RESUMEN
Cariogenic biofilms have a matrix rich in exopolysaccharides (EPS), mutans and dextrans, that contribute to caries development. Although several physical and chemical treatments can be employed to remove oral biofilms, those are only partly efficient and use of biofilm-degrading enzymes represents an exciting opportunity to improve the performance of oral hygiene products. In the present study, a member of a glycosyl hydrolase family 66 from Flavobacterium johnsoniae (FjGH66) was heterologously expressed and biochemically characterized. The recombinant FjGH66 showed a hydrolytic activity against an early EPS-containing S. mutans biofilm, and, when associated with a α-(1,3)-glucosyl hydrolase (mutanase) from GH87 family, displayed outstanding performance, removing more than 80% of the plate-adhered biofilm. The mixture containing FjGH66 and Prevotella melaninogenica GH87 α-1,3-mutanase was added to a commercial mouthwash liquid to synergistically remove the biofilm. Dental floss and polyethylene disks coated with biofilm-degrading enzymes also degraded plate-adhered biofilm with a high efficiency. The results presented in this study might be valuable for future development of novel oral hygiene products.
Asunto(s)
Biopelículas , Dextranasa , Flavobacterium , Glicósido Hidrolasas , Streptococcus mutans , Biopelículas/crecimiento & desarrollo , Dextranasa/metabolismo , Dextranasa/genética , Flavobacterium/enzimología , Flavobacterium/genética , Streptococcus mutans/enzimología , Streptococcus mutans/genética , Glicósido Hidrolasas/metabolismo , Glicósido Hidrolasas/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Hidrólisis , Biotecnología/métodosRESUMEN
We cloned and characterized a gene encoding isocitrate lyase from the methylotrophic yeast Pichia pastoris. This gene was isolated from a P. pastoris genomic library using a homologous PCR hybridization probe, amplified with two sets of degenerate primers designed from conserved regions in yeast isocitrate lyases. The cloned gene was sequenced and consists of an open reading frame of 1563 bp encoding a protein of 551 amino acids. The molecular mass of the protein is calculated to be 60.6 kDa with high sequence similarity to isocitrate lyase from other organisms. There is a 64% identity between amino acid sequences of P. pastoris Icl and Saccharomyces cerevisiae Icl. Northern blot analyses showed that, as in S. cerevisiae, the steady-state ICL1 mRNA levels depend on the carbon source used for cell growth. Expression in P. pastoris of the dextranase gene (dexA) from Penicillium minioluteum under control of the ICL1 promoter proved that P(ICL1) is a good alternative for the expression of heterologous proteins in this methylotrophic yeast. The sequence presented here has been deposited in the EMBL data library under Accession No. AJ272040.
Asunto(s)
Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/genética , Isocitratoliasa/genética , Pichia/genética , Transcripción Genética/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Clonación Molecular , Dextranasa/genética , Dextranasa/metabolismo , Proteínas Fúngicas/metabolismo , Isocitratoliasa/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Insercional , Pichia/metabolismo , Regiones Promotoras Genéticas/fisiología , Alineación de SecuenciaRESUMEN
The dexC cDNA, which is expressed in dextran-containing medium by the filamentous fungus Penicillium minioluteum, was cloned and sequence characterized. The cDNA sequence comprises 1859 bp plus a poly (A) tail, coding for a predicted protein of 597 amino acids. The genomic counterpart was isolated by PCR, finding three introns in its sequence. The dexC gene was located by Southern blot in the same 9-kb fragment that the previously isolated dextranase-encoding gene (dexA). Sequence analysis revealed that the deduced DexC protein belongs to glycosyl hydrolase family 13, showing a high sequence identity (58%) with Aspergillus parasiticus alpha-1,6-glucosidase. In addition, the high sequence identity (51%) between DexC protein and oligo-1,6-glucosidase of Bacillus cereus, with three-dimensional (3D) structure determined, leads us to proposed a 3D model for the structural core of DexC protein.
Asunto(s)
Proteínas Fúngicas , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Penicillium/enzimología , Penicillium/genética , alfa-Glucosidasas/química , alfa-Glucosidasas/genética , Secuencia de Aminoácidos , Aspergillus/enzimología , Bacillus/enzimología , Secuencia de Bases , Southern Blotting , Dominio Catalítico , Clonación Molecular , Secuencia Conservada , ADN/metabolismo , ADN Complementario/metabolismo , Bases de Datos Factuales , Dextranasa/metabolismo , Glucosidasas/metabolismo , Intrones , Modelos Genéticos , Modelos Moleculares , Datos de Secuencia Molecular , Familia de Multigenes , Oligo-1,6-Glucosidasa/metabolismo , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Differences in glycosylation between the natural alpha-1,6 glucan-6-glucanohydrolase from Penicillium minioluteum and the heterologous protein expressed in the yeast Pichia pastoris were analyzed. Glycosylation profiling was carried out using fluorophore-assisted carbohydrate electrophoresis and amine absorption high-performance liquid chromatography (NH(2)-HPLC) in combination with matrix-assisted laser desorption-time of flight-mass spectrometry. Both microorganisms produce only oligomannosidic type structures, but the oligosaccharide population differs in both enzymes. The native enzyme has mainly short oligosaccharide chains ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2), of which Man(8)GlcNAc(2) was the most represented oligosaccharide. The oligosaccharides linked to the protein produced in P. pastoris range from Man(7)GlcNAc(2) up to Man(14)GlcNAc(2), with Man(8)GlcNAc(2) and Man(9)GlcNAc(2) being the most abundant structures. In both enzymes the first glycosylation site (Asn(5)) is always glycosylated. However, Asn(537) and Asn(540) are only partially glycosylated in an alternate manner.
Asunto(s)
Dextranasa/genética , Dextranasa/metabolismo , Penicillium/enzimología , Pichia/genética , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Dextranasa/química , Glicosilación , Datos de Secuencia Molecular , Penicillium/genética , Pichia/enzimología , Análisis de Secuencia de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The regulation of dextranase (dexA) gene expression in the filamentous fungus Penicillium minioluteum grown on different carbon sources was studied. Growth in the presence of dextran leads to high expression of the dextranase enzyme, but growth in starch, glucose, glycerol, lactose and sorbitol did not. Dextran induced dexA gene expression at the mRNA level. However, in cultures containing dextran plus glucose or glycerol, the transcript was detected 24 h later than in the case where dextran was the only carbon source. When the glucose or glycerol concentration in the dextran-containing medium was kept at about 1% (w/v), no dextranase-transcripts were detected. It was found that both glucose and glycerol inhibited enzyme synthesis, because 1% (w/v) addition of both carbon sources to dextran-growing cultures was able to abolish the inducing effect of dextran. Our results suggest that dextran utilization responds to both specific induction and to glucose and glycerol repression, providing evidence that P. minioluteum dexA expression is regulated by the carbon source at the transcriptional level.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Dextranasa/genética , Penicillium/genética , Northern Blotting , Dextranasa/biosíntesis , Dextranasa/metabolismo , Regulación Fúngica de la Expresión Génica , Glucosa/metabolismo , Glicerol/metabolismo , Penicillium/enzimología , Penicillium/metabolismo , Reacción en Cadena de la Polimerasa , ARN de Hongos/análisis , ARN Mensajero/análisisRESUMEN
The DEX gene encoding an extracellular dextranase was isolated from the genomic DNA library of Penicillium minioluteum by hybridization using the dextranase cDNA as a probe. Comparison of the gene and cDNA sequences revealed that the DEX gene does not contain introns. Amino acid sequences comparison of P. minioluteum dextranase with other reported dextranases reveals a significant homology (29% identity) with a dextranase from Arthrobacter sp. CB-8. The DEX gene fragment encoding a mature protein of 574 amino acids was expressed in the methylotrophic yeast Pichia pastoris by using the SUC2 gene signal sequence from Saccharomyces cerevisiae under control of the alcohol oxidase-1 (AOX1) promoter. Over 3.2 g/l of enzymatically active dextranase was secreted into the medium after induction by methanol. The yeast product was indistinguishable from the native enzyme in specific activity and the N-terminus of both proteins were identical.