Structure and expression of a gene coding for thermostable alpha-glucosidase with a broad substrate specificity from Bacillus sp. SAM1606.
We cloned an alpha-glucosidase gene from thermophilic Bacillus sp. SAM1606 to overexpress it in Escherichia coli transformants. Deletion of the 5'-noncoding region as well as expression of the alpha-glucosidase gene under the control of the icp promotor of the insecticidal crystal protein gene from Bacillus thuringiensis subsp. sotto enhanced the enzyme productivity to 23.5 U/ml, which was 12,000-fold higher than that obtained by the strain SAM1606. The open reading frame corresponding to the alpha-glucosidase encoded 587 amino acid residues including a residue coded by the initiation codon TTG, and the molecular mass of the alpha-glucosidase from N-terminal serine was calculated to be 68,886 Da. Sequence analysis revealed that the SAM1606 alpha-glucosidase belonged to the alpha-amylase family. The SAM1606 alpha-glucosidase showed extremely high sequence identity (62-65%) to the Bacillus cereus and Bacillus thermoglucosidasius oligo-1,6-glucosidases, which were 72% identical to each other. Sequence identity in the suggested active site regions were essentially the same (80-82%) among these three enzymes. However, the substrate specificity of the SAM1606 alpha-glucosidase was significantly different from those of the oligo-1,6-glucosidases. The thermostability of these three alpha-glucosidases could be correlated with the increase in the number of proline residues, whose occurrence was predicted at beta turns and coils in the enzymes.
Nakao
M
M
Institute for Biomedical Research, Suntory Ltd., Osaka, Japan.
Nakayama
T
T
Kakudo
A
A
Inohara
M
M
Harada
M
M
Omura
F
F
Shibano
Y
Y
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli; D005798:Genes, Bacterial; D015698:Genomic Library; D006358:Hot Temperature; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013816:Thermodynamics; D000520:alpha-Glucosidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D017931:DNA Primers; D004269:DNA, Bacterial; D000520:alpha-Glucosidases