Expression and mutation of soybean β‐amylase in Escherichia coli

Abstract

The cDNA clones corresponding to soybean P-amylase mRNA were isolated and sequenced. The cDNA contained an open-reading frame composed of 496 amino acids. The comparison of the amino acid sequence deduced from the cDNA with the N-terminal peptide sequence from mature enzyme proved that P-amylase had no leader sequence. Employing the cDNA, the P-amylase was directly synthesized in Escherichia coli by the expression vector pKK233-2 controlled by the tac promoter. The enzyme activity detected in E. coli lysate drastically increased with a lower cultivation temperature, and the total activity and specific activity of the enzyme in E. coli lysate cultured at 13°C was 130-fold and 280-fold, respectively, the value at 37°C. The enzyme produced in E. coli was purified by the affhity column chromatography of cyclomaltohexaose-immobilized Sepharose 6B. Employing the established expression and purification system of the enzyme, the functional ionizable groups in the active site were searched. His93, involving an imidazole, and Asp348, involving a carboxylate, in the highly conserved regions within the P-amylases were replaced by Arg (H93R) and Asn (D348N) by site-directed mutagenesis, respectively. All P-amylases, including the non-mutant and mutant @-amylases, produced in E. coli exhibited lower V,, values than that of P-amylase isolated conventionally from soybean seeds. Especially the V,,, value of [H93R]P-amylase was reduced drastically compared to that of the non-mutant; however, none of them lost their enzyme activities completely. Therefore, neither His93 nor Asp348 may participate in the catalytic reaction directly. 7] and Clostridiurn themzosulfurogenes [ 81 were cloned and sequenced. The highly conserved amino acid sequences found among them suggest that those regions may assemble the active center of P-amylase reaction [9, 101. Although the crystallographic study of soybean P-amylase is in progress [ll], the relationship between the primary structure and the catalytic reaction still remains unclear. The thiol group of the cysteine residue [12, 131 or two functional ionizable groups with pKa 3.5, such as a carboxylate of a Glu or Asp residue, and pK, 8.2, such as an &-amino group of Lys or an imidazole group of His [14], were speculated to be involved in the active site. Recently, using affinity labeling of soybean Pamylase with 2,3-epoxy propyl a-D-glucopyranoside, Glul86 was shown to be involved in the active site [15]. Also the direct contributions of cysteine residues to the catalytic reacCorrespondence to C . Fukazawa, Genetic Engineering Laboratory, National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Kannondai, Tsukuba, Ibaraki 305, JapanFar: +81 0298 38 7996.Enzymes. a-Amylase, 1,4-a-r>-glucan glucanohydrolase (EC 3.2.1.1); P-amylase, 1,4-a-D-ghCan maltohydrolase (EC 3.2.1.2).Note. The novel nucleotide sequence data published here have been deposited with the GenBank EMBL sequence data bank and are available under the accession number X71419. The novel amino acid sequence d...

Residues essential for catalytic activity of soybean β‐amylase

“…To identify the residues essential for catalytic activity of soybean P-amylase, we have tried to mutate cysteinyl, aspartyl, and glutamyl residues located in the highly conserved regions. Throughout these experiments, we have demonstrated that substitution of AsplOl and that of Glu186 of soybean P-amylase by neutral and acidic amino acids resulted in the complete elimination of the enzymic activity but did not alter the binding ability of a substrate analogue nor the circular dichroic spectra, as determined using an Escherichia coli expression system [4].…”

“…As to aspartyl and glutamyl residues, AsplOl, Glu186, Glu345, and Asp348 of soybean P-amylase are contained in the above regions. Since mutation of Asp348 has already been shown not to affect the kinetic parameters [4], the targets for the mutation were focused on the AsplOl, Glu186, and Glu345 residues (Table 1). On the other hand, as to cysteinyl residues, the Cys95 in region I is highly conserved in every P-amylase known to date.…”

“…The plasmid, pBAMl9, involving soybean P-amylase cDNA and its expression plasmid, pBAMSO, were constructed previously [4]. Oligonucleotide site-directed mutagenesis for replacement of AsplOl by Glu (DlOlE) or Asn (DlOlN) and for replacement of Cys95 with Ser (C95S) was carried out on the Xbal-Sac1 fragment derived from the insertion of pBAM19 DNA ( Fig.…”

“…The expression and purification of recombinant non-mutant P-amylase and its mutants were carried out according to the procedure described previously [4] with slight modification. Briefly, E. coli JM105 harboring each expression plasmid described above was grown at 23°C in Luria broth supplemented with ampicillin (50 pg/ml).…”

“…Highly conserved regions involving the mutagenic amino acid residues. The amino acid sequences of P-amylases originating from (A) Bacillus cereus [16], (B) Bacillus circulans [17], (C) Bacillus polymyxu [18,191, (D) Clostridiurn thermosulfurogenes [20], (E) Arubidopsis thalianu 1211, (F) barley [22], (G) sweet potato [23], and (H) soybean [4] were aligned to facilitate visual comparison of the similarities. The residues identical to those of soybean P-amylase are denoted by asterisks.…”