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1.
Biomedicines ; 11(4)2023 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-37189810

RESUMO

The protein p53 is a well-known tumor suppressor that plays a crucial role in preventing cancer development [...].

2.
Biomedicines ; 10(7)2022 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-35884795

RESUMO

The members of the p53 family comprise p53, p63, and p73, and full-length isoforms of the p53 family have a tumor suppressor function. However, p53, but not p63 or p73, has a high mutation rate in cancers causing it to lose its tumor suppressor function. The top and second-most prevalent p53 mutations are missense and nonsense mutations, respectively. In this review, we discuss possible drug therapies for nonsense mutation and a missense mutation in p53. p63 and p73 activators may be able to replace mutant p53 and act as anti-cancer drugs. Herein, these p63 and p73 activators are summarized and how to improve these activator responses, particularly focusing on p53 gain-of-function mutants, is discussed.

3.
Molecules ; 27(12)2022 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-35745070

RESUMO

Our previous findings have shown that the chlorophyllides composites have anticancer activities to breast cancer cell lines (MCF-7 and MDA-MB-231). In the present study, microarray gene expression profiling was utilized to investigate the chlorophyllides anticancer mechanism on the breast cancer cells lines. Results showed that chlorophyllides composites induced upregulation of 43 and 56 differentially expressed genes (DEG) in MCF-7 and MDA-MB-231 cells, respectively. In both cell lines, chlorophyllides composites modulated the expression of annexin A4 (ANXA4), chemokine C-C motif receptor 1 (CCR1), stromal interaction molecule 2 (STIM2), ethanolamine kinase 1 (ETNK1) and member of RAS oncogene family (RAP2B). Further, the KEGG annotation revealed that chlorophyllides composites modulated DEGs that are associated with the endocrine system in MCF-7 cells and with the nervous system in MDA-MB-231 cells, respectively. The expression levels of 9 genes were validated by quantitative reverse transcription PCR (RT-qPCR). The expression of CCR1, STIM2, ETNK1, MAGl1 and TOP2A were upregulated in both chlorophyllides composites treated-MCF-7 and MDA-MB-231 cells. The different expression of NLRC5, SLC7A7 and PKN1 provided valuable information for future investigation and development of novel cancer therapy.


Assuntos
Neoplasias da Mama , Clorofilídeos , Sistema y+L de Transporte de Aminoácidos , Mama , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Detecção Precoce de Câncer , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células MCF-7 , Proteínas rap de Ligação ao GTP
4.
Biomolecules ; 11(8)2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34439782

RESUMO

Chlorophyllides can be found in photosynthetic organisms. Generally, chlorophyllides have a-, b-, c-, d-, and f-type derivatives, and all chlorophyllides have a tetrapyrrole structure with a Mg ion at the center and a fifth isocyclic pentanone. Chlorophyllide a can be synthesized from protochlorophyllide a, divinyl chlorophyllide a, or chlorophyll. In addition, chlorophyllide a can be transformed into chlorophyllide b, chlorophyllide d, or chlorophyllide f. Chlorophyllide c can be synthesized from protochlorophyllide a or divinyl protochlorophyllide a. Chlorophyllides have been extensively used in food, medicine, and pharmaceutical applications. Furthermore, chlorophyllides exhibit many biological activities, such as anti-growth, antimicrobial, antiviral, antipathogenic, and antiproliferative activity. The photosensitivity of chlorophyllides that is applied in mercury electrodes and sensors were discussed. This article is the first detailed review dedicated specifically to chlorophyllides. Thus, this review aims to describe the definition of chlorophyllides, biosynthetic routes of chlorophyllides, purification of chlorophyllides, and applications of chlorophyllides.


Assuntos
Técnicas Biossensoriais/métodos , Química Farmacêutica/métodos , Clorofila/análogos & derivados , Clorofilídeos/síntese química , Aditivos Alimentares/química , Protoclorifilida/metabolismo , Anti-Infecciosos/síntese química , Anti-Infecciosos/farmacologia , Antineoplásicos Fitogênicos/biossíntese , Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/farmacologia , Antivirais/síntese química , Antivirais/farmacologia , Técnicas Biossensoriais/instrumentação , Clorofila/biossíntese , Clorofila/farmacologia , Clorofilídeos/biossíntese , Clorofilídeos/farmacologia , Técnicas Eletroquímicas , Aditivos Alimentares/metabolismo , Humanos , Luz , Estrutura Molecular , Fotossíntese/fisiologia , Plantas/química , Plantas/metabolismo
5.
Plants (Basel) ; 10(4)2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918943

RESUMO

The fungus Antrodia cinnamomea has been used as a folk medicine for various diseases, especially cancer. When A. cinnamomea is cultured on the original host, an endangered woody plant Cinnamomum kanehirai Hayata, the fungus produces more active ingredients, but its growth is slow. Here, C. kanehirai leaf ethanol extract (KLEE) was used as a substitute for C. kanehirai wood to culture A. cinnamomea on solid medium to shorten the culture period and produce active metabolites en masse. The antioxidant activities of methanol extracts from A. cinnamomea cultured on KLEE (MEAC-KLEE) were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect, reducing power, and ferrous ion-chelating effect, and the effective concentration (EC50) values were 0.27, 0.74, and 0.37 mg mL-1, respectively. MEAC-KLEE exhibited specific anti-proliferative activity against a non-small-cell lung cancer cell line (A549) by Annexin V assay. A secondary metabolite (2,4-dimethoxy-6-methylbenzene-1,3-diol, DMMB) present in the extract (MEAC-KLEE) was purified by high-performance liquid chromatography (HPLC) and identified by nuclear magnetic resonance (NMR) spectra. DMMB exhibited moderate antioxidant activity against DPPH radicals and reducing power, with EC50 values of 12.97 and 25.59 µg mL-1, respectively, and also induced apoptosis in A549 cells. Our results provide valuable insight into the development of DMMB for nutraceutical biotechnology.

6.
PLoS One ; 16(4): e0250565, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33930043

RESUMO

The purity of chlorophylls plays one of the key role for the production of chlorophyllides. We have designed a facile method for chlorophyll purification by twice solvent extraction. Twice extraction causes the loss of chlorophylls, but the purity of total chlorophylls can be enhanced 182%. Then, the purified chlorophylls can be converted to relatively pure chlorophyllides facilely. The results show that higher purity of chlorophyllides could be obtained when purified chlorophylls (ethanol-hexane extract) was used as starting materials than that of crude chlorophylls (ethanol-only extract). In biocompatibility test, the results showed that the prepared chlorophyllides can be applied as biomaterials. When the prepared chlorophyllides were applied to anticancer tests, they were active both in MCF7 and MDA-MB-231 (multidrug resistant breast cancer cells) cell lines. In addition, the results suggested that the prepared chlorophyllides could be a potential candidate of combination therapy with doxorubicin to breast cancers.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Clorofila/isolamento & purificação , Clorofilídeos/farmacologia , Resistência a Múltiplos Medicamentos/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Clorofila/química , Clorofila/farmacologia , Clorofilídeos/biossíntese , Clorofilídeos/química , Doxorrubicina/efeitos adversos , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Células MCF-7 , Linfócitos T Citotóxicos/efeitos dos fármacos
7.
Molecules ; 26(5)2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33673610

RESUMO

Generally, bacteriochlorophyllides were responsible for the photosynthesis in bacteria. Seven types of bacteriochlorophyllides have been disclosed. Bacteriochlorophyllides a/b/g could be synthesized from divinyl chlorophyllide a. The other bacteriochlorophyllides c/d/e/f could be synthesized from chlorophyllide a. The chemical structure and synthetic route of bacteriochlorophyllides were summarized in this review. Furthermore, the potential applications of bacteriochlorophyllides in photosensitizers, immunosensors, influence on bacteriochlorophyll aggregation, dye-sensitized solar cell, heme synthesis and for light energy harvesting simulation were discussed.


Assuntos
Bactérias/metabolismo , Clorofilídeos/biossíntese , Clorofilídeos/química , Complexos de Coordenação/química , Técnicas Biossensoriais , Vias Biossintéticas , Heme/química , Heme/metabolismo , Fármacos Fotossensibilizantes/química , Fotossíntese , Energia Solar
8.
Front Pharmacol ; 11: 575704, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33328984

RESUMO

Cordyceps militaris (C. militaris) is a fungus with a long history of widespread use in folk medicine, and its biological and medicinal functions are well studied. A crucial pharmacological effect of C. militaris is immunomodulation. In this review, we catalog the immunomodulatory effects of different extracts of C. militaris, namely total extracts, polysaccharides and cordycepin. Total extracts obtained using water or 50% ethyl alcohol and polysaccharides from C. militaris were discovered to tend to promote type 1 immunity, whereas total extracts obtained using 70-80% ethyl alcohol and cordycepin from C. militaris were more likely to promote type 2 immunity. This article is the first to classify the immunomodulatory effects of different extracts of C. militaris. In addition, we discovered a relationship between different segments or extracts and differing types of immunity. This review can provide the readers a comprehensive understanding on the immunomodulatory effects of the precious folk medicine and guidance on its use for both health people and those with an immunodeficiency.

9.
Appl Biochem Biotechnol ; 191(1): 112-124, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31956956

RESUMO

Esterases are widely used in the food industry. Here, a new thermophilic bacterium, Geobacillus thermodenitrificans PS01, was isolated and the esterase-encoding gene est1 was cloned, sequenced, and recombinant expressed in Escherichia coli Tuner (DE3). The highest activity of recombinant Est1 was detected at pH 8.0, and 40 °C and the extreme stability was observed at pH 6-9 over 30 days at 4 °C. In particular, Est1 can hydrolyze short- to medium-chain (C2-C10) triglycerides and p-nitrophenyl esters (C2-C12) and was not inhibited by most metal ions. Kinetic parameters of p-nitrophenyl butyrate hydrolysis under optimal conditions were determined: Km, 22.76 µM; kcat, 10,415 s-1; and kcat/Km, 457.53 µM-1 s-1. The outstanding specification of Est1 indicates its potential for use in industrial applications.


Assuntos
Proteínas de Bactérias , Esterases , Geobacillus/enzimologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Esterases/biossíntese , Esterases/química , Esterases/genética , Esterases/isolamento & purificação , Geobacillus/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
10.
Food Chem ; 306: 125300, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31562927

RESUMO

Chlorophyll is a valuable bioactive compound, which is used as a natural food coloring agent and a photosensitizer for photodynamic therapy because of its antioxidant properties, antimutagenic ability, and near-infrared fluorescence. However, chlorophyll is unstable when it comes to retaining its antioxidant activity, when exposed to oxygen, high temperature, or light environments. To enhance the stability of chlorophyll, a polymer encapsulation method was proposed. Polycaprolactone (PCL) was employed to encapsulate the chlorophyll, and the particles size of the composites was controlled through droplet microfluidics. The composites (chlorophyll-encapsulated PCL particles) were characterized through UV-VIS spectrometry, SEM, optical microscopy, and light exposure. The particles were spherical, with diameters adjustable from 68 to 247 µm. Additionally, the chlorophyll-encapsulated PCL particles exhibited considerably prolonged chlorophyll stability. The solid microparticle is more convenient for storage and transportation, and have great potential for application in the food industry.


Assuntos
Clorofila/química , Poliésteres/química , Microfluídica/métodos , Tamanho da Partícula
11.
Artigo em Inglês | MEDLINE | ID: mdl-31379971

RESUMO

Chlorophyllide (chlide) is a natural catabolic product of chlorophyll (Chl), produced through the activity of chlorophyllase (chlase). The growth inhibitory and antioxidant effects of chlide from different plant leaf extracts have not been reported. The aim of this study is to demonstrate that chlide in crude extracts from leaves has the potential to exert cytotoxic effects on cancer cell lines. The potential inhibitory and antioxidant effects of chlide in crude extracts from 10 plant leaves on breast cancer cells (MCF7 and MDA-MB-231), hepatocellular carcinoma cells (Hep G2), colorectal adenocarcinoma cells (Caco2), and glioblastoma cells (U-118 MG) were studied using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and DPPH (1,1-diphenyl-2-picrylhydrazyl) assays. The results of the MTT assay showed that chlide in crude extracts from sweet potato were the most effective against all cancer cell lines tested. U-118 MG cells were the most sensitive, while Caco2 cells were the most resistant to the tested crude extracts. The cytotoxic effects of chlide and Chl in crude extracts from sweet potato and of commercial chlorophyllin (Cu-chlin), in descending order, were as follows: chlide > Chl > Cu-chlin. Notably, the IC50 of sweet potato in U-118 MG cells was 45.65 µg/mL while those of Chl and Cu-chlin exceeded 200 µg/mL. In the DPPH assay, low concentrations (100 µg/mL) of chlide and Cu-chlin from crude extracts of sweet potato presented very similar radical scavenging activity to vitamin B2. The concentration of chlide was negatively correlated with DPPH activity. The current study was the first to demonstrate that chlide in crude extracts from leaves have potential cytotoxicity in cancer cell lines. Synergism between chlide and other compounds from leaf crude extracts may contribute to its cytotoxicity.

12.
J Microbiol Methods ; 160: 20-28, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30890401

RESUMO

Astaxanthin possesses various biological properties and is used in the animal and fish feed, food, and beverage industries. In this study, we derived zeaxanthin biosynthesis genes (crtE, crtB, crtI, crtY, and crtZ) from Erwinia uredovora and crtW from Agrobacterium aurantiacum. We fused inducible and constitutive promoters to astaxanthin biosynthesis genes to construct a novel plasmid (dubbed PTP3-6) that can effectively enhance free-form astaxanthin (FFAX) production. The PTP3-6 plasmid contains one T7 promoter, driving IPTG inducible crtW expression, and three constitutive promoters (isolated from E. uredovora) driving expression of the other zeaxanthin biosynthesis genes. Escherichia coli BL21 (DE3) cells carrying the PTP3-6 plasmid produced 8.3 mg/g dry cell weight astaxanthin, which is 69.4-fold higher than has been previously reported. Using multiple promoter fusions of astaxanthin biosynthesis genes could be applied in other hosts to enhance astaxanthin production. FFAX was identified in recombinant E. coli cells through ultra-performance liquid chromatography-mass spectrometry.


Assuntos
Plasmídeos/biossíntese , Agrobacterium/genética , Clonagem Molecular , Escherichia coli/genética , Genes Bacterianos , Pantoea/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Xantofilas/isolamento & purificação
13.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 6): 351-354, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29870019

RESUMO

The Staphylococcus epidermidis lipase (SeLip, GehC) can be used in flavour-compound production via esterification in aqueous solution. This study reports the crystallization and crystallographic analysis of recombinant GehC (rGehC; Lys303-Lys688) with a molecular weight of 43 kDa. rGehC was crystallized at 293 K using PEG 10 000 as a precipitant, and a 99.9% complete native data set was collected from a cooled crystal at 77 K to a resolution of 1.9 Šwith an overall Rmerge value of 7.3%. The crystals were orthorhombic and belonged to space group P212121, with unit-cell parameters a = 42.07, b = 59.31, c = 171.30 Å, α = ß = γ = 90°. Solvent-content calculations suggest that there is likely to be one lipase subunit in the asymmetric unit.


Assuntos
Lipase/química , Staphylococcus epidermidis/enzimologia , Água , Sequência de Aminoácidos , Cristalografia/métodos , Esterificação , Lipase/genética , Lipase/metabolismo , Soluções/metabolismo , Staphylococcus epidermidis/genética , Água/metabolismo
14.
Plant Mol Biol ; 95(1-2): 181-197, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28840447

RESUMO

KEY MESSAGE: In this present study, we introduce a fundamental framework and provide information regarding the possible roles of GDSL-type esterase/lipase gene family in Arabidopsis. GDSL-type esterases/lipases are hydrolytic enzymes with multifunctional properties such as broad substrate specificity, regiospecificity, and stereoselectivity. In this study, we identified 105 GDSL-type esterase/lipase genes in Arabidopsis thaliana by conducting a comprehensive computational analysis. Expression studies indicated that GDSL-type lipase proteins showed varied expression patterns. Phylogenetic tree analysis indicated that AtGELP (Arabidopsis thaliana GDSL-type esterase/lipase protein) gene family was divided into four clades. The phylogenetic analysis, combined with protein motif architectures, and expression profiling were used to predict the roles AtGELP genes. To investigate the physical roles of the AtGELP gene family, we successfully screened 88 AtGELP T-DNA knockout lines for 54 AtGELP genes from 199 putative SALK T-DNA mutants. Transgenic plants of AtGELP genes were used to elucidate the phenotypic characteristics in various developmental stages or stress conditions. Our results suggest that the AtGELP genes have diverse physical functions such as affecting the germination rate and early growth of seedlings subjected to high concentrations of glucose, or being involved in biotic stress responses.


Assuntos
Arabidopsis/enzimologia , Arabidopsis/genética , Esterases/genética , Genoma de Planta , Lipase/genética , Arabidopsis/crescimento & desenvolvimento , Cromossomos de Plantas/genética , DNA Bacteriano/genética , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Genes de Plantas , Mutagênese Insercional/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Filogenia , Duplicações Segmentares Genômicas , Alinhamento de Sequência
15.
J Agric Food Chem ; 65(9): 1874-1886, 2017 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-28234464

RESUMO

Antodia cinnamomea, a precious brown-rot fungus endemic to Taiwan, has pharmaceutical applications due to its diverse array of metabolites. The terpenoids found in A. cinnamomea contribute to its most important bioactivities. We identified several terpenoid compounds in A. cinnamomea and revealed that their content in mycelium and fruiting body were significantly different. Using next-generation sequencing and an in-house transcriptome database, we identified several terpene synthase (TPS) candidates. After sequence analysis and functional characterization, 10 out of 12 candidates were found to have single or multiple terpene synthesis functions. Most of the terpenoid compounds were found to confer important bioactivities. RT-PCR results showed a positive correlation between terpene synthase expression pattern and terpenoid content. In addition, we identified several modification enzyme candidates that may be involved in the postmodification of terpenoid compounds with a genomic DNA scaffold, and a putative genetic network.


Assuntos
Antrodia/metabolismo , Carpóforos/metabolismo , Redes Reguladoras de Genes , Micélio/genética , Terpenos/metabolismo , Antrodia/genética , Antrodia/crescimento & desenvolvimento , Carpóforos/genética , Carpóforos/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Transcriptoma
16.
Molecules ; 21(8)2016 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-27472309

RESUMO

Recombinant Chlamydomonas reinhardtii chlorophyllase 1 (CrCLH1) that could catalyze chlorophyll hydrolysis to chlorophyllide and phytol in vitro was successfully expressed in Escherichia coli. The recombinant CrCLH1 was immobilized through covalent binding with a cubic (3-aminopropyl) triethoxysilane (APTES) coating on magnetic iron oxide nanoparticles (MIONPs), which led to markedly improved enzyme performance and decreased biocatalyst costs for potential industrial application. The immobilized enzyme exhibited a high immobilization yield (98.99 ± 0.91 mg/g of gel) and a chlorophyllase assay confirmed that the immobilized recombinant CrCLH1 retained enzymatic activity (722.3 ± 50.3 U/g of gel). Biochemical analysis of the immobilized enzyme, compared with the free enzyme, showed higher optimal pH and pH stability for chlorophyll-a hydrolysis in an acidic environment (pH 3-5). In addition, compared with the free enzyme, the immobilized enzyme showed higher activity in chlorophyll-a hydrolysis in a high temperature environment (50-60 °C). Moreover, the immobilized enzyme retained a residual activity of more than 64% of its initial enzyme activity after 14 cycles in a repeated-batch operation. Therefore, APTES-coated MIONP-immobilized recombinant CrCLH1 can be repeatedly used to lower costs and is potentially useful for the industrial production of chlorophyll derivatives.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Chlamydomonas reinhardtii/enzimologia , Clorofila/química , Compostos Férricos/química , Proteínas de Algas/química , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Biocatálise , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/genética , Fenômenos Eletromagnéticos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Nanopartículas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
17.
Int J Pharm ; 510(2): 493-500, 2016 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-26780124

RESUMO

Silver nanoparticle (Ag NP)-loaded chitosan composites have numerous biomedical applications; however, fabricating uniform composite microparticles remains challenging. This paper presents a novel microfluidic approach for single-step and in situ synthesis of Ag NP-loaded chitosan microparticles. This proposed approach enables obtaining uniform and monodisperse Ag NP-loaded chitosan microparticles measuring several hundred micrometers. In addition, the diameter of the composites can be tuned by adjusting the flow on the microfluidic chip. The composite particles containing Ag NPs were characterized using UV-vis spectra and scanning electron microscopy-energy dispersive X-ray spectrometry data. The characteristic peaks of Ag NPs in the UV-vis spectra and the element mapping or pattern revealed the formation of nanosized silver particles. The results of antibacterial tests indicated that both chitosan and composite particles showed antibacterial ability, and Ag NPs could enhance the inhibition rate and exhibited dose-dependent antibacterial ability. Because of the properties of Ag NPs and chitosan, the synthesized composite microparticles can be used in several future potential applications, such as bactericidal agents for water disinfection, antipathogens, and surface plasma resonance enhancers.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Quitosana/química , Nanopartículas Metálicas/química , Prata/química , Prata/farmacologia , Bactérias/efeitos dos fármacos , Microfluídica/métodos , Microscopia Eletrônica de Varredura/métodos , Tamanho da Partícula , Espectrometria por Raios X/métodos , Raios Ultravioleta
18.
Biotechnol Appl Biochem ; 63(3): 371-7, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-25828734

RESUMO

Bacteriopheophorbide a (BPheid a) is used as a precursor for bacteriochlorin a (BCA), which can be used for photodynamic therapy in both in vitro and in vivo biochemical applications. This study successfully isolated and expressed a photosynthetic bacterium (Cyanothece sp. ATCC 51142) chlorophyllase called CyanoCLH, which can be used as a biocatalyst for the production of a BCA precursor by degrading bacteriochlorophyll a (BChl a). Substrate specificity and enzyme kinetic analyses were performed and the results verified that the recombinant CyanoCLH preferred hydrolyzing BChl a to produce bacteriochlorophyllide a (BChlide a), which can be converted to BPheid a by removing magnesium ion. The recombinant CyanoCLH was cloned and expressed in Escherichia coli BL-21 (DE3), and its molecular weight was 54.7 kDa. The deduced amino acid sequence of the recombinant CyanoCLH comprised a unique lipase-motif GHSLG, which differs from the GHSRG sequence of other plants and lacks a histidine of the typical and conserved catalytic triad Ser-Asp-His. The recombinant CyanoCLH was subjected to biochemical analyses, and the results indicated that its optimal pH and temperature were 7.0 and 60 °C, respectively.


Assuntos
Bacterioclorofila A/biossíntese , Hidrolases de Éster Carboxílico/metabolismo , Cyanothece/enzimologia , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Clonagem Molecular , Cyanothece/genética , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato
19.
J Agric Food Chem ; 63(43): 9496-503, 2015 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-26478543

RESUMO

Natural chlorophyll metabolites have exhibited physiological activity in vitro. In this study, a recombinant chlorophyllase1 gene from Chlamydomonas reinhardtii (CrCLH1) was isolated and characterized. Recombinant CrCLH1 can perform chlorophyll dephytylation and produce chlorophyllide and phytol. In a transient assay, the subcellular localization of CrCLH1-green fluorescent protein was determined to be outside the chloroplast. Biochemical analyses of the activity of recombinant CrCLH1 indicated that its optimal pH value and temperature are 6.0 and 40 °C, respectively. Enzyme kinetic data revealed that the recombinant CrCLH1 had a higher catalytic efficiency for chlorophyll a than for chlorophyll b and bacteriochlorophyll a. According to high-performance liquid chromatography analysis of chlorophyll hydrolysis, recombinant CrCLH1 catalyzed the conversion of chlorophyll a to pheophorbide a at pH 5. Therefore, recombinant CrCLH1 can be used as a biocatalyst to produce chlorophyllide derivatives.


Assuntos
Hidrolases de Éster Carboxílico/química , Chlamydomonas reinhardtii/enzimologia , Clorofilídeos/química , Biocatálise , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/genética , Cinética , Estrutura Molecular
20.
J Nat Prod ; 78(7): 1556-62, 2015 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-26125648

RESUMO

Antrodia cinnamomea is a scarce, epiphyte, host-specific, brown-rot fungus that produces diverse bioactive compounds with potent biological activity. Natural wild-type fruiting bodies of A. cinnamomea are rare and highly valued, but their artificial culture poses challenges. Triterpenoids are a group of secondary metabolites that contribute to the bioactivities of A. cinnamomea. 2,3-Oxidosqualene cyclase (OSC) is a key enzyme in triterpenoid biosynthesis, which converts 2,3-oxidosqualene (OS) into polycyclic triterpenoids. In this study, we isolated a 2,3-oxidosqualene cyclase gene from A. cinnamomea with degenerate primers and designated it as AcOSC. The full length AcOSC cDNA was subcloned into a yeast expression vector, and AcOSC activity was confirmed. RT-PCR results showed that AcOSC expression was highest in the wild-type fruiting body and correlated with a higher concentration of triterpenoids. Agrobacterium-mediated gene transformation was conducted to enhance the triterpenoid synthesis capacity of the cultured mycelium. Metabolite profiling was conducted by LC-MS/MS and principal component analysis (PCA). The compositions and contents of metabolites in the AcOSC transgenic lines were different from those in the wild-type mycelium and vector control. The levels of two important triterpenoids, dehydrosulphurenic acid (DSA) and dehydroeburicoic acid (DEA), were increased in A. cinnamomea oxidosqualene cyclase overexpression strains compared to controls. In summary an Agrobacterium-mediated gene transformation procedure was established that successfully increased the level of transgene expression and enhanced the triterpenoid content in cultured A. cinnamomea.


Assuntos
Antrodia/genética , Transferases Intramoleculares/isolamento & purificação , Triterpenos/metabolismo , Antrodia/química , Carpóforos/química , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Estrutura Molecular , Micélio , Esqualeno/análogos & derivados , Esqualeno/química , Taiwan
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