Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Appl Environ Microbiol ; 86(8)2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32033954

RESUMO

Cytophaga hutchinsonii cells can bind to the surface of insoluble cellulose and degrade it by utilizing a novel cell contact-dependent mechanism, in which the outer membrane proteins may play important roles. In this study, the deletion of a gene locus, chu_1165, which encodes a hypothetical protein with 32% identity with TlpB, a disulfide oxidoreductase in Flavobacterium psychrophilum, caused a complete cellulolytic defect in C. hutchinsonii Further study showed that cells of the Δ1165 strain could not bind to cellulose, and the levels of many outer membrane proteins that can bind to cellulose were significantly decreased. The N-terminal region of CHU_1165 is anchored to the cytoplasmic membrane with five predicted transmembrane helices, and the C-terminal region is predicted to stretch to the periplasm and has a similar thioredoxin (Trx) fold containing a Cys-X-X-Cys motif that is conserved in disulfide oxidoreductases. Recombinant CHU_1165His containing the Cys-X-X-Cys motif was able to reduce the disulfide bonds of insulin in vitro Site-directed mutation showed that the cysteines in the Cys-X-X-Cys motif and at residues 106 and 108 were indispensable for the function of CHU_1165. Western blotting showed that CHU_1165 was in an oxidized state in vivo, suggesting that it may act as an oxidase to catalyze disulfide bond formation. However, many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of the cysteine in these proteins did not affect cellulose degradation, indicating that CHU_1165 may have an indirect or pleiotropic effect on the function of these outer membrane proteins.IMPORTANCE Cytophaga hutchinsonii can rapidly digest cellulose in a contact-dependent manner, in which the outer membrane proteins may play important roles. In this study, a hypothetical protein, CHU_1165, characterized as a disulfide oxidoreductase, is essential for cellulose degradation by affecting the cellulose binding ability of many outer membrane proteins in C. hutchinsonii Disulfide oxidoreductases are involved in disulfide bond formation. However, our studies show that many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of cysteine did not affect their function, indicating that CHU_1165 did not facilitate the formation of a disulfide bond in these proteins. It may have an indirect or pleiotropic effect on the function of these outer membrane proteins. Our study provides an orientation for exploring the proteins that assist in the appropriate conformation of many outer membrane proteins essential for cellulose degradation, which is important for exploring the novel mechanism of cellulose degradation in C. hutchinsonii.

2.
J Ind Microbiol Biotechnol ; 46(11): 1479-1490, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31321576

RESUMO

The crystalline region of cellulose is the main barrier to the utilization of crystalline cellulose. Cytophaga hutchinsonii actively digests the crystalline region of cellulose by an unknown mechanism. Transposon mutagenesis was done to identify a novel gene locus chu_1557, which is required for efficient disruption of the crystalline region of cellulose, and the absence of CHU_1557 resulted in decreased glucose assimilation efficiency. The defect of the mutant in the disruption of the crystalline region of cellulose was partially retained by additional glucose or pre-culturing the mutant in a low glucose concentration medium which could improve its glucose absorption efficiency. These results suggested that extracellular glucose has important roles in the disruption of crystalline cellulose by C. hutchinsonii. Further study showed that the expression of an outer membrane protein CHU_3732 was downregulated by the absence of CHU_1557 in a low glucose concentration medium. CHU_3732 was involved in uptake of glucose and its expression was induced by a low concentration of glucose. CHU_3732 was predicted to be a porin, so we inferred that it may work as a glucose transport channel in the outer membrane. Based on these results, we deduced that CHU_1557 played a role in the process of glucose assimilation and its disruption affected the expression of other proteins related to glucose transportation such as CHU_3732, and then affected the cell growth in a low glucose concentration medium and disruption of the crystalline region of cellulose.


Assuntos
Celulose/metabolismo , Cytophaga/metabolismo , Glucose/metabolismo , Proteínas de Membrana/metabolismo , Cytophaga/genética , Proteínas de Membrana/genética , Mutagênese
3.
ACS Appl Mater Interfaces ; 11(19): 17467-17474, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31021073

RESUMO

Graphitic carbon nitride (g-C3N4) as a metal-free nanozyme has attracted huge attention for catalytic applications. However, the catalytic activity of pure g-C3N4 causes very moderate H2O2 activation. Herein, a novel three-dimensional (3D) branched carbon nitride nanoneedle (3DBC-C3N4) nanozyme has been proposed to overcome such shortcoming. This unique 3D branched structure of 3DBC-C3N4 facilitated effective mass transfer during catalytic reaction and induced a lightning rodlike effect to accelerate electron collection at the tip area for H2O2 activation. With improved H2O2 activation for hydroxyl radical (•OH) generation, 3DBC-C3N4 showed excellent peroxidase-like activity toward 3,3',5,5'-tetramethylbenzidine oxidation in the presence of H2O2. As for H2O2, the Vmax value of 3DBC-C3N4 was found to be 20 times higher than that of natural horseradish peroxidase. Moreover, the 3D branched structure of 3DBC-C3N4 offered large interface for the reversible conjugation of single-stranded DNA, which enhanced the colorimetric sensitivity. Moreover, 3DBC-C3N4 exhibited high sensitivity toward oxytetracycline detection, with the detection limit and quantitative limit of 1 and 50 µg/L, respectively.

4.
Appl Microbiol Biotechnol ; 102(15): 6593-6611, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29876607

RESUMO

Cytophaga hutchinsonii, belonging to Bacteroidetes, is speculated to use a novel cell-contact mode to digest cellulose. In this study, we identified a histone-like protein HU, CHU_2750, in C. hutchinsonii, whose transcription could be induced by crystalline but not amorphous cellulose. We constructed a CHU_2750-deleted mutant and expressed CHU_2750 in Escherichia coli to study the gene's functions. Our results showed that although the deletion of CHU_2750 was not lethal to C. hutchinsonii, the mutant displayed an abnormal filamentous morphology, loose nucleoid, and obvious defects in the degradation of crystalline cellulose and cell motility. Further study indicated that the mutant displayed significantly decreased cell surface and intracellular endoglucanase activities but with ß-glucosidase activities similar to the wild-type strain. Analyses by real-time quantitative PCR revealed that the transcription levels of many genes involved in cellulose degradation and/or cell motility were significantly downregulated in the mutant. In addition, we found that CHU_2750 was important for biofilm formation of C. hutchinsonii. The main extracellular components of the biofilm were analyzed, and the results showed that the mutant yielded significantly less exopolysaccharide but more extracellular DNA and protein than the wild-type strain. Collectively, our findings demonstrated that CHU_2750 is important for cellulose degradation, cell motility, and biofilm formation of C. hutchinsonii by modulating transcription of certain related genes, and it is the first identified transcriptional regulator in these processes of C. hutchinsonii. Our study shed more light on the mechanisms of cellulose degradation, cell motility, and biofilm formation by C. hutchinsonii.


Assuntos
Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Celulose/metabolismo , Cytophaga/genética , Proteínas de Ligação a DNA/genética , Bacteroidetes/genética , Metabolismo dos Carboidratos , Celulase/metabolismo , Cytophaga/metabolismo , Escherichia coli/genética , Reação em Cadeia da Polimerase
5.
Front Microbiol ; 9: 632, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29666619

RESUMO

Cytophaga hutchinsonii is a gliding Gram-negative bacterium in the phylum Bacteroidetes with the capability to digest crystalline cellulose rapidly, but the mechanism is unclear. In this study, deletion of chu_0125, encoding a homolog of the peptidoglycan-associated lipoprotein (Pal), was determined to prevent degradation of the crystalline region of cellulose. We found that the chu_0125 deletion mutant grew normally in regenerated amorphous cellulose medium but displayed defective growth in crystalline cellulose medium and increased the degree of crystallinity of Avicel. The endoglucanase and ß-glucosidase activities on the cell surface were reduced by 60 and 30% without chu_0125, respectively. Moreover, compared with the wild type, the chu_0125 deletion mutant was found to be more sensitive to some harmful compounds and to release sixfold more outer membrane vesicles (OMVs) whose protein varieties were dramatically increased. These results indicated that CHU_0125 played a critical role in maintaining the integrity of the outer membrane. Further study showed that the amounts of some outer membrane proteins were remarkably decreased in the chu_0125 deletion mutant. Western blotting revealed that CHU_3220, the only reported outer membrane protein that was necessary and specialized for degradation of the crystalline region of cellulose, was largely leaked from the outer membrane and packaged into OMVs. We concluded that the deletion of chu_0125 affected the integrity of outer membrane and thus influenced the localization of some outer membrane proteins including CHU_3220. This might be the reason why deletion of chu_0125 prevented degradation of the crystalline region of cellulose.

6.
FEMS Microbiol Lett ; 364(20)2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28961729

RESUMO

Cytophaga hutchinsonii, an aerobic soil bacterium which could degrade cellulose, produces yellow flexirubin pigments. In this study, fabZ, annotated as a putative ß-hydroxyacyl-(acyl carrier protein) (ACP) dehydratase gene, was identified by insertional mutation and gene deletion as an essential gene for flexirubin pigment synthesis. The availability of a FabZ mutant that fails to produce flexirubin allowed us to investigate the biological role of the pigment in C. hutchinsonii. Loss of flexirubin made the FabZ mutant more sensitive to UV radiation, oxidative stress and alkaline stress than the wild type.


Assuntos
Cytophaga/genética , Enoil-CoA Hidratase/metabolismo , Polienos/metabolismo , Celulose/metabolismo , Enoil-CoA Hidratase/genética , Deleção de Genes , Mutagênese Insercional , Estresse Oxidativo , Raios Ultravioleta
7.
Front Microbiol ; 8: 140, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28210251

RESUMO

Cytophaga hutchinsonii can rapidly digest crystalline cellulose without free cellulases or cellulosomes. Its cell-contact cellulose degradation mechanism is unknown. In this study, the four ß-glucosidase (bgl) genes in C. hutchinsonii were singly and multiply deleted, and the functions of these ß-glucosidases in cellobiose and cellulose degradation were investigated. We found that the constitutively expressed BglB played a key role in cellobiose utilization, while BglA which was induced by cellobiose could partially make up for the deletion of bglB. The double deletion mutant ΔbglA/bglB lost the ability to digest cellobiose and could not thrive in cellulose medium, indicating that ß-glucosidases were important for cellulose degradation. When cultured in cellulose medium, a small amount of glucose accumulated in the medium in the initial stage of growth for the wild type, while almost no glucose accumulated for ΔbglA/bglB. When supplemented with a small amount of glucose, ΔbglA/bglB started to degrade cellulose and grew in cellulose medium. We inferred that glucose might be essential for initiating cellulose degradation, and with additional glucose, C. hutchinsonii could partially utilize cellulose without ß-glucosidases. We also found that there were both cellulose binding cells and free cells when cultured in cellulose. Since direct contact between C. hutchinsonii cells and cellulose is necessary for cellulose degradation, we deduced that the free cells which were convenient to explore new territory in the environment might be fed by the adherent cells which could produce cello-oligosaccharide and glucose into the environment. This study enriched our knowledge of the cellulolytic pathway of C. hutchinsonii.

8.
Appl Microbiol Biotechnol ; 101(5): 1919-1926, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27822737

RESUMO

Cytophaga hutchinsonii is a gram-negative bacterium that can efficiently degrade crystalline cellulose by a novel strategy without cell-free cellulases or cellulosomes. Genomic analysis implied that C. hutchinsonii had endoglucanases and ß-glucosidases but no exoglucanases which could processively digest cellulose and produce cellobiose. In this study, BglA was functionally expressed in Escherichia coli and found to be a ß-glucosidase with wide substrate specificity. It can hydrolyze pNPG, pNPC, cellobiose, and cellodextrins. Moreover, unlike most ß-glucosidases whose activity greatly decreases with increasing length of the substrate chains, BglA has similar activity on cellobiose and larger cellodextrins. The K m values of BglA on cellobiose, cellotriose, and cellotetraose were calculated to be 4.8 × 10-2, 5.6 × 10-2, and 5.3 × 10-2 mol/l, respectively. These properties give BglA a great advantage to cooperate with endoglucanases in C. hutchinsonii in cellulose degradation. We proposed that C. hutchinsonii could utilize a simple cellulase system which consists of endoglucanases and ß-glucosidases to completely digest amorphous cellulose into glucose. Moreover, BglA was also found to be highly tolerant to glucose as it retained 40 % activity when the concentration of glucose was 100 times higher than that of the substrate, showing potential application in the bioenergy industry.


Assuntos
Celulose/metabolismo , Cytophaga/enzimologia , Escherichia coli/metabolismo , beta-Glucosidase/genética , beta-Glucosidase/metabolismo , Celobiose/biossíntese , Celulose/análogos & derivados , Cytophaga/metabolismo , Dextrinas/metabolismo , Escherichia coli/genética , Glucose/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Tetroses/metabolismo
9.
Appl Environ Microbiol ; 83(1)2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27742681

RESUMO

Cytophaga hutchinsonii is a Gram-negative bacterium that can efficiently degrade crystalline cellulose by a unique mechanism different from the free cellulase or cellulosome strategy. In this study, chu_3220, encoding the hypothetical protein CHU_3220 (205 kDa), was identified by insertional mutation and gene deletion as the first gene essential for degradation of the crystalline region but not the amorphous region of cellulose by C. hutchinsonii A chu_3220 deletion mutant was defective in the degradation of crystalline cellulose and increased the degree of crystallinity of Avicel PH101 but could still degrade amorphous cellulose completely. CHU_3220 was found to be located on the outer surface of the outer membrane and could bind to cellulose. It contains 15 PbH1 domains and a C-terminal domain (CHU_C) that was proved to be critical for the localization of CHU_3220 on the cell surface and the function of CHU_3220 in crystalline cellulose degradation. Moreover, the degradation of crystalline cellulose was intact-cell dependent and inhibited by NaN3 Further study showed that chu_3220 was induced by cellulose and that the endoglucanase activity on the cell surface was significantly reduced without chu_3220 Real-time PCR revealed that the transcription of most genes encoding endoglucanases located on the cell surface was decreased in the chu_3220 deletion mutant, indicating that chu_3220 might also play a role in the regulation of the expression of some endoglucanases. IMPORTANCE: Cytophaga hutchinsonii could efficiently degrade crystalline cellulose with a unique mechanism without cellulosomes and free cellulases. It lacks proteins that are thought to play important roles in disruption of the crystalline region of cellulose, including exoglucanases, lytic polysaccharide monooxygenases, expansins, expansin-like proteins, or swollenins, and most of its endoglucanases lack carbohydrate binding modules. The mechanism of the degradation of crystalline cellulose is still unknown. In this study, chu_3220 was identified as the first gene essential for the degradation of the crystalline region but not the amorphous region of cellulose. CHU_3220 is a high-molecular-weight protein located on the outer surface of the outer membrane and could bind to cellulose. We proposed that CHU_3220 might be an essential component of a protein complex on the cell surface in charge of the decrystallization of crystalline cellulose. The degradation of crystalline cellulose by C. hutchinsonii was not only dependent on intact cells but also required the energy supplied by the cells. This was obviously different from other known cellulose depolymerization system. Our study has shed more light on the novel strategy of crystalline cellulose degradation by C. hutchinsonii.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Celulose/metabolismo , Cytophaga/metabolismo , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Celulase/metabolismo , Celulose/química , Cristalização , Cytophaga/genética , Deleção de Genes , Mutagênese Insercional , Reação em Cadeia da Polimerase em Tempo Real
10.
Oncotarget ; 7(32): 52317-52328, 2016 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-27419626

RESUMO

We used oxidative isotope-coded affinity tags (OxICAT) to investigate the global redox status of proteins in human papillomavirus (HPV)-related cervical cancer cells, in order to identify a potential target for gene therapy. Voltage-dependent anion channel 1 (VDAC1) was found to be highly oxidized in HPV-positive cervical cancer cells. VDAC1 expression correlated significantly with the invasion of cervical cancer, the grade of cervical intraepithelial neoplasia (CIN) and the expression of HPV16 E7 in CIN. Knockdown of VDAC1 in cell lines increased the rate of apoptosis, while overexpression of the VDAC1 (respectively) partly reversed the effect. Thus, VDAC1 may promote the malignant progression of HPV-related disease, and treatments designed to suppress VDAC1 could prevent the progression of HPV-induced cervical disease.


Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias do Colo do Útero/patologia , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Carcinógenos/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/virologia , Feminino , Humanos , Oxirredução , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/complicações , Proteômica/métodos , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologia
11.
Appl Microbiol Biotechnol ; 99(22): 9617-23, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26169628

RESUMO

Cytophaga hutchinsonii is a Gram-negative bacterium that can degrade crystalline cellulose efficiently with an unknown strategy. Genomic analysis suggested it lacks exoglucanases which are found in many cellulolytic organisms and most of the cellulases in C. hutchinsonii lack recognizable carbohydrate-binding modules (CBMs). CHU_1280, speculated to be an endoglucanase belonging to glycoside hydrolase family 9 (GH9) in C. hutchinsonii, was functionally expressed in Escherichia coli, and evidence was presented suggesting that it may be a processive endoglucanase. In the absence of Ca(2+), CHU_1280 was inactive. But in the presence of Ca(2+), it had a specific activity of 600 U/µmol with carboxymethyl cellulose (CMC) as the substrate. With Ca(2+), CHU_1280 hydrolyzed regenerated amorphous cellulose (RAC) with nearly 80 % of the reducing ends appearing in the soluble fraction, suggesting it degraded cellulose in a processive way. CHU_1280 could bind to cellulose without recognizable CBMs and its binding ability was also Ca(2+)-dependent. Ca(2+) could stabilize the catalytic domain at high temperature, but the denaturation temperature of the whole protein was decreased. C. hutchinsonii might have an exoglucanase-independent cellulases system which included endoglucanases, processive endoglucanases, and ß-glucosidases.


Assuntos
Cálcio/metabolismo , Celulase/genética , Celulase/metabolismo , Celulose/metabolismo , Cytophaga/enzimologia , Cytophaga/genética , Ativadores de Enzimas/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura Ambiente
12.
FEMS Microbiol Lett ; 362(14)2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26066317

RESUMO

Cytophaga hutchinsonii glides rapidly over surfaces by an unknown mechanism without flagella and type IV pili and it can degrade crystalline cellulose efficiently by a novel mechanism. Tn4351 transposon mutagenesis was used to identify a new gene, CHU_1798, essential for colony spreading on agar surfaces. Further study showed that disruption of CHU_1798 caused non-spreading colonies on both soft and hard agar surfaces and individual cells were partially deficient in gliding on glass surfaces. The CHU_1798 mutant could digest cellulose as long as the cells were in direct contact with the cellulose, but it could not degrade cellulose powder buried in the agar plate. Scanning electron microscopy showed that individual mutant cells arranged irregularly on the cellulose fiber surface at an early stage of incubation, but later showed a regular parallel arrangement when there were plenty of cells and could spread along the cellulose fibers. These results suggest that CHU_1798 plays an important role in the motility of C. hutchinsonii and provide insight into the relation between cell motility and cellulose degradation.


Assuntos
Cytophaga/genética , Cytophaga/fisiologia , Genes Bacterianos , Ágar , Celulose/metabolismo , Elementos de DNA Transponíveis , Movimento , Mutagênese , Mutação
13.
Appl Environ Microbiol ; 80(15): 4511-8, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24837387

RESUMO

Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium which was reported to use a novel contact-dependent strategy to degrade cellulose. It was speculated that cellooligosaccharides were transported into the periplasm for further digestion. In this study, we reported that most of the endoglucanase and -glucosidase activity was distributed on the cell surface of C. hutchinsonii.Cellobiose and part of the cellulose could be hydrolyzed to glucose on the cell surface. However, the cell surface cellulolytic enzymes were not sufficient for cellulose degradation by C. hutchinsonii. An outer membrane protein, CHU_1277, was disrupted by insertional mutation. Although the mutant maintained the same endoglucanase activity and most of the -glucosidase activity,it failed to digest cellulose, and its cellooligosaccharide utilization ability was significantly reduced, suggesting that CHU_1277 was essential for cellulose degradation and played an important role in cellooligosaccharide utilization. Further study of cellobiose hydrolytic ability of the mutant on the enzymatic level showed that the -glucosidase activity in the outer membrane of the mutant was not changed. It revealed that CHU_1277 played an important role in assisting cell surface -glucosidase to exhibit its activity sufficiently. Studies on the outer membrane proteins involved in cellulose and cellooligosaccharide utilization could shed light on the mechanism of cellulose degradation by C. hutchinsonii.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Celulose/metabolismo , Cytophaga/metabolismo , Oligossacarídeos/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Membrana Celular/enzimologia , Membrana Celular/genética , Membrana Celular/metabolismo , Celulase/genética , Celulase/metabolismo , Cytophaga/enzimologia , Cytophaga/genética , Mutagênese Insercional , beta-Glucosidase/genética , beta-Glucosidase/metabolismo
14.
Appl Microbiol Biotechnol ; 98(15): 6679-87, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24652064

RESUMO

Cytophaga hutchinsonii is a Gram-negative gliding bacterium which can efficiently degrade crystalline cellulose by an unknown strategy. Genomic analysis suggests the C. hutchinsonii genome lacks homologs to an obvious exoglucanase that previously seemed essential for cellulose degradation. One of the putative endoglucanases, CHU_2103, was successfully expressed in Escherichia coli JM109 and identified as a processive endoglucanase with transglycosylation activity. It could hydrolyze carboxymethyl cellulose (CMC) into cellodextrins and rapidly decrease the viscosity of CMC. When regenerated amorphous cellulose (RAC) was degraded by CHU_2103, the ratio of the soluble to insoluble reducing sugars was 3.72 after 3 h with cellobiose and cellotriose as the main products, indicating that CHU_2103 was a processive endoglucanase. CHU_2103 could degrade cellodextrins of degree of polymerization ≥3. It hydrolyzed p-nitrophenyl ß-D-cellodextrins by cutting glucose or cellobiose from the non-reducing end. Meanwhile, some larger-molecular-weight cellodextrins could be detected, indicating it also had transglycosylation activity. Without carbohydrate-binding module (CBM), CHU_2103 could bind to crystalline cellulose and acted processively on it. Site-directed mutation of CHU_2103 demonstrated that the conserved aromatic amino acid W197 in the catalytic domain was essential not only for its processive activity, but also its cellulose binding ability.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Celulase/química , Celulase/metabolismo , Cytophaga/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Celulase/genética , Celulose/análogos & derivados , Celulose/metabolismo , Cytophaga/química , Cytophaga/genética , Dextrinas/metabolismo , Estabilidade Enzimática , Cinética , Especificidade por Substrato
15.
Prep Biochem Biotechnol ; 43(5): 431-44, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23581779

RESUMO

Porous epichlorohydrin cross-linked alginate beads (ECAB) were prepared by the following method. Na-alginate solution containing Na2SO4 was introduced dropwise into CaCl2 solution to simultaneously form CaSO4 precipitate and Ca-alginate gel beads. The resultant beads were cross-linked with epichlorohydrin and then thoroughly washed with ethylenediamine tetraacetic acid (EDTA) solution to remove CaSO4. The structural features of porous ECAB were assessed with scanning electron microscopy (SEM) and experiments on water content and adsorption of bovine serum albumin (BSA). The results showed that macroporous ECAB can be obtained when the mass ratio of sodium sulfate to sodium alginate is 4:1. The adsorption behavior of the macroporous ECAB was well described by the Langmuir isotherm with maximum adsorption capacity equal to 740 mg BSA/g dry weight in 50 mM Na2HPO4-citric acid buffer (pH 4.0). BSA was more effectively adsorbed by macroporous ECAB at around pH 3 and the mechanism of the adsorption of BSA to the ECAB was ion exchange. Finally, experiments of a concentration of 1 mg/mL BSA using macroporous ECAB were performed.


Assuntos
Alginatos/química , Reagentes para Ligações Cruzadas/química , Epicloroidrina/química , Adsorção , Cloreto de Cálcio/química , Sulfato de Cálcio/química , Ácido Edético/química , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Concentração de Íons de Hidrogênio , Porosidade , Soroalbumina Bovina/química , Soluções/química , Sulfatos/química , Propriedades de Superfície
16.
Int J Biol Macromol ; 49(4): 688-92, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21763718

RESUMO

The one-pot synthesis and characterization of cross-linked quaternized chitosan microspheres (CQCM) as a protein adsorbent are presented. First of all, chitosan particles were prepared by spray drying method, and then they were quaternized and cross-linked in turn with glycidyltrimethylammonium (GTMAC) chloride and glutaraldehyde in isopropanol containing 10% water in one-pot. The effect of the reaction temperature, reaction time and the amounts of added GTMAC and glutaraldehyde on the protein adsorption ability of CQCM was investigated. The adsorption behavior of the CQCM prepared in the optimum synthetic conditions was well described by the Langmuir isotherm with maximum adsorption capacity equal to 1424 mg BSA/g dry weight. The particle size ranged from 7.6 to 48.9 µm. The mechanism of adsorption-desorption of BSA to the CQCM was ion-exchange. Finally, the extraction of soybean peroxidase from crude soybean peroxidase solution using the CQCM was performed.


Assuntos
Química Orgânica/métodos , Quitosana/síntese química , Reagentes para Ligações Cruzadas/química , Microesferas , Soroalbumina Bovina/metabolismo , Adsorção , Animais , Bovinos , Compostos de Epóxi/química , Glutaral/química , Tamanho da Partícula , Peroxidase/metabolismo , Compostos de Amônio Quaternário/química , Soja/enzimologia , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura Ambiente , Fatores de Tempo
17.
Wei Sheng Wu Xue Bao ; 51(3): 393-401, 2011 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-21604554

RESUMO

OBJECTIVE: To study the bactericidal effect and the possible mechanisms of the three components system [soybean peroxidases (SBP)-hydrogen peroxide (H2O2)-potassium iodide (KI), SBP-H2O2-KI]. METHODS: The inhibition and bactericidal effect of SBP-H2O2-KI system to bacteria was detected by OD600 and the number of live bacteria (CFU). The sensitivity was tested by comparing the minimum inhibitory concentration (MIC) of bacterial cultures before and after cultured under sub-lethal dose of SBP-H2O2-KI system. Oxidizing activity groups were detected with physical and chemical methods in order to explain the bactericidal mechanisms of SBP-H2O2-KI system. RESULTS: SBP-H2O2-KI ternary system had rapid and high efficient bactericidal effect to a variety of bacterial strains in just several minutes. The MICs had no significant changes when bacterial cultures continuously cultured in sub-lethal dose of SBP-H2O2-KI system, and no resistance/tolerance mutant strains could be isolated from them. Both physical and chemical test results showed that no hydroxyl radical produced in SBP- H2O2-KI reaction system, chemical test results showed that no superoxide anion but a singlet oxygen and iodine produced in SBP-H2O2-KI reaction system. CONCLUSION: These results suggested that singlet oxygen and iodine or the iodine intermediate state may possible be the main sterilization factors for SBP-H2O2-KI system, and hydroxyl radical and superoxide anion not. In addition, the both characteristics of SBP-H2O2-KI system: rapid and high efficient bactericidal effect, and bacteria difficultly resisting to it, indicated it would have a good potential application in medical and plant protection area.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Peroxidase/farmacologia , Iodeto de Potássio/farmacologia , Testes de Sensibilidade Microbiana/métodos , Soja/enzimologia
18.
Langmuir ; 25(4): 2363-8, 2009 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-19161266

RESUMO

In the pH 2.6 and 5.2 systems, soybean peroxidase (SBP) (isoelectric point, pI 3.9) has positive and negative charge, respectively. In order to acquire detailed knowledge on the role played by electrostatics in the denaturation of proteins, a comparison of anionic surfactant sodium dodecyl sulfate (SDS), nonionic surfactant nonaethylene glycol monododecyl ether [C12H25O(CH2CH2O)9H] (AEO9), and cationic surfactant cetyltrimethylammonium bromide (CTAB) for the influences on the activity and structure of soybean peroxidase (SBP) was carried out by measuring the activity, far-UV circular dichrosm, fluorescence, and electronic absorption spectra of SBP in the pH 2.6 and 5.2 systems at 30 degrees C. In the pH 2.6 systems, the interaction of SDS with SBP results in an increase in the fluorescence intensity with a red shift of the emission maximum of the tryptophan fluorescence and a blue shift of the Soret band. In the meantime, the alpha-helix of SBP is unfolded and the activity of SBP is lost irreversibly. In pH 5.2 systems, the fluorescence spectra features of SBP are similar to those in pH 2.6 systems with increasing SDS concentration, but a red shift of Soret band as well as an alteration of the tertiary structure of SBP occurs, and the lost activity is recoverable. The electrostatic interactions between SBP and SDS play an important role in the denaturation of SBP. The effects of AEO9 and CTAB in pH 2.6 and 5.2 systems on the activity and spectral features of SBP are similar to that of SDS in pH 5.2 systems, but AEO9 is prone to unfold the beta-sheet of SBP in pH 2.6 systems. The electrostatic interactions of CTAB with SBP are not the primary elements for denaturation of SBP, which distinctly differ from those of SDS. These results can be useful with respect to wide applications of the surfactants in the separation and purification of proteins.


Assuntos
Peroxidase/metabolismo , Soja/efeitos dos fármacos , Soja/enzimologia , Tensoativos/química , Tensoativos/farmacologia , Cetrimônio , Compostos de Cetrimônio/química , Compostos de Cetrimônio/farmacologia , Ativação Enzimática/efeitos dos fármacos , Polidocanol , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Estrutura Secundária de Proteína , Dodecilsulfato de Sódio/química , Dodecilsulfato de Sódio/farmacologia
19.
Bioresour Technol ; 100(6): 2077-81, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19038543

RESUMO

A novel reductive compound with molecular weight of about 1000Da, named Pc reducer, was purified from the liquid culture of a white-rot basidiomycete Phanerochaetechrysosporium. It was likely to have an alkene-ester structure according to Fourier-transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) spectra. Pc reducer reduced the hydroxyl radical HO(*) and the stable nitroxide radical under certain conditions. It inhibited the repolymerization of the products from the oxidation of phenolic lignin-model compounds by reducing certain intermediate radicals. The activity of manganese peroxidases was promoted by Pc reducer at certain concentrations. Pc reducer could also weaken the repolymerization of fragments from the oxidation of Na-lignosulfonate by lignin peroxidases and manganese peroxidases. It has potential ability to improve the ligninolytic efficiency of peroxidases in P. chrysosporium.


Assuntos
Lignina/metabolismo , Phanerochaete/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Espectroscopia de Ressonância Magnética , Peso Molecular , Oxirredução , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier
20.
Electrophoresis ; 28(18): 3174-7, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17854120

RESUMO

A novel sodium bis(2-ethylhexyl) sulfosuccinate-PAGE (AOT-PAGE) system which delivers high resolution and sensitivity for small peptides with molecular masses of 0.8-17 kDa is described. Small peptides migrate more slowly and are less prone to leakage than in conventional SDS-PAGE, thus allowing for the in-gel detection with CBB R 250 of 0.5 mug of peptide. The system is also compatible with electroblotting, activity staining in renatured gels, and the peptide analysis by MALDI-MS. AOT-PAGE is simpler, more rapid, and cheaper than the generally adopted Tricine-SDS-PAGE method.


Assuntos
Ácido Dioctil Sulfossuccínico/química , Eletroforese em Gel de Poliacrilamida/métodos , Peptídeos/isolamento & purificação , Peso Molecular , Peptídeos/química , Sensibilidade e Especificidade
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA