RESUMEN
In this study, protease Pph_Pro1 from Pseudoalteromonas phenolica, possessing extracellular proteolytic activity and salt tolerance, was investigated for cloning, expression, and purification purposes. Through optimization, it was determined that optimum soluble recombinant expression was achieved when Pph_Pro1 was co-expressed with the pTf16 vector chaperone in LB medium supplemented with CaCl2. Pph_Pro1 was purified using osmotic shock and immobilized metal-affinity chromatography (IMAC). Isolated Pph_Pro1 activity was measured as 0.44 U/mg using casein as a substrate. Interestingly, Pph_Pro1 displayed halophilic, alkaliphilic, and unexpected thermostable properties. Furthermore, it was resistant to several hydrophilic and hydrophobic organic solvents. Substrate specificity and kinetic values such as Km and Vmax were determined with casein, bovine serum albumin (BSA), and algal waste protein as substrates, indicating that the Pph_Pro1 protease enzyme had a greater affinity for casein. Based on the remarkable characteristics of this Pph_Pro1 protease enzyme, it can potentially be utilized in many biotechnological industries.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Péptido Hidrolasas/genética , Pseudoalteromonas/enzimología , Proteínas Recombinantes de Fusión/genética , Proteínas Algáceas/química , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/aislamiento & purificación , Cloruro de Calcio/farmacología , Caseínas/química , Cromatografía de Afinidad , Clonación Molecular , Medios de Cultivo/química , Medios de Cultivo/farmacología , Pruebas de Enzimas , Estabilidad de Enzimas , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Cinética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Péptido Hidrolasas/biosíntesis , Péptido Hidrolasas/aislamiento & purificación , Proteolisis , Pseudoalteromonas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Salinidad , Tolerancia a la Sal/fisiología , Albúmina Sérica Bovina/química , Especificidad por SustratoRESUMEN
n-Butanol is considered as the next-generation biofuel, because its physiochemical properties are very similar to fossil fuels and it could be produced by Clostridia under anaerobic culture. Due to the difficulties of strict anaerobic culture, a host which can be used with facultative environment was being searched for n-butanol production. As an alternative, Shewanella oneidensis MR-1, which is known as facultative bacteria, was selected as a host and studied. A plasmid containing adhE2 encoding alcohol dehydrogenase, various CoA transferases (ctfAB, atoAD, pct, and ACT), and acs encoding acetyl-CoA synthetase were introduced and examined to S. oneidensis MR-1 to produce n-butanol. As a result, ctfAB, acs, and adhE2 overexpression in S. oneidensis-pJM102 showed the highest n-butanol production in the presence of 2% of N-acetylglucosamine (NAG), 0.3% of butyrate, and 0.1 mM of IPTG for 96 h under microaerobic condition. When more NAG and butyrate were fed, n-butanol production was enhanced, producing up to 160 mg/L of n-butanol. When metal ions or extra electrons were added to S. oneidensis-pJM102 for n-butanol production, metal ion as electron acceptor or supply of extra electron showed no significant effect on n-butanol production. Overall, we made a newly engineered S. oneidensis that could utilize NAG and butyrate to produce n-butanol. It could be used in further microaerobic condition and electricity supply studies.