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 SubstratoRESUMO
Chitinase F1 (ChiF1) isolated from the alkaliphilic Nocardiopsis sp. strain F96 is a family 18 chitinase that hydrolyzes chitin, an insoluble beta-1,4-linked polymer of N-acetylglucosamine. Crystals of recombinant ChiF1 with molecular weight of 33 000 Da were grown to a suitable size for X-ray structure analysis using 18%(w/v) polyethylene glycol 8000, 200 mM zinc acetate dehydrate and 100 mM sodium cacodylate buffer pH 6.5. Diffraction data were collected at SPring-8 and show that the crystals belong to the trigonal space group P3(1)12 or P3(2)12, with unit-cell parameters a = 56.0, c = 179.5 A, and diffract X-rays beyond 1.2 A resolution. Crystallographic analysis was carried out using the multiwavelength anomalous diffraction (MAD) method using zinc as the anomalous scatter. The binding of Zn atoms was clarified from the Bijvoet and dispersive Patterson functions, which gave prominent zinc-zinc self-vectors on the Harker section.
Assuntos
Actinomycetales/enzimologia , Quitinases/química , Quitinases/isolamento & purificação , Cristalização , Cristalografia por Raios X , Concentração de Íons de HidrogênioRESUMO
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 TempoRESUMO
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 GenéticasRESUMO
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.
