RESUMEN
The chitinase A (ChiA)-coding gene of Pseudomonas sp. BK1, which was isolated from a marine red alga Porphyra dentata, was cloned and expressed in Escherichia coli. The structural gene consists of 1602 bp encoding a protein of 534 amino acids, with a predicted molecular weight of 55,370 Da. The deduced amino acid sequence of ChiA showed low identity (less than 32%) with other bacterial chitinases. The ChiA was composed of multiple domains, unlike the arrangement of domains in other bacterial chitinases. Recombinant ChiA overproduced as inclusion bodies was solubilized in the presence of 8 M urea, purified in a urea-denatured form and re-folded by removing urea. The purified enzyme showed maximum activity at pH 5.0 and 40 degrees C. It exhibited high activity towards glycol chitosan and glycol chitin, and lower activity towards colloidal chitin. The enzyme hydrolyzed the oligosaccharides from (GlcNAc)4 to (GlcNAc)6, but not GlcNAc to (GlcNAc)3. The results suggest that the ChiA is a novel enzyme, with different domain structure and action mode from bacterial family 18 chitinases.
Asunto(s)
Quitinasas/aislamiento & purificación , Quitinasas/metabolismo , Oligosacáridos/metabolismo , Pseudomonas/enzimología , Quitina/metabolismo , Quitinasas/genética , Clonación Molecular , Pseudomonas/genética , Especificidad por SustratoRESUMEN
A gene (pagA) encoding beta-agarase from Pseudomonas sp. SK38 was cloned and expressed in Escherichia coli. The structural gene consists of 1011 bp encoding 337 amino acids with a predicted molecular weight of 37326 and has a signal peptide of 18 amino acids. The deduced amino acid sequence showed 57% and 58% homology to beta-agarase from Pseudoalteromonas atalntica and Aeromonas sp., respectively. The recombinant enzyme was purified and biochemically characterized. The enzyme had maximum activity at pH 9 and 30 degrees C. It was stable at pHs from 8 to 9 and below 37 degrees C.
Asunto(s)
Genes Bacterianos , Glicósido Hidrolasas/biosíntesis , Glicósido Hidrolasas/genética , Pseudomonas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Datos de Secuencia Molecular , Pseudomonas/enzimología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Homología de Secuencia de AminoácidoRESUMEN
Aqualysin I is a subtilisin-type serine protease secreted into the culture medium by Thermus aquaticus YT-1. It is first produced as a large precursor that consists of a signal peptide, an N-terminal pro-domain, the mature protease domain and a C-terminal pro-domain. To investigate whether the N-terminal pro-domain supplied in trans as an independent peptide plays an important role in the folding and secretion of the protease, the N-terminal pro-domain in E. coli has been expressed independent of the mature domain with or without the C-terminal pro-domain using an expression system with separate promoters and signal peptides. Protease assay and SDS-PAGE clearly showed that the N-terminal pro-domain plays an essential role in guiding the proper folding in trans of the enzymatically active conformation of aqualysin I. The N-terminal amino acid sequences of the purified enzymes were identical and had no signal peptides. These results indicate that independently expressed domains are secreted into the periplasmic space before the N-terminal pro-domain-assisted folding of the mature domain.