RESUMEN
The effect of organic solvents on alkaline proteases was assessed for native, recombinant, and metagenomically derived alkaline proteases. Their stability and the effects of physicochemical parameters were studied in the presence of hexane. The native enzyme was comparatively more resistant against the organic solvents than the recombinant counterparts. On the other hand, the metagenomically derived alkaline protease was minimally resistant against solvents. A similar trend was apparent for the stability of enzyme in organic solvents. The novelty of this study lies in the fact that the majority of the studies on the solvent tolerance have focused on the mesophilic enzymes, while those from the haloalkaliphilic bacteria have received little attention. The comparative tolerance of the native, recombinant, and metagenomic alkaline proteases against the organic solvent has practical importance. The phylogenetic relatedness among the various protease sequences will be described.
Asunto(s)
Hexanos , Péptido Hidrolasas , Estabilidad de Enzimas , Filogenia , Solventes/farmacología , CatálisisRESUMEN
Metagenomics has opened new horizon to unlock the biotechnological potential for novel enzymes. An alkaline protease gene was obtained from the total environmental DNA extracted from a saline habitat. After cloning and sequencing, it was identified that the protease gene related to uncultivable bacteria (HM219181). The protease was over expressed at 6h of induction with optimum induction at 1mM IPTG and 27°C. The purified enzyme was characterized with respect to various factors; temperature, pH, NaCl and chemical denaturant. The sequence analysis indicated a hydrophobic tendency of the protein, while the predicted 3D structure indicated the enzyme as a serine protease.
Asunto(s)
Proteínas Bacterianas/genética , Endopeptidasas/genética , Metagenoma , Secuencia de Aminoácidos , Bacillus/enzimología , Bacillus/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Clonación Molecular , Endopeptidasas/biosíntesis , Endopeptidasas/química , Estabilidad de Enzimas , Escherichia coli , Expresión Génica , Concentración de Iones de Hidrógeno , Metagenómica , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Plásmidos/genética , Plásmidos/aislamiento & purificación , Desnaturalización Proteica , Proteolisis , Tolerancia a la Sal , Cloruro de Sodio/química , Microbiología del Suelo , Urea/químicaRESUMEN
Thermostable alkaline proteases from two haloalkaliphilic bacteria, Oceanobacillus iheyensis O.M.A(1)8 (EU680961) and Haloalkaliphilic bacterium O.M.E(1)2 (EU680960) were studied for enzymatic properties and amino acid sequences in comparative manner. The bacteria were isolated from salt enriched soil located in Okha, Coastal Gujarat, India. The unique aspect of the study was that alkaline protease from Haloalkaliphilic bacterium O.M.A(1)8 optimally catalyzed the reaction over a wide range of temperature, 50-90°C, with a half-life of 36 h at 90°C. The molecular weights of O.M.A(1)8 and O.M.E(1)2 were 35 kDa and 25 kDa, respectively. The enzyme secretion was over the broader range of pH 8-11, with an optimum at 11. The alkaline proteases from the two haloalkaliphilic strains isolated from the same site reflected quite different characteristics features. To the best of our knowledge, we have not come across with any such report on the thermal stability of alkaline proteases from haloalkaliphiles. Amino acid sequences for both enzymes were deduced from the nucleotide sequences of their corresponding genes followed by the analysis of physico-chemical properties of the enzymes.