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1.
Extremophiles ; 10(3): 251-5, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16601914

RESUMO

Alkaliphilic Nocardiopsis sp. strain F96 produced three beta-1,3-glucanase isozymes of different molecular masses (BglF1, BglF2 and BglF3). The N-terminal amino acid sequences of BglFs indicated that these isozymes were the products of a single gene. The beta-1,3-glucanase gene (bglF) was cloned from the chromosomal DNA of strain F96. The bglF gene encoded a polypeptide of 270 amino acids including a signal sequence. The deduced amino acid sequence of mature BglF exhibited the highest homology to those of glycoside hydrolase (GH) family 16 beta-1,3-glucanases, suggesting that the enzyme belonged to the GH family 16. The mature region of bglF gene was functionally expressed in Escherichia coli. The optimum pH and temperature of purified recombinant BglF were pH 9.0 and 70 degrees C, respectively. This enzyme efficiently hydrolyzed insoluble beta-1,3-glucans and showed the highest activity toward a beta-1,3-1,4-glucan rather than beta-1,3-glucans. These results suggested that BglF would be a novel beta-1,3-glucanse. Mutational analysis revealed that Glu123 and Glu128 should be the catalytic residues of BglF.


Assuntos
Actinomycetales/enzimologia , Glucana 1,3-beta-Glucosidase/metabolismo , Actinomycetales/genética , Álcalis , Sequência de Aminoácidos , Sequência Conservada , Glucana 1,3-beta-Glucosidase/química , Glucana 1,3-beta-Glucosidase/genética , Glucanos/metabolismo , Hidrólise , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
2.
Acta Crystallogr D Biol Crystallogr ; 60(Pt 11): 2081-3, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15502334

RESUMO

Chitosanase from Bacillus sp. strain K17 (ChoK) belongs to glycoside hydrolase family 8 and exhibits subclass II specificity. The purified protein is structurally stable over a wide pH range (3-10), but is active in a much narrower pH range (4.5-7.5), with optimal activity around pH 6.0. The protein has been successfully crystallized at two different pH values corresponding to the active and inactive states. The crystals diffract to 1.5 and 2.0 A resolution, respectively.


Assuntos
Bacillus/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Bacillus/classificação , Cristalização , Cristalografia por Raios X , Ativação Enzimática , Estabilidade Enzimática , Concentração de Íons de Hidrogênio
3.
J Mol Biol ; 343(3): 785-95, 2004 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-15465062

RESUMO

Crystal structures of chitosanase from Bacillus sp. K17 (ChoK) have been determined at 1.5 A resolution in the active form and at 2.0 A resolution in the inactive form. This enzyme belongs to the family GH-8, out of 93 glycoside hydrolase families, and exhibits the substrate specificity of subclass II chitosanase. The catalytic site is constructed on the scaffold of a double-alpha(6)/alpha(6)-barrel, which is formed by six repeating helix-loop-helix motifs. This structure is quite different from those of the GH-46 chitosanases and of GH-5. Structural comparison with CelA (a cellulase belonging to the same family GH-8) suggests that the proton donor Glu122 is conserved, but the proton acceptor is the inserted Glu309 residue, and that the corresponding Asp278 residue in CelA is inactivated in ChoK. The four acidic residues, Asp179, Glu309, Asp183 and Glu107, can be involved in substrate recognition through interactions with the amino groups of the glucosamine residues bound in the -3, -2, -1 and +1 sites, respectively. The hydrophobic Trp235, Trp166, Phe413 and Tyr318 residues are highly conserved for binding of the hexose rings at the -3, -2, +1 and +2 sites, respectively. These structural features indicate that enzymes in GH-8 can be further divided into three subfamilies. Different types of chitosanases are discussed in terms of convergent evolution from different structural ancestors.


Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Evolução Molecular , Glicosídeo Hidrolases/classificação , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Estrutura Secundária de Proteína , Alinhamento de Sequência , Especificidade por Substrato
4.
Nucleic Acids Symp Ser (Oxf) ; (48): 167-8, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-17150531

RESUMO

Alkaliphilic Bacillus sp. strain J813 produces a novel chitinase (chitinase J). The gene encoding chitinase J (chij) was cloned and sequenced. Deduced amino acid sequence revealed that Chij contained a family 18 catalytic domain, a fibronectin type III-like domain and a chitin-binding domain. Analysis of deletion derivatives indicated that the chitin-binding domain was important for binding to chitin and it enhanced the hydrolysis of insoluble chitin. The subsites existing in the catalytic domain of Chij was thought to bind to insoluble chitosan, although Chij did not hydrolyze chitosan. Some amino acid-substituted mutants were prepared and characterized, suggesting that Glu198 should be the catalytic residue of Chij.


Assuntos
Bacillus/enzimologia , Quitinases/genética , Quitinases/metabolismo , Deleção de Genes , Ligação Competitiva , Quitinases/química , Quitosana/metabolismo , Clonagem Molecular , Processamento de Proteína Pós-Traducional , Especificidade por Substrato , Fatores de Tempo
5.
Nucleic Acids Res Suppl ; (3): 317-8, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14510508

RESUMO

The gene encoding a novel beta-1,3-glucanase was cloned from alkaliphilic Nocardiopsis sp. F96 and sequenced. The gene contained an open reading frame of 936 bp. The deduced amino acid sequence of the beta-1,3-glucanase exhibited highest homology to those of family 16 glucanases, suggesting that the enzyme belonged to family 16. The beta-1,3-glucanase gene was functionally expressed in Escherichia coli.


Assuntos
Proteínas de Escherichia coli , Genes Bacterianos , Bactérias Gram-Positivas/genética , Proteínas de Membrana/genética , Proteínas Quinases/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Dados de Sequência Molecular , Fases de Leitura Aberta , Regiões Promotoras Genéticas
6.
Nucleic Acids Res Suppl ; (2): 227-8, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12903188

RESUMO

The gene encoding a novel chitosanase from Bacillus sp. strain K17 was cloned and sequenced. The nucleotide sequence of the gene contained an open reading frame corresponded to a protein of 453 amino acids. The deduced amino acid sequence of the K17 chitosanase exhibited the highest homology to those of family 8 glycanases, suggesting that the enzyme belonged to family 8.


Assuntos
Bacillus/enzimologia , Glicosídeo Hidrolases/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , Escherichia coli/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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