RESUMO
Hydroxymethyltransferase (SHMT) and tryptophanase (TPase) are key enzymes in biosynthesis of L-tryptophan. We constructed three recombinant plasmids, including pET-SHMT, pET-TPase, and pET-ST for over-expression or co-expression of SHMT and TPase in Escherichia coli BL21 (DE3). The SDS-PAGE analysis showed that the recombinant proteins of 47 kDa and 50 kDa were expressed of pET-SHMT and pET-TPase, respectively. As compared to the host stain, the enzyme activity of SHMT and TPase was increased by 6.4 and 8.4 folds, respectively. Co-expression of both recombinant proteins, 47 kDa and 50 kDa, was also successful by using pET-ST and the enzyme activities were enhanced by 6.1 and 6.9 folds. We designed two pathways of dual-enzymatic synthesis of L-tryptophan by using these recombinant strains as source of SHMT and TPase. In the first pathway, the pET-SHMT carrying strain was used to catalyze synthesis of L-serine, which was further transformed into L-tryptophan by the pET-TPase expressing strain. These two steps sequentially took place in different bioreactors. In the second pathway, the pET-ST carrying strain, in which two enzymes were co-expressed, was used to catalyze simultaneously two steps in a single bioreactor. HPLC analysis indicated a high yield of 41.5 g/L of L-tryptophan was achieved in the first pathway, while a lower yield of 28.9 g/L was observed in the second pathway. In the first pathway, the calculated conversion rates for L-glycine and indole were 83.3% and 92.5%, respectively. In the second pathway, a comparable conversion rate, 82.7%, was achieved for L-glycine, while conversion of indole was much lower, only 82.9%.
Assuntos
Escherichia coli , Genética , Metabolismo , Regulação Bacteriana da Expressão Gênica , Fisiologia , Regulação Enzimológica da Expressão Gênica , Fisiologia , Vetores Genéticos , Genética , Glicina Hidroximetiltransferase , Genética , Plasmídeos , Genética , Proteínas Recombinantes de Fusão , Genética , Farmacologia , Recombinação Genética , Genética , Triptofano , Triptofanase , GenéticaRESUMO
Se examinaron en ratas, mediante voltametría, los efectos de la diabetes inducida por estreptozotocina o de la hiperglucemia, sobre la liberción de dopamina y serotonina en el estriado in vivo. En el estado diabético agudo se observó un incremento del 67% en la liberación de dopamina, mientras que el incremento observado en la diabetes crónica fue menor (19%). En las ratas a los 3 días de inducida la diabetes la señal electroquímica correspondiente a serotonina aumentó en un 62%, efecto que desapareció en la diabetes crónica. Luego de la inyección de L-triptófano en ratas normales, se detectó una disminución del 45% en la dopamina estriatal liberada y un aumento del 25% en la liberación de serotonina. Este incremento fue máximo a los 90 min más. Los animales crónicamente diabéticos mstraron una disminución significativa en la liberación de dopamina y serotonina estriatal luego de inyectar triptófano. Los efectos de la hiperflucemia en ratas no diabéticas fueron una disminución (52%) de la liberación de dopamina, y un incremento (304%) de la liberación de serotonina. Estos cambios indican que el estado diabético no tratado se asocia con una disminución progresiva de la liberación de neurotrasmisor, y que las modificaciones emocionales observadas en la disponibilidad de dopamina y serotonina en sinapsis centrales