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1.
Int J Biol Macromol ; 175: 254-261, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33561459

RESUMO

The efficiency of enzymatic cyclodextrin production using cyclodextrin glycosyltransferases (CGTases) is limited by product inhibition. In this study, maltose binding site 2 (MBS2) of the ß-CGTase from Bacillus circulans STB01 was modified to decrease product inhibition. First, two point mutants were prepared at position 599 (A599V and A599N). Then, two double mutants incorporating alanine at position 633 (A599N/Y633A and A599V/Y633A) were prepared. Finally, the entire MBS2 region was replaced by that of the α-CGTase from Paenibacillus macerans JFB05-01 to form multipoint mutant MBS2 ߠ→ α. All five mutants exhibited mixed-type product inhibition, although both the competitive and uncompetitive components of this inhibition were decreased. The total cyclization activities of A599N, A599V and A599V/Y633A were 15.6%, 76.8% and 70.9% lower than that of the wild-type, respectively, while that of A599N/Y633A was 22.4% higher. Among the mutants, only MBS2 ߠ→ α showed catalytic efficiency (kcat/Km) comparable with that of the wild-type. Moreover, A599N, A599N/Y633A and MBS2 ߠ→ α produced cyclodextrin yields 13.1%, 15.8% and 19.7% greater than that of the wild-type, respectively. These results suggest that A599N, A599N/Y633A and MBS2 ߠ→ α may be more suitable than the wild-type for cyclodextrin production.


Assuntos
Bacillus/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Maltose/metabolismo , Bacillus/genética , Proteínas de Bactérias/química , Sítios de Ligação/genética , Ciclização/genética , Ciclodextrinas/metabolismo , Cinética , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida/métodos , Mutação/genética , Paenibacillus/genética , Especificidade por Substrato/genética , beta-Ciclodextrinas/química
2.
J Agric Food Chem ; 68(47): 13791-13797, 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33166453

RESUMO

Disulfide bonds play crucial roles in thermostabilization, recognition, or activation of proteins. They are vital in maintaining the respective conformations of globular structures, thereby enhancing thermostability. Bioinformatic approaches provide practical strategies to build disulfide bonds based on structural information. We constructed nine mutants by rational analysis of the 1,4-α-glucan branching enzyme (EC 2.4.1.18) from Geobacillus thermoglucosidans STB02, which catalyzes the synthesis of α-1,6-glucosidic bonds by acting on α-(1,4) and/or α-(1,6) glucosidic linkages. Four of the mutations enhanced thermostability, and five of them had adverse or negligible effects on stability. Circular dichroism spectra and intrinsic fluorescence analysis showed that introducing disulfide bonds might only affect secondary structures. The results also demonstrated that the distances of Cα carbons and thiol groups, as well as the sequence between the two cysteines, need to be considered when designing disulfide bonds.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana , Geobacillus , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Bacillaceae , Dissulfetos , Estabilidade Enzimática , Geobacillus/genética , Glucanos
3.
Food Chem Toxicol ; 135: 110934, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31682931

RESUMO

The novel commensal strain of Bacteroides fragilis HCK-B3 isolated from a healthy Chinese donor was discovered beneficial effects of attenuating lipopolysaccharides-induced inflammation. In order to contribute to the development of natural next-generation probiotic strains, the safety assessment was carried out with in vitro investigations of its morphology, potential virulence genes and antimicrobial resistance, and an in vivo acute toxicity study based on both healthy and immunosuppressed mice by cyclophosphamide injection. Consequently, the potential virulence genes in the genome of B. fragilis HCK-B3 have yet been identified as toxicity-associated. The absence of plasmids prevents the possibility of transferring antibiotic resistance features to other intestinal commensals. No intracorporal pathogenic properties were observed according to the body weight, hematological and liver parameters, cytokine secretions and tissue integrity. In addition, B. fragilis HCK-B3 performed alleviations on part of the side effects caused by the cyclophosphamide treatment. Thus, the novel strain of B. fragilis HCK-B3 was confirmed to be non-toxigenic and did not display adverse effects in both healthy and immune-deficient mice at a routinely applicable dose.


Assuntos
Bacteroides fragilis/patogenicidade , Probióticos/toxicidade , Animais , Antibacterianos/farmacologia , Bacteroides fragilis/efeitos dos fármacos , Bacteroides fragilis/genética , Citocinas/metabolismo , Resistência Microbiana a Medicamentos/genética , Resistência Microbiana a Medicamentos/fisiologia , Ilhas Genômicas , Masculino , Camundongos , Anotação de Sequência Molecular , Projetos Piloto , Medição de Risco
4.
PLoS One ; 7(7): e36978, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22815679

RESUMO

BACKGROUND: The gut of most insects harbours nonpathogenic microorganisms. Recent work suggests that gut microbiota not only provide nutrients, but also involve in the development and maintenance of the host immune system. However, the complexity, dynamics and types of interactions between the insect hosts and their gut microbiota are far from being well understood. METHODS/PRINCIPAL FINDINGS: To determine the composition of the gut microbiota of two lepidopteran pests, Spodoptera littoralis and Helicoverpa armigera, we applied cultivation-independent techniques based on 16S rRNA gene sequencing and microarray. The two insect species were very similar regarding high abundant bacterial families. Different bacteria colonize different niches within the gut. A core community, consisting of Enterococci, Lactobacilli, Clostridia, etc. was revealed in the insect larvae. These bacteria are constantly present in the digestion tract at relatively high frequency despite that developmental stage and diet had a great impact on shaping the bacterial communities. Some low-abundant species might become dominant upon loading external disturbances; the core community, however, did not change significantly. Clearly the insect gut selects for particular bacterial phylotypes. CONCLUSIONS: Because of their importance as agricultural pests, phytophagous Lepidopterans are widely used as experimental models in ecological and physiological studies. Our results demonstrated that a core microbial community exists in the insect gut, which may contribute to the host physiology. Host physiology and food, nevertheless, significantly influence some fringe bacterial species in the gut. The gut microbiota might also serve as a reservoir of microorganisms for ever-changing environments. Understanding these interactions might pave the way for developing novel pest control strategies.


Assuntos
Bactérias/classificação , Biodiversidade , Intestinos/microbiologia , Lepidópteros/microbiologia , Metagenoma , Ração Animal/microbiologia , Animais , Bactérias/genética , Hibridização in Situ Fluorescente , Larva/crescimento & desenvolvimento , Larva/microbiologia , Lepidópteros/crescimento & desenvolvimento , Metagenoma/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Análise de Sequência de RNA , Análise Espaço-Temporal
5.
FEMS Microbiol Ecol ; 80(3): 659-70, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22356591

RESUMO

Although hematophagous black flies are well-known socioeconomic pests and vectors of disease agents, their associated bacteria are poorly known. A systematic analysis of the bacterial community associated with freshly emerged adult black flies of four North American species, using cultivation-independent molecular techniques, revealed 75 nonsingleton bacterial phylotypes. Although 17 cosmopolitan phylotypes were shared among host species, each fly species had a distinct bacterial profile. The bacterial composition, however, did not correlate strongly with the host phylogeny but differed between male and female flies of the same species from the same habitat, demonstrating that a group of insects have a gender-dependent bacterial community. In general, female flies harbor a less diverse bacterial community than do males. The anatomical locations of selected bacteria were revealed using fluorescence in situ hybridization. Understanding the physiological function of the associated bacterial community could provide clues for developing novel pest-management strategies.


Assuntos
Bactérias/isolamento & purificação , Caracteres Sexuais , Simuliidae/microbiologia , Animais , Bactérias/classificação , DNA Bacteriano/genética , Ecossistema , Feminino , Hibridização in Situ Fluorescente , Masculino , América do Norte , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Especificidade da Espécie
6.
Plant Mol Biol ; 77(6): 609-17, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22038094

RESUMO

Wood identification is extremely important in the modern forest industry. It also has significant applications in forensics, as well as in archeology and ecological research. In this study, five universal primer pairs amplifying chloroplast noncoding sequences of 300-1,200 bp were designed. Sequencing these amplicons in combination can lead to reliable identification of logs and wood products to cultivar, ecotype, or even the falling population. These primer pairs work on both gymnosperms and angiosperm trees. They also are potentially applicable to accurately identify shrubs and herbaceous species. In addition, a wood DNA purification method is proposed in which N-phenacylthiazolium bromide (PTB) is used to increase the quality and quantity of extracted DNA. This method was first validated using air-dried timber disks from three different tree species that were felled 4 years ago. The sapwood and outer heartwood provided the best locations for DNA extraction. The method was also successfully applied to extract DNA from the recalcitrant processed white oak wood, randomly selected staves of wine barrels. The single nucleotide polymorphism detected on the oak DNA sequences showed correlation to their geographical origins.


Assuntos
DNA de Cloroplastos/genética , Reação em Cadeia da Polimerase/métodos , Árvores/classificação , Árvores/genética , Tiazóis
7.
Insect Biochem Mol Biol ; 41(4): 273-81, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21255649

RESUMO

In the animal kingdom, carotenoids are usually absorbed from dietary sources and transported to target tissues. Despite their general importance, the uptake mechanism is still poorly understood. Here we report the "red crop" phenomenon, an accumulation of α- and ß-carotene in crystalline inclusions in the enlarged foregut of the polyphagous Spodoptera larvae feeding on some potentially toxic plant leaves. The carotene crystals give the insect foregut a distinctive orange-red color. The crystals are embedded in a homogenous lawn of the bacterium Enterococcus casseliflavus, but the carotene seems to be selectively taken from the food plant. Caterpillars which fail to develop these carotene crystals exhibit a high mortality or fail to develop to adulthood. The crystallization of carotene and the enlargement of the foregut thus appears to manifest a multiple-step physiological adaptation of the insects to toxic food plants.


Assuntos
Carotenoides/química , Carotenoides/metabolismo , Plantas Comestíveis/parasitologia , Plantas Tóxicas/parasitologia , Spodoptera/fisiologia , Animais , Cristalização , Comportamento Alimentar , Mucosa Intestinal/metabolismo , Intestinos/química , Larva/química , Larva/crescimento & desenvolvimento , Larva/fisiologia , Spodoptera/química , Spodoptera/crescimento & desenvolvimento , beta Caroteno/química , beta Caroteno/metabolismo
8.
Wei Sheng Wu Xue Bao ; 42(4): 478-83, 2002 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-12557556

RESUMO

Many evidences show bachteria that can be cultured in lab are only the minority of germs existing in nature. Most of bacteria, containing rich valuable gene resource, are unknown for they are uncultured. To research these uncultured bacteria, Directly extract crude DNA from farm soil, separate 16S rDNA mixture amplified by PCR from the crude DNA, sequence it and compare the sequences with the records of NCBI to classify the germs. The significant microbe community difference from respective soil, which have possibility in developing a new tool for researching soil bacteria diversity were finded.


Assuntos
Bactérias/classificação , DNA Bacteriano/genética , Filogenia , RNA Ribossômico 16S/genética , Microbiologia do Solo , Bactérias/genética , Bactérias/isolamento & purificação , Sequência de Bases , DNA Bacteriano/isolamento & purificação , Dados de Sequência Molecular , Análise de Sequência de DNA
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