calculated, and a lower KM value was obtained for the immobilized enzyme compared to the free enzyme. The obtained immobilization parameters by a simple adsorption method and the significant operational stability indicate promising applications in different fields. 相似文献
15.
Basri M Samsudin S Ahmad MB Razak CN Salleh AB 《Applied biochemistry and biotechnology》1999,81(3):205-217
Lipase from Candida rugosa was immobilized by entrapment on poly(N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate)(poly[VP-co-HEMA]) hydrogel, and divinylbenzene was the crosslinking agent.
The immobilized enzymes were used in the esterification reaction of oleic acid and butanol in hexane. The activities of the
immobilized enzymes and the leaching ability of the enzyme from the support with respect to the different compositions of
the hydrogels were investigated. The thermal, solvent, and storage stability of the immobilized lipases was also determined.
Increasing the percentage of composition of VP from 0 to 90, which corresponds to the increase in the hydrophilicity of the
hydrogels, increased the activity of the immobilized enzyme. Lipase immobilized on VP(%):HEMA(%) 90∶10 exhibited the highest
activity. Lipase immobilized on VP(%):HEMA(%) 50∶50 showed the highest thermal, solvent, storage, and operational stability
compared to lipase immobilized on other compositions of hydrogels as well as the native lipase. 相似文献
16.
Ah-Reum Park Hye-Jung Kim Jung-Kul Lee Deok-Kun Oh 《Applied biochemistry and biotechnology》2010,160(8):2236-2247
We expressed a putative β-galactosidase from Sulfolobus acidocaldarius in Escherichia coli and purified the recombinant enzyme using heat treatment and Hi-Trap ion-exchange chromatography. The resultant protein gave
a single 57-kDa band by SDS-PAGE and had a specific activity of 58 U/mg. The native enzyme existed as a dimer with a molecular
mass of 114 kDa by gel filtration. The maximum activity of this enzyme was observed at pH 5.5 and 90 oC. The half-lives of
the enzyme at 70, 80, and 90 oC were 494, 60, and 0.2 h, respectively. The hydrolytic activity with p-nitrophenyl(pNP) substrates followed the order p-nitrophenyl-β-d-fucopyranoside > pNP-β-d-glucopyranoside > pNP-β-d-galactopyranoside > pNP-β-d-mannopyranoside > pNP-β-d-xylopyranoside, but not toward aryl-α-glycosides or pNP-β-l-arabinofuranoside. Thus, the enzyme was actually a β-glycosidase. The β-glycosidase exhibited transglycosylation activity
with pNP-β-d-galactopyranoside, pNP-β-d-glucopyranoside, and pNP-β-d-fucopyranoside in decreasing order of activity, in the reverse order of its hydrolytic activity. The hydrolytic activity
was higher toward cellobiose than toward lactose, but the transglycosylation activity was lower with cellobiose than with
lactose. 相似文献
17.
Yan Sun X. -H. Jin X. -Y. Dong K. Yu X. Z. Zhou 《Applied biochemistry and biotechnology》1996,56(3):331-339
A polymerized liposome (PLS) was prepared using a synthesized phosphatidylethanolamine with a diacetylene moiety that showed
a reversibly precipitable property on addition and removal of salt. To prepare a soluble-insoluble immobilized enzyme, chymotrypsin
was covalently immobilized on the outer surface of the PLS. The carbodiimide method was employed for the enzyme immobilization.
Coupling was rapid and nearly complete at a weight ratio of enzyme to the PLS of < 0.12. The immobilized enzyme showed favorable
activity yields for both low-and high-mol-wt substrates, i.e., 90 ±9% forN-benzoyl-L-tyrosine ethyl ester and 59 ±5% for casein up to an enzyme coupling density of 0.38 g/g-PLS. The immobilized enzyme
was reusable and more stable at high temperature and long-term incubation than the native enzyme. 相似文献
18.
Li Yang Jing Shi Cuijie Chen Shaoming Wang Liande Zhu Wenlin Xie Liping Guo 《Electrophoresis》2009,30(20):3527-3533
A new method for high‐sensitive determination of glutamate was developed and evaluated based on CE by using dual‐enzyme co‐immobilized capillary microreactor combined with substrate recycling. The capillary microreactor was prepared by covalently co‐immobilizing glutamate dehydrogenase (GDH) and glutamic pyruvic transaminase (GPT) on the inner surface of a capillary and was characterized by SEM, ultraviolet‐visible spectroscopy, and fluorescence spectroscopy. The GDH‐GPT co‐immobilized capillary microreactor showed great stability and reproducibility. The apparent Km for glutamate with GDH‐GPT coupled reaction was determined to be 0.61±0.06 mM but 2.56±0.24 mM when only GDH was immobilized. Glutamate determination was based on on‐column monitoring UV absorption at 340 nm of the reaction product reduced nicotinamide adenine dinucleotide, of which peak area was directly related to the glutamate concentration. The response of the present co‐immobilized GDH‐GPT assay for glutamate is greatly enhanced over single enzyme system, and a 15.7‐fold improvement in sensitivity was obtained. The detection limit of the proposed method is 0.15 μM glutamate (S/N=3). Selectivity for glutamate is good over most of the 20 amino acids. Finally, this method was successfully applied to determine the glutamate content in rat plasma and serum samples. 相似文献
19.
Purified hydrogenase fromDesulfovibrio desulfuricans was immobilized either by entrapment or absorption onto porous neutral and charged acrylamide beads. Surface absorption and
crosslinking on the beads resulted in a high hydrogenase activity and a good immobilization coefficient compared to the enzyme
and whole cells entrapped in the same matrix. Maximum enzyme activity (citrate-phosphate buffer) was shifted to pH 6.5 upon
immobilization in contrast to 6.0 for the free enzyme and the range of 6–7 for whole cells. Both the purified enzyme and whole
cells were most active when held in neutral matrices. Immobilization improved the temperature stability (65‡C) and long term
storage (4‡C) of the hydrogenase activity of both the purified enzyme and whole cells. 相似文献
20.
This paper deals with the immobilization of alkaline phosphatase by physical entrapment within colloidal particles produced by inverse microemulsion polymerization. Functionality has been imparted to the nanoparticle surface by copolymerization of acrylamide (the main monomer),N,N-methylene-bis-acrylamide (the cross-linking agent) with eitherN-acryloyl-1,6-diaminohexane (an amine promoter) or acrylic acid (a carboxylic acid promoter). The effect of the functional comonomers on the size and zeta potential of the reactive latexes has been studied. Integrity of the immobilized enzyme has been ascertained from its catalytic activity towards hydrolysis ofp-nitrophenylphosphate. 相似文献