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1.
Enzyme Microb Technol ; 133: 109461, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31874681

RESUMO

The lipase from Thermomyces lanuginosus (TLL) has been immobilized on octyl-agarose beads via interfacial activation under 16 different conditions (changing the immobilization pH, the ionic strength, the presence of additives like calcium, phosphate or glycerol) and using a low loading (1 mg/g support). Then, the properties of the different biocatalysts have been evaluated: stability at pH 7.0 and 70 °C and activity versus p-nitro phenyl propionate, triacetin and R- and S- methyl mandelate. Results clearly indicate that the immobilization conditions determine the final enzyme properties, altering enzyme stability (by 10 folds), activity (by 8 folds using R- methyl mandelate) and specificity (VR/VS changed from 0.7 to 2.3 using mandelate esters). For instance, the enzymes immobilized at pH 7.0 using 5 mM buffer were the most stable preparations, while the presence of 250 mM sodium phosphate greatly decreased the final enzyme stability. The biocatalyst stability of TLL increased with increasing NaCl in the immobilization buffer at pH 5. Fluorescence studies confirmed that the conformation of the different immobilized enzymes were different, despite being a physical and reversible immobilization method. Thus, the immobilization of TLL on octyl agarose beads under different conditions produced biocatalysts with different properties, the optimal condition depends on the studied reaction and condition.


Assuntos
Ascomicetos/enzimologia , Células Imobilizadas/enzimologia , Glioxilatos/química , Lipase/metabolismo , Sefarose/química , Biocatálise , Estabilidade Enzimática , Cinética
2.
J Agric Food Chem ; 67(46): 12720-12729, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31652059

RESUMO

Many current controlled-release fertilizers (CRFs) are coated with nonbiodegradable polymers that can contribute to microplastic pollution. Here, coatings of self-immolative poly(ethyl glyoxylate) (PEtG) capped with a carbamate and blended with polycaprolactone (PCL) or poly(l-lactic acid) (PLA) were evaluated. They were designed to depolymerize and release fertilizers in the vicinity of plant roots, where the pH is lower than that in the surrounding environment. PEtG/PCL coatings exhibited significant temperature and pH effects, requiring 18 days at pH 5 and 30 °C, compared to 77 days at pH 7 and 22 °C, to reach 15% mass loss. Plant roots were also effective in triggering coating degradation. Spray-coating and melt-coating were explored, with the latter being more effective in providing pellets that retained urea prior to polymer degradation. Finally, PEtG/PCL-coated pellets promoted plant growth to a similar degree or better than currently available CRFs.


Assuntos
Composição de Medicamentos/métodos , Fertilizantes/análise , Glioxilatos/química , Poliésteres/química , Agrostis/crescimento & desenvolvimento , Preparações de Ação Retardada , Concentração de Íons de Hidrogênio , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Solubilidade
3.
Int J Biol Macromol ; 138: 234-243, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31315021

RESUMO

Polygalacturonase (PG) from Aspergillus niger was immobilized using glyoxyl, vinylsulfone or glutaraldehyde-activated supports. The use of supports pre-activated with glutaraldehyde presented the best results. The immobilization of PG on glutaraldehyde-supports was studied under different conditions: at pH 5 for 24 h; at pH 5, 6.5 or 8 for 3 h and then incubated at pH 8 for 24 h; at pH 8 in the presence of 300 mM NaCl for 24 h, to prevent ion exchange. The immobilization under all conditions showed a significant increase in the enzyme thermal stability under inactivation conditions at pH 4-10. As a result, at temperatures over 70 °C or pH values over 7, the immobilized PG maintained significant levels of activity while the free PG was fully inactivated. The immobilization conditions presented a clear effect on enzyme activity, thermostability and operational stability, suggesting that the different conditions permitted to get immobilized PG having different orientations. Varying the immobilization protocol it is possible to achieve high activity or stability, and the optimal biocatalyst depends on the conditions where it will be utilized. The immobilized PG biocatalysts could be reused 10 times without a significant decrease in enzyme activity and offered very linear reaction courses.


Assuntos
Aspergillus niger/enzimologia , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Poligalacturonase/química , Poligalacturonase/metabolismo , Aldeídos/química , Biocatálise , Celulose/metabolismo , Ativação Enzimática , Estabilidade Enzimática , Glioxilatos/química , Concentração de Íons de Hidrogênio , Microesferas , Pectinas/metabolismo , Sefarose/química
4.
Biochim Biophys Acta Proteins Proteom ; 1867(9): 741-747, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31202001

RESUMO

The lipase from Pseudomonas fluorescens (PFL) has been immobilized on glyoxyl-octyl agarose and compared to the enzyme immobilized on octyl-agarose. Thus, PFL was immobilized at pH 7 on glyoxyl-octyl support via lipase interfacial activation and later incubated at pH 10.5 for 20 h before reduction to get some enzyme-support covalent bonds. This permitted for 70% of the enzyme molecules to become covalently attached to the support. This biocatalyst was slightly more stable than the octyl-PFL at pH 5, 7 and 9, or in the presence of some organic solvents (stabilization factor no higher than 2). The presence of phosphate anions produced enzyme destabilization, partially prevented by the immobilization on glyoxyl-octyl (stabilization factor became 4). In contrast, the presence of calcium cations promoted a great PFLstabilization, higher in the case of the glyoxyl-octyl preparation (that remained 100% active when the octyl-PFL preparations had lost 20% of the activity). However, it is in the operational stability where the new biocatalyst showed the advantages: in the hydrolysis of 1 M triacetin in 60% 1.4 dioxane, the octyl biocatalyst released >60% of the enzyme in the first cycle, while the covalently attached enzyme retained its full activity after 5 reaction cycles.


Assuntos
Proteínas de Bactérias/química , Enzimas Imobilizadas/química , Glioxilatos/química , Lipase/química , Pseudomonas fluorescens/enzimologia , Sefarose/química , Estabilidade Enzimática , Concentração de Íons de Hidrogênio
5.
Food Chem ; 295: 259-266, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174757

RESUMO

New cauliflower-like phloroglucinol-glyoxylic acid resin microspheres (PGRMs) with controllable diameters and tuneable surface roughness were prepared using a one-step environmentally-friendly method without a catalyst. The PGRMs obtained exhibited a rough surface, narrow size distribution, and excellent adsorption capacity for polar compounds. The PGRMs were employed as an adsorbent for solid phase extraction (SPE) of kinetin (KT) and 6-benzyladenine (6-BA) in cucumbers and demonstrated better extraction recoveries and purification efficiency than phloroglucin-formaldehyde resin and common commercial adsorbents. Our PGRMs-SPE-HPLC method showed good linearity (r ≥ 0.9997) ranging from 0.04 to 4.00 µg/g for KT and 6-BA, and recoveries at three spiked concentration ranged from 77.8% to 104.4% with RSDs ≤ 6.8%. This PGRMs-SPE-HPLC method was applied successfully to determine of KT and 6-BA in cucumbers.


Assuntos
Compostos de Benzil/análise , Cucumis sativus/química , Cinetina/análise , Reguladores de Crescimento de Planta/análise , Purinas/análise , Extração em Fase Sólida/métodos , Adsorção , Compostos de Benzil/isolamento & purificação , Brassica/química , Cromatografia Líquida de Alta Pressão , Análise de Alimentos/instrumentação , Análise de Alimentos/métodos , Glioxilatos/química , Cinetina/isolamento & purificação , Microesferas , Tamanho da Partícula , Floroglucinol/química , Reguladores de Crescimento de Planta/isolamento & purificação , Purinas/isolamento & purificação , Extração em Fase Sólida/instrumentação , Propriedades de Superfície
6.
Environ Sci Process Impacts ; 21(6): 1038-1051, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31124553

RESUMO

At pH 4.0, hydrous manganese oxide (HMO) oxidizes mandelic acid by two mole-equivalents of electrons, yielding phenylglyoxylic acid and benzaldehyde. These intermediates, in turn, are oxidized by two mole-equivalents of electrons to the same ultimate oxidation product, benzoic acid. The four compounds of the "reaction set" just described are conveniently monitored using capillary electrophoresis (CE) and HPLC. Extents of adsorption are negligible and their sum exhibits mass balance. Concentrations of phenylglyoxylic acid, benzaldehyde, and benzoic acid can therefore be used to calculate mole-equivalents delivered to HMO for comparison with experimentally-determined dissolved MnII concentrations. Semi-log plots (ln[substrate] versus time) and numerical analysis can also be used to explore rates of oxidation of the functional groups represented, i.e. an α-hydroxycarboxylic acid, an α-ketocarboxylic acid, and an aldehyde. Inserting a -CH2- group between the benzene ring and the functional groups just described yields a new reaction set comprised of phenyllactic acid, phenylpyruvic acid, and phenylacetaldehyde, plus the C-1 ultimate oxidation product, phenylacetic acid. At pH 4, mass balance for phenyllactic acid oxidation fell short by ∼9%. Phenyllactic acid was oxidized 2.7-times more slowly than mandelic acid, while phenylpyruvic acid was oxidized 12.7-times faster than phenylglyoxylic acid. Unlike benzaldehyde, oxidation rates for phenylacetaldehyde were too fast to measure. Under pH 4.0 conditions, this reaction set approach was used to explore the acceleratory effects of increases in HMO loading and inhibitory effects of 500 µM phosphate and pyrophosphate additions. Mandelic acid and phenyllactic acid were oxidized by HMO far more slowly at pH 7.0 than at pH 4.0. At pH 7.0, 2 mM MOPS and phosphate buffers did not yield appreciable dissolved MnII, despite oxidation of organic substrate. 2 mM pyrophosphate, in contrast, solubilized HMO-bound MnII and MnIII.


Assuntos
Glioxilatos/química , Ácidos Mandélicos/química , Compostos de Manganês/química , Óxidos/química , Adsorção , Benzaldeídos/química , Cinética , Oxirredução , Ácidos Fenilpirúvicos/química
7.
Int J Biol Macromol ; 133: 412-419, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31004647

RESUMO

Ficin extract has been aminated using ethylenediamine and carbodiimide to transform all exposed carboxylic groups into amino groups, retaining around 80% of activity versus benzoyl-d,l-arginine p-nitroanilide hydrochloride (BANA) and 90% versus casein. This aminated enzyme was then immobilized on glyoxyl agarose beads. After optimization of the immobilization protocol (immobilization at pH 10 for just 1 h), the new biocatalyst was compared to that obtained using the non-aminated enzyme. Activity versus BANA was lower, but was higher versus casein. The new biocatalyst was more stable than the reference mainly at pH 7. The new biocatalyst permitted to have a more linear course and a higher hydrolysis yield of casein at 75 °C. Moreover, the activity of the new preparations was significantly higher than the reference or the free enzyme in 8 M urea, at pH 7 and 55 °C. The enzyme in an overloaded biocatalyst exhibited a much higher specific activity versus casein (75% of the low loaded biocatalysts) than the non-aminated enzyme (only 30%), suggesting a more appropriate enzyme orientation that decreased steric hindrances. Finally, the enzyme was reused for 5 cycles of casein hydrolysis at 40 °C and pH 7 without any decrease in enzyme activity.


Assuntos
Caseínas/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Ficina/química , Ficina/metabolismo , Glioxilatos/química , Sefarose/química , Aminação , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Temperatura
8.
Int J Biol Macromol ; 131: 989-997, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30917914

RESUMO

Lipase B from Candida antarctica (CALB), lipase from Rhizomucor miehei (RML) and phospholipase Lecitase Ultra (LEU) were immobilized via interfacial activation and their stabilities were compared. Immobilized CALB was much more stable than immobilized RML or LEU. That meant that, if they were coimmobilized, after the inactivation of the least stable lipases, CALB should be discarded even though it may maintain full activity. This could be solved by sequential coimmobilization on octyl-glyoxyl (OCGLX). First, CALB was immobilized on OCGLX getting some covalent bonds between most of the CALB molecules and the support. Then, after reduction of CALB immobilized on OCGLX, RML or LEU can be immobilized on the support via interfacial activation. These enzymes could be released from the support just by using detergents, without affecting CALB activity. After optimization of the lipase desorption conditions, the bi-combilipases CALB/RML and CALB/LEU or the triple-combilipase CALB/RML/LEU could be submitted to several cycles of immobilized biocatalyst inactivation, desorption and enzyme reloading keeping the activity of the immobilized CALB almost intact. This way, by using OCGLX and a stepwise immobilization protocol, discarding all coimmobilized lipases when one becomes inactivated is no longer required. Thus, the most stable ones can be reused in several cycles.


Assuntos
Enzimas Imobilizadas , Glioxilatos/química , Lipase/química , Sefarose/química , Biocatálise , Candida/enzimologia , Detergentes/farmacologia , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática , Proteínas Fúngicas/química , Cinética
9.
Anal Chim Acta ; 1061: 122-133, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-30926030

RESUMO

A facile and efficient method for the determination of triazine herbicides in tomato samples was developed by employing 3-aminophenol-glyoxylic acid resin microspheres as a solid-phase extraction adsorbent followed by high performance liquid chromatography analysis. These resin microspheres were synthesized by a simple green precipitation polymerization method, and a range of functional groups (hydroxyl, amino, carboxylic group) were introduced through the 3-aminophenol and glyoxylic acid components. The as-prepared resin microspheres were characterized by scanning electron microscopy, Fourier transform infrared spectrometry, and thermal gravimetric analyzer. The resin microspheres exhibited a good adsorption rate, large adsorption amount, and short adsorption equilibrium time (almost in ∼5 min). Under the optimal extraction and determination conditions, a good linearity was obtained in the range of 0.025-7.5 µg g-1 (r2 ≥ 0.9997) for atraton, ametryn, and prometryn. The limits of detection of atraton, ametryn, and prometryn were 0.57, 0.75, and 1.06 µg kg-1, respectively. The intra-day and inter-day precisions expressed as relative standard deviations were in the ranges of 1.8-3.2% and 1.7-4.1%, respectively. In addition, the recoveries at three spiked levels ranged from 85.1 to 97.7% with the relative standard deviation ≤ 5.4% (n = 3). This novel method is simple and accurate and has proved to be a reliable alternative method for the determination of triazine herbicides in tomato samples.


Assuntos
Aminofenóis/química , Contaminação de Alimentos/análise , Glioxilatos/química , Herbicidas/análise , Lycopersicon esculentum/química , Triazinas/análise
10.
Bioorg Chem ; 83: 55-62, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30342386

RESUMO

A new coordination polymer Zn(II) with thiosemicarbazone glyoxalic acid H2GAT was obtained in this study. According to the X-ray diffraction data, the coordination of the Zn(II) ion is carried out by one sulfur atom, in the thiol form, one nitrogen atom of the azomethine group and two oxygen atoms of the carboxylate groups, one of which belongs to neighbouring complex molecule. The oxygen atom of the water molecule completes Zn(II) ion environment to a distorted square-pyramidal structure. The binding of the monomer complex into polimer occurs through the bridge oxygen atom of carboxylate group. This complex is effective inhibitor of the α-glycosidase, butyrylcholinesterase (BChE), cytosolic carbonic anhydrase I and II isoforms (hCA I and II), and acetylcholinesterase enzymes (AChE) enzymes with Ki values of 1.45 ±â€¯0.23 µM for hCA I, 2.04 ±â€¯0.11 µM for hCA II, 3.47 ±â€¯0.88 µM for α-glycosidase, 0.47 ±â€¯0.10 µM for BChE, and 0.58 ±â€¯0.13 µM for AChE, respectively.


Assuntos
Complexos de Coordenação/farmacologia , Inibidores Enzimáticos/farmacologia , Glioxilatos/farmacologia , Polímeros/farmacologia , Tiossemicarbazonas/farmacologia , Zinco/farmacologia , Acetilcolinesterase/metabolismo , Butirilcolinesterase/metabolismo , Anidrase Carbônica I/antagonistas & inibidores , Anidrase Carbônica I/metabolismo , Anidrase Carbônica II/antagonistas & inibidores , Anidrase Carbônica II/metabolismo , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/metabolismo , Glioxilatos/química , Humanos , Estrutura Molecular , Polímeros/química , Relação Estrutura-Atividade , Tiossemicarbazonas/química , Zinco/química
11.
J Mol Graph Model ; 87: 22-29, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30472634

RESUMO

The RRKM calculation and bonding evolution theory analysis coupled with quantum theory of atoms in molecules have been used to investigate kinetics and molecular mechanism of gas phase thermal decomposition of methyl benzoylformate. The pressure-dependent rate coefficients, by applying different collisional efficiency values, indicated that the atmospheric pressure is in high-pressure limit of fall-off curve and low-pressure limit rate coefficients are in the range of 10-13-10-12 cm3 molec-1 s-1. Temperature dependence of high-pressure limiting of rate coefficient over the temperature range 733 ±â€¯20% K was estimated to be k∞RRKM(CBS-QB3)=2.92×1013s-1exp(227.4kJmol-1/RT) and k∞RRKM(PBE1PBE)=2.67×1013s-1exp(232.8kJmol-1/RT). Topological analysis of electron localization function and electron density at the B3LYP/6-311G(d,p) level reveal that the reaction can be occurred as going through seven turning points defined as methyl benzoylformate 8-CF†FF†TSFC†[CF†]-0: methyl benzoate + carbon monoxide. The molecular mechanism can be categorized in three fundamental sections A) heterolytic rupture of O3C8 bond and detachment of methoxy part; B) formation of O3C7 bond via donation bond formation mechanism; and C) heterolytic rupture of C7C8 bond and detachment of carbon monoxide. The electron density ρ(3,-1)(r) in the region of bond forming and breaking increases and decreases along the reaction course to reflect bond strengthening and weakening, respectively. AIM parameters revealed that so long as chemical bonds are unformed/ruptured, interactions at related BCPs are covalence in nature, otherwise the nature of chemical bonds are strong shared covalence.


Assuntos
Glioxilatos/química , Ácidos Mandélicos/química , Transição de Fase , Teoria Quântica , Termodinâmica , Cinética , Modelos Químicos
12.
Molecules ; 23(12)2018 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-30513981

RESUMO

Alcalase was immobilized on glyoxyl 4% CL agarose beads. This permitted to have Alcalase preparations with 50% activity retention versus Boc-l-alanine 4-nitrophenyl ester. However, the recovered activity versus casein was under 20% at 50 °C, as it may be expected from the most likely area of the protein involved in the immobilization. The situation was different at 60 °C, where the activities of immobilized and free enzyme became similar. The chemical amination of the immobilized enzyme or the treatment of the enzyme with glutaraldehyde did not produce any significant stabilization (a factor of 2) with high costs in terms of activity. However, the modification with glutaraldehyde of the previously aminated enzyme permitted to give a jump in Alcalase stability (e.g., with most than 80% of enzyme activity retention for the modified enzyme and less than 30% for the just immobilized enzyme in stress inactivation at pH 7 or 9). This preparation could be used in the hydrolysis of casein at pH 9 even at 67 °C, retaining around 50% of the activity after 5 hydrolytic cycles when the just immobilized preparation was almost inactive after 3 cycles. The modified enzyme can be reused in hydrolysis of casein at 45 °C and pH 9 for 6 cycles (6 h) without any decrease in enzyme activity.


Assuntos
Caseínas/metabolismo , Enzimas Imobilizadas/química , Glutaral/química , Subtilisinas/química , Subtilisinas/metabolismo , Reagentes para Ligações Cruzadas/química , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Etilenodiaminas/química , Glioxilatos/química , Concentração de Íons de Hidrogênio , Hidrólise , Sefarose/química , Temperatura
13.
Bioconjug Chem ; 29(10): 3285-3292, 2018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30234289

RESUMO

Glyoxylate-mediated transamination (GT) is a classic, potentially general, and N-terminus-specific protein modification method useful for the preparation of bioconjugates. However, there is a lack of information on whether and how readily a particular N-terminal amino acid (in the context of a peptide chain) can be converted to the 2-oxoacyl moiety under GT conditions. Here, we conducted a systematic investigation of GT using membrane-bound dipeptide arrays that include all the 400 possible dipeptide combinations of the 20 genetically encoded amino acids. This colorimetric method offers a convenient way to assess the GT reaction tendency of N-terminal residues by the naked eye. It also provides interesting information about the effect of the second residues on GT, which has not been reported previously. In addition, we also designed a proteomics approach to study GT in solution using tryptic peptide mixtures, which not only confirmed many of our findings in peptide array assays but also revealed potential side reaction products. Taken together, our studies will make the future use of GT for protein modification in a much more predictable way.


Assuntos
Glioxilatos/química , Peptídeos/química , Proteômica , Aminação , Biomimética , Colorimetria/métodos , Misturas Complexas , Dipeptídeos/química , Mapeamento de Peptídeos , Tripsina/química
14.
Life Sci ; 207: 80-89, 2018 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-29852189

RESUMO

AIMS: The cytotoxic response of an intermediate metabolite glyoxylate (Glx) on colon carcinoma has been evaluated in vitro. MAIN METHODS: The anti-proliferative effect of Glx was assessed on HT-29 and HCT-116 cells by performing MTT assay as well as beta-hexosaminidase assay. Evaluation of apoptotic event of Glx treated cells was measured by flow cytometry using annexin-V/PI staining. The mitochondrial membrane potential and level of ROS were estimated using DiOC6(3)/CCCP and DCFH-DA method, respectively. The assessment of catalase, LDH and IDH were performed. KEY FINDINGS: The results of MTT assay indicated that treatment with Glx significantly inhibited the proliferation of HT-29 and HCT-116 cells. Beta-hexosaminidase assay also confirmed the inhibition of cellular viability. The dose-dependent Glx treatment indicated lowering the colony forming ability of HT-29 and HCT-116 cells. Flow cytometric data demonstrated the significant increment of late apoptotic event after Glx treatment. In addition, substantial LDH activity was noticed in both the colon cancer cells whereas the IDH activity was unaltered after extra-cellular addition of Glx. Further, dissipation of mitochondrial membrane potential and subsequently elevated ROS generation was also detected in the Glx treated colon cancer cells. However, gradual elevation of catalase activities indicated that Glx treatment on colon cancer cells exhibit oxidative stress. SIGNIFICANCE: This study depicts that supra-physiological concentration of Glx inhibits the proliferation of colon cancer cells due to oxidative stress.


Assuntos
Carcinoma/metabolismo , Neoplasias do Colo/metabolismo , Glioxilatos/química , Estresse Oxidativo , Antineoplásicos/farmacologia , Apoptose , Carcinoma/tratamento farmacológico , Catalase/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Células HCT116 , Células HT29 , Humanos , Potencial da Membrana Mitocondrial , Espécies Reativas de Oxigênio/metabolismo
15.
J Biotechnol ; 278: 34-38, 2018 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-29738785

RESUMO

l-phenylglycine is a rare non-proteinogenic amino acid, which only occurs in a few natural compounds, such as the streptogramin antibiotics pristinamycin I and virginiamycin S or the bicyclic peptide antibiotic dityromycin. Here we report on the biochemical characterization of the aminotransferase PglE that catalyzes the transamination from phenylglyoxylate to l-phenylglycine, which represents the final reaction step during phenylglycine biosynthesis. Enzyme assays with the purified PglE enzyme revealed that l-phenylalanine is used as an amino group donor for the transamination reaction, leading to the formation of phenylpyruvate, which may re-enter phenylglycine biosynthesis as a precursor. Based on these results, we postulate a novel l-phenylglycine biosynthetic pathway.


Assuntos
Proteínas de Bactérias/metabolismo , Glicina/análogos & derivados , Streptomyces/enzimologia , Transaminases/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Glicina/análise , Glicina/química , Glicina/metabolismo , Glioxilatos/química , Glioxilatos/metabolismo , Ácidos Mandélicos/química , Ácidos Mandélicos/metabolismo , Redes e Vias Metabólicas , Streptomyces/genética , Streptomyces/metabolismo , Transaminases/química , Transaminases/genética
16.
Macromol Rapid Commun ; 39(11): e1800173, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29700924

RESUMO

The temperature-dependent depolymerization of self-immolative poly(ethyl glyoxylate) (PEtG) capped with triphenylmethyl (trityl) groups is studied and its potential application for smart packaging is explored. PEtGs with four different trityl end-caps are prepared and found to undergo depolymerization to volatile products from the solid state at different rates depending on temperature and the electron-donating substituents on the trityl aromatic rings. Through the incorporation of hydrophobic dyes including Nile red and IR-780, the depolymerization is visualized as a color change of the dye as it changes from a dispersed to aggregated state. The ability of this platform to provide information on thermal history through an easily readable signal makes it promising in smart packaging applications for sensitive products such a food and other cargo that is susceptible to degradation.


Assuntos
Glioxilatos/química , Polímeros/química , Indóis/química , Oxazinas/química , Embalagem de Produtos , Espectrofotometria , Temperatura
17.
J Inorg Biochem ; 183: 84-93, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29604496

RESUMO

Two unspecific peroxygenases (UPO, EC 1.11.2.1) from the basidiomycetous fungi Marasmius rotula and Marasmius wettsteinii oxidized steroids with hydroxyacetyl and hydroxyl functionalities at C17 - such as cortisone, Reichstein's substance S and prednisone - via stepwise oxygenation and final fission of the side chain. The sequential oxidation started with the hydroxylation of the terminal carbon (C21) leading to a stable geminal alcohol (e.g. cortisone 21-gem-diol) and proceeded via a second oxygenation resulting in the corresponding α-ketocarboxylic acid (e.g. cortisone 21-oic acid). The latter decomposed under formation of adrenosterone (4-androstene-3,11,17-trione) as well as formic acid and carbonic acid (that is in equilibrium with carbon dioxide); fission products comprising two carbon atoms such as glycolic acid or glyoxylic acid were not detected. Protein models based on the crystal structure data of MroUPO (Marasmius rotula unspecific peroxygenase) revealed that the bulky cortisone molecule suitably fits into the enzyme's access channel, which enables the heme iron to come in close contact to the carbons (C21, C20) of the steroidal side chain. ICP-MS analysis of purified MroUPO confirmed the presence of magnesium supposedly stabilizing the porphyrin ring system.


Assuntos
Corticosteroides/química , Corticosteroides/metabolismo , Oxigenases de Função Mista/metabolismo , Catálise , Glicolatos/química , Glioxilatos/química , Espectrometria de Massas , Oxirredução , Especificidade por Substrato
18.
Nat Commun ; 9(1): 91, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29311556

RESUMO

The development of metabolic approaches towards understanding the origins of life, which have focused mainly on the citric acid (TCA) cycle, have languished-primarily due to a lack of experimentally demonstrable and sustainable cycle(s) of reactions. We show here the existence of a protometabolic analog of the TCA involving two linked cycles, which convert glyoxylate into CO2 and produce aspartic acid in the presence of ammonia. The reactions proceed from either pyruvate, oxaloacetate or malonate in the presence of glyoxylate as the carbon source and hydrogen peroxide as the oxidant under neutral aqueous conditions and at mild temperatures. The reaction pathway demonstrates turnover under controlled conditions. These results indicate that simpler versions of metabolic cycles could have emerged under potential prebiotic conditions, laying the foundation for the appearance of more sophisticated metabolic pathways once control by (polymeric) catalysts became available.


Assuntos
Dióxido de Carbono/química , Glioxilatos/química , Modelos Químicos , Origem da Vida , Ácido Oxaloacético/química , Ácido Pirúvico/química , Amônia/química , Ácido Aspártico/química , Descarboxilação , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Cinética , Malonatos/química , Redes e Vias Metabólicas , Oxirredução
19.
Arch Microbiol ; 200(5): 719-727, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29380014

RESUMO

A link between carbon and nitrogen metabolism is important for serving as metabolic ancillary reactions. Here, we identified and characterized the alanine dehydrogenase gene in Aphanothece halophytica (ApalaDH) that is involved in alanine assimilation/dissimilation. Functional analysis revealed that ApalaDH encodes a bifunctional protein catalyzing the reversible reaction of pyruvate to L-alanine via its pyruvate reductive aminase (PvRA) activity, the reaction of L-alanine to pyruvate via its alanine oxidative dehydrogenase activity, and the non-reversible reaction of glyoxylate to glycine via its glyoxylate reductive aminase (GxRA) activity. Kinetic analysis showed the lowest affinity for pyruvate followed by L-alanine and glyoxylate with a Km of 0.22 ± 0.02, 0.72 ± 0.04, and 1.91 ± 0.43 mM, respectively. ApalaDH expression was upregulated by salt. Only PvRA and GxRA activities were detected in vivo and both activities increased about 1.2- and 2.7-fold upon salt stress. These features implicate that the assimilatory/dissimilatory roles of ApAlaDH are not only selective for L-alanine and pyruvate, but also, upon salt stress, can catabolize glyoxylate to generate glycine.


Assuntos
Alanina Desidrogenase/genética , Proteínas de Bactérias/genética , Cianobactérias/enzimologia , Alanina/química , Alanina Desidrogenase/biossíntese , Alanina Desidrogenase/química , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Cianobactérias/genética , Indução Enzimática , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Glioxilatos/química , Concentração de Íons de Hidrogênio , Cinética , Ácido Pirúvico/química , Tolerância ao Sal , Especificidade por Substrato
20.
Appl Microbiol Biotechnol ; 102(2): 773-787, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29177938

RESUMO

Sucrose synthases (SuSys) have been attracting great interest in recent years in industrial biocatalysis. They can be used for the cost-effective production of uridine 5'-diphosphate glucose (UDP-glucose) or its in situ recycling if coupled to glycosyltransferases on the production of glycosides in the food, pharmaceutical, nutraceutical, and cosmetic industry. In this study, the homotetrameric SuSy from Acidithiobacillus caldus (SuSyAc) was immobilized-stabilized on agarose beads activated with either (i) glyoxyl groups, (ii) cyanogen bromide groups, or (iii) heterogeneously activated with both glyoxyl and positively charged amino groups. The multipoint covalent immobilization of SuSyAc on glyoxyl agarose at pH 10.0 under optimized conditions provided a significant stabilization factor at reaction conditions (pH 5.0 and 45 °C). However, this strategy did not stabilize the enzyme quaternary structure. Thus, a post-immobilization technique using functionalized polymers, such as polyethyleneimine (PEI) and dextran-aldehyde (dexCHO), was applied to cross-link all enzyme subunits. The coating of the optimal SuSyAc immobilized glyoxyl agarose with a bilayer of 25 kDa PEI and 25 kDa dexCHO completely stabilized the quaternary structure of the enzyme. Accordingly, the combination of immobilization and post-immobilization techniques led to a biocatalyst 340-fold more stable than the non-cross-linked biocatalyst, preserving 60% of its initial activity. This biocatalyst produced 256 mM of UDP-glucose in a single batch, accumulating 1 M after five reaction cycles. Therefore, this immobilized enzyme can be of great interest as a biocatalyst to synthesize UDP-glucose.


Assuntos
Acidithiobacillus/enzimologia , Enzimas Imobilizadas/metabolismo , Glucosiltransferases/metabolismo , Glicosiltransferases/metabolismo , Uridina Difosfato Glucose/biossíntese , Proteínas de Bactérias/metabolismo , Biocatálise , Biotecnologia , Brometo de Cianogênio/química , Estabilidade Enzimática , Glicômica , Glioxilatos/química , Concentração de Íons de Hidrogênio , Multimerização Proteica , Sefarose/química , Temperatura
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