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1.
Arginase isolated from beef liver was covalently attached to a polyacrylamide bead support bearing carboxylic groups activated
by a water-soluble carbodiimide. The most favorable carbodiimide wasN-cyclohexyl-Nt’-(methyl-2-p-nitrophenyl-2-oxoethyl) aminopropyl carbodiimide methyl bromide, but for practical purposes,N-cyclohexyl-Nt’-morpholinoethyl carbodiimide methyl tosylate was used. The optimal conditions for the coupling procedure were
determined. The catalytic activity of the immobilized arginase was 290–340 U/g solid or 2.9–3.4 U/mL wet gel. The pH optimum
for the catalytic activity was pH 9.5, the apparent temperature maximum was at 60°C and Kmapp was calculated to be 0.37M L-arginine. Immobilization markedly improved the conformational stability of arginase. At 60°C,
the pH for maximal stability was found to be 8.0. The immobilized arginase was used for the production of L-ornithine and
D-arginine. 相似文献
2.
N. N. Ugarova 《Moscow University Chemistry Bulletin》2010,65(3):139-143
The results of Luciola mingrelica firefly luciferase stabilization by genetic engineering methods are reviewed. The Cys62, Cys146, and Cys164 to Ser mutant
enzymes with an enhanced thermostability and lower sensitivity to dithiothreitol were obtained by site-directed mutagenesis.
The double mutant G216N, A217L was obtained, which displayed a higher thermostability and resistance to DMSO in comparison
with WT luciferase. Random mutagenesis of the gene region encoding residues 1–225 and subsequent screening of the mutants
resulted in the production of the mutant MT8 with a higher thermostability, as well as mutants MT3 and MT4 with higher resistance
to dimethyl sulfoxide. The mutant 4TS was obtained by the method of directed evolution of the gene site encoding residues
130–390, which was shown to contain eight replacements after four cycles of mutagenesis and had two-fold higher specific activity,
eight-fold lower K
m
value for ATP, and stability at 42°C, which was 65-fold higher that of WT luciferase. The stabilization mechanism of this
mutant is discussed. 相似文献
3.
4.
The synthetic IgG‐binding domain (Z domain) of staphylococcal protein A catalyzes the oxidation of coelenterazine to emit light like a coelenterazine‐utilizing luciferase. The Z domain derivatives (ZZ‐gCys, Z‐gCys and Z‐domain) were purified and the luminescence properties were characterized by comparing with coelenterazine‐utilizing luciferases, including Renilla luciferase, Gaussia luciferase and the catalytic 19 kDa protein of Oplophorus luciferase. Three Z domain derivatives showed luminescence activity with coelenterazine and the order of the initial maximum intensity of luminescence was ZZ‐gCys (100%) > Z‐gCys (36.8%) > Z‐domain (1.1%) > bovine serum albumin (BSA; 0.9%) > staphylococcal protein A (0.1%) and the background value of coelenterazine (0.1%) in our conditions. The luminescence properties of ZZ‐gCys showed the similarity to that of Gaussia luciferase, including the luminescence pattern, the emission spectrum, the stimulation by halogen ions and nonionic detergents and the substrate specificity for coelenterazine analogues. In contrast, the luminescence properties of Z‐gCys were close to the catalytic 19 kDa protein of Oplophorus luciferase. The catalytic region of the Z domain for the luminescence reaction might be different from the IgG‐binding region of the Z domain. 相似文献
5.
M. I. Koksharov D. V. Smirnova S. G. Abbasova N. N. Ugarova 《Moscow University Chemistry Bulletin》2011,66(4):241-246
A plasmid encoding a fusion protein (4TS-bccp87) composed of a thermostable mutant of the Luciola mingrelica firefly luciferase (4TS) and 87 carboxy-terminal amino acid residues of the biotin-binding domain (bccp87) from E. coli was constructed using genetic-engineering techniques. It was established that fusion-protein expression in BL21(DE3) E. coli resulted in 60% of the biotinylated form. The catalytic properties, thermostability, and bioluminescence spectra of the fusion
protein were shown to be similar to that of the initial luciferase. The possibility of using the streptavidin-biotinylated
luciferase complex for defining the Salmonella typhimurium cell concentration in the range from 104 to 5 × 106 CFU/ml by enzyme immunoassay was shown. 相似文献
6.
Li Gaoxiang Huang Jiayu Kou Xiufen Zhang Shuzheng 《Applied biochemistry and biotechnology》1982,7(5):325-341
Glucoamylase (EC 3.2.1.3) was immobilized to alkylamine porous glass with glutaraldehyde. The choice and pretreatment of carrier
and conditions for immobilization have been investigated. The immobilized enzyme contained about 4.0–8.0% protein and its
activity was about 1000–1700 U/g. Some characteristics of the immobilized enzyme and the native enzyme have been comparatively
investigated. The optimum temperature and the pH stability of the preparation were almost identical to the native one. However,
the optimum pH of bound glucoamylase shifted 1.3 pH units toward the alkaline side compared to the native one. The Michaelis
constant(K
m
) of bound glucoamylase for soluble starch was about four times higher than that of the native enzyme, whileK
m
values for maltose approached those of the native material. At 45‡C the half-life of IMG was 104 days under operational conditions.
Alkaline protease, α-amylase, asparaginase, and penicillin acylase were also chemically coupled to porous glass by the same
method and high relative activities were obtained. 相似文献
7.
Elena A. Markvicheva Svetlana V. Kuptsova Tatyana Yu. Mareeva Alexander A. Vikhrov Tamara N. Dugina Svetlana M. Strukova Yury N. Belokon Konstantin A. Kochetkov Ekaterine N. Baranova Vitali P. Zubov Denis Poncelet Virinder S. Parmar Rajesh Kumar Lev D. Rumsh 《Applied biochemistry and biotechnology》2000,88(1-3):145-157
A one-step mild method for entrapping animal cells and enzymes in macroporous composite poly (N-vinyl caprolactam)-calcium alginate (PVCL-CaAlg) hydrogels is described. Some properties of immobilized enzymes, such as
thermal and storage stabilities and stability in water/organic media were investigated. Composite PVCL-CaAlg gels were successfully
applied to immobilize a number of proteases, namely, trypsin, α-chymotrypsin, carboxypeptidase B, and thrombin. Thermal stability
of the immobilized preparations obtained by entrapment in hydrogel beads allowed us to use them at 65–80†C, while the native
enzymes were completely inactivated at 50–55°C. Various applications of enzymes and cells immobilized in beads weredemonstrated.
Immobilized trypsin and carboxypeptidase B were applied to prepare human insulin from recombinant proinsulin. The hydrogel
beads with entrapped α-chymotrypsin were used in enantioselective hydrolysis of Shiff's base of D,L-phenylalanine ethyl ester
(SBPH) in acetonitrile/water medium. Thrombin immobilized in PVCL-based hydrogel films was shown to be a promising compound
for wound treatment. To prepare pure preparations of monoclonal antibodies (MAb) several hybridoma cell lines, including hybridoma
cell lines producing MAb to interleukin-2, were successfully cultivated in the hydrogel beads. 相似文献
8.
Lin Ma Xu-Ting Wang De-Lin You Shuang Tang Zhong-Li Huang Yu-Hua Cheng 《Applied biochemistry and biotechnology》1996,56(3):223-233
In this article, the immobilization of prostaglandin synthetase onn-alkyl or aryl amino-agar beads by hydrophobic adsorption is reported. The effects of different hydrophobic groups in the
agar beads, pH of buffer, concentration of salts on the adsorption of prostaglandin synthetase, and the properties of immobilized
prostaglandin synthetase were also studied. The results showed that 20–35 mg of microsome containing PG synthetase (protein
content 8–15 mg) could be adsorbed on each gram ofn-dodecylamino-agar beads after suction drying the gel in the buffer of pH 5.5 (containing 0.5 mol/L KC1), 0.1 mol/L citric-phosphate
at 4‡C. The remaining immobilized enzyme activity was over 80%. The optimum pH of immobilized PG synthetase is 8.0, similar
to that of the native enzymes. The thermostability of immobilized PG synthetase in the buffer containing 0.5 mol/L KC1 was
increased. Immobilized PG synthetase was used as a catalyst of synthesis of prostaglandin E1. The preservation of activity after 10 working cycles was 86.2%. 相似文献
9.
Konsoula Z Liakopoulou-Kyriakides M Perysinakis A Chira P Afendra A Drainas C Kyriakidis DA 《Applied biochemistry and biotechnology》2008,149(2):99-108
A hyperthermophilic α-amylase encoding gene from Pyrococcus woesei was transferred and expressed in Xanthomonas campestris ATCC 13951. The heterologous α-amylase activity was detected in the intracellular fraction of X. campestris and presented similar thermostability and catalytic properties with the native P. woesei enzyme. The recombinant α-amylase was found to be stable at 90 °C for 4 h and within the same period it retained more than
50% of its initial activity at 110 °C. Furthermore, X. campestris transformants produced similar levels of recombinant α-amylase activity regardless of the carbon source present in the growth
medium, whereas the native X. campestris α-amylase production was highly dependent on starch availability and it was suppressed in the presence of glucose or other
reducing sugars. On the other hand, xanthan gum yield, which appeared to be similar for both wild type and recombinant X. campestris strains, was enhanced at higher starch or glucose concentrations. Evidence presented in this study supports that X. campestris is a promising cell factory for the co-production of recombinant hyperthermophilic α-amylase and xanthan gum. 相似文献
10.
Phenylalanine dehydrogenase (l-PheDH) from Sporosarcina ureae was immobilized on DEAE-cellulose, modified initially with 2-amino-4,6-dichloro-s-triazine followed by hexamethylenediamine
and glutaraldehyde. The highest activity of immobilized PheDH was determined as 95.75 U/g support with 56% retained activity.
The optimum pH value of immobilized l-PheDH was shifted from pH 10.4 to 11.0. The immobilized l-PheDH showed activity variations close to the maximum value in a wider temperature range of 45–55 °C, whereas it was 40 °C
for the native enzyme. The pH and the thermal stability of the immobilized l-PheDH were also better than the native enzyme. At pH 10.4 and 25 °C, K
m values of the native and the immobilized l-PheDH were determined as K
m Phe = 0.118, 0.063 mM and K
m NAD+ = 0.234, 0.128 mM, respectively. Formed NADH at the exit of packed bed reactor column was detected by the flow-injection
analysis system. The conversion efficiency of the reactor was found to be 100% in the range of 5–600 μM Phe at 9 mM NAD+ with a total flow rate of 0.1 mL/min. The reactor was used for the analyses of 30 samples each for 3 h per day. The half-life
period of the reactor was 15 days. 相似文献
11.
Cunha AG Fernández-Lorente G Bevilaqua JV Destain J Paiva LM Freire DM Fernández-Lafuente R Guisán JM 《Applied biochemistry and biotechnology》2008,146(1-3):49-56
Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, predictable decay rates
and improved economics. This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase (YLL) by physical adsorption and covalent attachment. The enzyme was adsorbed on octyl–agarose and octadecyl–sepabeads
supports by hydrophobic adsorption at low ionic strength and on MANAE–agarose support by ionic adsorption. CNBr–agarose was
used as support for the covalent attachment immobilization. Immobilization yields of 71, 90 and 97% were obtained when Y. lipolytica lipase was immobilized into octyl–agarose, octadecyl–sepabeads and MANAE–agarose, respectively. However, the activity retention
was lower (34% for octyl–agarose, 50% for octadecyl–sepabeads and 61% for MANAE–agarose), indicating that the immobilized
lipase lost activity during immobilization procedures. Furthermore, immobilization by covalent attachment led to complete
enzyme inactivation. Thermal deactivation was studied at a temperature range from 25 to 45°C and pH varying from 5.0 to 9.0
and revealed that the hydrophobic adsorption on octadecyl–sepabeads produced an appreciable stabilization of the biocatalyst.
The octadecyl–sepabeads biocatalyst was almost tenfold more stable than free lipase, and its thermal deactivation profile
was also modified. On the other hand, the Y. lipolytica lipase immobilized on octyl–agarose and MANAE–agarose supports presented low stability, even less than the free enzyme. 相似文献
12.
Candida rugosa lipase was encapsulated within a chemically inert sol–gel support prepared by polycondensation with tetraethoxysilane and
octyltriethoxysilane in the presence of β-cyclodextrin-based polymer. The catalytic activity of the encapsulated lipases was
evaluated both in the hydrolysis of p-nitrophenylpalmitate and the enantioselective hydrolysis of racemic Naproxen methyl ester. It has been observed that the
percent activity yield of the encapsulated lipase was 65 U/g, which is 7.5 times higher than that of the covalently immobilized
lipase. The β-cyclodextrin-based encapsulated lipases had higher conversion and enantioselectivity compared with covalently
immobilized lipase. The study confirms an excellent enantioselectivity (E >300) for the encapsulated lipase with an enantiomeric excess value of 98% for S-naproxen. 相似文献
13.
A Novel Streptavidin–luciferase Fusion Protein: Preparation,Properties and Application in Hybridization Analysis of DNA 下载免费PDF全文
Daria V. Smirnova Maya Y. Rubtsova Vitaly G. Grigorenko Natalia N. Ugarova 《Photochemistry and photobiology》2017,93(2):541-547
A streptavidin–luciferase fusion protein comprising the thermostable mutant form of firefly luciferase Luciola mingrelica and minimal core streptavidin was constructed. The streptavidin–luciferase fusion was mainly produced in a tetrameric form with high luciferase and biotin‐binding activities. It was shown that fusion has the same Km values for ATP and luciferin and the bioluminescence spectra as initial luciferase. The linear dependence of the bioluminescence signal on the content of the fusion was observed within the range of 10?18–10?13 mol per well. Successful application of obtained fusion in a biospecific bioluminescence assay based on biotin–streptavidin interactions was demonstrated by the example of a specific DNA hybridization analysis. A DNA hybridization analysis for Escherichia coli cells identification was developed using unique for these cells gadB fragment encoding glutamate decarboxylase. The amplified biotinylated GadB fragments were hybridized with the immobilized oligonucleotide probes; then, the biotin in the DNA duplexes was detected using the streptavidin–luciferase fusion protein. To reach the high sensitivity of the assay, we optimized the conditions of the assay. It was shown that the use of Pluronic for plate modification resulted in a significant reduction in the DNA detection limit which finally was 0.4 ng per well. 相似文献
14.
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17.
Gaffney Alexea M. Markov Sergei A. Gunasekaran M. 《Applied biochemistry and biotechnology》2001,91(1-9):185-193
The effectiveness of photosynthetic free-living and polyurethane foam (PU) immobilized Anabaena variabilis cells for, removal of orthophosphate (P) from water in batch cultures and in a photobioreactor was studied. Immobilization
in PU foams was found to have a positive effect on P uptake by cyanobacteria in batch cultures. The efficiency of P uptake
by immobilized cells was higher than by free-living cells. A laboratory scale photobioreactor was constructed for removal
of P from water by the immobilized cyanobacteria. The photobioreactor was designed so that the growth medium (water) from
a reservoir was pumped through a photobioreactor column with immobilized cyanobacteria and back to the reservoir. This created
a closed system in which it was possible to measure P uptake. No leakage of cells into the photobioreactor medium reservoir
was observed during the operation. The immobilized cells incorporated into a photobioreactor column removed P continuously
for about 15 d. No measurable uptake was demonstrated after this period. Orthophosphate uptake efficiency of 88–92% was achieved
by the photobioreactor. 相似文献
18.
Enhancing enzymatic properties by the immobilization method 总被引:4,自引:0,他引:4
Effects of some immobilized carriers on enzymatic properties have been studied. The following results were obtained: (1) When
cholinesterase was immobilized on the hydrophobic carrier with either α-naphthylamine, benzylamine, orp-methylbenzylamine groups, the affinities of immobilized cholinesterase for toxic organophosphors, GB (Isopnopy 1-methylphophonofluoridate)
and Vx [o-ethyl-S-(2-diisopnoylomino-thyl) methyl phosphonothiolate], were enhanced 60–90 times and 700–1200 times, respectively,
whereas the thermal stability of the immobilized cholinesterase increased to 110%. Approximately 82–88% activity of the immobilized
cholinesterase remained after continuously operating for 8 h; and (2) Lipase was immobilized on the carrier that was made
up of 6% polyethylenimine, 1% alginate gel, and 1% glutaraldehyde. The initial reaction rate of the esterification of lauric
acid with lauric alcohol catalyzed by this kind of immobilized lipase was increased 21 times, as compared to lipase powder.
About 72% esterification activity of lipase remained after continuous operating for 10 d. 相似文献
19.
A. M. Aliev U. A. Mamedova Kh. R. Samedov A. A. Sarydzhanov R. Yu. Agaeva 《Russian Journal of Physical Chemistry A, Focus on Chemistry》2011,85(2):288-292
ZSM-5 high-silica zeolite was obtained from metakaolinite, Dzhenranchel’sk volcanic ash, and silica gel at T = 150–220°C, pH 9–13, and τ = 48–240 h with the use of an organic structure-forming additive, butanediol-1,4, in an alkaline
solution. Optimum conditions for the synthesis of ZSM-5 zeolite were found (T = 200°C, pH 10, τ = 144 h). The catalytic properties of its H-form in vapor-phase esterification of acetic acid (I) with ethanol (II) were studied at 140–180°C and a I: II molar ratio from 1 to 2. Synthesized HZSM-5 showed high activity and selectivity in this reaction. 相似文献
20.
Kirillova TN Gerasimova MA Nemtseva EV Kudryasheva NS 《Analytical and bioanalytical chemistry》2011,400(2):343-351
The paper investigates an application of luminescent bioassays to monitor the toxicity of organic halides. Effects of xanthene
dyes (fluorescein, eosin Y, and erythrosin B), used as model compounds, on bioluminescent reactions of firefly Luciola mingrelica, marine bacteria Photobacterium leiognathi, and hydroid polyp Obelia longissima were studied. Dependence of bioluminescence quenching constants on the atomic weight of halogen substituents in dye molecules
was demonstrated. Bacterial bioluminescence was shown to be most sensitive to heavy halogen atoms involved in molecular structure;
hence, it is suitable for construction of sensors to monitor toxicity of halogenated compounds. Mechanisms of bioluminescence
quenching—energy transfer processes, collisional interactions, and enzyme–dye binding—were considered. Changes of bioluminescence
(BL) spectra in the presence of the dyes were analyzed. Interactions of the dyes with enzymes were studied using fluorescence
characteristics of the dyes in steady-state and time-resolved experiments. The dependences of fluorescence anisotropy of enzyme-bound
dyes, the average fluorescence lifetime, and the number of exponential components in fluorescence decay on the atomic weight
of halogen substituents were demonstrated. The results are discussed in terms of “dark effect of heavy halogen atom” in the
process of enzyme–dye binding; hydrophobic interactions were assumed to be responsible for the effect. 相似文献