Expression in<i>Escherichia coli</i>of cDNA Encoding Barley<i>β</i>-Amylase and Properties of Recombinant<i>β</i>-Amylase

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  • Expression in Escherichia coli of cDNA Encoding Barley .BETA.-Amylase and Properties of Recombinant .BETA.-Amylase.

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To express the cloned β-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 β-amylase activity that produced β-maltose from soluble starch. The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h. 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 incubation. But Western blot analysis found that one β-amylase having a molecular weight of about 56, 000 was synthesized. The recombinant β-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 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 recombiant 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. The pI of recombiant barley β-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, some post-translational reaction(s) might happen after protein synthesis in E. coli cells, and this modification might cause the differences in the pI and the Km values for maltohexaose and maltoheptaose between the barley and the recombinant β-amylases.

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