RESUMEN
A genomic laccase gene and cDNA were cloned from the white-rot fungi Ganoderma lucidum TR6. The genomic laccase gene contained 2086 bp with nine introns. The laccase cDNA had an open reading frame of 1563 bp. The deduced mature protein consisted of 520 amino acids. Both the genomic laccase gene and cDNA were expressed in the Pichia pastoris GS115. Laccase activities could be detected in transformants with laccase cDNA but not in transformants with genomic laccase gene. The highest activity value reached 685.8 U L(-1). The effects of temperature, pH and nitrogen source on laccase expression in P. pastoris were analyzed. The recombinant laccase was purified and the molecular mass was 73.4 KDa, a little bigger than native laccase. The optimal pH and temperature were specific at pH 3.5 and special range from 60 to 90 °C. The laccase was stable at pH 7.0 and temperature range of 20-30 °C. The Km and Vm values of this recombinant laccase for ABTS were 0.521 mM and 19.65 mM min(-1), respectively.
Asunto(s)
Lacasa/metabolismo , Pichia/genética , Reishi/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , ADN de Hongos/química , ADN de Hongos/genética , Estabilidad de Enzimas , Expresión Génica , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/efectos de la radiación , Vectores Genéticos , Concentración de Iones de Hidrógeno , Intrones , Cinética , Lacasa/química , Lacasa/genética , Lacasa/aislamiento & purificación , Datos de Secuencia Molecular , Peso Molecular , Nitrógeno/metabolismo , Sistemas de Lectura Abierta , Pichia/enzimología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Reishi/genética , Análisis de Secuencia de ADN , TemperaturaRESUMEN
A field experiment was conducted to study the effects of different nitrogen forms ((NH2)2CO-N, NO(3-)-N, NH(4+)-N, and NO(3-)-NH4+) and their ratios on the yield formation, quality, and nutrient (N, P, and K) absorption of broccoli (Brassica oleracea). Fertilization with NO(3-)-N increased the accumulation of nitrate and soluble sugars in ball flower. When the NO(3-)-N: NH(4+)-N ratio was ranged from 3:7 to 5:5 and from 5:5 to 7:3, respectively, the accumulation of nitrate in the ball flower was reduced, while the soluble sugars content was promoted. Fertilization with (NH2)2CO-N and NH(4+)-N was conducive to the improvement of Vc content in the ball flower. N fertilization increased the accumulation of N, P, and K in plants, with the highest contents of N, P, and K observed at rosette stage. Throughout the entire growth period, NH(4+)-N fertilization improved the plant N content, whereas NO(3-)-N fertilization improved the plant K content. At different growth stages, the effects of different N sources on plant P content varied. As compared with applying single N forms, the NO(3-)-N:NH(4+)-N ratio ranged from 3:7 to 5:5 could improve the yield significantly. It was suggested that a combined application of NO(3-)-N and NH(4+)-N with an appropriate ratio could improve the productivity, quality, and economic return of broccoli.