EXPRESSION IN ESCHERICHIA-COLI OF CDNA-ENCODING BARLEY BETA-AMYLASE AND PROPERTIES OF RECOMBINANT BETA-AMYLASE

To express the cloned beta-amylase cDNA in Escherichia coli under cont rol of the tac promoter, a plasmid pBETA92 was constructed. The plasmi d consisted of 6312 bp. An extract of E. coli JM109 harboring pBETA92 had beta-amylase activity that produced beta-maltose from soluble star ch. The enzyme production started in the Logarithmic phase, increased linearly, and reached a maximum after 12 h. The recombinant barley bet a-amylase gave two major (pI 5.33 and 5.63) and four minor (pI 5.20, 5 .36, 5.80, and 6.13) activity bands on isoelectric focusing, and their pIs didn't change throughout the incubation. But Western blot analysi s found that one beta-amylase having a molecular weight of about 56,00 0 was synthesized. The recombinant beta-amylase was purified from the cells by consecutive column chromatography. The purified enzyme gave a single band of protein on SDS-PAGE but showed heterogeneity on isoele ctric focusing. The N-terminal amino acid sequence showed that the rec ombinant beta-amylase lacked four amino acids at positions 2-5 (Glu-Va l-Asn-Val) when compared with the presumed amino acid sequence of barl ey beta-amylase. Therefore, the recombiant beta-amylase consisted of 5 31 amino acids, and its molecular weight was calculated to be 59,169. The N-terminal amino acid sequence of the recombinant beta-amylase and the nucleotide sequence of the junction position in plasmid pBETA92 i ndicated that GTG (Val-5 in the case of barley beta-amylase) at positi ons 27-29 from the SD sequence (AGGA) was the translation initiation c odon. The properties of the recombinant beta-amylase were almost the s ame as those of barley beta-amylase except for the pI and the K-m valu es for maltohexaose and maltoheptaose. The pI of recombiant barley bet a-amylase calculated by Genetyx Version 9 based on the presumed amino acid sequence was 5.60, but the real pIs were 5.20-6.13. Therefore, so me post-translational reaction(s) might happen after protein synthesis in E. coli cells, and this modification might cause the differences i n the pI and the K-m values for maltohexaose and maltoheptaose between the barley and the recombinant beta-amylases.