Large-Scale Production of Glaciozyma antarctica Antifreeze Protein 1 (Afp1) by Fed-Batch Fermentation of Pichia pastoris

Antifreeze proteins (AFPs) are unique proteins that have an affinity toward ice. Due to their potential applications in the food and medical industries, many attempts have been made to produce AFPs in large quantities. In this study, recombinant Afp1 from Glaciozyma antarctica, a psychrophilic yeast, was overexpressed in a methylotrophic yeast, Pichia pastoris, followed by 5-L fermenter expression. The highest yield of Afp obtained from the fermentation was 39.5 mg/L when the cells were cultivated at and at pH 5.0. Afp1 was produced as a glycoprotein (55 kDa) based on gel staining using a glycoprotein kit. Antifreeze activities of the recombinant Afp1 were exhibited through thermal hysteresis (TH) and recrystallization inhibition (RI) where the highest TH value recorded was at at 10 mg/mL. This value is higher when compared to the recombinant Afp1 produced in Escherichia coli () as well as the native antifreeze protein from G. antarctica (). Both TH and RI activities increased when higher protein concentrations were used. Effects of temperature on stability showed that Afp1 had lost its activity after being incubated at a temperature higher than . The cryoprotective effects of Afp1 on cellulases showed that the treated cellulase retained up to 20% of its activity following several cycles of freeze-thawing. This indicates that Afp1 might act as a cryoprotective agent and has the potential for use in biotechnology applications.