共查询到20条相似文献,搜索用时 31 毫秒
1.
Yu-Guang Li Hong-Shuai GaoWang-Liang Li Jian-Min Xing Hui-Zhou Liu 《Bioresource technology》2009,100(21):5092-5096
In situ cell separation and immobilization of bacterial cells for biodesulfurization were developed by using superparamagnetic Fe3O4 nanoparticles (NPs). The Fe3O4 NPs were synthesized by coprecipitation followed by modification with ammonium oleate. The surface-modified NPs were monodispersed and the particle size was about 13 nm with 50.8 emu/g saturation magnetization. After adding the magnetic fluids to the culture broth, Rhodococcus erythropolis LSSE8-1 cells were immobilized by adsorption and then separated with an externally magnetic field. The maximum amount of cell mass adsorbed was about 530 g dry cell weight/g particles to LSSE8-1 cells. Analysis showed that the nanoparticles were strongly absorbed to the surface and coated the cells. Compared to free cells, the coated cells not only had the same desulfurizing activity but could also be easily separated from fermentation broth by magnetic force. Based on the adsorption isotherms and Zeta potential analysis, it was believed that oleate-modified Fe3O4 NPs adsorbed bacterial cells mainly because of the nano-size effect and hydrophobic interaction. 相似文献
2.
Muqing Zheng Zhiguo Su Xiaoyuan Ji Guanghui Ma Ping Wang Songping Zhang 《Journal of biotechnology》2013
Efficient dynamic interactions among cofactor, enzymes and substrate molecules are of primary importance for multi-step enzymatic reactions with in situ cofactor regeneration. Here we showed for the first time that the above dynamic interactions could be significantly intensified by exerting an external alternating magnetic field on magnetic nanoparticles-supported multi-enzymatic system so that the inter-particle collisions due to Brownian motion of nanoparticles could be improved. To that end, a multienzyme system including glutamate dehydrogenase (GluDH), glucose dehydrogenase (GDH) and cofactor NAD(H) were separately immobilized on silica coated Fe3O4 magnetic nanoparticles with an average diameter of 105 nm, and the effect of magnetic field strength and frequency on the kinetics of the coupled bi-enzyme reaction was investigated. It was found that at low magnetic field frequency (25 Hz and 100 Hz), increasing magnetic field strength from 9.8 to 161.1 Gs led to only very slight increase in reaction rate of the coupled bi-enzyme reaction expressed by glucose consumption rate. At higher magnetic field of 200 Hz and 500 Hz, reaction rate increased significantly with increase of magnetic field strength. When the magnetic field frequency was kept at 500 Hz, the reaction rate increased from 3.89 μM/min to 8.11 μM/min by increasing magnetic field strength from 1.3 to 14.2 Gs. The immobilized bi-enzyme system also showed good reusability and stability in the magnetic field (500 Hz, 14.2 Gs), that about 46% of original activity could be retained after 33 repeated uses, accounting for totally 34 days continuous operation. These results demonstrated the feasibility in intensifying molecular interactions among magnetic nanoparticle-supported multienzymes by using nano-magnetic stirrer for efficient multi-step transformations. 相似文献
3.
Cofactor-dependent multi-step enzymatic reactions generally require dynamic interactions among cofactor, enzyme and substrate molecules. Maintaining such molecular interactions can be quite challenging especially when the catalysts are tethered to solid state supports for heterogeneous catalysis for either biosynthesis or biosensing. The current work examines the effects of the pattern of immobilization, which presumably impacts molecular interactions on the surface of solid supports, on the reaction kinetics of a multienzymic system including glutamate dehydrogenase, glucose dehydrogenase and cofactor NAD(H). Interestingly, particle collision due to Brownian motion of nanoparticles successfully enabled the coupled reactions involving a regeneration cycle of NAD(H) even when the enzymes and cofactor were immobilized separately onto superparamagnetic nanoparticles (124 nm). The impact of particle motion and collision was evident in that the overall reaction rate was increased by over 100% by applying a moderate alternating magnetic field (500 Hz, 17 Gs), or using additional spacers, both of which could improve the mobility of the immobilized catalysts. We further observed that integrated immobilization, which allowed the cofactor to be placed in the molecular vicinity of enzymes on the same nanoparticles, could enhance the reaction rate by 1.8 fold. These results demonstrated the feasibility in manipulating molecular interactions among immobilized catalyst components by using nanoscale fabrication for efficient multienzymic biosynthesis. 相似文献
4.
In this work, lipase from Arthrobacter sp. was immobilized by sol–gel encapsulation to improve its catalytic properties. Various silanizing agents including vinyl-trimethoxy silane, octyl-trimethoxy silane, γ-(methacryloxypropyl)-trimethoxy silane (MAPTMS) and tetraethoxysilane (TEOS) were chosen as the precursors. Among them, MAPTMS was for the first time utilized to encapsulate lipases, and the prepared enzyme by copolymerization of MAPTMS and TEOS exhibited the highest activity in both the hydrolysis of p-nitrophenyl palmitate and the asymmetric acylation of 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one. The effects of various immobilization parameters were investigated. Under the optimum conditions of MAPTMS/TEOS = 1/1 (mol/mol), water/silane molar ratio (R value) = 20 and lipase loading = 0.01 g/mL sol, the total activity of the immobilized enzyme reached up to 13.6-fold of the free form. Moreover, the encapsulated lipase exhibited higher thermal stability than the free form and retained 54% of the original activity after uses for 60 d. Enantioselectivity of enzyme was also improved with an E value of 150 after encapsulation from 85 for the free form. 相似文献
5.
In this study, we synthesized magnetic nanoparticles (MNPs) by co-precipitation method. After that, silica coating with tetraethyl orthosilicate (TEOS) (SMNPs), amine functionalization of silica coated MNPs (ASMNPs) by using 3-aminopropyltriethoxysilane (APTES) were performed, respectively. After activation with glutaraldehyde (GA) of ASMNPs, human carbonic anhydrase (hCA I) was immobilized on ASMNPs. The characterization of nanoparticles was performed by transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD) and vibrating sample magnetometer (VSM). The immobilization conditions such as GA concentration, activation time of support with GA, enzyme amount, enzyme immobilization time were optimized. In addition of that, optimum conditions for activity, kinetic parameters (Km, Vmax, kcat, kcat/Km), thermal stability, storage stability and reusability of immobilized enzyme were determined.The immobilized enzyme activity was optimum at pH 8.0 and 25 °C. The Km value of the immobilized enzyme (1.02 mM) was higher than the free hCA I (0.48 mM). After 40 days incubation at 4 °C and 25 °C, the immobilized hCA I sustained 89% and 85% of its activity, respectively. Also, it sustained 61% of its initial activity after 13 cycles. Such results revealed good potential of immobilized enzyme for various applications. 相似文献
6.
Immobilization of yeast alcohol dehydrogenase on magnetic nanoparticles for improving its stability 总被引:1,自引:0,他引:1
Yeast alcohol dehydrogenase (YADH) was immobilized covalently on Fe3O4 magnetic nanoparticles (10.6 nm) via carbodiimide activation. The immobilization process did not affect the size and structure of magnetic nanoparticles. The YADH-immobilized magnetic nanoparticles were superparamagnetic with a saturation magnetization of 61 emu g–1, only slightly lower than that of the naked ones (63 emu g–1). Compared to the free enzyme, the immobilized YADH retained 62% activity and showed a 10-fold increased stability and a 2.7-fold increased activity at pH 5. For the reduction of 2-butanone by immobilized YADH, the activation energies within 25–45 °C, the maximum specific activity, and the Michaelis constants for NADH and 2-butanone were 27 J mol–1, 0.23 mol min–1 mg–1, 0.62 mM, and 0.43 M, respectively. These results indicated a structural change of YADH with a decrease in affinity for NADH and 2-butanone after immobilization compared to the free enzyme. 相似文献
7.
Elham Afzali Touba Eslaminejad Sahar Amirpour-Rostami 《Biocatalysis and Biotransformation》2019,37(3):201-209
The main aim of this study was to synthesize the superparamagnetic nanoparticles coated by alginate/chitosan/β-cyclodextrin to purify α-amylase. Isolated bacteria were identified by morphological, biochemical and taxonomic molecular studies. FTIR- spectrometer, VSM, X-ray instruments and Malvern Zetasizer were used to characterize nanoparticles characteristics. The morphological structures and the elemental composition of the nanoparticles were studied by using FESEM and EDS, respectively. The molecular weight of enzyme was determined using SDS-PAGE, and the enzyme activity detected by zymographic analysis. FTIR studies showed the presence of Fe–O–Fe in the Fe3O4 and verified the interaction between chitosan, β-cyclodextrin and alginate. The saturation magnetization for superparamagnetic and coated superparamagnetic nanoparticles was indicated 39 and 1.9?emu?g?1, respectively. The maximum intensity of the XRD peak indicated the presence of the Fe3O4. FESEM and EDS analysis showed that the nanoparticles were regular and spherical in shape and corresponded to the Fe and O elements. Enzyme purification by synthesized nanoparticles was achieved 13.84?U?mg?1; purification fold of 3.50. The molecular weight of α-amylase was about 22?kDa. The highest activity of α-amylase was observed at 70?°C, pH 9.3 and Ca2+-independent. As a conclusion, the coated superparamagnetic nanoparticles showed more applications in enzyme purification comparing to the conventional methods. 相似文献
8.
Homodimeric thymidine phosphorylase from Escherichia coli (TP, E.C. 2.4.2.4) was immobilized on solid support with the aim to have a stable and recyclable biocatalyst for nucleoside synthesis. Immobilization by ionic adsorption on amine-functionalized agarose and Sepabeads® resulted in a very high activity recovery (>85%). To prevent undesirable leakage of immobilized enzyme away from the support, the ionic preparations were cross-linked with aldehyde dextran (MW 20 kDa) and the influence of the dextran oxidation degree on the resulting biocatalyst activity was evaluated. Although in all cases the percentage of expressed activity after immobilization drastically decreased (≤25%), this procedure allowed to obtain an active catalyst which resulted up to 6-fold and 3-fold more stable than the soluble (non immobilized) enzyme and the just adsorbed (non cross-linked) counterpart, respectively, at pH 10 and 37 °C. No release of the enzyme from the support could be observed. Covalent immobilization on aldehyde or epoxy supports was generally detrimental for enzyme activity. Optimal TP preparation, achieved by immobilization onto Sepabeads® coated with polyethyleneimine and cross-linked, was successfully used for the one-pot synthesis of 5-fluoro-2′-deoxyuridine starting from 2′-deoxyuridine or thymidine (20 mM) and 5-fluorouracil (10 mM). In both cases, the reaction proceeded at the same rate (3 μmol min−1) affording 62% conversion in 1 h. 相似文献
9.
Husain Q Ansari SA Alam F Azam A 《International journal of biological macromolecules》2011,49(1):37-43
The present study demonstrates the immobilization of Aspergillus oryzae β galactosidase on native zinc oxide (ZnO) and zinc oxide nanoparticles (ZnO-NP) by simple adsorption mechanism. The binding of enzyme on ZnO-NP was confirmed by Fourier transform-infrared spectroscopy and atomic force microscopy. Native ZnO and ZnO-NP showed 60% and 85% immobilization yield, respectively. Soluble and immobilized enzyme preparations exhibited similar pH-optima at pH 4.5. ZnO-NP bound β galactosidase retained 73% activity at pH 7.0 while soluble and ZnO adsorbed enzyme lost 68% and 53% activity under similar experimental conditions, respectively. There was a marked broadening in temperature-activity profile for ZnO-NP adsorbed β galactosidase; it showed no difference in temperature-optima between 50 °C and 60 °C. Moreover, ZnO-NP adsorbed β galactosidase retained 53% activity after 1 h incubation with 5% galactose while the native ZnO- and soluble β galactosidase exhibited 35% and 28% activity under similar exposure, respectively. Native ZnO and ZnO-NP adsorbed β galactosidase retained 61% and 75% of the initial activity after seventh repeated use, respectively. It was noticed that 54%, 63% and 71% milk lactose was hydrolyzed by soluble, ZnO adsorbed and ZnO-NP adsorbed β galactosidase in batch process after 9 h while whey lactose was hydrolyzed to 61%, 68% and 81% under similar experimental conditions, respectively. In view of its easy production, improved stability against various denaturants and excellent reusability, ZnO-NP bound β galactosidase may find its applications in constructing enzyme-based analytical devices for clinical, environmental and food technology. 相似文献
10.
Qing Dong 《Carbohydrate research》2010,345(11):1622-1626
Uridine diphosphate glucose (UDP-Glc) serves as a glucosyl donor in many enzymatic glycosylation processes. This paper describes a multiple enzyme, one-pot, biocatalytic system for the synthesis of UDP-Glc from low cost raw materials: maltodextrin and uridine triphosphate. Three enzymes needed for the synthesis of UDP-Glc (maltodextrin phosphorylase, glucose-1-phosphate thymidylytransferase, and pyrophosphatase) were expressed in Escherichia coli and then immobilized individually on amino-functionalized magnetic nanoparticles. The conditions for biocatalysis were optimized and the immobilized multiple-enzyme biocatalyst could be easily recovered and reused up to five times in repeated syntheses of UDP-Glc. After a simple purification, approximately 630 mg of crystallized UDP-Glc was obtained from 1 l of reaction mixture, for a moderate yield of around 50% (UTP conversion) at very low cost. 相似文献
11.
Chia-Hung KuoYung-Chuan Liu Chieh-Ming J. ChangJiann-Hwa Chen Cheng ChangChwen-Jen Shieh 《Carbohydrate polymers》2012,87(4):2538-2545
Magnetic Fe3O4-chitosan nanoparticles are prepared by the coagulation of an aqueous solution of chitosan with Fe3O4 nanoparticles. The characterization of Fe3O4-chitosan is analyzed by FTIR, FESEM, and SQUID magnetometry. The Fe3O4-chitosan nanoparticles are used for the covalent immobilization of lipase from Candida rugosa using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling agents. The response surface methodology (RSM) was employed to search the optimal immobilization conditions and understand the significance of the factors affecting the immobilized lipase activity. Based on the ridge max analysis, the optimum immobilization conditions were immobilization time 2.14 h, pH 6.37, and enzyme/support ratio 0.73 (w/w); the highest activity obtained was 20 U/g Fe3O4-chitosan. After twenty repeated uses, the immobilized lipase retains over 83% of its original activity. The immobilized lipase shows better operational stability, including wider thermal and pH ranges, and remains stable after 13 days of storage at 25 °C. 相似文献
12.
In the present study, iron oxide magnetite nanoparticles, prepared through a co-precipitation method, were coated with phosphonic acid or iminodicarboxylic acid derivatives of calix[4]arene to modulate their surfaces with different acidic groups. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through sol–gel encapsulation. The catalytic activities and enantioselectivities of the two encapsulated lipases in the hydrolysis reaction of (R/S)-naproxen methyl ester and (R/S)-2-phenoxypropionic acid methyl ester were assessed. The results showed that the activity and enantioselectivity of the lipase were improved when the lipase was encapsulated in the presence of calixarene-based additives; the encapsulated lipase with the phosphonic acid derivative of calix[4]arene had an excellent rate of enantioselectivity against the (R/S)-naproxen methyl and (R/S)-2-phenoxypropionic acid methyl esters, with E = 350 and 246, respectively, compared to the free enzyme. The encapsulated lipases (Fe-Calix-N(COOH)) and (Fe-Calix–P) showed good loading ability and little loss of enzyme activity, and the stability of the catalyst was very good; they only lost 6–11% of the enzyme’s activity after five batches. 相似文献
13.
Ok-Jae Sohn Chun-Kwang Kim Jong Il Rhee 《Biotechnology and Bioprocess Engineering》2008,13(6):716-723
Glucose oxidase (GOD) and lactate dehydrogenase (LDH) were immobilized onto magnetic nanoparticles, viz. Fe3O4, via carbodiimide and glutaraldehyde. The immobilization efficiency was largely dependent upon the immobilization time and
concentration of glutaraldehyde. The magnetic nanoparticles had a mean diameter of 9.3 nm and were superparamagnetic. The
immobilization of GOD and LDH on the nanoparticles slightly decreased their saturation magnetization. However, the FT-IR spectra
showed that GOD and LDH were immobilized onto the nanoparticles by different binding mechanisms, the reason for which was
not well explained. The optimum pH values of the immobilized GOD and LDH were changed to 8 and 10, respectively. The free
and immobilized enzyme kinetic parameters (Km and Vmax) were determined by Michaelis-Menten enzyme kinetics. The Km values for free and immobilized GOD were 0.168 and 0.324 mM,
respectively, while those for free and immobilized LDH were 0.19 and 0.163 mM for NAD, and 2.976 and 4.785 mM for lactate,
respectively. High operational stability was observed, with more than 80% of the initial enzyme activity being retained for
the immobilized GOD up to 12 h and for the immobilized LDH up to 24 h. The immobilized GOD was applied to a sequential injection
analysis system for the application of bioprocess monitoring. 相似文献
14.
Immobilization of lipases on alkyl silane modified magnetic nanoparticles: effect of alkyl chain length on enzyme activity 总被引:1,自引:0,他引:1
Background
Biocatalytic processes often require a full recycling of biocatalysts to optimize economic benefits and minimize waste disposal. Immobilization of biocatalysts onto particulate carriers has been widely explored as an option to meet these requirements. However, surface properties often affect the amount of biocatalysts immobilized, their bioactivity and stability, hampering their wide applications. The aim of this work is to explore how immobilization of lipases onto magnetite nanoparticles affects their biocatalytic performance under carefully controlled surface modification.Methodology/Principal Findings
Magnetite nanoparticles, prepared through a co-precipitation method, were coated with alkyl silanes of different alkyl chain lengths to modulate their surface hydrophobicity. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through hydrophobic interaction. Enzyme activity was assessed by catalytic hydrolysis of p-nitrophenyl acetate. The activity of immobilized lipases was found to increase with increasing chain length of the alkyl silane. Furthermore, the catalytic activities of lipases immobilized on trimethoxyl octadecyl silane (C18) modified Fe3O4 were a factor of 2 or more than the values reported from other surface immobilized systems. After 7 recycles, the activities of the lipases immobilized on C18 modified nanoparticles retained 65%, indicating significant enhancement of stability as well through hydrophobic interaction. Lipase immobilized magnetic nanoparticles facilitated easy separation and recycling with high activity retaining.Conclusions/Significance
The activity of immobilized lipases increased with increasing alkyl chain length of the alkyl trimethoxy silanes used in the surface modification of magnetite nanoparticles. Lipase stability was also improved through hydrophobic interaction. Alkyl silane modified magnetite nanoparticles are thus highly attractive carriers for enzyme immobilization enabling efficient enzyme recovery and recycling. 相似文献15.
Lin Xu Hao-ra Su Gui-Rong Sun Yan Wang Shang-jing Guo Xiao-ru Zhang Shu-sheng Zhang Shi-chao Xing 《Journal of biotechnology》2013
In the present study, a gold nanoparticle-modified gold electrode (nanogold electrode) was used to develop a novel fluorescein electrochemical DNA biosensor based on a target-induced conformational change. The nanogold electrode was obtained by electrodepositing gold nanoparticles onto a bare gold electrode. This modification not only immobilized probe oligonucleotides, but also adsorbed fluorescein onto the surface of the gold nanoparticles to form an “arch-like” structure. This article compares the electrochemical signal changes caused by the hybridization of “arch-like” DNA on nanogold electrode and linear DNA on bare gold electrode. The results showed that the adsorption effect of nanogold can enhance the sensitivity of the sensor. The linear range of target ssDNA is from 2.0 × 10−9 M to 2.0 × 10−8 M with a correlation coefficient of 0.9956 and detection limit (3σ) of 7.10 × 10−10 M. Additionally, the specificity and hybridization response of this simple sensor were investigated. 相似文献
16.
Biodesulfurization of Dibenzothiophene by Microbial Cells Coated with Magnetite Nanoparticles 总被引:4,自引:2,他引:2
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Microbial cells of Pseudomonas delafieldii were coated with magnetic Fe3O4 nanoparticles and then immobilized by external application of a magnetic field. Magnetic Fe3O4 nanoparticles were synthesized by a coprecipitation method followed by modification with ammonium oleate. The surface-modified Fe3O4 nanoparticles were monodispersed in an aqueous solution and did not precipitate in over 18 months. Using transmission electron microscopy (TEM), the average size of the magnetic particles was found to be in the range from 10 to 15 nm. TEM cross section analysis of the cells showed further that the Fe3O4 nanoparticles were for the most part strongly absorbed by the surfaces of the cells and coated the cells. The coated cells had distinct superparamagnetic properties. The magnetization (δs) was 8.39 emu · g−1. The coated cells not only had the same desulfurizing activity as free cells but could also be reused more than five times. Compared to cells immobilized on Celite, the cells coated with Fe3O4 nanoparticles had greater desulfurizing activity and operational stability. 相似文献
17.
Rudakov YO Shumyantseva VV Bulko TV Suprun EV Kuznetsova GP Samenkova NF Archakov AI 《Journal of inorganic biochemistry》2008,102(11):2020-2025
Stoichiometry of the electrocatalytical cycle of cytochrome P450 2B4 was studied in kinetic mode according to bielectrode scheme. Graphite screen-printed electrodes with immobilized cytochrome P450 2B4 were used as the operating electrode (at the potential E0′ = −450 mV) and electrodes, modified with cytochrome c (E0′ = −50 mV) or Prussian Blue (E0′ = 0), as measuring electrodes (for H2O2) and Clark-type electrode (for O2). Benzphetamine N-demethylation rate was 17 ± 3 nmol/nmol of enzyme/min, peroxide production was 4.8 ± 0.7 nmol/nmol of enzyme/min (substrate-free system), 3.3 ± 0.6 nmol/nmol of enzyme/min (0.5 mM benzphetamine), the oxygen consumption rate by Р450 2В4 was 19.4 ± 0.6 nmol/nmol of enzyme/min (in the presence of benzphetamine), 4.8 ± 0.4 nmol/nmol of enzyme/min (without substrate). Based on stoichiometry of P450 electrocatalysis adequacy of electrochemical reduction and P450-monooxygenase system was revealed. 相似文献
18.
The catalytic activity of gold nanoparticles (AuNPs) on a luminol–H2O2 chemiluminescence (CL) system is found to be greatly enhanced after its crosslinking aggregation induced by immunoreaction. Based on this observation, a one-step homogeneous non-stripping CL metalloimmunoassay was designed. In the presence of corresponding antigen (Ag), the immunoreaction caused the aggregation of antibody (Ab)-modified AuNPs, and these crosslinking aggregated AuNPs could catalyze luminol–H2O2 CL reaction to produce a much stronger CL signal than dispersed Ab-modified AuNPs. The assay, including immunoreaction and detection, can be accomplished in homogeneous solution. In the assay, no tedious and strict stripping of metal nanoparticles, difficult synthesis of labels, multiple steps of immunoreactions and washings, and complicated magnetic separation process were required. The detection limit of human immunoglobulin G (IgG, 3σ) was estimated to be as low as 3.2 × 10−11 g ml−1. The sensitivity was increased by two orders of magnitude over that of other AuNP-based CL immunoassay. The current CL metalloimmunoassay offers the advantages of being simple, cheap, rapid, and sensitive. 相似文献
19.
In the present work nanoparticles (NPs) of pepsin were generated in an aqueous solution using high-intensity ultrasound, and were subsequently immobilized on low-density polyethylene (PE) films, or on polycarbonate (PC) plates, or on microscope glass slides. The pepsin NPs coated on the solid surfaces have been characterized by HRSEM, TEM, FTIR, XPS and DLS. The amount of enzyme introduced on the substrates, the leaching properties, and the catalytic activity of the immobilized enzyme on the three surfaces are compared. Catalytic activities of pepsin deposited onto the three solid surfaces as well as free pepsin, without sonication, and free pepsin NPs were compared at various pH levels and temperatures using a hemoglobin assay. Compared to native pepsin, pepsin coated onto PE showed the best catalytic activity in all the examined parameters. Pepsin immobilized on glass exhibited better activity than the native enzyme, especially at high temperatures. Enzyme activity of pepsin immobilized on PC was no better than native enzyme activity at all temperatures at pH 2, and only over a narrow pH range at 37 °C was the activity improved over the native enzyme. A remarkable observation is that immobilized pepsin on all the surfaces was still active to some extent even at pH 7, while free pepsin was completely inactive. The kinetic parameters, Km and Vmax were also calculated and compared for all the samples. Relative to the free enzyme, pepsin coated PE showed the greatest improvement in kinetic parameters (Km = 15 g/L, Vmax = 719 U/mg versus Km = 12.6 g/L and Vmax = 787 U/mg, respectively), whereas pepsin coated on PC exhibited the most unfavorable kinetic parameters (Km = 18 g/L, Vmax = 685 U/mg). The values for the anchored enzyme-glass were Km = 19 g/L, Vmax = 763 U/mg. 相似文献
20.
Direct electrochemical transfer of electrons to the enzyme provides an excellent method of driving the catalytic reactions of cytochrome P450 enzymes that form a superfamily of vital heme enzymes involved in biological monooxygenation reactions. Covalent attachment of N-(1-pyrenyl) maleimide (pyrene maleimide) to the bacterial cytochrome P450, CYP101 has been carried out and the conjugated enzyme was shown to be specifically immobilized onto the glassy carbon electrode through the pyrene group. The electrode immobilized pyrene-conjugated enzyme showed quasi-reversible electrochemistry with a midpoint potential at −330 ± 10 mV versus Ag/AgCl. The unconjugated enzyme that did not have specific linkage with the pyrene maleimide was non-specifically adsorbed on the electrode surface and the electrochemical response was much weaker than that observed in case of the conjugated enzyme, though the midpoint potential was almost unchanged. The pyrene maleimide bound CYP101 was found to have surface coverage of 1.35 ± 0.3 × 10−10 mol/cm2 and the heterogeneous rate of electron transfer was found to be 0.21 ± 0.02 s−1, which is larger than that for the unconjugated enzyme. The pyrene maleimide linked immobilized enzyme was oriented to the electrode so that efficient electron transfer takes place from the electrode to the immobilized enzyme. The oxygenase activity of the immobilized conjugated enzyme was assayed from the enhancement of catalytic current in presence of oxygen and the natural substrate camphor. Mass spectrometric studies also showed enhanced formation of hydroxycamphor by electrochemically driven catalysis in the pyrene maleimide linked immobilized CYP101. 相似文献