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Cloning and expression of a thermostable exo-α-1,4-glucosidase gene fromBacillus stearothermophilusATCC12016 inEscherichia coli

✍ Scribed by Yukio Takii; Katsuya Daimon; Yuzuru Suzuki

Publisher
Springer
Year
1992
Tongue
English
Weight
856 KB
Volume
38
Category
Article
ISSN
1432-0614

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✦ Synopsis

The gene coding for a thermostable exo-alpha-1,4-glucosidase (alpha-glucoside glucohydrolase: EC 3.2.1.20) of Bacillus stearothermophilus ATCC 12016 was cloned within a 2.8-kb AvaI fragment of DNA using the plasmid pUC19 as a vector and Escherichia coli JM109 as a host. E. coli with the hybrid plasmid accumulated exo-alpha-1,4-glucosidase mainly in the cytoplasm. The level of enzyme production was about sevenfold higher than that observed for B. stearothermophilus. The cloned enzyme coincided absolutely with the B. stearothermophilus enzyme in its relative molecular mass (62,000), isoelectric point (5.0), amino-terminal sequence of 15 residues (Met-Lys-Lys-Thr-Trp-Trp-Lys-Glu-Gly-Val-Ala-Tyr-Gln-Ile-Tyr-), the temperature dependency of its activity and stability, and its antigenic determinants.

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