Monto H. Kumagai

Rapid, high-level expression of glycosylated rice α-amylase in transfected plants by an RNA viral vector.

Tobamoviral vectors have been developed for the heterologous expression of glycoproteins in plants. The rice alpha-amylase gene (OS103) was placed under the transcriptional control of a tobamovirus subgenomic promoter in a RNA viral vector. One to two weeks after inoculation, transfected Nicotiana benthamiana plants accumulated glycosylated alpha-amylase to levels of at least 5% total soluble protein. The 46kDa recombinant enzyme was purified, and its structural and biological properties were analyzed. Post-translational modifications of the secreted protein were compared to rice alpha-amylase expressed in amylolytic strains of Pichia pastoris and Saccharomyces cerevisiae. Endo-H analysis revealed that the alpha-amylase was moderately glycosylated in transfected plants and hyperglycosylated in yeast.

Expression and secretion of rice α-amylase by Saccharomyces cerevisiae

Abstract We report the high level expression and secretion of rice α-amylase isozyme by Saccharomyces cerevisiae . Transcription of this gene was under control of the yeast enolase promoter. The synthesized protein had an approximate molecular size of 45 kDa and a pI of approx 4.7 to 5.0. The rice α-amylase signal peptide was recognized and efficiently processed by yeast and the active, glycosylated enzyme was secreted into the culture media. This enzyme was purified to homogeneity by affinity chromotography and its enzymatic properties were characterized. The K m and V max were found to be similar to those of α-amylase from other organisms. The high level of secretion observed in these studies may be due to the unique features of the rice signal peptide and/or to the glycosylation of the recombinant enzyme.

Characteristics of a chimeric enzyme engineered from two rice α-amylase isozymes

Abstract A chimeric enzyme, engineered from two rice α-amylase isozymes, Amy1A and Amy3D, showed unique characteristics in soluble-starch and maltoheptaose hydrolysis. Effects of pH on soluble-starch hydrolysis and the thermostability of the chimeric enzyme were similar to those of the isozyme Amy3D. The previous study revealed that Amy1A shows high activity in soluble-starch hydrolysis and low activity in oligosaccharide degradation, while Amy3D shows low activity in soluble-starch hydrolysis and high activity in oligosaccharide degradation. The chimeric enzyme showed high activities in both soluble-starch hydrolysis and oligosaccharide degradation. These results suggest that protein modules of highly homologous enzymes are interchangeable, and that a novel enzyme with unique characteristics can be obtained by creating a chimeric enzyme.