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α-Amylases of B. amyloliquefaciens and a mutant strain were purified through a series of four steps and the purity of enzymes was checked by electrophoresis. Differences were found among the enzymes with respect to pH, temperature, stability, Km and Vmax and binding of Ca. The degradation ability of wild type α-amylase on starch granules from various botanical sources (potato, sweet potato, corn, wheat and rice) were examined by scanning electron microscopy and by kinetic assays. Differences in enzyme activity on raw granules were noted. The gene coding mature enzyme was amplified by PCR and expressed in E. coli under trc and T7 promoter.

β-Conglycinin (7S globulin), one of the major storage proteins of soybean, is a trimeric protein composed of α (67 kDa), α′ (71 kDa) and β (50 kDa) subunits and exhibits molecular heterogeneity. It is synthesized on the endoplasmic reticulum and transported to the protein storage vacuole (PSV). In this study, we examined a vacuolar sorting determinant (VSD) of the β subunit using seeds of transgenic Arabidopsis accumulating deletion derivatives of β subunit. We found that the β subunit was transported to the matrix and the globoid compartment of the PSV through distinct pathways, and that a VSD to the matrix resides in the C-terminal 10 amino acids. We further verified that the C-terminal 10 amino acids of β subunit are sufficient to transport green fluorescent protein to the PSV matrix in soybean seed cells by using transient expression system. The results of this study show a transport mechanism for the β subunit similar to that of the α′ subunit [Plant J., 34 (2003) 647].

The degradation abilities of α-amylase from Bacillus amyloliquefaciens and β-amylases from Bacillus cereus and soybean on raw starch granules from various botanical sources (potato, sweet potato, wheat, rice and corn) were examined by scanning electron microscopy. All the amylases showed different degradation patterns on starch granules. The α-amylase was more efficient than the β-amylases. α-Amylase showed both centrifugal and centripetal hydrolysis on corn, rice and wheat granules, but only centrifugal hydrolysis on potato granules. On the other hand, β-amylase moved very slowly on granules. The kinetic assays which explain the release of maltose were carried out at 12, 18 and 24 h. The rice granules were found to be the best substrate for enzymic hydrolysis by α and β-amylases. In addition, While Bacillus cereus β-amylase hydrolyzed corn granules efficiently at 45°C; soybean β-amylase was 60% less active than bacterial β-amylase at the same temperature.

Chimeric genes composed of the class I patatin promoter, A1aB1b preproglycinin cDNA and its modified versions (IV+4Met and V+4Met) containing four contiguous methionine codons and nopaline synthase gene terminator were inserted into the genome of potato by Agrobacterium-mediated transformation. Transcripts of the chimeric genes were detected specifically in tubers. Translational products were observed in parenchyma cells of the tubers of potato plants transformed with the normal and modified glycinin cDNAs. This distribution is consistent with the expression specificity of the patatin promoter. The expression levels of the normal and the modified glycinins were 0.2–1.0% of the total soluble tuber proteins. Both the normal and the modified glycinins expressed in the tubers were present as proglycinin trimers. These results indicate that the modifications in IV+4Met and V+4Met did not disrupt any stage in the biosynthetic process of glycinin in potato tubers.