RESUMEN
An agar-degrading bacterium, Catenovulum sp. X3, was isolated from the seawater of Shantou, China. A novel ß-agarase gene agaXa was cloned from the strain Catenovulum sp. X3. The gene agaXa consists of 1,590 bp and encodes a protein of 529 amino acids, with only 40 % amino acid sequence identity with known agarases. AgaXa should belong to the glycoside hydrolase family GH118 based on the amino acid sequence similarity. The molecular mass of the recombinant AgaXa (rAgaXa) was estimated to be 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had a maximal agarase activity at 52 °C and pH 7.4 and was stable over pH 5.0 ~ 9.0 and at temperatures below 42 °C. The K m and V max for agarose were 10.5 mg/ml and 588.2 U/mg, respectively. The purified rAgaXa showed endolytic activity on agarose degradation, yielding neoagarohexaose, neoagarooctaose, neoagarodecaose, and neoagarododecaose as the end products. The results showed that AgaXa has potential applications in agar degradation for the production of oligosaccharides with various bioactivities.
Asunto(s)
Agar/metabolismo , Alteromonadaceae/enzimología , Alteromonadaceae/genética , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Alteromonadaceae/aislamiento & purificación , China , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Glicósido Hidrolasas/química , Glicósido Hidrolasas/aislamiento & purificación , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Peso Molecular , ARN Ribosómico 16S/genética , Agua de Mar/microbiología , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , TemperaturaRESUMEN
Maltoporin (LamB) is a family of outer membrane proteins. There has been no report of immunological characteristics of LamB in the Vibrio species so far. In this study, lamB genes from eight Vibrio strains were cloned and sequenced. The bioinformatics analysis indicated that sequence similarities of LamB proteins were ranged from 46.7% to 81.1%. Further, the result showed that their antigenic epitopes were highly conserved implying that LamB might be a shared antigen among Vibrios. The Western blot of rabbit sera against recombinant LamB from V. alginolyticus ATCC 33787 with cell lysate of 18 Vibrio strains showed cross-recognition. Bands observed on cell lysate of Vibrio strains immunoblotted with the anti-LamB sera ranged between 40 and 49 kDa. The Whole-cell ELISA assay further confirmed that the antisera of recombinant LamB recognized the tested Vibrio strains indicating the surface-exposed of LamB. Finally, the cross-protective property of recombinant LamB was evaluated through vaccination and subsequent challenge with heterogeneous virulent Vibrio strains in zebrafish. Recorded relative percent survival (RPS) of the vaccinated group varied from 54.1% to 77.8%, showing that zebrafish were protected from Vibrio infection after immunization with LamB protein. The cumulative evidences in this study suggested that LamB was a conserved antigen among tested Vibrio species and might be a potentially versatile vaccine candidate for the prevention of Vibriosis.