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1.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(5): 714-719, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31762243

RESUMO

OBJECTIVE: To select and identify the bacterium which highly produces protease and ß-D-glucosidase from 72 strains of Shuidouchi from Sichuan, and to provide evidence for further research on its nutritional value and fermentation strain exploiting. METHODS: Casein degradation test and pNPG chemical test were applied respectively to detect the capacity to produce protease and ß-D-glucosidase of each strain. Characteristics of morphology, biochemistry, 16S rRNA and MALDI-TOF-MS were used to identify the fermentation strain, which genetic stability, curves of growth and enzyme producing were also obtained. RESULTS: The strain with the highest enzyme activity of ß-D-glucosidase (0.084 U/L) among the top 10 strains for producing protease was selected as the fermentation strain and was identified as Bacillus subtilis, which curves of growth and enzyme producing conformed as well. The result of genetic stability showed that capacity of enzyme producing was stable until the 10th generation. CONCLUSIONS: The fermentation strain which highly produced protease and ß-D-glucosidase was selected from 72 strains of shuidouchi from Sichuan and was identified as Bacillus subtilis.


Assuntos
Bacillus subtilis/enzimologia , Glucosidases/biossíntese , Peptídeo Hidrolases/biossíntese , Alimentos de Soja/microbiologia , China , Fermentação , RNA Ribossômico 16S
2.
Bioprocess Biosyst Eng ; 42(3): 345-354, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30523449

RESUMO

Maltooligosyl trehalose trehalohydrolase (MTHase, EC 3.2.1.141) catalyzes the release of trehalose, a novel food ingredient, by splitting the α-1,4-glucosidic linkage adjacent to the α-1,1-glucosidic linkage of maltooligosyl trehalose. However, the high-yield preparation of recombinant MTHase has not yet been reported. In this study, a codon-optimized synthetic gene encoding Sulfolobus acidocaldarius MTHase was expressed in Escherichia coli. In initial expression experiments conducted using pET-24a (+) and E. coli BL21 (DE3), the MTHase activity was 10.4 U/mL and a large amount of the expression product formed inclusion bodies. The familiar strategies, including addition of additives, co-expression with molecular chaperones, and expression with a fusion partner, failed to enhance soluble MTHase expression. Considering the intermolecular disulfide bond of MTHase, expression was investigated using a system comprising plasmid pET-32a (+) and host E. coli Origami (DE3), which is conducive to cytoplasmic disulfide bond formation. The MTHase activity increased to 55.0 U/mL, a 5.3-fold increase. Optimization of the induction conditions in a 3-L fermentor showed that when the lactose was fed at 0.2 g/L/h beginning at an OD600 of 40 and the induction temperature was maintained at 30 °C, the MTHase activity reached a maximum of 204.6 U/mL. This is the first report describing a systematic effort to obtain high-efficiency MTHase production. The high yield obtained using this process provides the basis for the industrial-scale production of trehalose. This report is also expected to be valuable in the production of other enzymes containing disulfide bonds.


Assuntos
Proteínas de Bactérias/biossíntese , Escherichia coli/metabolismo , Expressão Gênica , Glucosidases/biossíntese , Sulfolobus acidocaldarius/genética , Proteínas de Bactérias/genética , Escherichia coli/genética , Glucosidases/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Sulfolobus acidocaldarius/enzimologia
3.
Plant Physiol Biochem ; 127: 223-230, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29614441

RESUMO

The glycoside hydrolase family 1 members Os4BGlu14, Os4BGlu16, and Os4BGlu18 were proposed to be rice monolignol ß-glucosidases. In vitro studies demonstrated that the Os4BGlu16 and Os4BGlu18 hydrolyze the monolignol glucosides coniferin and syringin with high efficiency compared to other substrates. The replacement of the conserved catalytic acid/base glutamate residue by a nonionizable glutamine residue in Os4BGlu14 suggested that it may be inactive as a ß-glucosidase. Here, we investigated the activities of Os4BGlu14, Os4BGlu16, and Os4BGlu18 in planta by recombinant expression of their genes in the Arabidopsis bglu45-2 (monolignol ß-glucosidase) mutant and analysis of monolignol glucosides by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MSMS). The bglu45-2 line exhibits elevated monolignol glucoside levels, but lower amounts of coniferin, syringin, and p-coumaryl alcohol glucoside were seen in Arabidopsis bglu45-2 rescued lines complemented by the Os4BGlu14, Os4BGlu16, and Os4BGlu18 genes. These data suggest that the bglu45-2 mutant has a broader effect on monolignols than previously reported and that the Os4BGlu14, Os4BGlu16 and Os4BGlu18 proteins act as monolignol ß-glucosidases to complement the defect. An OsBGlu16-GFP fusion protein localized to the cell wall. This apoplastic localization and the effect of these enzymes on monolignol glucoside levels suggest monolignol glucosides from the vacuole may meet the monolignol ß-glucosidases, despite their different localization.


Assuntos
Arabidopsis , Glucosidases , Mutação , Oryza/genética , Proteínas de Plantas , Plantas Geneticamente Modificadas , Arabidopsis/enzimologia , Arabidopsis/genética , Glucosidases/biossíntese , Glucosidases/genética , Oryza/enzimologia , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética
4.
World J Microbiol Biotechnol ; 34(3): 40, 2018 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-29468428

RESUMO

Extremophilic microorganisms are a rich source of enzymes, the enzymes which can serve as industrial catalysts that can withstand harsh processing conditions. An example is thermostable ß-glucosidases that are addressing a challenging problem in the biodiesel industry: removing steryl glucosides (SGs) from biodiesel. Steryl glucosidases (SGases) must be tolerant to heat and solvents in order to function efficiently in biodiesel. The amphipathic nature of SGs also requires enzymes with an affinity for water/solvent interfaces in order to achieve efficient hydrolysis. Additionally, the development of an enzymatic process involving a commodity such as soybean biodiesel must be cost-effective, necessitating an efficient manufacturing process for SGases. This review summarizes the identification of microbial SGases and their applications, discusses biodiesel refining processes and the development of analytical methods for identifying and quantifying SGs in foods and biodiesel, and considers technologies for strain engineering and process optimization for the heterologous production of a SGase from Thermococcus litoralis. All of these technologies might be used for the production of other thermostable enzymes. Structural features of SGases and the feasibility of protein engineering for novel applications are explored.


Assuntos
Biotecnologia/métodos , Glucosidases/biossíntese , Glucosidases/química , Biocombustíveis , Celulases/biossíntese , Celulases/química , Celulases/genética , Estabilidade Enzimática , Glucosidases/genética , Temperatura Alta , Hidrólise , Engenharia de Proteínas , Solventes/química , Soja
5.
Appl Microbiol Biotechnol ; 97(13): 6009-18, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23563988

RESUMO

Streptococcus alactolyticus strain FGM, isolated from chicken cecum, was used to increase the extract yield of polysaccharides during Astragalus membranaceus fermentation. It was previously demonstrated that polysaccharides from fermented A. membranaceus by S. alactolyticus had some similar properties to those from A. membranaceus in terms of its ability to help heal hepatic fibrosis in rats and modulate immunopotentiation of broiler chicken. However, methods to increase the yield of the polysaccharides during fermentation of A. membranaceus are not well understood. In this paper, we investigated the involvement of uridine diphosphate (UDP)-glucose 4-epimerase (galE) and glucan-1,6-α-glucosidase (dexB) during A. membranaceus fermentation through real-time reverse transcription quantitative PCR. The galE and dexB genes of S. alactolyticus were cloned by homology-based cloning and the genome walking method for the first time, and the 3D structure of dexB was analyzed by Swiss-PdbViewer 4.0.1 software. The expression of both the galE and dexB genes in A. membranaceus fermentation was studied using the determined ideal reference gene ldh for transcript normalization. The results showed that these two genes were both highly induced and peaked after 12 h of fermentation. The expression level of galE was stepwise increased from 48 to 72 h, while dexB transcripts were markedly increased at 60 h and decreased by 72 h. These data suggested that dexB and galE of S. alactolyticus strain FGM were involved in the regulation of A. membranaceus fermentation and they might play some roles in the increase of polysaccharides.


Assuntos
Astragalus propinquus/química , Perfilação da Expressão Gênica , Glucosidases/biossíntese , Polissacarídeos/metabolismo , Streptococcus/enzimologia , Streptococcus/metabolismo , UDPglucose 4-Epimerase/biossíntese , Animais , Ceco/microbiologia , Galinhas/microbiologia , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Fermentação , Glucosidases/química , Glucosidases/genética , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Streptococcus/genética , Streptococcus/isolamento & purificação , UDPglucose 4-Epimerase/química , UDPglucose 4-Epimerase/genética
6.
Protein Expr Purif ; 88(1): 20-5, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23201279

RESUMO

Escherichia coli is considered one of the most appropriate hosts for the production of recombinant proteins. However, its usage is undermined by its inability to efficiently secrete proteins into the extracellular medium. We selected two cellulolytic enzymes with potential biofuel applications, ß-1,4-endoglucanase (Endo5A) and ß-1,4-glucosidase (Gluc1C), and determined the genetic and environmental parameters for their optimal secretion into culture medium. Endo5A and Gluc1C were fused with the hyperosmotically inducible periplasmic protein of E. coli, OsmY, and their activities in the extracellular, periplasmic and cytoplasmic fractions were monitored. Most of the endoglucanase activity (0.15 µmol min(-1) ml(-1)) and ß-glucosidase activity (2.2 µmol min(-1) ml(-1)) in the extracellular fraction was observed at 16 h post-induction. To reduce the overall cost, we expressed Endo5A and Gluc1C together either via a synthetic operon or through a bifunctional chimeric protein. Both systems efficiently secreted the enzymes, as evident from the functional activities and protein profiles on SDS-PAGE gels. The enzymes secreted via a synthetic operon showed higher activities (0.14 µmol min(-1) ml(-1) for endoglucanase and 2.4 µmol min(-1) ml(-1) for ß-glucosidase) as compared to the activities shown by the- bifunctional chimera (0.075 µmol min(-1) ml(-1) for endoglucanase and 2.0 µmol min(-1)ml(-1) for ß-glucosidase). The cellulase secretion system developed here has potential for use in the production of lignocellulosic biofuels.


Assuntos
Celulase/metabolismo , Escherichia coli/enzimologia , Glucosidases/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Reatores Biológicos , Celulase/biossíntese , Celulase/genética , Celulase/isolamento & purificação , Citoplasma/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Matriz Extracelular/enzimologia , Glucosidases/biossíntese , Glucosidases/genética , Glucosidases/isolamento & purificação , Periplasma/enzimologia , Proteínas Periplásmicas de Ligação/biossíntese , Proteínas Periplásmicas de Ligação/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
7.
Rev. colomb. biotecnol ; 13(1): 66-72, jul. 2011. tab, graf, ilus
Artigo em Espanhol | LILACS | ID: lil-600575

RESUMO

Las beta-glucosidasas son enzimas que poseen actividad hidrolitica y transferasa o transglucosidasa. Tienen diversas aplicaciones; en la biosintesis de oligosacaridos, produccion de etanol utilizando residuos agricolas y en la industria de vinos. La aplicacion industrial, sin embargo, requiere estabilidad a temperaturas elevadas, por lo que los microorganismos termofilos tienen gran interes. El proposito de esta investigacion es el de optimizar el medio de cultivo anaerobio de bacterias termofilas, para aumentar la produccion de beta-glucosidasas. Esta enzima es producida por tres aislados bacterianos: FT3, 2B y P5 los cuales fueron aislados de la region andina de Bolivia. El aislado bacteriano FT3 mostro una actividad beta-glucosidasa de 0,35 [UI/mL]. Se tomaron como variables dentro de la optimizacion del medio de cultivo las fuentes de nitrogeno y de carbono, y el pH. Asi tambien se probaron dos sistemas de cultivo: celulas libres y encapsuladas. Empleando extracto de levadura como fuente de nitrogeno se obtuvo una actividad de 0,52 [UI/mL]. En la optimizacion del pH del medio de cultivo se obtuvo una actividad de 0,81 [UI/mL] a pH 5. Como fuente de carbono se eligieron los hidrolizados de paja de trigo y paja de quinoa lleg¨¢ndose a obtener actividades de 1,27 y 1,34 [UI/mL] respectivamente. Se establecio que la localizacion celular de la enzima beta-glucosidasa es extracelular y presenta estabilidad hasta una temperatura de 80 ºC y un pH de 7.


The beta-glucosidases possess hydrolytic and transferase activity or transglucosidase. They have various applications; such as biosynthesis of oligosaccharides, production of ethanol using agricultural residues and wine industry. However for industrial application, stability to high temperatures is needed. Therefore a great interesting in the thermophile microorganism study exist. The purpose of this research is to optimize the culture medium of thermophilic anaerobic bacteria to increase the production of beta-glucosidase. This enzyme is produced by three isolate bacterial FT3, 2B and P5 which were isolated from the Andean region of Bolivia. FT3 isolate showed beta-glucosidase activity of 0.35 [IU/mL]. In regards to the optimization of culture medium variables such as nitrogen source, carbon source and pH were taken into account and also the combination with free and encapsulated bacterial cells. Yeast extract was the selected source of nitrogen obtaining an activity of 0.52 [IU/ mL]. The optimal pH was 5 obtaining an activity of 0.81 [IU/mL]. The selected carbon source was the hydrolyzed wheat straw and quinoa straw obtaining activities of 1.27 and 1.34 [IU/mL], respectively. The cellular localization of beta-glucosidase enzyme is extracellular and provides stability to temperature of 80 ºC and stability at pH 7.


Assuntos
Glucosidases/análise , Glucosidases/biossíntese , Glutationa Transferase/análise , Glutationa Transferase/biossíntese , Glutationa Transferase/classificação , Glutationa Transferase/farmacologia , Glutationa Transferase/química , Glutationa Transferase/síntese química , Glutationa Transferase/ultraestrutura , Oligossacarídeos/isolamento & purificação , Oligossacarídeos/análise , Oligossacarídeos/genética , Oligossacarídeos/química , Oligossacarídeos/síntese química , Oligossacarídeos/ultraestrutura , Oligossacarídeos
8.
Protein Expr Purif ; 73(1): 90-8, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20406687

RESUMO

The native beta-d-glucan exohydrolase isoenzyme ExoI from barley seedlings, designated HvExoI, was the first GH3 glycoside hydrolase, for which a crystal structure was determined. A precise understanding of relationships between structure and function in this enzyme has been gained by structural and enzymatic studies. To allow testing of hypotheses gained from these studies, an efficient system for expression of HvExoI in Pichia pastoris was developed using a codon-optimized cDNA. Protein expression at a temperature of 20 degrees C yielded a recombinant enzyme, designated rHvExoI, which had molecular masses of 70-110 kDa due to heavy glycosylation at Asn221, Asn498 and Asn600, the three sites of N-glycosylation in native HvExoI. Most of the N-linked carbohydrate could be removed from rHvExoI, resulting in N-deglycosylated rHvExoI with a substantially decreased molecular mass of 67 kDa. rHvExoI was able to hydrolyse barley (1,3;1,4)-beta-D-glucan, laminarin and lichenans. The catalytic efficiency value k(cat)/K(M) of rHvExoI with barley (1,3;1,4)-beta-D-glucan was similar to that reported for native HvExoI. Further, laminaribiose, cellobiose and gentiobiose were formed through transglycosylation reactions with 4-nitrophenyl beta-D-glucoside and barley (1,3;1,4)-beta-D-glucan. Overall, the biochemical properties of rHvExoI were similar to those reported for native HvExoI, although differences were seen in thermostabilities and hydrolytic rates of certain beta-linked glucosides.


Assuntos
Clonagem Molecular/métodos , Glucosidases/biossíntese , Hordeum/enzimologia , Pichia/genética , Sequência de Aminoácidos , Cromatografia em Camada Delgada , Códon , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Glucosidases/química , Glucosidases/genética , Glicosilação , Hordeum/genética , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos , Pichia/metabolismo , Polissacarídeos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato , Temperatura Ambiente , Tripsina/metabolismo
9.
Nucleic Acids Res ; 35(12): e88, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17576673

RESUMO

The extreme thermoacidophiles of the genus Sulfolobus are among the best-studied archaea but have lacked small, reliable plasmid vectors, which have proven extremely useful for manipulating and analyzing genes in other microorganisms. Here we report the successful construction of a series of Sulfolobus-Escherichia coli shuttle vectors based on the small multicopy plasmid pRN1 from Sulfolobus islandicus. Selection in suitable uracil auxotrophs is provided through inclusion of pyrEF genes in the plasmid. The shuttle vectors do not integrate into the genome and do not rearrange. The plasmids allow functional overexpression of genes, as could be demonstrated for the beta-glycosidase (lacS) gene of S. solfataricus. In addition, we demonstrate that this beta-glycosidase gene could function as selectable marker in S. solfataricus. The shuttle plasmids differ in their interruption sites within pRN1 and allowed us to delineate functionally important regions of pRN1. The orf56/orf904 operon appears to be essential for pRN1 replication, in contrast interruption of the highly conserved orf80/plrA gene is tolerated. The new vector system promises to facilitate genetic studies of Sulfolobus and to have biotechnological uses, such as the overexpression or optimization of thermophilic enzymes that are not readily performed in mesophilic hosts.


Assuntos
Vetores Genéticos/química , Plasmídeos/genética , Sulfolobus acidocaldarius/genética , Sulfolobus solfataricus/genética , Replicação do DNA , Elementos de DNA Transponíveis , Escherichia coli/genética , Dosagem de Genes , Genes Reporter , Glucosidases/biossíntese , Glucosidases/genética , Modelos Genéticos , Plasmídeos/biossíntese , Sulfolobus acidocaldarius/crescimento & desenvolvimento , Transformação Genética
10.
J Biotechnol ; 127(2): 199-210, 2007 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-16959351

RESUMO

A regulative two-component system previously identified in Pseudoalteromonas haloplanktis TAC125 was used to construct a cold inducible expression system that is under the control of l-malate. Performances of the inducible system were tested for both psychrophilic and mesophilic protein production using two "difficult" proteins as control. The results obtained demonstrated that both psychrophilic beta-galactosidase and yeast alpha-glucosidase are produced in a fully soluble and catalytically competent form. Optimal conditions for protein production, including growth temperature, growth medium and l-malate concentration were also investigated. Under optimized conditions yields of 620 and 27 mg/l were obtained for beta-galactosidase and alpha-glucosidase, respectively.


Assuntos
Temperatura Baixa , Regulação Bacteriana da Expressão Gênica , Pseudoalteromonas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Catálise , DNA Bacteriano , Indução Enzimática , Vetores Genéticos/genética , Genoma Bacteriano , Glucosidases/biossíntese , Glucosidases/química , Glucosidases/genética , Saccharomyces cerevisiae/enzimologia , beta-Galactosidase/biossíntese , beta-Galactosidase/genética
11.
Artigo em Inglês | MEDLINE | ID: mdl-16511316

RESUMO

Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 A, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 A.


Assuntos
Glucosidases/química , Clonagem Molecular , Cristalização/métodos , Cristalografia por Raios X , Escherichia coli/metabolismo , Glucosidases/biossíntese , Glucosidases/isolamento & purificação , Rauwolfia/enzimologia
12.
Protein Expr Purif ; 48(2): 205-14, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16542852

RESUMO

Hyper-expression of a secretory exoglucanase, Exg, encoded by the cex gene of Cellulomonas fimi was previously shown to saturate the SecYEG pathway and result in dramatic cell death of recombinant Escherichia coli (Z.B. Fu, K.L. Ng, T.L. Lam, W.K.R. Wong, Cell death caused by hyper-expression of a secretory exoglucanase in Esherichia coli, Protein Expr. Purif. 42 (2005) 67-77). We propose here that the cell lysate ratio (Pre/Mat RQ) of the unprocessed precursor Exg protein (Pre-Exg) and its processed mature product (Mat-Exg) reflects the capacity of E. coli to secrete Exg. A Pre/Mat RQ of 20/80, designated the "Critical Value," was an important threshold measurement. A rise in the Pre/Mat RQ triggered a mass killing effect. The use of various secretion signal peptides did not improve the viability of cells expressing high levels of Pre-Exg under strong tac promoter control. However, use of the weaker vegG promoter in conjunction with a change in start codon of the spa leader sequence from ATG to TTG in a pM1vegGcexL plasmid construct resulted in a high level (0.9 U ml(-1)) of excreted Exg in shake-flask cultures. This was 50% higher than the best result obtained from plasmid construct lacUV5par8cex, using the lacUV5 promoter and the ompA leader sequence. Variations in the excreted Exg activities were attributable to differences in the Pre/Mat RQ values of the induced cultures harboring pM1vegGcexL and lacUV5par8cex. These values were 18/82 and 10/90, respectively. Employing fed-batch cultivation in two-liter fermentors, an induced JM101(pM1vegGcexL) culture yielded 4.5 U ml(-1) of excreted Exg, which was over six fold greater that previously reported. Our results illustrate the successful application of the Pre/Mat RQ ratio as a guide to the attainment of a maximum level of secreted/excreted Exg.


Assuntos
Escherichia coli/genética , Expressão Gênica , Glucosidases/biossíntese , Glucosidases/genética , Morte Celular , Fermentação , Glucosidases/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
13.
Mycol Res ; 107(Pt 4): 421-7, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12825514

RESUMO

The effect of Trichoderma atroviride, T. harzianum, T. longibrachiatum, Clonostachys rosea and Bacillus subtilis isolates applied to wheat seeds against Rhizoctonia cerealis disease of seedlings was investigated under controlled greenhouse conditions. Most Trichoderma isolates significantly reduced the incidence of disease compared with the infected control. Bacillus subtilis was also effective against sharp eyespot, although less active than Trichoderma spp. Interactions between the antagonistic microorganisms and the cereal pathogenic fungus in dual culture experiments on agar growth medium were also studied. Almost all tested antagonists showed competitive activity against R. cerealis: inhibition of its mycelial growth and hyphal interaction. The production of extracellular beta-N-acetylhexosaminidase, chitin 1,4-beta-chitobiosidase, glucan 1,3-beta-glucosidase and protease activity by the tested microorganisms in the presence of cell walls of R. cerealis was then determined. All isolates showed glucosaminidase and chitobiosidase activity. They also produced glucosidase activity, except B. subtilis, whereas only C. rosea, B. subtilis and one isolate of T. harzianum showed detectable levels of protease activity.


Assuntos
Bacillus subtilis/fisiologia , Basidiomycota/fisiologia , Gliocladium/fisiologia , Trichoderma/fisiologia , Triticum/microbiologia , Antibiose , Bacillus subtilis/enzimologia , Basidiomycota/citologia , Basidiomycota/patogenicidade , Biodegradação Ambiental , Parede Celular/metabolismo , Endopeptidases/biossíntese , Endopeptidases/metabolismo , Indução Enzimática , Gliocladium/enzimologia , Glucosidases/biossíntese , Glucosidases/metabolismo , Hexosaminidases/biossíntese , Hexosaminidases/metabolismo , Plântula/citologia , Plântula/microbiologia , Trichoderma/enzimologia
14.
Biotechnol Bioeng ; 82(3): 313-20, 2003 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-12599258

RESUMO

Membrane-coupled bioreactors (MBRs) offer many benefits compared to conventional biological wastewater treatment systems; however, their performance characteristics are poorly understood. Laboratory-scale MBRs were used to study bacterial adaptations in physiology and community structure. MBRs were fed a mixture of starch, gelatin, and polyoxyethylene-sorbitan monooleate to simulate the polysaccharide, protein, and lipid components of municipal wastewater. Physiological adaptations were detected by measuring ectoenzyme activity while structural dynamics were studied by denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments. As cell biomass accumulated in the MBRs, pollutant removal efficiency initially improved and then stabilized with respect to effluent concentrations of chemical oxygen demand, protein, and carbohydrate. Comparison of the MBR effluent to filtered reactor fluid indicated that a portion of the observed pollutant removal was due to filtration by the membrane rather than microbial activity. The rates of ectoenzyme-mediated polysaccharide (alpha-glucosidase) and protein (leucine aminopeptidase) hydrolysis became relatively constant once pollutant removal efficiency stabilized. However, the maximum rate of lipid hydrolysis (heptanoate esterase) concomitantly increased more than 10-fold. Similarly, alpha-glucosidase and leucine aminopeptidase ectoenzyme affinities were relatively constant, while the heptanoate esterase affinity increased more than 30-fold. Community analysis revealed that a substantial community shift occurred within the first 7 days of operation. A Flavobacterium-like bacterial population dominated the community (>50% of total band intensity) and continued to do so for the remainder of the experiment.


Assuntos
Adaptação Fisiológica/fisiologia , Bactérias Aeróbias/fisiologia , Reatores Biológicos/microbiologia , Hidrolases/biossíntese , Membranas Artificiais , Aminopeptidases/biossíntese , Bactérias Aeróbias/genética , Bactérias Aeróbias/crescimento & desenvolvimento , Bactérias Aeróbias/metabolismo , Biodegradação Ambiental , Metabolismo dos Carboidratos , Cidades , Ectogênese/fisiologia , Ativação Enzimática , Esterases/biossíntese , Glucosidases/biossíntese , Resíduos Industriais/prevenção & controle , Proteínas/metabolismo , Especificidade por Substrato , Ultrafiltração/instrumentação , Ultrafiltração/métodos , Eliminação de Resíduos Líquidos/métodos , Poluentes Químicos da Água/metabolismo
15.
Mar Environ Res ; 54(1): 1-19, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12148942

RESUMO

A seasonal study of the quantitative and qualitative distribution of heterotrophic bacterial community was carried out in the Adriatic Sea between April 1995 and January 1996, in order to evaluate its spatial and temporal variability and metabolic potential in the degradation processes of organic matter. The culturable bacteria (CFU) ranged between 0.1 and 22% of total bacterioplankton with a maximum percentage in surface samples of coastal zones. Their distribution was generally affected by the prevailing hydrological conditions. At the coastal stations about 44-75% of CFU variance could be explained by river runoff. The changes in the composition of heterotrophic bacterial community showed a seasonal succession of main bacterial groups, with a prevalence of Gram negative, non fermenting bacteria in the cold period (April-January) and an increase of Vibrionaccae and pigmented bacteria in summer. The seasonal variations were more important at the stations influenced by rivers than offshore. The bacterial community showed a greater versatility for organic polymers hydrolysis in the offshore station than in the coastal areas. Over 60% of all isolated heterotrophic bacteria expressed peptidase, lipase and phosphatase ectoenzymes activities, in all seasons and showed an increasing trend in warm period (in July October). The alpha- and beta-glucosidase potentials of bacteria were lower (20% on average) and showed different pattern during the year. These results suggest different role of the bacterial community in the decomposition of organic matter in the Adriatic Sea. Since only 20% of bacterial strains expressed glucosidase activity, carbohydrate-rich polymers such as mucilage might accumulate.


Assuntos
Bactérias/enzimologia , Microbiologia da Água , Monitoramento Ambiental , Glucosidases/biossíntese , Glucosidases/farmacologia , Hidrólise , Itália , Lipase/biossíntese , Lipase/farmacologia , Compostos Orgânicos/análise , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/farmacologia , Polímeros/metabolismo , Estações do Ano , Temperatura Ambiente
16.
Planta ; 215(1): 51-9, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12012241

RESUMO

When seedlings of barley (Hordeum vulgare L.) were transferred from a natural light/dark cycle into darkness, (1-->3,1-->4)-beta- D-glucan endohydrolase (EC 3.2.1.73) activity in leaf extracts increased 3- to 4-fold after 2 days. Activity decreased to normal levels within a day if the light/dark cycle was restored. Although there are two (1-->3,1-->4)-beta-D-glucan endohydrolase isoenzymes in barley, the increased enzyme activity in dark-grown seedlings was attributable entirely to increases in isoenzyme EI. Northern hybridization analyses confirmed that mRNA transcripts encoding (1-->3,1-->4)-beta-D-glucan endohydrolase isoenzyme EI accumulated in the leaves of dark-incubated seedlings; no isoenzyme EII mRNA was detected. Activity of beta-D-glucan glucohydrolases also increased 10-fold after 2 days of dark treatment. The latter, broad-specificity enzymes release glucose from (1-->3,1-->4)-beta-D-glucans and from beta-D-oligoglucosides released by (1-->3,1-->4)-beta-D-glucan endohydrolases. Consistent with the activity patterns of these enzymes, the (1-->3,1-->4)-beta-D-glucan content of leaf cell walls decreased by about 30% when barley seedlings were transferred into darkness. Soluble sugars in the leaves decreased by about 60% during the same period. Because no measurable leaf elongation was detected during the various light/dark treatments, the enzymes were unlikely to be participating in wall loosening and cell elongation. Instead, the results suggest that cell wall (1-->3,1-->4)-beta-D-glucans can be re-mobilized in the non-elongating, dark-incubated leaves and the glucose so generated could serve as an energy source under conditions of sugar depletion.


Assuntos
Glicosídeo Hidrolases/biossíntese , Hordeum/genética , Folhas de Planta/genética , Sequência de Bases , Northern Blotting , Western Blotting , Parede Celular/metabolismo , Clorofila/metabolismo , Escuridão , Indução Enzimática/efeitos da radiação , Glucosidases/biossíntese , Glucosidases/genética , Glicosídeo Hidrolases/genética , Hordeum/enzimologia , Isoenzimas/biossíntese , Isoenzimas/genética , Luz , Dados de Sequência Molecular , Folhas de Planta/enzimologia , Homologia de Sequência do Ácido Nucleico
17.
Mikrobiol Z ; 62(2): 19-26, 2000.
Artigo em Russo | MEDLINE | ID: mdl-10872283

RESUMO

Different glucose concentrations have been studied for their effect on biosynthesis of bacterio- and proteolytic enzymes in Streptomyces recifensis var. lyticus 2435 and strains II-29 and 2P-15 obtained from the latter. It has been established that synthesis of enzymes by the parent strain 2435 was subjected to catabolite repression: synthesis of proteolytic and staphylolytic enzymes was the most sensitive to glucose, synthesis of glucosidases was less sensitive. The role of catabolite repression in regulation of synthesis of bacteriolytic enzymes to the latter in strain II-29 and rifamycin-resistant mutant 2P-15 is essentially decreased. As compared with the initial strains, the high level of activity of lytic enzymes in the strain 2P-15 is achieved at higher glucose concentrations in the fermentation medium, synthesis of staphylolysins and glucosideases are inhibited inconsiderably at 2% and 4% content of glucose, in medium respectively and it does not decrease under further increase of glucose concentration.


Assuntos
Glucose/metabolismo , Mutação/fisiologia , Peptídeo Hidrolases/biossíntese , Streptomyces/enzimologia , Meios de Cultura , Relação Dose-Resposta a Droga , Endotoxinas/biossíntese , Glucosidases/biossíntese , Concentração de Íons de Hidrogênio , Staphylococcus , Streptomyces/crescimento & desenvolvimento
18.
Oral Microbiol Immunol ; 14(2): 122-6, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10219172

RESUMO

Capnocytophaga gingivalis was grown with proteins (albumin, collagen, mucin and hemoglobin) as carbon and energy sources in chemostat culture. The mu max (0.34 h-1) and biomass yield (0.96 g.l-1) were as high with hemoglobin (3 g.l-1) as with glucose (3 g.l-1) (20). Albumin, collagen and mucin also supported an increased mu max, or yield or both, in comparison with basal (tryptone/thiamine) medium. In steady-state, trypsin-like protease specific activity increased 3- to 5-fold in the presence of albumin, collagen and hemoglobin: whereas the greatest increase (21-fold) in alpha-glucoside activity was in the presence of mucin. There were significant, but less substantial changes in other hydrolytic enzymes (aminopeptidase, acid and alkaline phosphatases). The bulk of the detected hydrolytic activity (> 66%) was associated with the cells. The data indicate that C. gingivalis regulates its production of hydrolytic enzymes in response to environmental conditions.


Assuntos
Proteínas de Bactérias/biossíntese , Capnocytophaga/enzimologia , Capnocytophaga/crescimento & desenvolvimento , Hidrolases/biossíntese , Fosfatase Ácida/biossíntese , Fosfatase Alcalina/biossíntese , Aminopeptidases/biossíntese , Análise de Variância , Meios de Cultura , Glucosidases/biossíntese , Serina Endopeptidases/biossíntese , Especificidade por Substrato
19.
Appl Environ Microbiol ; 64(10): 3615-9, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9758775

RESUMO

The expression of genes encoding enzymes involved in xylan degradation and two endoglucanases involved in cellulose degradation was studied at the mRNA level in the filamentous fungus Aspergillus niger. A strain with a loss-of-function mutation in the xlnR gene encoding the transcriptional activator XlnR and a strain with multiple copies of this gene were investigated in order to define which genes are controlled by XlnR. The data presented in this paper show that the transcriptional activator XlnR regulates the transcription of the xlnB, xlnC, and xlnD genes encoding the main xylanolytic enzymes (endoxylanases B and C and beta-xylosidase, respectively). Also, the transcription of the genes encoding the accessory enzymes involved in xylan degradation, including alpha-glucuronidase A, acetylxylan esterase A, arabinoxylan arabinofuranohydrolase A, and feruloyl esterase A, was found to be controlled by XlnR. In addition, XlnR also activates transcription of two endoglucanase-encoding genes, eglA and eglB, indicating that transcriptional regulation by XlnR goes beyond the genes encoding xylanolytic enzymes and includes regulation of two endoglucanase-encoding genes.


Assuntos
Aspergillus niger/genética , Celulase/genética , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica , Glucosidases/genética , Transativadores/genética , Transativadores/metabolismo , Transcrição Genética , Aspergillus niger/metabolismo , Celulase/biossíntese , Celulose/metabolismo , Regulação Enzimológica da Expressão Gênica , Genes Fúngicos , Glucosidases/biossíntese , Mutação , Xilanos/metabolismo
20.
J Bacteriol ; 180(13): 3400-4, 1998 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9642194

RESUMO

A newly identified bglH gene coding for a phospho-beta-glucosidase of Lactobacillus plantarum was isolated and expressed in Escherichia coli. The sequence analysis of the cloned DNA fragment showed an open reading frame encoding a 480-amino-acid protein with a calculated molecular mass of 53 kDa. The bglH gene was shown to be expressed on a monocistronic transcriptional unit. Its transcription was repressed 10-fold in L. plantarum cells grown on glucose compared to the beta-glucoside salicin as a sole carbon source. A catabolite-responsive element (CRE) spanning from -3 to +11 with respect to the transcriptional start point was found, and its functionality was assessed by mutational analysis. In vitro and in vivo DNA binding experiments suggested the occurrence of a DNA-protein complex at the CRE site, which would mediate glucose repression of bglH expression.


Assuntos
Repressão Enzimática , Glucosidases/biossíntese , Lactobacillus/genética , Sequência de Aminoácidos , Sequência de Bases , Álcoois Benzílicos/metabolismo , Clonagem Molecular , Sequência Consenso , Genes Bacterianos , Glucose/metabolismo , Glucosidases/química , Glucosidases/genética , Glucosídeos , Lactobacillus/crescimento & desenvolvimento , Lactobacillus/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , RNA Mensageiro/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Genética
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