RESUMO
Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from Halobacterium salinarum NRC-1 was cloned and successfully expressed in Haloferax volcanii. The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using p-nitrophenyl valerate as substrate (KM = 78 µM, kcat = 0.67 s-1). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, n-hexane, n-heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications.
Assuntos
Carboxilesterase , Clonagem Molecular , Halobacterium salinarum , Proteínas Recombinantes , Carboxilesterase/genética , Carboxilesterase/metabolismo , Carboxilesterase/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Halobacterium salinarum/enzimologia , Halobacterium salinarum/genética , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Concentração de Íons de Hidrogênio , Cinética , Estabilidade Enzimática , Proteínas Arqueais/genética , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , TemperaturaRESUMO
The enzymatic conversion of lignocellulosic biomass to fermentable sugars is determined by the enzymatic activity of cellulases; consequently, improving enzymatic activity has attracted great interest in the scientific community. Cocktails of commercial cellulase often have low ß-glucosidase content, leading to the accumulation of cellobiose. This accumulation inhibits the activity of the cellulolytic complex and can be used to determine the enzymatic efficiency of commercial cellulase cocktails. Here, a novel codon optimized ß-glucosidase gene (B-glusy) from Trichoderma reesei QM6a was cloned and expressed in three strains of Escherichia coli (E. coli). The synthetic sequence containing an open reading frame (ORF) of 1491 bp was used to encode a polypeptide of 497 amino acid residues. The ß-glucosidase recombinant protein that was expressed (57 kDa of molecular weight) was purified by Ni agarose affinity chromatography and visualized by SDS-PAGE. The recombinant protein was better expressed in E. coli BL21 (DE3), and its enzymatic activity was higher at neutral pH and 30 °C (22.4 U/mg). Subsequently, the ß-glucosidase was immobilized using magnetite nano-support, after which it maintained >65% of its enzymatic activity from pH 6 to 10, and was more stable than the free enzyme above 40 °C. The maximum immobilization yield had enzyme activity of 97.2%. In conclusion, ß-glucosidase is efficiently expressed in the microbial strain E. coli BL21 (DE3) grown in a simplified culture medium.
Assuntos
Enzimas Imobilizadas , Escherichia coli , Proteínas Fúngicas , Expressão Gênica , Hypocreales/genética , Nanopartículas de Magnetita/química , beta-Glucosidase , Estabilidade Enzimática , Enzimas Imobilizadas/biossíntese , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Hypocreales/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , beta-Glucosidase/biossíntese , beta-Glucosidase/química , beta-Glucosidase/genética , beta-Glucosidase/isolamento & purificaçãoRESUMO
OBJECTIVE: The present report describes the enzymatic acylation of umbelliferone with different vinyl esters as acyl donors biocatalyzed by the commercial lipase Novozym® 435, and the investigation for their antibacterial activity against ATCC and clinical strains isolated from hospital infection sites. RESULTS: The umbelliferone esters (1-5) were synthesized through the acylation reaction of 7-hydroxy-2H-chromen-2-one with different long chain vinyl esters catalyzed by the lipase Novozym 435. The reaction conditions were: 10% Novozym 435; tetrahydrofuran:acetone (3:1) for the reactions with acetate, propionate and butyrate vinyl esters 50-90% conversion, and (9:1) for decanoate and laurate vinyl esters 10-15% conversion; acyl donor/umbelliferone molar ratio of 10:1 and 60 °C. All the umbelliferone esters were characterized NMR and (HRMS). The antibacterial activity of the products were tested using the broth microdilution method in order to determine the minimum inhibitory concentration (MIC). The results displayed by 7-laurate and 7-decanoate-umbelliferone esters showed the highest antibacterial potential, with 1 mM inhibitory activity for ATCC 33591, a methicillin and oxacillin resistant Staphylococcus aureus strain. They were also able to inhibit gram-negative bacterial strains, such as Pseudomonas aeruginosa (MIC 0.5 mM) and Klebsiella pneumoniae (MIC 1 mM). In addition, 7-laurate- and 7-decanoate-umbelliferone esters were able to inhibit all clinical strains (MIC 1 mM; except 7-laurate-umbelliferone in which MIC 0.5 mM against 55a). CONCLUSIONS: This is the first study performing the biocatalysis of umbelliferone followed by the purification of the products and the antibacterial evaluation.
Assuntos
Infecções Bacterianas/tratamento farmacológico , Ésteres/farmacologia , Lipase/química , Umbeliferonas/farmacologia , Antibacterianos/síntese química , Antibacterianos/farmacologia , Infecções Bacterianas/microbiologia , Biocatálise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Ésteres/síntese química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/patogenicidade , Lipase/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Transdução de Sinais/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Umbeliferonas/síntese químicaRESUMO
Despite the proven therapeutic role of capsaicin in human health, its usage is still hampered by its high pungency. In this sense, nonpungent capsaicin analogues as olvanil are a feasible alternative to the unpleasant sensations produced by capsaicin while maintaining a similar pharmacological profile. Olvanil can be obtained by a lipase-catalyzed chemoenzymatic process. In the present work, recombinant Candida antarctica lipase B (CALB) was expressed in Pichia pastoris and subsequently immobilized by cross-linked enzyme aggregate (CLEA) methodology for the synthesis of olvanil. The CALB-CLEAs were obtained directly from the fermentation broth of P. pastoris without any purification step in order to assess the role of the contaminant proteins of the crude extract as co-feeders. The CALB-CLEAs were also bioimprinted to enhance the catalytic performance in olvanil synthesis. When CALB was precipitated with isopropanol, the obtained CALB-CLEAs exhibited the highest activity in the synthesis of olvanil, regardless of the glutaraldehyde concentration. The maximum product synthesis was found at 72 hr obtaining 6.8 g L-1 of olvanil with a reaction yield of 16%. When CALB was bioimprinted with olvanil, the synthesis was enhanced 1.3 times, reaching 10.7 g L-1 of olvanil at 72 hr of reaction with a reaction yield of 25%. Scanning electron microscopy images indicated different morphologies of the CLEAs depending on the precipitating agent and the template used for bioimprinting. Recombinant CALB-CLEAs obtained directly from the fermentation broth are a suitable alternative to commercial enzymatic preparations for the synthesis of olvanil in organic medium.
Assuntos
Biotecnologia/métodos , Capsaicina/análogos & derivados , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Lipase/química , Lipase/metabolismo , Proteínas Recombinantes/metabolismo , Capsaicina/síntese química , Capsaicina/metabolismo , Reagentes de Ligações Cruzadas/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Fermentação , Proteínas Fúngicas/genética , Glutaral/química , Lipase/genética , Microscopia Eletrônica de Varredura , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
A recombinant L-arabinose isomerase from Enterococcus faecium DBFIQ E36 was immobilized onto multifunctional epoxide supports by chemical adsorption and onto a chelate-activated support via polyhistidine-tag, located on the N-terminal (N-His-L-AI) or on the C-terminal (C-His-L-AI) sequence, followed by covalent bonding between the enzyme and the support. The results were compared to reversible L-AI immobilization by adsorption onto charged agarose supports with improved stability. All the derivatives presented immobilization yields of above 75%. The ionic interaction established between agarose gels containing monoaminoethyl-N-aminoethyl structures (MANAE) and the enzyme was the most suitable strategy for L-AI immobilization in comparison to the chelate-activated agarose. In addition, the immobilized biocatalysts by ionic interaction in MANAE showed to be the most stable, retaining up to 100% of enzyme activity for 60 min at 60 °C and with Km values of 28 and 218 mM for MANAE-N-His-L-AI and MANAE-C-His-L-AI, respectively.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Proteínas de Bactérias/metabolismo , Enterococcus faecium/enzimologia , Hexoses/biossíntese , Aldose-Cetose Isomerases/genética , Proteínas de Bactérias/genética , Biocatálise , Biotecnologia , Enterococcus faecium/genética , Estabilidade Enzimática , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
The 2'-N-deoxyribosyltransferases [NDT; EC 2.4.2.6] are a group of enzymes widely used as biocatalysts for nucleoside biosynthesis. In this work, the molecular cloning, expression and purification of a novel NDT from Lactobacillus animalis (LaNDT) have been reported. On the other hand, biocatalyst stability has been significantly enhanced by multipoint covalent immobilization using a hetero-functional support activated with nickel-chelates and glyoxyl groups. The immobilized enzyme could be reused for more than 300â¯h and stored during almost 3 months without activity loss. Besides, the obtained derivative (Ni2+-Gx-LaNDT) was able to biosynthesize 88â¯mg floxuridine/g biocatalyst after 1â¯h of reaction. In this work, a green bioprocess by employing an environmentally friendly methodology was developed, which allowed the obtaining of a compound with proven anti-tumor activity. Therefore, the obtained enzymatic biocatalyst meets the requirements of high activity, stability, and short reaction times needed for low-cost production in a future preparative application.
Assuntos
Clonagem Molecular/efeitos dos fármacos , Enzimas Imobilizadas/metabolismo , Lactobacillus/enzimologia , Transferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Lactobacillus/química , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transferases/química , Transferases/genéticaRESUMO
A Trametes versicolor laccase was functionally expressed on the membrane surface of Saccharomyces cerevisiae EBY100. Laccase expression was increased 6.57-fold by medium optimization and surpassed production by the native strain. Maximal laccase and biomass production reached 19â735 ± 1719 Ug-1 and 6.22 ± 0.53 gL-1 respectively, after 2 d of culture. Optimum oxidization of all substrates by laccase was observed at pH 3. Laccase showed high affinity towards substrates used with Km (mM) and Vmax (µmol min-1) values of 0.57 ± 0.0047 and 24.55 ± 0.64, 1.52 ± 0.52 and 9.25 ± 1.78, and 2.67 ± 0.12 and 11.26 ± 0.75, were reported for ABTS, 2, 6-DMP and GUA, respectively. EDTA and NaN3 displayed none competitive inhibition towards laccase activity. The optimum temperature for activity was 50 °C; however, the enzyme was stable over a wide range of temperatures (25-70 °C). The biologically immobilized laccase showed high reusability towards phenolic substrates and low reusability with non-phenolic substrates. High affinity for a diversity phenolic compounds and great ethanol tolerance substantiates this laccase/yeast biocatalyst potential for application in the production of bioethanol.
Assuntos
Técnicas de Visualização da Superfície Celular , Enzimas Imobilizadas/metabolismo , Expressão Gênica , Lacase/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Meios de Cultura/química , Estabilidade Enzimática , Enzimas Imobilizadas/genética , Concentração de Íons de Hidrogênio , Lacase/química , Lacase/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Especificidade por Substrato , Temperatura , Trametes/enzimologia , Trametes/genéticaRESUMO
In previous work, a new lipase and its cognate foldase were identified and isolated from a metagenomic library constructed from soil samples contaminated with fat. This new lipase, called LipG9, is a true lipase that shows specific activities that are comparable to those of well-known industrially-used lipases with high activity against long-chain triglycerides. In the present work, LipG9 was co-expressed and co-immobilized with its foldase, on an inert hydrophobic support (Accurel MP1000). We studied the performance of this immobilized LipG9 (Im-LipG9) in organic media, in order to evaluate its potential for use in biocatalysis. Im-LipG9 showed good stability, maintaining a residual activity of more than 70% at 50 °C after incubation in n-heptane (log P 4.0) for 8 h. It was also stable in polar organic solvents such as ethanol (log P -0.23) and acetone (log P -0.31), maintaining more than 80% of its original activity after 8 h incubation at 30 °C. The synthesis of ethyl esters was tested with fatty acids of different chain lengths in n-heptane at 30 °C. The best conversions (90% in 3 h) were obtained for medium and long chain saturated fatty acids (C8, C14 and C16), with the maximum specific activity, 29 U per gram of immobilized preparation, being obtained with palmitic acid (C16). Im-LipG9 was sn-1,3-specific. In the transesterification of the alcohol (R,S)-1-phenylethanol with vinyl acetate and the hydrolysis of the analogous ester, (R,S)-1-phenylethyl acetate, Im-LipG9 showed excellent enantioselectivity for the R-isomer of both substrates (E> 200), giving an enantiomeric excess (ee) of higher than 95% for the products at 49% conversion. The results obtained in this work provide the basis for the development of applications of LipG9 in biocatalysis.
Assuntos
Proteínas de Bactérias/química , Enzimas Imobilizadas/química , Lipase/química , Proteínas de Bactérias/genética , Biocatálise , Enzimas Imobilizadas/genética , Ésteres , Ácidos Graxos/química , Lipase/genética , MetagenomaRESUMO
Nonexponential distance dependence of the apparent electron-transfer (ET) rate has been reported for a variety of redox proteins immobilized on biocompatible electrodes, thus posing a physicochemical challenge of possible physiological relevance. We have recently proposed that this behavior may arise not only from the structural and dynamical complexity of the redox proteins but also from their interplay with strong electric fields present in the experimental setups and in vivo (J. Am Chem. Soc. 2010, 132, 5769-5778). Therefore, protein dynamics are finely controlled by the energetics of both specific contacts and the interaction between the protein's dipole moment and the interfacial electric fields. In turn, protein dynamics may govern electron-transfer kinetics through reorientation from low to high donor-acceptor electronic coupling orientations. Here we present a combined computational and experimental study of WT cytochrome c and the surface mutant K87C adsorbed on electrodes coated with self-assembled monolayers (SAMs) of varying thickness (i.e., variable strength of the interfacial electric field). Replacement of the positively charged K87 by a neutral amino acid allowed us to disentangle protein dynamics and electron tunneling from the reaction kinetics and to rationalize the anomalous distance dependence in terms of (at least) two populations of distinct average electronic couplings. Thus, it was possible to recover the exponential distance dependence expected from ET theory. These results pave the way for gaining further insight into the parameters that control protein electron transfer.
Assuntos
Substituição de Aminoácidos , Citocromos c/química , Citocromos c/metabolismo , Mutação , Adsorção , Animais , Citocromos c/genética , Eletrodos , Transporte de Elétrons , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica , Eletricidade Estática , Propriedades de SuperfícieRESUMO
This paper presents two immobilization methods for the intracellular invertase (INVA), from Zymomonas mobilis. In the first method, a chimeric protein containing the invertase INVA, fused through its C-terminus to CBDCex from Cellulomonas fimi was expressed in Escherichia coli strain BL21 (DE3). INVA was purified and immobilized on crystalline cellulose (Avicel) by means of affinity, in a single step. No changes were detected in optimal pH and temperature when INVA-CBD was immobilized on Avicel, where values of 5.5 and 30 degrees C, respectively, were registered. The kinetic parameters of the INVA-CBD fusion protein were determined in both its free form and when immobilized on Avicel. Km and Vmax were affected with immobilization, since both showed an increase of up to threefold. Additionally, we found that subsequent to immobilization, the INVA-CBD fusion protein was 39% more susceptible to substrate inhibition than INVA-CBD in its free form. The second method of immobilization was achieved by the expression of a 6xHis-tagged invertase purified on Ni-NTA resin, which was then immobilized on Nylon-6 by covalent binding. An optimal pH of 5.5 and a temperature of 30 degrees C were maintained, subsequent to immobilization on Nylon-6 as well as with immobilization on crystalline cellulose. The kinetic parameters relating to Vmax increased up to 5.7-fold, following immobilization, whereas Km increased up to 1.7-fold. The two methods were compared showing that when invertase was immobilized on Nylon-6, its activity was 1.9 times that when immobilized on cellulose for substrate concentrations ranging from 30 to 390 mM of sucrose.
Assuntos
Enzimas Imobilizadas/química , Enzimas Imobilizadas/isolamento & purificação , Expressão Gênica , Zymomonas/enzimologia , beta-Frutofuranosidase/química , beta-Frutofuranosidase/isolamento & purificação , Caprolactama/análogos & derivados , Caprolactama/química , Celulose/química , Estabilidade Enzimática , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Polímeros/química , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismoRESUMO
The recombinant invertase INVB (re-INVB) from Zymomonas mobilis was immobilized on microbeads of Nylon-6, by means of covalent bonding. The enzyme was strongly and successfully bound to the support. The activity of the free and immobilized enzyme was determined, using 10% (w/v) sucrose, at a temperature ranging between 15 and 60 degrees C and a pH ranging between 3.5 and 7. The optimal pH and temperature for the immobilized enzyme were 5.5 and 25 degrees C, respectively. Immobilization of re-INVB on Nylon-6 showed no significant change in the optimal pH, but a difference in the optimal temperature was evident, as that for the free enzyme was shown to be 40 degrees C. The values for kinetic parameters were determined as: 984 and 98 mM for Kappm of immobilized and free re-INVB, respectively. Kappcat values for immobilized and free enzymes were 6.1x10(2) and 1.2x10(4) s(-1), respectively, and immobilized re-INVB showed Vappmax of 158.73 micromol h min(-1) mg(-1). Immobilization of re-INVB on Nylon-6 enhanced the thermostability of the enzyme by 50% at 30 degrees C and 70% at 40 degrees C, when compared to the free enzyme. The immobilization system reported here may have future biotechnological applications, owing to the simplicity of the immobilization technique, the strong binding of re-INVB to the support and the effective thermostability of the enzyme.
Assuntos
Proteínas de Bactérias/química , Enzimas Imobilizadas/química , Zymomonas/enzimologia , beta-Frutofuranosidase/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caprolactama/análogos & derivados , Caprolactama/química , Estabilidade Enzimática , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Membranas Artificiais , Polímeros/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismoRESUMO
Genetically modified acetylcholinesterase (AChE) from Drosophila melanogaster (dm) and from commercial sources, Electric eel (ee), Bovine erythrocites (be) and Human erythrocites (he), were investigated as biological receptors for the detection of methamidophos pesticide based on inhibition studies. Most engineered variant of AChE from dm showed enhanced sensitivity toward methamidophos pesticide. Among 24 dmAChE variants tested, 12 presented a sensitivity comparable to the commercially available eeAChE, but higher than AChEs from be and he. Four were found more sensitive and six others were insensitive to methamidophos insecticide. The D375G,Y370F,Y374A,F376L mutant was the most sensitive, with a ki value of 2.2 X 10(6) mol(-1) L min(-1), three orders of magnitude higher than eeAChE (1.1 X 10(3) mol(-1) L min(-1)). The sensor constructed with genetically modified enzyme showed better characteristics with respect to detection limit and sensitivity compared with those using commercial eeAChE. Differential pulse polarography and chronoamperometry were used as electrochemical techniques to characterize the AChE biosensors. The lower detection limit of 1 ppb was obtained with D375G,Y370F,Y374A,F376L mutant of dmAChE, compared to 90 ppb for the commercial eeAChE. This study may stimulate scientists to develop more sensitive and selective procedures for organophosphorus insecticides detection by using engineered variant of dmAChE.