[Cloning and expression of the gene encoding novel alpha-amylase from Sulfolobus shibatae in E. coli]

A novel alpha-amylase gene was amplified from Sulfolobus shibatae by using PCR technique. The amplified 1.7 kb DNA fragment was inserted into an expression vector pBV220 to yield the recombinant plasmid pSBAM. The novel alpha-amylase gene in pSBAM was expressed in E. coli. The production of the novel alpha-amylase activity reached over 8 units/100 mL of the culture. The molecular weight of this enzyme was about 61 kD by SDS-PAGE. The expressed novel alpha-amylase protein in E. coli DH5 alpha accounted for about 20% of the total protein in the recombinant cell. The cooperative action of the novel alpha-amylase and the maltooligosyltrehalose synthase from Sulfolobus shibatae was investigated and trehalose was detected by using HPLC analysis when using amylose and partial starch hydrolysates as substrates.