RESUMO
A genomic library of the hyperthermophilic archaeon Sulfolobus solfataricus strain MT4 was constructed in Escherichia coli using a cloning vector not designed for heterologous gene expression. One positive clone exhibiting acquired thermophilic acetylesterase activity was directly detected by an in situ plate assay using a colony staining procedure with the chromogenic substrate beta-naphthyl acetate. The plasmid isolated from the clone contained a 3.3 kb genomic fragment from S. solfataricus and a full-length esterase coding sequence could be identified. Expression of the active thermostable esterase in E. coli was independent of isopropyl-beta-D-thiogalactopyranoside and of the kind of vector, suggesting that the archaeal esterase gene was controlled by fortuitous bacterial-like sequences present in its own 5' flanking region, not by the bacterial lac promoter or other serendipitous vector-located sequences. The protein, partially purified by thermoprecipitation of the host proteins at high temperature and gel exclusion chromatography, showed a homo-tetrameric structure with a subunit of molecular mass of 32 kDa which was in perfect agreement with that deduced from the cloned gene. The same protein was revealed in S. solfataricus cell extracts, thus demonstrating its functional occurrence in vivo under the cell culture conditions tested. The recombinant enzyme exhibited high thermal activity and thermostability with optimal activity between pH 6.5 and 7.0. The hydrolysis of p-nitrophenyl esters of fatty acids (from C(2) to C(8)) allowed the enzyme to be classified as a short length acyl esterase.
Assuntos
Hidrolases de Éster Carboxílico/genética , Sulfolobus/genética , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sequência de Bases , Carboxilesterase , Hidrolases de Éster Carboxílico/metabolismo , Clonagem Molecular , DNA Arqueal/química , DNA Arqueal/genética , Estabilidade Enzimática , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Genes Arqueais/genética , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sulfolobus/enzimologiaRESUMO
Two strains (O(alpha) and X(2)) of the hyperthermophilic crenarchaeon Sulfolobus solfataricus strain MT4 were selected and isolated for their ability to grow on xylan. O(alpha) and X(2), grown on media containing oat spelt xylan and birchwood xylan as the sole nutrient source, respectively, produced the same thermostable xylanase that was demonstrated to be inducible in xylan cultures. In an oat spelt medium, S. solfataricus O(alpha) underwent interesting morphological changes in the cell envelope, exhibiting mobile appendages not present in the typical coccal shape. The enzyme was prevalently membrane associated and showed a molecular mass of approximately 57.0 kDa. It was also highly thermostable, with a half-life of 47 min at 100 degrees C, and exhibited an optimal temperature and pH of 90 degrees C and 7.0, respectively. Xylo-oligosaccharides were the enzymatic products of xylan hydrolysis, and the smallest degradation product was xylobiose, thus indicating that the enzyme was an endoxylanase. The enzyme was able to bind weakly to crystalline cellulose (Avicel) and more strongly to insoluble xylan in a substrate amount-and temperature-dependent manner.