Enhanced Production of Acid α-glucosidase by Using Protein Stabilizer in Transgenic Rice Cell Cultures

초록

Acid α-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes glycogen to glucose. In this study, GAA was produced by using RAmy3D promoter in transgenic rice cells. Since RAmy3D promoter is induced by sugar starvation, proteases were secreted and accumulated in medium during production phase. Secreted proteases degraded GAA, leading to low activity. In order to protect GAA during cultures, protein stabilizer could be used in production phase. First, 1 μM and 10 μM 1-deoxynojirimycin (DNJ) was supplemented as a substrate analogue. In addition, 0.5 g/L polyvinylpyrrolidone (PVP) which is an amphiphillic polymer could be used as a protein stabilizer. As an another stabilizer, 5 g/L pluronic F-68 (PF-68) that is nonionic surfactant was used to protect the cells or target protein. As a result, these supplements did not show any negative effects on relative viability and growth of the cells. Production of GAA with protein stabilizer was higher than that of control. Especially, production level of GAA at day 6 with PF-68 was 1.55-fold higher than that without PF-68. In conclusion, protein stabilizer could enhance the level of GAA in production phase by protecting degradation of GAA regardless of growth and viability of the cells.