RESUMO
The cyclodextrin glycosyltransferase (CGTase) is an important enzyme for cyclodextrin (CD) production, and is also widely used in the biotechnology, food, and pharmaceuticals industries. Secretory CGTase production by recombinant Komagataella phaffii using defined medium is a promising approach because of low cost, less impurity protein. It was found that no CGTase was expressed using traditional defined medium (basal salt medium [BSM]) because of pH value decreasing significantly. CGTase was expressed by recombinant K. phaffii through pH maintenance in range of 5.5-7.0. ß-CGTase activity increased to 122.0 U/mL after optimization of glycerol, phosphate buffer, pH value, ammonium sulfate, temperature, methanol, and additives based on BSM, establishing a modified defined medium. These results showed that it was necessary to establish recombinant K. phaffii-based special defined medium although the same host cell used for different heterologous protein expression.
Assuntos
Meios de Cultura , Glucosiltransferases/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomycetales/metabolismo , Sulfato de Amônio/metabolismo , Sulfato de Amônio/farmacologia , Biotecnologia , Meios de Cultura/química , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Glucosiltransferases/análise , Glucosiltransferases/genética , Glicerol/metabolismo , Glicerol/farmacologia , Concentração de Íons de Hidrogênio , Metanol/metabolismo , Metanol/farmacologia , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Saccharomycetales/genética , TemperaturaRESUMO
Cyclodextrin glycosyltransferase (CGTase) catalyzes the conversion of starch into cyclodextrin (CD), which is widely applied in food, pharmaceutical, cosmetic, and agricultural industries. For efficient production of CD, high yield of CGTase with good characteristics is necessary. In this study, the cgt gene from Bacillus pseudalcaliphilus was expressed in Komagataella phaffii after codon optimization and expression vector selection. The ß-CGTase activity in the transformant reached 3885.1UmL-1, which is the highest value reported so far, at 28°C, 6% inoculum ratio, and 1.5% methanol addition following 24h of incubation. The recombinant CGTase showed high specific activity at 80°C without any γ-CGTase activity, and had good stability in a wide pH and temperature range. These results demonstrate that the recombinant CGTase could have potential industrial applications.
