RESUMO
In multienzymes cascade reaction, the inter-enzyme spacing is supposed to be a factor affecting the cascade activity. Here, a simple and efficient Y-shaped DNA scaffold is assembled using two partially complementary DNA single strands on magnetic microspheres, which is used to coimmobilize glucose oxidase (GOD) and horseradish peroxidase (HRP). As a result, on poly(vinyl acetate) magnetic microspheres (PVAC), GOD/HRP-DNA@PVAC multienzyme system is obtained, which can locate GOD and HRP accurately and control the inter-enzyme distance precisely. The distance between GOD and HRP is regulated by changing the length of DNA strand. It showed that the cascade activity is significantly distance-dependent. Moreover, the inter-enzyme spacing is not the closer the better, and too short distance would generate steric hindrance between enzymes. The cascade activity reached the maximum value of 967 U mg-1 at 13.6 nm, which is 3.5 times higher than that of free enzymes. This is ascribed to the formation of substrate channeling.
Assuntos
Enzimas Imobilizadas , Glucose Oxidase , Peroxidase do Rábano Silvestre , Microesferas , DNARESUMO
Here, lipase encapsulation is constructed by locking enzyme molecules in nanomolecular cages on the surface of SH-PEI@PVAC magnetic microspheres. To improve the encapsulation efficiency in enzyme loading, the thiol group is efficiently modified on the grafted polyethyleneimine (PEI) using 3-mercaptopropionic acid. N2 adsorption-desorption isotherms reveal the existence of mesoporous molecular cages on the microsphere surface. The robust immobilizing strength of carriers to lipase demonstrates the successful encapsulation of enzymes in nanomolecular cages. The encapsulated lipase shows high enzyme loading (52.9 mg/g) and high activity (51.4 U/mg). Different sizes of molecular cages are established, and the cage size showed important effects on lipase encapsulation. It shows that enzyme loading is low at a small size of molecular cages, which is attributed to that the nanomolecular cage is too small to house lipase. The investigation in lipase conformation suggests that the encapsulated lipase retains its active conformation. Compared with the adsorbed lipase, the encapsulated lipase shows higher thermal stability (4.9 times) and higher resistance to denaturants (5.0 times). Encouragingly, the encapsulated lipase shows high activity and reusability in lipase-catalyzed synthesis of propyl laurate, suggesting the potential application value of encapsulated lipase.
Assuntos
Enzimas Imobilizadas , Lipase , Lipase/metabolismo , Enzimas Imobilizadas/metabolismo , Microesferas , Adsorção , Estabilidade EnzimáticaRESUMO
To establish cholyglycine (CG) detection via enzyme multiplied immunoassay technique (EMIT), glucose-6-phosphate dehydrogenase (G6PD) was used as a reporter enzyme to prepare hapten-enzyme conjugate. Gel electrophoresis and UV scanning demonstrated that G6PD was successfully labeled with cholyglycine and CG-G6PD conjugate was obtained. Furthermore, the effects of various parameters on the preparation of CG-G6PD conjugates were investigated. Consequently, CG amount, NADH, D-glucose-6-phosphate (G6P), phosphate buffer and the pH, and ionic strength of solution had important effects on the residual activity of CG-G6PD. Moreover, CG amount, the pH, and G6P played important roles in changing CG labeling location on G6PD. Using the CG-G6PD conjugate as test kit, the cholyglycine-EMIT calibration curve was established, which could be employed in clinical detection of cholyglycine. This study provides some valuable information for preparing hapten-G6PD conjugates. This article is protected by copyright. All rights reserved.
RESUMO
To establish cholyglycine (CG) detection via enzyme-multiplied immunoassay technique (EMIT), glucose-6-phosphate dehydrogenase (G6PD) was used as a reporter enzyme to prepare hapten-enzyme conjugate. Gel electrophoresis and UV scanning demonstrated that G6PD was successfully labeled with cholyglycine, and CG-G6PD conjugate was obtained. Furthermore, the effects of various parameters on the preparation of CG-G6PD conjugates were investigated. Consequently, CG amount, nicotinamide adenine dinucleotide, d-glucose-6-phosphate (G6P), phosphate buffer and the pH, and ionic strength of solution had important effects on the residual activity of CG-G6PD. Moreover, CG amount, the pH, and G6P played important roles in changing CG labeling location on G6PD. Using the CG-G6PD conjugate as test kit, the cholyglycine-EMIT calibration curve was established, which could be employed in clinical detection of cholyglycine. This study provides some valuable information for preparing hapten-G6PD conjugates.
Assuntos
Glucosefosfato Desidrogenase/metabolismo , Ácido Glicocólico/análise , Ácido Glicocólico/metabolismo , Leuconostoc mesenteroides/enzimologiaRESUMO
Thermomyces lanuginosus lipase (Lipozyme TLIM)-catalyzed esterification of L-ascorbic acid was studied. It was suggested that Lipozyme TLIM was a suitable biocatalyst for enzymatic esterification of L-ascorbic acid. Three solvents were investigated for the reaction, and acetone was found to be a suitable reaction medium. Furthermore, it was found that water activity could notably affect the conversion. Moreover, pH memory of Lipozyme TLIM lipase for catalyzing L-ascorbic acid esterification in acetone was observed and the effect of pH on the reaction was estimated. In addition, the influences of other parameters such as substrate mole ratio, enzyme loading, and reaction temperature and reusability of lipase on esterification of L-ascorbic acid were also analyzed systematically and quantitatively. Kinetic characterization of Lipozyme TLIM showed that K(m,a) and V(max) were 80.085 mM and 0.747 mM min(-1), respectively. As a result, Lipozyme TLIM-catalyzed esterification of L: -ascorbic acid gave a maximum conversion of 99%.
Assuntos
Ascomicetos/enzimologia , Ácido Ascórbico/análogos & derivados , Lipase/química , Acetona/química , Ácido Ascórbico/química , Biotransformação , Catálise , Cromatografia Líquida de Alta Pressão , Concentração de Íons de Hidrogênio , Cinética , Modelos Químicos , Solventes/química , Temperatura , Fatores de Tempo , Água/químicaRESUMO
To improve the performance of lipase in biosynthesis of benzyl cinnamate, a new immobilized lipase by entrapping enzyme into nano-molecular cages was designed. Consequently, the entrapped lipase showed a robust immobilization, which diminished the leakage of lipase notably in use. Moreover, the entrapped lipase exhibited higher activity (57.1 U/mg) than free lipase (50.0 U/mg), demonstrating that the native conformation of lipase was not destroyed during immobilization. Compared with the adsorbed lipase (half-life 40.7 min) and free lipase (half-life 29.8 min), the entrapped lipase (half-life 85.3 min) increased the stability by about 2-3 times. Furthermore, the entrapped lipase was applied in biosynthesis of benzyl cinnamate, where it showed excellent activity and re-usability. After 7 cycles, the yield of benzyl cinnamate catalyzed by the entrapped lipase remained 70.2%, while the yield catalyzed by the adsorbed lipase was only about 10%. These results indicated that the nano-molecular cages could inhibit denaturation of lipase and maintain its activity well.
Assuntos
Enzimas Imobilizadas , Lipase , Compostos de Benzil , Cinamatos , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Lipase/metabolismoRESUMO
We systematically and quantitatively investigated the structure and thermodynamics of G-quadruplexes of RNAs and corresponding DNAs of the same sequences under molecular crowding conditions that mimic the high osmotic stress induced by the numerous molecules inside of living cells. Structural analyses demonstrated that various telomere RNA sequences folded into parallel-stranded G-quadruplexes in a manner independent of the surrounding conditions with different cations under both dilute and molecular crowding conditions. In contrast, DNA G-quadruplexes showed structural polymorphism. Moreover, we demonstrated that the G-quadruplexes of the RNA sequences were more stable than those of the same DNA sequences. These results show that a single and robust RNA G-quadruplex structure can exist in a manner independent of the sequence and surrounding conditions. To confirm this, we studied a guanine-rich sequence located in the 5'-untranslated region of human bcl-2 mRNA that is thought to play a role in translation. The results revealed a stable parallel G-quadruplex that formed under all conditions tested. For example, a bcl-RNA G-quadruplex in the presence of 5 mM KCl [free energy change at 25 degrees C (DeltaG degrees (25)) of -5.42 kcal/mol] was more stable than its corresponding DNA G-quadruplex (DeltaG degrees (25) = -2.31 kcal/mol). Our results further indicated that water molecules binding to the 2'-OH group of RNA G-quadruplexes play a critical role in their formation and stability.
Assuntos
DNA/química , Quadruplex G , Água/química , Sequência de Bases , Cátions/química , Células , DNA/genética , Guanina/química , Humanos , Mutagênese , Polimorfismo Genético , Telômero/química , Telômero/genética , TermodinâmicaRESUMO
Here, PEI@PMMA microspheres were prepared by grafting polyethyleneimine (PEI) on poly(methyl methacrylate) (PMMA) magnetic microspheres and successfully used to immobilize lipase. The results showed that PEI@PMMA microspheres had strongly adsorbed lipase (49.1â¯mg/g microsphere) via electrostatic attraction. To prevent lipase shedding, the adsorbed lipase was further crosslinked with PEI on microspheres using glutaraldehyde as crosslinker. Consequently, PEI-crosslinked lipase (2.14 U/mg) exhibited 2.6 times and 1.4 times higher activity respectively than the directly covalent lipase (0.82 U/mg) and the crosslinked lipase aggregates (1.57 U/mg), which was close to the activity of adsorbed lipase (2.20 U/mg). Conformational analysis from FTIR spectroscopy showed that PEI-crosslinked lipase retained its natural structure well. And the α-helix structure seemed to play a key role in enhancing lipase activity. Furthermore, the effects of various parameters on crosslinking reaction were investigated. Also, PEI-crosslinked lipase revealed higher pH and thermal stability. The Michaelis constant (Km) was increased and the optimum temperature of lipase was widened observably after crosslinking with PEI on PEI@PMMA magnetic microspheres.
Assuntos
Reagentes de Ligações Cruzadas/química , Lipase/química , Polietilenoimina/química , Adsorção , Candida/enzimologia , Reagentes de Ligações Cruzadas/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Lipase/metabolismo , Tamanho da Partícula , Polietilenoimina/metabolismo , Propriedades de SuperfícieRESUMO
Recent studies in mice suggested that KLF5 (Kruppel like factor 5), a zinc-finger transcription factor, plays an important role in skeletal muscle development and regeneration. As an important factor in the process of muscle development, KLF5 participates in the regulation of the cell cycle, cell survival, and cell dryness under different environmental conditions, but it is not clear whether KLF5 participates in muscle atrophy. Therefore, we investigated whether KLF5 can regulate the atrophy of chicken satellite cells in vitro and examined its mechanism of action. qPCR showed that KLF5 gene knockdown promoted the expression of key genes in muscle atrophy. Subsequently, we sequenced and analyzed the transcriptomes of KLF5 silenced and control cells, and we showed that the differentially expressed genes were mainly enriched in 10 signaling pathways (P<0.05), with differential gene and enrichment analyses indicating that the Wnt signaling pathways are extremely important. In conclusion, our results indicate that KLF5 may regulate the atrophy of chicken skeletal muscle through the Wnt/ß-catenin signaling pathway.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Fatores de Transcrição Kruppel-Like/fisiologia , Músculo Esquelético/patologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Animais , Atrofia/genética , Células Cultivadas , Galinhas , MasculinoRESUMO
Magnetic DEAE-GMA-EDMA microspheres were prepared via suspension polymerization and used for the immobilization of Candida rugosa lipase by ion exchange. The effect of pH values on the immobilization of lipase was investigated. Resolution of (+/-)-menthol in the hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate was performed by immobilized lipase-catalyzed enantioselective esterification with propionic anhydride as acyl donor. The effects of pH condition at lipase immobilization on the conversion and enantioselectivity were investigated. As a result, pH memory of the immobilized lipase for catalyzing (+/-)-menthol resolution in the ionic liquid was observed. Better conversion and the best enantioselectivity were obtained with the immobilized lipase prepared at pH 5.0. Under the condition, (-)-menthyl propionate with enantiomeric excess of >90% was obtained. Moreover, the enantioselectivity of the immobilized lipase decreased gradually with increasing pH value.
Assuntos
Enzimas Imobilizadas/metabolismo , Lipase/metabolismo , Mentol/metabolismo , Adsorção , Candida/enzimologia , Concentração de Íons de Hidrogênio , Íons , Magnetismo , Microesferas , SoluçõesRESUMO
Response surface methodology was successfully applied to optimize lipase-catalyzed enantioselective esterification of (±)-menthol. The effects of various reaction conditions, including reaction time, temperature, enzyme loading, substrate molar ratio and water activity, were investigated. A Central Composite Rotatable Design was employed to search for the optimal conversion of (±)-menthol and enantiomeric excess. A quadratic polynomial regression model was used to analyze the experimental data at a 95% confidence level (p<0.05). The analysis confirmed that reaction temperature, enzyme loading and reaction time were the significant factors affecting the conversion of (±)-menthol. Moreover, reaction temperature, enzyme loading, substrate molar ratio and reaction time were found to affect the enantiomeric excess significantly. The coefficient of determination of these two models was found to be 0.980 and 0.967, respectively. Two sets of optimum reaction conditions were established and the verified experimental trials were performed for validating the optimum points. Under the optimum conditions, the conversion of (±)-menthol and the enantiomeric ratio exceeded 53% and 40%, respectively.
RESUMO
Response surface methodology was successfully applied to optimize lipase-catalyzed regioselective esterification of pyridoxine (PN). Effects of various reaction conditions, including reaction temperature, time, enzyme loading, substrate molar ratio, and water activity, were investigated. A central composite design was employed to search for the optimal conversion of PN. A quadratic polynomial regression model was used for analysis of the experimental data at a 95% level (p < 0.05). The analysis confirmed that the water activity was the most significant factor affecting the conversion of PN. It was also suggested that the conversion was strongly affected by independent variables of temperature, time, substrate molar ratio, and water activity as well as interaction terms of temperature and enzyme loading/substrate molar ratio/water activity, time and enzyme loading/substrate molar ratio, substrate molar ratio, and water activity. The coefficient of determination of the model was found to be 0.963. Three sets of optimum reaction conditions were established, and the verified experimental trials were performed for validating the optimum points. A scale-up experiment was also done under the first set of optimal conditions.
Assuntos
Acilação , Lipase/química , Piridoxina/química , Vitamina B 6/química , Cinética , Modelos Químicos , EstereoisomerismoRESUMO
In this work, the new approach to synthesis of benzyl cinnamate by enzymatic esterification of cinnamic acid with benzyl alcohol is optimized by response surface methodology. The effects of various reaction conditions, including temperature, enzyme loading, substrate molar ratio of benzyl alcohol to cinnamic acid, and reaction time, are investigated. A 5-level-4-factor central composite design is employed to search for the optimal yield of benzyl cinnamate. A quadratic polynomial regression model is used to analyze the experimental data at a 95% confidence level (P<0.05). The coefficient of determination of this model is found to be 0.9851. Three sets of optimum reaction conditions are established, and the verified experimental trials are performed for validating the optimum points. Under the optimum conditions (40°C, 31mg/mL enzyme loading, 2.6:1 molar ratio, 27h), the yield reaches 97.7%, which provides an efficient processes for industrial production of benzyl cinnamate.
Assuntos
Álcool Benzílico/metabolismo , Compostos de Benzil/metabolismo , Cinamatos/metabolismo , Lipase/metabolismo , Esterificação , TemperaturaRESUMO
In this paper, Lipozyme TLIM-catalyzed synthesis of ethyl cinnamate through esterification of cinnamic acid with ethanol was studied. In order to increase the yield of ethyl cinnamate, several media, including acetone, isooctane, DMSO and solvent-free medium, were investigated in this reaction. The reaction showed a high yield by using isooctane as reaction medium, which was found to be much higher than the yields reported previously. Furthermore, several parameters such as shaking rate, water activity, reaction temperature, substrate molar ratio and enzyme loading had important influences on this reaction. For instance, when temperature increased from 10 to 50 °C, the initial reaction rate increased by 18 times and the yield of ethyl cinnamate increased by 6.2 times. Under the optimum conditions, lipase-catalyzed synthesis of ethyl cinnamate gave a maximum yield of 99%, which was of general interest for developing industrial processes for the preparation of ethyl cinnamate.
Assuntos
Cinamatos/química , Cinamatos/síntese química , Enzimas Imobilizadas/química , Esterificação/fisiologiaRESUMO
Poly(vinyl acetate-acrylamide) microspheres with an average diameter of 2-4µm were successfully prepared and characterized via SEM and FTIR. Then the microspheres were modified with epoxy groups through reacting with epichlorohydrin and used as carriers to covalently immobilize Candida rugosa lipase. The results revealed that agitation played an important role on epoxy activation and the immobilization ratio increased with the increase of the epoxy density. On the other hand, the specific activity of the immobilized lipase as well as the activity recovery declined gradually with the increase in the immobilization ratio from 72% to 93%, which were attributed to the steric hindrance effects caused by enzyme overloading. When epoxy density was 76µmol/g microsphere, the activity recovery reached the maximum at 47.5%, and the activity of the immobilized lipase was 261.3U/g microsphere. Moreover, the thermal stability of the immobilized lipase was much better than that of the free one, which indicated potential applications of the immobilized lipase.
Assuntos
Acrilamida/química , Candida/enzimologia , Enzimas Imobilizadas/metabolismo , Resinas Epóxi/química , Lipase/metabolismo , Microesferas , Polivinil/química , Estabilidade Enzimática , Enzimas Imobilizadas/química , Ácidos Graxos/metabolismo , Concentração de Íons de Hidrogênio , Lipase/química , Espectroscopia de Infravermelho com Transformada de Fourier , TemperaturaRESUMO
In this study, lipase catalysis was successfully applied in synthesis of benzyl cinnamate through esterification of cinnamic acid with benzyl alcohol. Lipozyme TLIM was found to be more efficient for catalyzing this reaction than Novozym 435. In order to increase the yield of benzyl cinnamate, several media, including acetone, trichloromethane, methylbenzene, and isooctane, were used in this reaction. The reaction showed a high yield using isooctane as medium. Furthermore, the effects of several parameters such as water activity, reaction temperature, etc, on this reaction were analyzed. It was pointed out that too much benzyl alcohol would inhibit lipase activity. Under the optimum conditions, lipase-catalyzed synthesis of benzyl cinnamate gave a maximum yield of 97.3%. Besides, reusable experiment of enzyme demonstrated that Lipozyme TLIM retained 63% of its initial activity after three cycles. These results were of general interest for developing industrial processes for the preparation of benzyl cinnamate.
Assuntos
Compostos de Benzil/síntese química , Cinamatos/síntese química , Cinamatos/metabolismo , Acetona , Catálise , Enzimas Imobilizadas , Esterificação/fisiologia , Proteínas Fúngicas , Lipase/metabolismo , Octanos , Temperatura , ÁguaRESUMO
Kinetics and thermodynamics of lipase-catalyzed esterification of l-ascorbic acid in acetone were investigated by using vinyl acetate as acyl donor. The results showed that l-ascorbic acid could generate inhibition effect on lipase activity. A suitable model, Ping-Pong Bi-Bi mechanism having substrate inhibition, was thus introduced to describe the enzymatic kinetics. Furthermore, the kinetic and thermodynamic parameters were calculated from a series of experimental data according to the kinetic model. The inhibition constant of L-ascorbic acid was also obtained, which seemed to imply that enhancing reaction temperature could depress the substrate inhibition. Besides, the activation energy values of the first-step and the second-step reaction were estimated to be 37.31 and 4.94 kJ/mol, respectively, demonstrating that the first-step reaction was the rate-limiting reaction and could be easily improved by enhancing temperature.
Assuntos
Ácido Ascórbico/biossíntese , Ácido Ascórbico/química , Eurotiales/enzimologia , Lipase/química , Ácido Ascórbico/metabolismo , Catálise , Enzimas Imobilizadas , Esterificação , Cinética , Lipase/antagonistas & inibidores , Modelos Químicos , Especificidade por Substrato , Termodinâmica , Compostos de Vinila/químicaRESUMO
Magnetic microspheres with carboxyl groups were prepared by copolymerization of vinyl acetate (VAC), acrylamide (AM), and acrylic acid (AA) in the presence of oleic acid-coated Fe(3)O(4) nanoparticles. Scanning electron microscope (SEM) photo showed that the average diameter of magnetic microspheres was about 400 nm. Also, FTIR spectra analysis indicated that monomers were successfully enfolded on the microspheres' surface. They were used as support to immobilize lipase via physical adsorption and covalent binding. To investigate the effect of the microsphere surface properties on lipase immobilization, a series of microspheres with different hydrophobic/hydrophilic surface characteristics were prepared by adjusting molar percentages of different monomers. The results showed that microspheres with different hydrophobicities/hydrophilicities had different immobilized ratios and different activity recovery. Compared with microspheres having hydrophilic characteristics, that with hydrophobic characteristics had a much higher lipase binding efficiency. However, this study further demonstrated that moderate hydrophobicity/hydrophilicity of microsphere surface was very important for elevating activity recovery. When AM (hydrophilic monomer) held 14.3% of total amount of monomers, the activity recovery was the highest (reaching 87%, 418 U/g support). Possible reasons for these observations were discussed and a supposed mechanism was speculated.
Assuntos
Enzimas Imobilizadas/metabolismo , Lipase/metabolismo , Magnetismo , Microesferas , Adsorção , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Poly(vinyl acetate-acrylamide) microspheres were synthesized in the absence or presence of isooctane via suspension polymerization and utilized as carriers to immobilize Candida rugosa lipase. When the hydrophobic/hydrophilic surface characteristics of the microspheres were modified by changing the ratio of vinyl acetate (hydrophobic monomer) to acrylamide (hydrophilic monomer) from 50:50 to 86:24, the immobilization ratio changed from 45% to 92% and the activity of the immobilized lipase increased from 202.5 to 598.0 U/g microsphere. Excessive lipase loading caused intermolecular steric hindrance, which resulted in a decline in lipase activity. The maximum specific activity of the immobilized lipase (4.65 U/mg lipase) was higher than that of free lipase (3.00 U/mg lipase), indicating a high activity recovery during immobilization.