Abstract
Polymerase chain reaction (PCR) amplification of mRNA from developing barley (cultivar Haruna two-rows) endosperm was used to clone and sequence full-length cDNA encoding β-amylase. The β-amylase cDNA was 1775 by in length . The β-amylase was deduced to be composed of 535 amino acid residues and its molecular weight was calculated to be 59, 610. To express the cloned -amylase cDNA in Escherichia coli under control of the tac promoter, a plasmid pBETA92 was constructed. The plasmid consisted of 6312 bp. The emzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 hr. The recombinant barley β-amylase gave two major (pI 5.43 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 cultivation. But Western blot analysis found that one β-amylase having a molecular weight of about 56, 000 was synthesized. The purified enzyme gave a single band of protein on SDS-PAGE but showed heterogeneity on isoelectric focusing . The N-terminal amino acid sequence showed that the recombinant β-amylase lacked four amino acids at positions 2-5 (Glu-Val-Asn-Val) when compared with the presumed amino acid sequence of barley β-amylase. Therefore, the recombinant β-amylase consisted of 531 amino acids, and its molecular weight was calculated to be 59, 169. The N-terminal amino acid sequence of the recombinant β-amylase and the nucleotide sequence of the junction position in plasmid pBETA92 indicated that GTG (Val-5 in the case of barley β-amylase) at positions 27-29 from the SD sequence (AGGA) was the translation initiation codon. The properties of the recombinant β-amylase were almost the same as those of barley β-amylase except for the pI and the Km values for maltohexaose and maltoheptaose.
