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71.
Double d-amino acid oxidases (dRtDAO and dTvDAO) were previously genetically constructed by linking the C-terminus of one subunit
of their corresponding native DAOs from Rhodosporidium toruloides and Trigonopsis variabilis (RtDAO and TvDAO) to the N-terminus of the other identical subunit. We have now immobilized these double DAOs and their native
counterparts onto streptavidin-coated magnetic beads through the interaction between biotin and streptavidin. The catalytic
efficiencies (kcat/KM) of immobilized DAOs toward d-alanine and cepharosporin C remained similar to those of their soluble forms, except the catalytic efficiency of immobilized
TvDAO toward d-alanine was decreased by 56%. After immobilization, the Tm value for RtDAO was shifted 15°C higher to 60°C, while those for dRtDAO, TvDAO and dTvDAO were increased by 5–8°C to 56,
60 and 60°C, respectively. In the presence of 10 mM H2O2, immobilized RtDAO, dRtDAO, TvDAO and dTvDAO exhibited half-lives of about 8, 10, 3 and 5 h, respectively, giving 16-, 10-,
6- and 7-fold greater stability than their soluble forms, respectively. Therefore, immobilization through biotin–streptavidin
affinity binding enhances the thermal and oxidative stability of native and double DAOs studied, especially RtDAO. The additive
stabilizing effect of subunit fusion and immobilization was more pronounced in the case of RtDAO than TvDAO. 相似文献
72.
Candida rugosa lipase (CRL) was applied in a non-solvent esterification reaction to yield twelve wax esters. All products were obtained
in nearly 100% yield for 10 h at 50°C when immobilized PEG2000-activated C. rugosa lipase was added to the reaction mixture. The surfactant had also a beneficial effect on the stability of the biocatalytic
preparation with 83% of its activity conserved after the seventh run of repeated batch reactions. 相似文献
73.
Biró E Németh AS Sisak C Feczkó T Gyenis J 《Journal of biochemical and biophysical methods》2008,70(6):1240-1246
Macro-, micro- and nanosized chitosan particles suitable as immobilization carriers were prepared by precipitation, emulsion cross-linking and ionic gelation methods, respectively. Effects of particle preparation parameters on particle size were investigated. Activities of β-galactosidase covalently attached to differently sized particles have been evaluated and compared. The highest activity was shown by the biocatalyst immobilized on nanoparticles obtained by means of the ionotropic gelation method with sodium sulphate as gelation agent. β-Galactosidase fixed on macro- and microspheres exhibited excellent storage stability in aqueous solution, with no more than 5% loss of activity after 3 weeks storage at 4 °C and pH 7.0. 相似文献
74.
Influence of phenolics on the sensitivity of free and immobilized bioluminescent Acinetobacter bacterium 总被引:1,自引:0,他引:1
Zaki S Abd-El-Haleem D Abulhamd A Elbery H Abuelreesh G 《Microbiological research》2008,163(3):277-285
In this work, the constructed bioluminescent Acinetobacter strain DF4/PUTK2 was employed to assess the toxicity of phenolic compounds and the 5 min EC50 values were calculated. The results of the DF4/PUTK2 assay were further evaluated by comparing with the results of the Vibrio fischeri luminescence inhibition assay. To develop a bioassay system appropriate to be used in continuous toxicity testing, strain DF4/PUTK2 was subjected for immobilization in microtiter plates into the matrices Ca-alginate, polyacrylamide, agar and agarose. After a choice of materials was tried, Ca-alginate was selected as the most suitable candidate material. Because, it could be stored at least 8 weeks at 4 °C, during which the ability of the bioreporter DF4/PUTK2 to detect the toxicity of phenolics was maintained. However, the stability of the bioluminescence for DF4/PUTK2 cells immobilized into agarose and agar was significantly less than that of cells stored in alginate suspensions. This study recommended that luxCDABE-marked Acinetobacter strain DF4/PUTK2 could be employed to assay the ecotoxicity of environmental samples contaminated with phenols. The host strain of the bioreporter DF4/PUTK2 is Acinetobacter strain DF4. It is known that members of the genus Acinetobacter are widespread in nature and also involved in biodegradation, leaching and removal of several organic and inorganic man-made hazardous wastes. 相似文献
75.
É. Lojou X. Luo M. Brugna N. Candoni S. Dementin M. T. Giudici-Orticoni 《Journal of biological inorganic chemistry》2008,13(7):1157-1167
We report the modification of gold and graphite electrodes with commercially available carbon nanotubes for immobilization
of Desulfovibrio fructosovorans [NiFe] hydrogenase, for hydrogen evolution or consumption. Multiwalled carbon nanotubes, single-walled carbon nanotubes (SWCNs),
and amine-modified and carboxyl-functionalized SWCNs were used and compared throughout. Two separate methods were performed:
covalent attachment of oriented hydrogenase by controlled architecture of carbon nanotubes at gold electrodes, and adsorption
of hydrogenase at carbon-nanotube-coated pyrolytic graphite electrodes. In the case of self-assembled carbon nanotubes at
gold electrodes, hydrogenase orientation based on electrostatic interaction with the electrode surface was found to control
the electrocatalytic process for H2 oxidation. In the case of carbon nanotube coatings on pyrolytic graphite electrodes, catalysis was controlled more by the
geometry of the nanotubes than by the orientation of the enzyme. Noticeably, shortened SWCNs were demonstrated to allow direct
electron transfer and generate high and quite stable current densities for H2 oxidation via adsorbed hydrogenase, despite having many carboxylic surface functions that could yield unfavorable hydrogenase
orientation for direct electron transfer. This result is attributable to the high degree of oxygenated surface functions in
addition to the length of shortened SWCNs that yields highly divided materials.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
76.
Vijayalakshmi A Tarunashree Y Baruwati B Manorama SV Narayana BL Johnson RE Rao NM 《Biosensors & bioelectronics》2008,23(11):1708-1714
Ion-selective field effect transistor (ISFET) is a robust platform to develop biosensors. A variety of methods are used including covalent attachment or polymer entrapment, to associate enzymes or antibodies to the gate surface of a FET. We have employed a novel method of retaining the enzyme molecules at the gate surface by immobilizing the enzyme on magnetic nickelferrite nanoparticles and applying a permanent magnet below the gate of the FET. We were able to estimate the triglyceride concentrations in the range of 0.1–1.5% by immobilizing a thermostable lipase on nanoparticles. Tributyrin, trioctanoate and triolein have given similar results. The reaction volume could be scaled down to 0.2 ml without a loss in slope or sensitivity. Ionic strength (>150 mM NaCl) has a strong influence on the sensitivity of the measurement. The advantages of this configuration of enzyme biosensor are reduction of mass transfer problems, increasing the amount of enzyme at the gate surface besides providing an opportunity to use a single FET device for multiple analyte detection. 相似文献
77.
A novel immobilization method based on oligonucleotide as linker has been developed for small molecule microarrays (SMMs) construction. The oligonucleotide tail was employed as a linker in solid-phase synthesis. Small molecules could be easily conjugated at the 5′ end of the oligonucleotide, previously modified with a functional group. Being a reactive species, the oligonucleotide was activated by UV irradiation, for the attachment of the conjugate to the slide surface. The method was successfully applied to structurally distinct small molecules, including biotin, antibiotic and drug. This immobilization strategy showed high efficiency, 1.1 fmol of small molecules in the spotting solution per spot gave a detectable signal (mean S/N = 10.9). The results suggest that it is very promising for exploring interaction between small molecules and proteins, and high throughput detecting the chemical compounds. 相似文献
78.
Sébastien Givry Vincent Prevot Francis Duchiron 《World journal of microbiology & biotechnology》2008,24(6):745-752
Consumption of hexoses/pentoses and production of lactic acid by Lactobacillus bifermentans were investigated in optimized culture medium and hemicellulosic hydrolyzates. The hydrolyzate used had the following composition
(expressed in gL−1): xylose 50 ± 5 gL−1; glucose 18 ± 3 gL−1; arabinose 29 ± 5 gL−1. The immobilization experiments were conducted with microbial cells entrapped in calcium alginate beads. The results indicate
that maximum concentrations of lactic acid were produced after 54 h of fermentation. All glucose and arabinose in wheat bran
hydrolyzate were consumed during fermentation. Only xylose was not completely consumed. The substrate consumption rate was
3.2 gh−1, 1.9 gh−1, 1.6 gh−1 respectively for glucose, arabinose, and xylose. The optimized culture condition gave a lactic acid concentration and metabolic
yield of 62.77 gL−1 and 0.83 gg−1. These parameters improved to 41.3 gL−1 and 0.47 gg−1 respectively, when cell free was used. 相似文献
79.
Chan-Su Rha Dae-Hee Lee Sung-Gun Kim Won-Ki Min Seong-Goo Byun Dae-Hyuk Kweon Nam Soo Han Jin-Ho Seo 《Journal of Molecular Catalysis .B, Enzymatic》2005,34(1-6):39-43
Bacillus macerans cyclodextrin glycosyltransferase (CGTase) fused with 10 lysine residues at its C-terminus (CGTK10ase) was immobilized onto a cation exchanger by ionic interaction and used to produce -cyclodextrin (CD) from soluble starch. Poly-lysine fused immobilization increased the Vm of the immobilized CGTase by 40% without a change in Km. The activation energies of thermal deactivation (Ea) were 41.4, 28.1, and 25.9 kcal mol−1, respectively, for soluble wild-type (WT) CGTase, soluble CGTK10ase, and immobilized CGTK10ase, suggesting destabilization of CGTase by poly-lysine fusion and immobilization onto a cation exchanger. Maximum -CD productivity of 539.4 g l−1 h−1 was obtained with 2% soluble starch solution which was constantly fed at a flow rate of 4.0 ml min−1 (D = 240 h−1) in a continuous operation mode of a packed-bed reactor. The operational half-life of the packed-bed enzyme reactor was estimated 12 days at 25 °C and pH 6.0. 相似文献
80.
The immobilization of papain on the mesoporous molecular sieve MCM‐48 (with a pore size of 6.2 nm in diameter) with the aid of glutaraldehyde, and the characteristics of this immobilized papain are described. The optimum conditions for immobilization were as follows: 20 mg native free enzyme/g of the MCM‐48 and 0.75 % glutaraldehyde, 2 h at 10–20 °C and pH 7.0. Under these optimum conditions for immobilization, the activity yield [%] of the immobilized enzyme was around 70 %. The influence of the pH on the activity of the immobilized enzyme was much lower compared to the free enzyme. The thermostability of the immobilized enzyme, whose half‐life was more than 2500 min, was greatly improved and was found to be significantly higher than that of the free enzyme (about 80 min). The immobilized enzyme also showed good operational stability, and the activity of the immobilized enzyme continued to maintain 76.5 % of the initial activity even after a 12‐day continuous operation. Moreover, the immobilized enzyme still exhibited good storage stability. From these results, papain immobilized on the MCM‐48 with the aid of glutaraldehyde, can be used as a high‐performance biocatalyst in biotechnological processing, in particular in industrial and medical applications. 相似文献