RESUMO
The reaction pattern of an endoglycanase from Paenibacillus cookii SS-24 (Pgl8A) was improved to facilitate chitosan oligosaccharide production. Based on the sequence alignment with chitosanase of a known structure, we performed site-directed mutagenesis of possible substrate-binding residues in Pgl8A. The mutants were expressed in Escherichia coli cells, and their cellulase and chitosanase activities were then characterised. Our results indicated that three amino acid residues (W139, W208 and E285) were important for the substrate specificity of Pgl8A. D156 and Y390 were also essential for the cellulase and chitosanase activities of Pgl8A. The products of chitosan degradation by W139A, W208A and E285Q mutants were different from those by the wild type. A chitosan pentamer accumulated following chitosan degradation by W139A, W208A and E285Q mutants. Thus, the mutants obtained in this study are potentially useful for the production of biofunctional chitosan oligosaccharides.
Assuntos
Quitosana/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Mutagênese Sítio-Dirigida , Oligossacarídeos/biossíntese , Paenibacillus/enzimologia , Sequência de Aminoácidos , Glicosídeo Hidrolases/química , Hidrólise , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
The expression levels of the cellulase and xylanase genes between the host strain and an xlnR disruptant were compared by quantitative RT-PCR (qPCR) to identify the genes controlled by XlnR-independent signaling pathway. The cellulose induction of the FI-carboxymethyl cellulase (cmc1) and FIb-xylanase (xynIb) genes was controlled by XlnR; in contrast, the cellulose induction of the FIII-avicelase (cbhI), FII-carboxymethyl cellulase (cmc2), and FIa-xylanase (xynIa) genes was controlled by an XlnR-independent signaling pathway. To gain deeper insight into the XlnR-independent signaling pathway, the expression profile of cbhI was analyzed as a representative target gene. Cellobiose together with 1-deoxynojirimycin (DNJ), a glucosidase inhibitor, induced cbhI the most efficiently among disaccharides composed of ß-glucosidic bonds. Furthermore, cellobiose with DNJ induced the transcription of cmc2 and xynIa, whereas cmc1 and xynIb were not induced. GUS reporter fusion analyses of truncated and mutated cbhI promoters revealed that three regions were necessary for effective cellulose-induced transcription, all of which contained the conserved sequence 5'-CCGN(2)CCN(7)G(C/A)-3' within the CeRE, which has been identified as the upstream activating element essential for expression of eglA in A. nidulans (Endo et al. 2008). The data therefore delineate a pathway in which A. aculeatus perceives the presence of cellobiose, thereby activating a signaling pathway that drives cellulase and hemicellulase gene expression under the control of the XlnR-independent regulation through CeRE.
Assuntos
Aspergillus/metabolismo , Celobiose/genética , Celulase/genética , Transdução de Sinais , Sequência de Bases , Celobiose/metabolismo , Celulase/metabolismo , Clonagem Molecular , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
An endoglycanase gene of Paenibacillus cookii SS-24 was cloned and sequenced. This Pgl8A gene had an open reading frame of 1,230 bp that encoded a putative signal sequence (31 amino acids) and mature enzyme (378 amino acids: 41,835 Da). The enzyme was most homologous to a ß-1,3-1,4-glucanase of Bacillus circulans WL-12 with 84% identity. The recombinant enzyme hydrolyzed carboxymethyl cellulose, swollen celluloses, chitosan and lichenan but not Avicel, chitin powder or xylan. With chitosan as the substrate, the optimum temperature and hydrolysis products of the recombinant enzyme varied at pH 4.0 and 8.0. This is the first report that characterizes chitosanase activity under different pH conditions.
Assuntos
Celulase/genética , Celulase/metabolismo , Paenibacillus/enzimologia , Paenibacillus/genética , Celulase/química , Celulose/metabolismo , Quitosana/metabolismo , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Estabilidade Enzimática , Glucanos/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , TemperaturaRESUMO
FI-Carboxymethylcellulase (cmc1; family 12) is one of the endoglucanases of Aspergillus aculeatus and consists of single polypeptide chain of 221 amino acids. The cmc1 gene was expressed in Aspergillus oryzae niaD300 (niaD-) under promoter 8142. The plasmid pCMG14 carrying the cmc1 gene at PstI site was used as a source of the gene (920 bp) and Aspergillus oryzae was successfully transformed by the plasmid pNAN-cmc1 (harboring cmc1 gene). The plasmid was integrated in Aspergillus oryzae niaD300 genome at niaD locus and the transformed fungus constitutively produced very high amounts of endoglucanases when grown on glucose, maltose, soluble starch and wheat bran.
Assuntos
Aspergillus oryzae/genética , Aspergillus/genética , Celulase/biossíntese , Clonagem Molecular , Aspergillus/enzimologia , Aspergillus oryzae/enzimologia , Aspergillus oryzae/metabolismo , Celulase/genética , Celulase/metabolismo , Regulação Fúngica da Expressão Gênica , Microbiologia Industrial , Transformação GenéticaRESUMO
Fructosyl-amino acid oxidase (FOD-F) from Fusarium oxysporum f. sp. raphani (NBRC 9972) is the enzyme catalyzing the oxidative deglycation of fructosyl-amino acids such as N(epsilon)-fructosyl N(alpha)-benzyloxycarbonyl-lysine (FZK) and fructosyl valine (FV), which are model compounds of the glycated proteins in blood. Wild-type FOD-F has high activities toward both substrates. We obtained a mutant FOD-F, which reacts with FZK but not with FV by random mutagenesis. One amino-acid substitution (K373R) occurred in the mutant FOD-F. In addition to K373R, K373W, K373M, K373T, and K373V, which were selected for optimization of the substitution at position K373, were purified and characterized. Kinetic analysis showed that the catalytic turnover for FV greatly decreased, whereas that for FZK did not. In consequence, the specificities toward FZK were increased in the mutant FOD-Fs. The relation between the substrate specificity of the mutant FOD-Fs and the position of the carboxyl group of the substrates was demonstrated using a series of the substrates having the carboxyl group at the different position. The mutant FOD-Fs are attractive candidates for developing an enzymatic measurement method for glycated proteins such as glycated albumin in serum. This study will be helpful to establish an easier and rapid clinical assay system of glycated albumin.
Assuntos
Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Fusarium/enzimologia , Especificidade por Substrato/genética , Aminoácido Oxirredutases/isolamento & purificação , Sequência de Aminoácidos , Substituição de Aminoácidos , Cinética , Lisina/análogos & derivados , Lisina/metabolismo , Dados de Sequência Molecular , Mutagênese , Mutação de Sentido Incorreto , Valina/análogos & derivados , Valina/metabolismoRESUMO
We showed by random mutagenesis that one-amino-acid substitution at Arg94 in fructosyl-amino acid oxidase from Ulocladium sp. JS-103 enhanced substrate specificity toward fructosyl valine (FV), a model compound of hemoglobin A(1c). Kinetic analysis showed that the specificity of the R94W mutant enzyme toward FV was 14-fold that of the wild-type enzyme. The mutant enzyme obtained will be useful in developing an enzymatic measurement method for hemoglobin A(1c).
Assuntos
Aminoácido Oxirredutases/genética , Substituição de Aminoácidos , Ascomicetos/genética , Proteínas Fúngicas/genética , Mutagênese/genética , Mutação de Sentido Incorreto , Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/metabolismo , Ascomicetos/química , Ascomicetos/enzimologia , Bioensaio/métodos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Hemoglobinas Glicadas/análise , Humanos , Especificidade por Substrato/genética , Valina/análogos & derivados , Valina/química , Valina/metabolismoRESUMO
A gene encoding exoglucanase (CBHII) of Streptomyces sp. M 23 was cloned and sequenced. The cbhII gene consisted of 1359 bp capable of encoding a polypeptide of 453 amino acids with a calculated molecular mass of 45,175 Da. The deduced amino acid sequence showed homology with those of cellulases belonging to family 6 of the glycosyl hydrolases. The cbhII gene was subcloned into the plasmid pSEV1 and expressed in Streptomyces lividans TK-24. The transformed cells were able to secrete the enzyme efficiently in an active form. The CBHII expressed by S. lividans TK-24 was purified to homogeneity by SDS-polyacrylamide gel and characterized. The recombinant CBHII was stable up to 50 degrees C and more than 30% of the original activity remained after heating at 100 degrees C for 10 min. The amino-terminal amino acid sequence of the recombinant CBHII was identified as GPAAPTARVD. These results agreed well with the properties of the authentic CBHII.
Assuntos
Clonagem Molecular , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Streptomyces/enzimologia , Streptomyces/genética , Sequência de Aminoácidos , Ativação Enzimática , Glicosídeo Hidrolases/análise , Dados de Sequência Molecular , Peso Molecular , Engenharia de Proteínas/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Análise de Sequência de ProteínaRESUMO
Family 19 chitinase from Aeromonas sp. No.10S-24 (72.6 kDa) is composed of two chitin-binding domains (ChBDs), two proline- and threonine-rich (PT-rich) linkers, and a catalytic domain. The purified enzyme was labile in a standard buffer condition and spontaneously degraded into a 46-kDa fragment upon storage at 4 degrees C. The N-terminal sequence of the 46-kDa fragment was found to correspond to the sequence of the C-terminal region of the second PT-rich linker, indicating that the 46-kDa fragment is produced by truncation of the two ChBDs and the two PT-rich linkers from the mature protein, and consists only of the catalytic domain. The hydrolytic activities toward insoluble and soluble substrates were significantly reduced by the truncation of two ChBDs. In addition, antifungal activity determined from the digestion rate of haustoria of powdery mildew was reduced by the ChBD truncation. Although the profile of the time-course of N-acetylglucosamine hexasaccharide [(GlcNAc)6] degradation catalyzed by the ChBD-truncated enzyme was similar to that of the mature enzyme protein, the specific activity of the ChBD-truncated enzyme determined from the rate of hexasaccharide degradation was lower than that of the mature enzyme. The two CBDs appear to be responsible for facilitating the hydrolytic reaction. The sugar residue affinities (binding free energy changes) at the individual subsites, (-2) (-1) (+1) (+2) (+3) (+4), were estimated by modeling the hexasaccharide hydrolysis by the mature and ChBD-truncated enzymes. The truncation of ChBDs was found to strongly affect the affinity at the (-1) site. This situation seems to result in the lower enzymatic activity of the ChBD-truncated enzyme toward the chitinous substrates.
Assuntos
Aeromonas/enzimologia , Quitina/metabolismo , Quitinases/química , Quitinases/metabolismo , Sequência de Aminoácidos , Quitina/química , Quitinases/isolamento & purificação , Hidrólise , Dados de Sequência Molecular , Polissacarídeos/química , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
We cloned the gene and corresponding cDNA for an extracellular Rhodotorula-lytic enzyme which has beta-1,3-mannase activity, tentatively named MAN5C, from Paecilomyces lilacinus. MAN5C showed a high homology score with the members of glycoside hydrolase family 5 in a domain search with the Pfam database, indicating that MAN5C is a novel and unique member of glycoside hydrolase family 5.
Assuntos
Paecilomyces/enzimologia , beta-Manosidase/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Mananas/metabolismo , Dados de Sequência Molecular , Paecilomyces/genética , Paecilomyces/metabolismo , Rhodotorula/genética , Rhodotorula/metabolismo , Alinhamento de Sequência , beta-Manosidase/isolamento & purificação , beta-Manosidase/metabolismoRESUMO
A family 19 chitinase gene from Aeromonas sp. No.10S-24 was cloned, sequenced, and expressed in Escherichia coli. From the deduced amino acid sequence, the enzyme was found to possess two repeated N-terminal chitin-binding domains, which are separated by two proline-threonine rich linkers. The calculated molecular mass was 70 391 Da. The catalytic domain is homologous to those of plant family 19 chitinases by about 47%. The enzyme produced alpha-anomer by hydrolyzing beta-1,4-glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N-acetylglucosamine hexasaccharide [(GlcNAc)6] was hydrolyzed by the chitinase, the second glycosidic linkage from the nonreducing end was predominantly split producing (GlcNAc)2 and (GlcNAc)4. The evidence from this work suggested that the subsite structure of the enzyme was (-2)(-1)(+1)(+2)(+3)(+4), whereas most of plant family 19 chitinases have a subsite structure (-3)(-2)(-1)(+1)(+2)(+3). Thus, the Aeromonas enzyme was found to be a novel type of family 19 chitinase in its structural and functional properties.
Assuntos
Aeromonas/metabolismo , Quitinases/química , Quitinases/classificação , Aeromonas/genética , Sequência de Aminoácidos , Sequência de Bases , Quitinases/genética , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Biblioteca Gênica , Vetores Genéticos , Glicosídeos/metabolismo , Hidrólise , Dados de Sequência Molecular , Oligossacarídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
Transformation systems for Aspergillus aculeatus has been developed, based on the use of the pyrithiamine resistance gene of Aspergillus oryzae and the orotidine-5'-monophosphate decarboxylase gene (pyrG) of Aspergillus nidulans. An A. aculeatus mutant which can be transformed effectively by the A. nidulans pyrG gene was isolated as a transformation host. This is the first report of transformation of A. aculeatus.
Assuntos
Aspergillus nidulans/genética , Aspergillus oryzae/genética , Aspergillus/genética , Orotidina-5'-Fosfato Descarboxilase/genética , Southern Blotting , Vetores Genéticos , Mutação , Protoplastos/citologia , Piritiamina/química , Transformação GenéticaRESUMO
For direct and efficient ethanol production from cellulosic materials, we constructed a novel cellulose-degrading yeast strain by genetically codisplaying two cellulolytic enzymes on the cell surface of Saccharomyces cerevisiae. By using a cell surface engineering system based on alpha-agglutinin, endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414 was displayed on the cell surface as a fusion protein containing an RGSHis6 (Arg-Gly-Ser-His(6)) peptide tag in the N-terminal region. EGII activity was detected in the cell pellet fraction but not in the culture supernatant. Localization of the RGSHis6-EGII-alpha-agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley beta-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and beta-glucosidase 1 from Aspergillus aculeatus No. F-50 were codisplayed on the cell surface. The resulting yeast cells could grow in synthetic medium containing beta-glucan as the sole carbon source and could directly ferment 45 g of beta-glucan per liter to produce 16.5 g of ethanol per liter within about 50 h. The yield in terms of grams of ethanol produced per gram of carbohydrate utilized was 0.48 g/g, which corresponds to 93.3% of the theoretical yield. This result indicates that efficient simultaneous saccharification and fermentation of cellulose to ethanol are carried out by a recombinant yeast cells displaying cellulolytic enzymes.
Assuntos
Celulose/metabolismo , Etanol/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Celulase/metabolismo , Celulose/química , Cromatografia Líquida de Alta Pressão , Meios de Cultura , Enzimas Imobilizadas , Fermentação , Genes Sintéticos , Engenharia Genética , Vetores Genéticos , Microscopia de Fluorescência , Fragmentos de Peptídeos/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
The cmc2 gene, coding for an endoglucanase 2 (CMC2) of Aspergillus aculeatus, was cloned using both genomic and cDNA libraries, and sequenced. The gene consists of 1230 bp encoding a protein of 410 amino acid residues with a molecular mass of 43,697 Da. The CMC2, composed of an N-terminal catalytic domain belonging to the family 5 of glycosyl hydrolases and a C-terminal cellulose-binding domain (CBD) belonging to the family I of CBDs, showed identity with other fungal endoglucanases, particularly with that of A. niger, A. nidulans, A. kawachii and A. aculeatus. The transcription of the cmc2 gene in A. aculeatus cells that were grown on different carbon sources was measured. Analysis by the ribonuclease protection assay revealed that expression of the cmc2 gene is induced by cellulose and some disaccharides and repressed by glucose.