RESUMEN
α-Amylase as an important industrial enzyme has been widely used in starch processing, detergent, and paper industries. To improve expression efficiency of recombinant α-amylase from Bacillus licheniformis (B. licheniformis), the α-amylase gene from B. licheniformis was optimized according to the codon usage of Pichia pastoris (P. pastoris) and expressed in P. pastoris. Totally, the codons encoding 305 amino acids were optimized in which a total of 328 nucleotides were changed and the G+C content was increased from 47.6 to 49.2%. The recombinants were cultured in 96-deep-well microplates and screened by a new plate assay method. Compared with the wild-type gene, the optimized gene is expressed at a significantly higher level in P. pastoris after methanol induction for 168 h in 5- and 50-L bioreactor with the maximum activity of 8100 and 11000 U/mL, which was 2.31- and 2.62-fold higher than that by wild-type gene. The improved expression level makes the enzyme a good candidate for α-amylase production in industrial use.
Asunto(s)
Bacillus/enzimología , Codón/genética , Pichia/genética , Proteínas Recombinantes/genética , alfa-Amilasas/genética , Bacillus/genética , Secuencia de Bases , Datos de Secuencia Molecular , Pichia/metabolismo , Ingeniería de Proteínas , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , alfa-Amilasas/análisis , alfa-Amilasas/química , alfa-Amilasas/metabolismoRESUMEN
The present study describes the gene cloning and high-level expression of an alkaline and thermostable lipase gene from Trichosporon coremiiforme V3. Nucleotide analysis revealed that this lipase gene has an open reading frame of 1,692 bp without any introns, encoding a protein of 563 amino acid residues. The lipase gene without its signal sequence was cloned into plasmid pPICZαA and overexpressed in Pichia pastoris X33. The maximum lipase activity of recombinant lipase was 5,000 U/ml, which was obtained in fed-batch cultivation after 168 h induction with methanol in a 50 L bioreactor. The purified lipase showed high temperature tolerance, and being stable at 60 °C and kept 45% enzyme activity after 1 h incubation at 70 °C. The stability, effects of metal ions and other reagents were also determined. The chain length specificity of the recombinant lipase showed high activity toward triolein (C18:1) and tripalmitin (C16:0).
Asunto(s)
Lipasa/genética , Lipasa/metabolismo , Trichosporon/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas , Clonación Molecular , Coenzimas/análisis , ADN de Hongos/química , ADN de Hongos/genética , Estabilidad de Enzimas , Expresión Génica , Lipasa/química , Lipasa/aislamiento & purificación , Metales/análisis , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Pichia/genética , Pichia/crecimiento & desarrollo , Pichia/metabolismo , Plásmidos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Temperatura , Trichosporon/genéticaRESUMEN
A gene encoding xylanase 2 mutant from Trichoderma reesei (T2C/T28C, named mxyn2) was cloned into the Pichia pastoris X33 strain using the vector pPICZαA. Recombinant Mxyn2p was functionally expressed in P. pastoris X33 and secreted into the supernatant. Real time qPCR demonstrated that an increase in gene copy number correlated with higher levels of expression. Supernatant from methanol induced cells was concentrated by ultrafiltration with a 10kDa cut off membrane, and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. Recombinant Mxyn2p protein had the highest activity at 75°C, while recombinant protein encoded by the "wild type" xylanase gene xyn2, also expressed in Pichia, was 20°C lower. The Mxyn2p enzyme retained more than 70% of its activity after incubation at 80°C for 10min. The effects of the optimal pH and temperature for higher expression levels in P. pastoris were also determined, 6.0 and 22°C, respectively. The maximum xylanase activity of Mxyn2p was 13,000nkat/mg (9.88g/l) in fed-batch cultivation after 168h induction with methanol in a 50l bioreactor.
Asunto(s)
Endo-1,4-beta Xilanasas , Proteínas Fúngicas , Expresión Génica , Pichia/metabolismo , Mutación Puntual , Trichoderma/enzimología , Endo-1,4-beta Xilanasas/biosíntesis , Endo-1,4-beta Xilanasas/química , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/aislamiento & purificación , Estabilidad de Enzimas , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Pichia/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Trichoderma/genéticaRESUMEN
A gene encoding Rhizopus oryzae lipase containing prosequence (ProROL) was cloned into the pPICZαA and electrotransformed into the Pichia pastoris X-33 strain. The lipase was functionally expressed and secreted in Pichia pastoris with a molecular weight of 35 kDa. The maximum lipase activity of recombinant lipase (rProROL) was 21,000 U/mL, which was obtained in a fed-batch cultivation after 168 h induction with methanol in a 50-L bioreactor. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut off membrane and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. The optimum pH and temperature of the rProROL were pH 9.0 and 40 °C, respectively. The lipase was stable from pH 4.0 to 9.0 and from 25 to 55 °C. The enzyme activity was enhanced by Ca(2+) and inhibited by Hg(2+) and Ag(+). The lipase showed high activity toward triglyceride-Tripalmitin (C16:0) and triglyceride-Trilaurin (C12:0).