Abstract: Objective:To express and purifyα-glucosidase gene from E.sakazakii. Methods:α-glucosidase gene was cloned from E.sakzakii genome DNA. Plasmid pET22b(+) containing α-glucosidase gene was constructed and transferred into competent Escherichia coli and α-glucosidase gene was overexpressed by the challenge of isopropylthio-β-D-glactoside(IPTG). The expression of α-glucosidase gene protein was one-step purified. Furthermore, the catalyse activity of α-glucosidase gene protein was preliminarily detected. Results:The recombinant Escherichia coli produced α-glucosidase gene protein in large quantities, accounting for 18% of total cell protein. The molecular weight of α-glucosidase gene protein was estimated to be 65 kD by sodium dodecyl sulfate-polyacrylamide gradient gel electrophresis (SDS-PAGE). It was found that imidazole at the concentration of 500 mmol/L could elute the α-glucosidase gene protein most efficiently and its purity higher than 90%. The α-glucosidase gene protein was preliminarily detected to have the activity of catalyse. Conclusions:The stable α-glucosidase gene overexpressing recombinant Escherichia coli can be constructed by the plasmid pET22b(+) containing α-glucosidase gene. The recombinant strain can produce α-glucosidase gene protein with catalyse activity and the product of which can be purified into higher activity.

Key words: Enterobacter.sakazakii； &alpha, -glucosidase； clone； expression；