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 control of the tac promoter, a plasmid pBETA92 was constructed. The plasmid consisted of 6312 bp. An extract of E. coli JM109 harboring pBETA92 had beta-amylase activity that produced beta-maltose from soluble starch. The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h. The recombinant barley beta-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 incubation. But Western blot analysis found that one beta-amylase having a molecular weight of about 56,000 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 isoelectric focusing. The N-terminal amino acid sequence showed that the recombinant beta-amylase lacked four amino acids at positions 2-5 (Glu-Val-Asn-Val) when compared with the presumed amino acid sequence of barley beta-amylase. Therefore, the recombinant beta-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 beta-amylase and the nucleotide sequence of the junction position in plasmid pBETA92 indicated that GTG (Val-5 in the case of barley beta-amylase) at positions 27-29 from the SD sequence (AGGA) was the translation initiation codon. The properties of the recombinant beta-amylase were almost the same as those of barley beta-amylase except for the pI and the Km values for maltohexaose and maltoheptaose.(ABSTRACT TRUNCATED AT 250 WORDS)
Yoshigi
N
N
Brewing Research Laboratories, Sapporo Breweries Ltd., Shizuoka, Japan.
Okada
Y
Y
Sahara
H
H
Koshino
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli; D005822:Genetic Vectors; D001467:Hordeum / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D001614:beta-Amylase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D004269:DNA, Bacterial; D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; D001614:beta-Amylase