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1.
Molecules ; 27(19)2022 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-36234786

RESUMO

Herein, we present a qualitative and quantitative analysis of the compositions of plasmalogens and phospholipids (PLs) in dried big head shrimp (Solenocera melantho), opossum shrimp (Neomysis awatschensis), mussel (Mytilus galloprovincialis), and sea cucumber (Apostichopus japonicus). We also analyze the fatty acid composition of the extracted lipids, phosphatidyl choline (PtdCho), and plasmalogen choline (PlsCho) from each sample. In big head shrimp, opossum shrimp, and mussel, phosphatidyl choline (PtdCho) was the most abundant PL at 1677.9, 1603, and 1661.6 mg/100 g of dried sample, respectively, whereas the most abundant PL in sea cucumber was PlsCho (206.9 mg/100 g of dried sample). In all four samples, plasmalogen ethanolamine (PlsEtn) was higher than phosphatidyl ethanolamine (PtdEtn). The content (mg/100 g of dried sample) of PlsCho was highest in mussel (379.0), and it was higher in big head shrimp (262.3) and opossum shrimp (245.6) than sea cucumber (206.9). The contents (mg/100 g of dried sample) of PlsEtn were in the order of mussel (675.4) > big head shrimp (629.5) > opossum shrimp (217.9) > sea cucumber (51.5). For analyzing the fatty acids at the sn-2 position of PlsCho, the consecutive treatment with phospholipase A1, solid phase extraction, thin-layer chromatography (TLC), and GC-FID were applied. The most abundant fatty acid was eicosapentaenoic acid (EPA, C20:5, n-3) in big head shrimp and sea cucumber, palmitoleic acid (C16:1, n-7) in opossum shrimp, and docosadienoic acid (C22:2, n-6) in mussel.


Assuntos
Bivalves , Pepinos-do-Mar , Animais , Colina , Ácido Eicosapentaenoico , Etanolaminas , Ácidos Graxos/análise , Espectroscopia de Ressonância Magnética , Gambás , Fosfatidilcolinas , Fosfolipases , Fosfolipídeos/análise , Plasmalogênios/química
2.
Anal Biochem ; 597: 113688, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32194075

RESUMO

Glutaric acid is a precursor of a plasticizer that can be used for the production of polyester amides, ester plasticizer, corrosion inhibitor, and others. Glutaric acid can be produced either via bioconversion or chemical synthesis, and some metabolites and intermediates are produced during the reaction. To ensure reaction efficiency, the substrates, intermediates, and products, especially in the bioconversion system, should be closely monitored. Until now, high performance liquid chromatography (HPLC) has generally been used to analyze the glutaric acid-related metabolites, although it demands separate time-consuming derivatization and non-derivatization analyses. To substitute for this unreasonable analytical method, we applied herein a gas chromatography - mass spectrometry (GC-MS) method with ethyl chloroformate (ECF) derivatization to simultaneously monitor the major metabolites. We determined the suitability of GC-MS analysis using defined concentrations of six metabolites (l-lysine, cadaverine, 5-aminovaleric acid, 2-oxoglutaric acid, glutamate, and glutaric acid) and their mass chromatograms, regression equations, regression coefficient values (R2), dynamic ranges (mM), and retention times (RT). This method successfully monitored the production process in complex fermentation broth.


Assuntos
Ésteres do Ácido Fórmico/metabolismo , Glutaratos/metabolismo , Lisina/metabolismo , Cromatografia Líquida de Alta Pressão , Fermentação , Ésteres do Ácido Fórmico/química , Cromatografia Gasosa-Espectrometria de Massas , Glutaratos/química , Lisina/química , Estrutura Molecular
3.
J Ind Microbiol Biotechnol ; 47(12): 1045-1057, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33259029

RESUMO

Psychrophilic bacteria, living at low and mild temperatures, can contribute significantly to our understanding of microbial responses to temperature, markedly occurring in the bacterial membrane. Here, a newly isolated strain, Pseudomonas sp. B14-6, was found to dynamically change its unsaturated fatty acid and cyclic fatty acid content depending on temperature which was revealed by phospholipid fatty acid (PLFA) analysis. Genome sequencing yielded the sequences of the genes Δ-9-fatty acid desaturase (desA) and cyclopropane-fatty acid-acyl-phospholipid synthase (cfa). Overexpression of desA in Escherichia coli led to an increase in the levels of unsaturated fatty acids, resulting in decreased membrane hydrophobicity and increased fluidity. Cfa proteins from different species were all found to promote bacterial growth, despite their sequence diversity. In conclusion, PLFA analysis and genome sequencing unraveled the temperature-related behavior of Pseudomonas sp. B14-6 and the functions of two membrane-related enzymes. Our results shed new light on temperature-dependent microbial behaviors and might allow to predict the consequences of global warming on microbial communities.


Assuntos
Ácidos Graxos Insaturados , Pseudomonas , Sequência de Aminoácidos , Bactérias/metabolismo , Sequência de Bases , Ciclopropanos , Escherichia coli/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Ácido Graxo Sintases/genética , Ácidos Graxos/análise , Ácidos Graxos Insaturados/metabolismo , Pseudomonas/metabolismo , Temperatura
4.
Bioprocess Biosyst Eng ; 43(5): 909-918, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31989256

RESUMO

Bacteria have evolved a defense system to resist external stressors, such as heat, pH, and salt, so as to facilitate survival in changing or harsh environments. However, the specific mechanisms by which bacteria respond to such environmental changes are not completely elucidated. Here, we used halotolerant bacteria as a model to understand the mechanism conferring high tolerance to NaCl. We screened for genes related to halotolerance in Halomonas socia, which can provide guidance for practical application. Phospholipid fatty acid analysis showed that H. socia cultured under high osmotic pressure produced a high portion of cyclopropane fatty acid derivatives, encoded by the cyclopropane-fatty acid-acyl phospholipid synthase gene (cfa). Therefore, H. socia cfa was cloned and introduced into Escherichia coli for expression. The cfa-overexpressing E. coli strain showed better growth, compared with the control strain under normal cultivation condition as well as under osmotic pressure (> 3% salinity). Moreover, the cfa-overexpressing E. coli strain showed 1.58-, 1.78-, 3.3-, and 2.19-fold higher growth than the control strain in the presence of the inhibitors furfural, 4-hydroxybenzaldehyde, vanillin, and acetate from lignocellulosic biomass pretreatment, respectively. From a practical application perspective, cfa was co-expressed in E. coli with the polyhydroxyalkanoate (PHA) synthetic operon of Ralstonia eutropha using synthetic and biosugar media, resulting in a 1.5-fold higher in PHA production than that of the control strain. Overall, this study demonstrates the potential of the cfa gene to boost cell growth and production even in heterologous strains under stress conditions.


Assuntos
Proteínas de Bactérias , Escherichia coli , Expressão Gênica , Metiltransferases , Microrganismos Geneticamente Modificados , Pressão Osmótica/efeitos dos fármacos , Cloreto de Sódio/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Cupriavidus necator/enzimologia , Cupriavidus necator/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Halomonas/enzimologia , Halomonas/genética , Metiltransferases/biossíntese , Metiltransferases/genética , Microrganismos Geneticamente Modificados/enzimologia , Microrganismos Geneticamente Modificados/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
5.
Int J Cancer ; 144(2): 389-401, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29978469

RESUMO

PIK3CA is a frequently mutated gene in cancer, including about ~15 to 20% of colorectal cancers (CRC). PIK3CA mutations lead to activation of the PI3K/AKT/mTOR signaling pathway, which plays pivotal roles in tumorigenesis. Here, we investigated the mechanism of resistance of PIK3CA-mutant CRC cell lines to gedatolisib, a dual PI3K/mTOR inhibitor. Out of a panel of 29 CRC cell lines, we identified 7 harboring one or more PIK3CA mutations; of these, 5 and 2 were found to be sensitive and resistant to gedatolisib, respectively. Both of the gedatolisib-resistant cell lines expressed high levels of active glycogen synthase kinase 3-beta (GSK3ß) and harbored the same frameshift mutation (c.465_466insC; H155fs*) in TCF7, which encodes a positive transcriptional regulator of the WNT/ß-catenin signaling pathway. Inhibition of GSK3ß activity in gedatolisib-resistant cells by siRNA-mediated knockdown or treatment with a GSK3ß-specific inhibitor effectively reduced the activity of molecules downstream of mTOR and also decreased signaling through the WNT/ß-catenin pathway. Notably, GSK3ß inhibition rendered the resistant cell lines sensitive to gedatolisib cytotoxicity, both in vitro and in a mouse xenograft model. Taken together, these data demonstrate that aberrant regulation of WNT/ß-catenin signaling and active GSK3ß induced by the TCF7 frameshift mutation cause resistance to the dual PI3K/mTOR inhibitor gedatolisib. Cotreatment with GSK3ß inhibitors may be a strategy to overcome the resistance of PIK3CA- and TCF7-mutant CRC to PI3K/mTOR-targeted therapies.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Morfolinas/farmacologia , Triazinas/farmacologia , Via de Sinalização Wnt/fisiologia , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Humanos , Camundongos , Mutação , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
6.
J Oleo Sci ; 73(4): 455-466, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38556280

RESUMO

In this study, the phospholipid species [i.e., phosphatidylethanolamine (PE), phosphatidylcholine (PC), and sphingomyelin (SM)] in human milk (HM) were compared according to their fatty acid (FA) composition. 34 HM samples were collected and classified into three groups (A < B < C) according to their fat content. Stearic acid (C18:0) was the main FA in PE, PC, and SM. The highest concentrations of arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosapentaenoic acid (DPA) were observed in PE, whereas docosahexaenoic acid (DHA) was predominant in SM. Although PC exhibited the highest total saturated FAs (SFAs) and PE contained the highest unsaturated FAs (UFAs), very long-chain SFAs and monounsaturated FAs (MUFAs) were preferentially distributed in SM. PC and SM had higher saturation compared to PE. Regarding the effect of the fat content of HM on the FA composition of the phospholipid species, a limited influence was observed on the composition of SFAs and MUFAs of PE, SM, and particularly PC. However, a more pronounced effect on the composition of polyunsaturated FAs (PUFAs) in phospholipids was observed, especially for linoleic acid (LA), α-linolenic acid (ALA), EPA, and DHA, indicating that the composition of FAs in the phospholipid species was probably affected by the maternal diet.


Assuntos
Ácidos Graxos , Fosfolipídeos , Humanos , Leite Humano , Ácido Linoleico , Ácidos Docosa-Hexaenoicos , Ácido Eicosapentaenoico , Fosfatidilcolinas , República da Coreia
7.
Toxics ; 10(5)2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35622614

RESUMO

Chemical accidents in rivers may be triggered by natural or anthropogenic causes and refer to the flow of large quantities of hazardous chemicals into rivers. In South Korea, domestic water is sourced from large rivers, such as the Nakdong River. However, owing to rapid industrialization, industrial facilities have become heavily concentrated in the middle and upper reaches of the Nakdong River. Therefore, severe problems could arise if harmful chemicals are leaked from industrial facilities into the river, and this contaminated river water is supplied to cities. Quantitative evaluation based on instrumental analysis during chemical accidents and prediction research based on modeling is actively being conducted however, research on the initial response is insufficient. Therefore, in this study, the variations in pH and EC were analyzed according to their chemical concentrations for seven chemicals. These seven chemicals are designated accident-preparedness substances that frequently cause chemical spills in South Korea. Additionally, we evaluated the possibility of identifying unknown substances by comparing the variations in pH and EC and statistics while diluting unknown substances. Thus, the potential of pH and EC as alternative indicators for detecting and identifying chemicals was evaluated in this study. NaF, NH4HF2, NaCN, and NH4OH were classified by comparing their spatial distributions in a pH-EC relation curve. However, H2SO4, HCl, and SOCl2 showed similar spatial distributions in the pH-EC curves and were difficult to identify. The results of this study provide information for chemical detection and identification using alternative sensors that permit easy and rapid field measurements in the event of a chemical spill and could be used as preliminary data for rapidly responding to accidents.

8.
Oncogene ; 41(14): 2026-2038, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35173310

RESUMO

The emergence of RAS/RAF mutant clone is the main feature of EGFR inhibitor resistance in KRAS wild-type colon cancer. However, its molecular mechanism is thought to be multifactorial, mainly due to cellular heterogeneity. In order to better understand the resistance mechanism in a single clone level, we successfully isolated nine cells with cetuximab-resistant (CR) clonality from in vitro system. All CR cells harbored either KRAS or BRAF mutations. Characteristically, these cells showed a higher EMT (Epithelial to mesenchymal transition) signature, showing increased EMT markers such as SNAI2. Moreover, the expression level of CXCL1/5, a secreted protein, was significantly higher in CR cells compared to the parental cells. In these CR cells, CXCL1/5 expression was coordinately regulated by SNAI2/NFKB and transactivated EGFR through CXCR/MMPI/EGF axis via autocrine singling. We also observed that combined cetuximab/MEK inhibitor not only showed growth inhibition but also reduced the secreted amounts of CXCL1/5. We further found that serum CXCL1/5 level was positively correlated with the presence of RAS/RAF mutation in colon cancer patients during cetuximab therapy, suggesting its role as a biomarker. These data indicated that the application of serum CXCL1/5 could be a potential serologic biomarker for predicting resistance to EGFR therapy in colorectal cancer.


Assuntos
Neoplasias Colorretais , Transição Epitelial-Mesenquimal , Cetuximab/uso terapêutico , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Quimiocina CXCL5/genética , Quimiocina CXCL5/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Humanos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética
9.
Int J Biol Macromol ; 167: 151-159, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33249160

RESUMO

Poly(3-hydroxybutyrate) (PHB) is a common polyhydroxyalkanoate (PHA) with potential as an alternative for petroleum-based plastics. Previously, we reported a new strain, Halomonas sp. YLGW01, which hyperproduces PHB with 94% yield using fructose. In this study, we examined the PHB production machinery of Halomonas sp. YLGW01 in more detail by deep-genome sequencing, which revealed a 3,453,067-bp genome with 65.1% guanine-cytosine content and 3054 genes. We found two acetyl-CoA acetyltransferases (Acetoacetyl-CoA thiolase, PhaA), one acetoacetyl-CoA reductase (PhaB), two PHB synthases (PhaC1, PhaC2), PHB depolymerase (PhaZ), and Enoyl-CoA hydratase (PhaJ) in the genome, along with two fructose kinases and fructose transporter systems, including the phosphotransferase system (PTS) and ATP-binding transport genes. We then examined the PHB production by Halomonas sp. YLGW01 using high-fructose corn syrup (HFCS) containing fructose, glucose, and sucrose in sea water medium, resulting in 7.95 ± 0.11 g/L PHB (content, 67.39 ± 0.34%). PHB was recovered from Halomonas sp. YLGW01 using different detergents; the use of Tween 20 and SDS yielded micro-sized granules with high purity. Overall, these results reveal the distribution of PHB synthetic genes and the sugar utilization system in Halomonas sp. YLGW01 and suggest a possible method for PHB recovery.


Assuntos
Meios de Cultura , Fermentação , Halomonas/metabolismo , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Açúcares/química , Açúcares/metabolismo , Biomassa , Vias Biossintéticas/genética , Biologia Computacional/métodos , Genoma Bacteriano , Halomonas/genética , Anotação de Sequência Molecular , Sequenciamento Completo do Genoma
10.
Polymers (Basel) ; 13(9)2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33925903

RESUMO

Arctic bacteria employ various mechanisms to survive harsh conditions, one of which is to accumulate carbon and energy inside the cell in the form of polyhydroxyalkanoate (PHA). Whole-genome sequencing of a new Arctic soil bacterium Pseudomonas sp. B14-6 revealed two PHA-production-related gene clusters containing four PHA synthase genes (phaC). Pseudomonas sp. B14-6 produced poly(6% 3-hydroxybutyrate-co-94% 3-hydroxyalkanoate) from various carbon sources, containing short-chain-length PHA (scl-PHA) and medium-chain-length PHA (mcl-PHA) composed of various monomers analyzed by GC-MS, such as 3-hydroxybutyrate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, 3-hydroxydodecenoic acid, 3-hydroxydodecanoic acid, and 3-hydroxytetradecanoic acid. By optimizing the PHA production media, we achieved 34.6% PHA content using 5% fructose, and 23.7% PHA content using 5% fructose syrup. Differential scanning calorimetry of the scl-co-mcl PHA determined a glass transition temperature (Tg) of 15.3 °C, melting temperature of 112.8 °C, crystallization temperature of 86.8 °C, and 3.82% crystallinity. In addition, gel permeation chromatography revealed a number average molecular weight of 3.6 × 104, weight average molecular weight of 9.1 × 104, and polydispersity index value of 2.5. Overall, the novel Pseudomonas sp. B14-6 produced a polymer with high medium-chain-length content, low Tg, and low crystallinity, indicating its potential use in medical applications.

11.
J Microbiol Biotechnol ; 31(2): 250-258, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33148940

RESUMO

Among various species of marine bacteria, those belonging to the genus Halomonas have several promising applications and have been studied well. However, not much information has been available on their antibiotic resistance. In our efforts to learn about the antibiotic resistance of strain Halomonas socia CKY01, which showed production of various hydrolases and growth promotion by osmolytes in previous study, we found that it exhibited resistance to multiple antibiotics including kanamycin, ampicillin, oxacillin, carbenicillin, gentamicin, apramycin, tetracycline, and spectinomycin. However, the H. socia CKY01 resistance pattern to kanamycin, gentamicin, apramycin, tetracycline, and spectinomycin differed in the presence of 10% NaCl and 1% NaCl in the culture medium. To determine the mechanism underlying this NaCl concentration-dependent antibiotic resistance, we compared four aminoglycoside resistance genes under different salt conditions while also performing time-dependent reverse transcription PCR. We found that the aph2 gene encoding aminoglycoside phosphotransferase showed increased expression under the 10% rather than 1% NaCl conditions. When these genes were overexpressed in an Escherichia coli strain, pETDuet-1::aph2 showed a smaller inhibition zone in the presence of kanamycin, gentamicin, and apramycin than the respective control, suggesting aph2 was involved in aminoglycoside resistance. Our results demonstrated a more direct link between NaCl and aminoglycoside resistance exhibited by the H. socia CKY01 strain.


Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Halomonas/efeitos dos fármacos , Cloreto de Sódio/metabolismo , Aminoglicosídeos/análise , Antibacterianos/análise , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Gentamicinas/farmacologia , Halomonas/genética , Halomonas/metabolismo , Canamicina/farmacologia , Canamicina Quinase/genética , Canamicina Quinase/metabolismo , Nebramicina/análogos & derivados , Nebramicina/farmacologia , Cloreto de Sódio/análise
12.
J Biotechnol ; 322: 21-28, 2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-32653639

RESUMO

Bacteria from the genus Halomonas hold promise in biotechnology as sources of salt-tolerant enzymes, biosurfactants, biopolymers, osmolytes, and as actors in bioremediation processes. In a previous work, we have identified Halomonas socia strain CKY01 having various hydrolase activities. Here, we aimed to study the survival strategies of marine bacteria. A deep genome sequencing study of H. socia CKY01 has revealed 4627 genes reaching 4,753,299 bp with 64 % of GC content. This strain produced polyhydroxybutyrate (PHB) having one gene clusters having phaC and phasin, and it has several genes responsible for the uptake, synthesis, and transport of the osmolytes such as betaine, choline, ectoine, carnitine, and proline in the bacterial genome. The addition of 60 mM glutamate, 60 mM proline and 60 mM ectoine enhanced growth 300.8 %, 294.2 % and 235.0 %, respectively, under 10 % saline conditions. In particular, ectoine and proline increased salt resistance and allowed cells to survive in more than 15 % NaCl. By combining experimental and genome sequencing data, we have investigated the importance of osmolytes on the survival of this Halomonas strain.


Assuntos
Genoma Bacteriano/genética , Halomonas , Tolerância ao Sal , Diamino Aminoácidos/farmacologia , Halomonas/efeitos dos fármacos , Halomonas/genética , Halomonas/fisiologia , Concentração Osmolar , Prolina/farmacologia , Salinidade , Tolerância ao Sal/efeitos dos fármacos , Tolerância ao Sal/fisiologia , Cloreto de Sódio/farmacologia , Sequenciamento Completo do Genoma
13.
Sci Total Environ ; 710: 136282, 2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-31923664

RESUMO

We evaluated the production of Spirulina sp. (microalgae)-derived biochars (SPAL-BCs) at different pyrolysis temperatures for the removal of an emerging water contaminant, tetracycline (TC). Physicochemical properties of SPAL-BCs were characterized and related with their capacity to adsorb TC. Increasing pyrolysis temperatures led to higher aromaticity, higher hydrophobicity, and higher specific surface area. In particular, SPAL-BC750 possessed the highest hydrophobicity, various strong crystallizations (i.e., calcite, hydroxyapatite, and rhenanite) and functional groups (i.e., CH2, CN, CO, and CO32-), which may be associated with high TC adsorption. SPAL-BC750 also presented the highest TC adsorption capacity (132.8 mg TC/g biochar) via batch experimentation because of hydrophobic, π-π interactions, functional groups, and metal complexation. The best fitting isotherm and kinetic models of TC adsorption by SPAL-BC750 were the Langmuir and pseudo-first order models, respectively. SPAL-BCs obtained as a by-product of pyrolysis may be an economical and potentially valuable adsorbent for aqueous antibiotic removal.


Assuntos
Microalgas , Spirulina , Adsorção , Antibacterianos , Carvão Vegetal , Temperatura , Tetraciclina , Poluentes Químicos da Água
14.
J Microbiol Biotechnol ; 30(5): 785-792, 2020 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-32482946

RESUMO

L-Theanine, found in green tea leaves has been shown to positively affect immunity and relaxation in humans. There have been many attempts to produce L-theanine through enzymatic synthesis to overcome the limitations of traditional methods. Among the many genes coding for enzymes in the L-theanine biosynthesis, glutamylmethylamide synthetase (GMAS) exhibits the greatest possibility of producing large amounts of production. Thus, GMAS from Methylovorus mays No. 9 was overexpressed in several strains including vectors with different copy numbers. BW25113(DE3) cells containing the pET24ma::gmas was selected for strains. The optimal temperature, pH, and metal ion concentration were 50°C, 7, and 5 mM MnCl2, respectively. Additionally, ATP was found to be an important factor for producing high concentration of L-theanine so several strains were tested during the reaction for ATP regeneration. Bakers yeast was found to decrease the demand for ATP most effectively. Addition of potassium phosphate source was demonstrated by producing 4-fold higher L-theanine. To enhance the conversion yield, GMAS was additionally overexpressed in the system. A maximum of 198 mM L-theanine was produced with 16.5 mmol/l/h productivity. The whole-cell reaction involving GMAS has greatest potential for scale-up production of L-theanine.


Assuntos
Proteínas de Bactérias/metabolismo , Carbono-Nitrogênio Ligases/metabolismo , Escherichia coli/metabolismo , Glutamatos/metabolismo , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Carbono-Nitrogênio Ligases/genética , Meios de Cultura/química , Meios de Cultura/metabolismo , Escherichia coli/genética , Engenharia Metabólica , Methylophilaceae/enzimologia , Methylophilaceae/genética
15.
Enzyme Microb Technol ; 133: 109446, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31874692

RESUMO

Glutaric acid is a C5 dicarboxylic acid that can be used as a building block for bioplastics. Although high concentrations of glutaric acid can be produced by fermentation or bioconversion, a large amount of α-ketoglutaric acid (α-KG) is necessary to accept the amine group from 5-aminovaleric acid. To decrease the demand for α-KG, we introduced l-glutamate oxidase (GOX) from Streptomyces mobaraensis in our previous system for cofactor regeneration in combination with a glutaric acid production system from 5-aminovaleric acid. To enhance glutaric acid production, critical factors were optimized such as the expression vector, pH, temperature, and cell ratio. As a result, the demand for α-KG was decreased by more than 6-fold under optimized conditions. Additionally, the effect of catalase was also demonstrated by blocking the degradation of α-KG to succinic acid because of the hydrogen peroxide. Finally, 468.5 mM glutaric acid was produced from 800 mM 5-aminovaleric acid using only 120 mM α-KG. Moreover, this system containing davBA, gabTD-nox, and gox can be applied to produce glutaric acid from L-lysine by reusing α-KG with GOX. This improved cofactor regeneration system has a potential to apply much larger production of glutaric acid.


Assuntos
Aminoácido Oxirredutases/metabolismo , Escherichia coli/enzimologia , Glutaratos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Catalase/metabolismo , Escherichia coli/genética , Fermentação , Engenharia Metabólica/métodos
16.
Enzyme Microb Technol ; 140: 109643, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32912695

RESUMO

Pipecolic acid, a non-proteinogenic amino acid, is a metabolite in lysine metabolism and a key chiral precursor in local anesthesia and macrolide antibiotics. To replace the environmentally unfriendly chemical production or preparation procedure of pipecolic acid, many biological synthetic routes have been studied for a long time. Among them, synthesis by lysine cyclodeaminase (LCD), encoded by pipA, has several advantages, including stability of enzyme activity and NAD+ self-regeneration. Thus, we selected this enzyme for pipecolic acid biosynthesis in a whole-cell bioconversion. To construct a robust pipecolic acid production system, we investigated important conditions including expression vector, strain, culture conditions, and other reaction parameters. The most important factor was the introduction of multiple pipA genes into the whole-cell system. As a result, we produced 724 mM pipecolic acid (72.4 % conversion), and the productivity was 0.78 g/L/h from 1 M l-lysine after 5 days. This is the highest production reported to date.


Assuntos
Amônia-Liases/genética , Escherichia coli/metabolismo , Ácidos Pipecólicos/metabolismo , Amônia-Liases/metabolismo , Biotransformação , Meios de Cultura/química , Escherichia coli/genética , Fermentação , Expressão Gênica , Lisina/análise , Lisina/metabolismo , Engenharia Metabólica , Metais/análise , Metais/metabolismo , Sequências de Repetição em Tandem , Fatores de Tempo
17.
Int J Biol Macromol ; 154: 929-936, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32198033

RESUMO

Polyhydroxyalkanoates (PHA), such as poly (3-hydroxybutyrate) (PHB), have emerged as potential alternatives to petroleum-based plastics and can be produced through the appropriate selection of marine bacteria that are already adapted to high salt and low temperature conditions without the requirement of antibiotic treatment. The present study, thus, aimed to screen and characterize thirteen PHA-producing microbial strains isolated from the Gwangalli beach in Busan, South Korea. Among them, Halomonas sp. YLGW01 produced the highest amount of PHB (94.6 ± 1.8% (w/w)) using fructose. Interestingly Halomonas sp. YLGW01 showed increase in cell size (8.39 ± 3.63 µm) with fructose as carbon source as compared to glucose (2.34 ± 0.44 µm). Fructose syrup was investigated as carbon source under unsterilized conditions and 95.26 ± 1.78% of PHB was produced. Overall, this strain showed the highest PHB contents in halotolerant bacteria.


Assuntos
Halomonas/metabolismo , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Carbono/metabolismo , Halomonas/classificação , Filogenia , República da Coreia , Microbiologia do Solo
18.
AMB Express ; 10(1): 64, 2020 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-32266584

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) strains are distinct from general Staphylococcus strains with respect to the composition of the membrane, ability to form a thicker biofilm, and, importantly, ability to modify the target of antibiotics to evade their activity. The agr gene is an accessory global regulator of gram-positive bacteria that governs virulence or resistant mechanisms and therefore an important target for the control of resistant strains. However, the mechanism by which agr impacts resistance to ß-lactam antibiotics remains unclear. In the present study, we found the Δagr mutant strain having higher resistance to high concentrations of ß-lactam antibiotics such as oxacillin and ampicillin. To determine the influence of variation in the microenvironment of cells between the parental and mutant strains, fatty acid analysis of the supernatant, total lipids, and phospholipid fatty acids were compared. The Δagr mutant strain tended to produce fewer fatty acids and retained lower amounts of C16, C18 fatty acids in the supernatant. Phospholipid analysis showed a dramatic increase in the hydrophobic longer-chain fatty acids in the membrane. To target membrane, we applied several surfactants and found that sorbitan monolaurate (Span20) had a synergistic effect with oxacillin by decreasing biofilm formation and growth. These findings indicate that agr deletion allows for MRSA to resist antibiotics via several changes including constant expression of mecA, fatty acid metabolism, and biofilm thickening.

19.
Bioresour Technol ; 302: 122872, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32014731

RESUMO

In this study, a heterogeneous catalyst prepared by pyrolysis of waste cork (Quercus suber) was used for the transesterification of waste cooking oil (WCO). Physicochemical properties of the synthesized biochar catalyst were studied using BET, SEM, FTIR, and XRD. The experiment results demonstrate that heterogeneous catalyst synthesized at 600 °C showed maximum fatty acids methyl esters (FAMEs) conversion (98%) at alcohol:oil (25:1), catalyst loading (1.5% w/v) and temperature 65 °C. Biodiesel produced from WCO (Canola oil) mainly composed of FAMEs in following order C18:1 > C18:2 > C16:0 > C18:0 > C20:0. Properties of produced biodiesel were analysed as cetane number (CN) 50.56, higher heating value (HHV) 39.5, kinematic viscosity (ʋ) 3.9, and density (ρ) 0.87.


Assuntos
Biocombustíveis , Carvão Vegetal , Catálise , Culinária , Esterificação , Óleos de Plantas
20.
Bioresour Technol ; 297: 122472, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31791917

RESUMO

The present study enlightens facile synthesis and characterization of magnetic biochar derived from waste banana pseudostem biomass for the removal of nitrofuran antibiotic 'furazolidone' (FZD). Brunauer-Emmett-Teller (BET), scanning electron microscopy (SEM), magnetic hysteresis, X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) revealed successful hybridization of magnetic nanocomposites with biochar (BPB600). The maximum adsorption capacity of magnetic BPB600 was 96.81% (37.86 mg g-1), which was significantly higher than the non-coated BPB600 (77.25%; 31.45 mg g-1). Adsorption kinetics data fitted well with pseudo-second order, and Elovich model demonstrating dominance of the chemisorption mechanism. Furthermore, the response surface methodology (RSM) was applied to evaluate the interactive effect of pH, temperature, and FZD concentration on adsorption. Therefore, the results of present study would provide an effective strategy to tackle antibiotic contaminants responsible for the antibiotic resistance genes or bacteria that decreases the therapeutic value of antibiotics.


Assuntos
Musa , Nanocompostos , Poluentes Químicos da Água , Adsorção , Carvão Vegetal , Furazolidona , Cinética , Fenômenos Magnéticos , Espectroscopia de Infravermelho com Transformada de Fourier
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