Structure and expression of a gene coding for thermostable α-glucosidase with a broad substrate specificity from Bacillus sp. SAM1606

We cloned an α-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 α-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 12000-fold higher than that obtained by the strain SAM1606. The open reading frame corresponding to the α-glucosidase encoded 587 amino acid residues including a residue coded by the initiation codon TTG, and the molecular mass of the α-glucosidase from N-terminal serine was calculated to be 68886Da. Sequence analysis revealed the SAM1606 α-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 α-glucosidase was significantly different from those of the oligo-1,6-glucosidases. The thermostability of these three α-glucosidases could be correlated with the increase in the number of proline residues, whose occurrence was predicted at β turns and coils in the enzymes.