RESUMEN
Pcal_0768 gene encoding an amylomaltase, a 4-α-glucanatransferase belonging to family 77 of glycosyl hydrolases, from Pyrobaculum calidifontis was cloned and expressed in Escherichia coli. The recombinant protein was produced in E. coli in soluble and active form. However, the expression level was not very high. Analysis of the mRNA of initial seven codons at the 5'-end of the gene revealed the presence of a hair pin like secondary structure. This secondary structure was removed by site directed mutagenesis, without altering the amino acids, which resulted in enhanced expression of the cloned gene. Recombinant Pcal_0768 exhibited optimal amylomaltase activity at 80 °C and pH 6.9. Under these conditions, the specific activity was 690 U/ mg. Recombinant Pcal_0768 was highly thermostable with a half-life of 6 h at 100 °C. It exhibited the highest kcat value among the characterized glucanotransferases. No metal ions were required for activity or stability of the enzyme. Recombinant Pcal_0768 was successfully employed in the synthesis of modified starch for producing thermoreversible gel. To the best of our knowledge, till now this is the most thermostable enzyme among the characterized amylomaltases. High thermostability and starch modification potential make it a novel and distinct amylomaltase.
Asunto(s)
Proteínas Arqueales , Clonación Molecular , Expresión Génica , Sistema de la Enzima Desramificadora del Glucógeno , Calor , Pyrobaculum , Proteínas Arqueales/biosíntesis , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/aislamiento & purificación , Estabilidad de Enzimas , Escherichia coli/enzimología , Escherichia coli/genética , Sistema de la Enzima Desramificadora del Glucógeno/biosíntesis , Sistema de la Enzima Desramificadora del Glucógeno/química , Sistema de la Enzima Desramificadora del Glucógeno/genética , Sistema de la Enzima Desramificadora del Glucógeno/aislamiento & purificación , Pyrobaculum/enzimología , Pyrobaculum/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Thermostable cyclodextrinase (Tk1770 CDase) from hyperthermophilic archaeon Thermococcus kodakarensis (KOD1) hydrolyzes cyclodextrins into linear dextrins. The sequence of Tk1770 CDase retrieved from UniProt was aligned with sequences of sixteen CD hydrolyzing enzymes and a phylogenetic tree was constructed using Bayesian inference. The homology model of Tk1770 CDase was constructed and optimized with Modeller v9.14 program. The model was validated with ProSA server and PROCHECK analysis. Four conserved regions and the catalytic triad consisting of Asp411, Glu437, and Asp502 of GH13 family were identified in catalytic site. Also an additional fifth conserved region downstream to the fourth region was also identified. The structure of Tk1770 CDase consists of an additional N'-domain and a helix-loop-helix motif that is conserved in all archaeal CD hydrolyzing enzymes. The N'-domain contains an extended loop region that forms a part of catalytic domain and plays an important role in stability and substrate binding. The docking of substrate into catalytic site revealed the interactions with different conserved residues involved in substrate binding and formation of enzyme-substrate complex.
Asunto(s)
Glicósido Hidrolasas/metabolismo , Thermococcus/enzimología , Secuencias de Aminoácidos , Dominio Catalítico , Simulación por Computador , Secuencia Conservada , Ciclodextrinas/metabolismo , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Modelos Moleculares , Simulación del Acoplamiento Molecular , Filogenia , Conformación Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Thermococcus/genéticaRESUMEN
Eleven children between 4 and 17 years of age underwent ligation of a patent ductus arteriosus during two medical camps in September 2003 and August 2004 at the Sonam Norboo Memorial District Hospital at Leh, Ladakh (11,400 feet above mean sea level). These children had a low mean arterial oxygen saturation while breathing room air. They were all acclimatized to high altitude. Surgery was performed under general anaesthesia with endotracheal intubation. Patients were extubated on the table. All children survived the operation and were discharged within 5-6 days following surgery. No surgical or anaesthetic complications were encountered. Simple cardiac surgical procedures such as ligation of a patent ductus arteriosus can be performed safely at high altitudes.