Transformed Escherichia coli with amylase gene from Bacillus subtilis

Recent Advances in Animal Nutrition in Australia, Volume 13 (2001) Starch can be degraded by amylase produced by the animal itself and by anaerobic and aerobic bacteria existing in animal digestive tracts and in the environment. Naturally occurring Escherichia coli has not been found to produce amylase, but if an amylase gene could be expressed in harmless E. coli the transformed bacterium might produce abundant amylase. Though it has been reported that amylase gene from Bacillus subtilis has been expressed in E. coli (Lin and Hsu 1997), the use of different species of B. subtilis and different primers can produce different characteristics of amylases. B. subtilis, E. coli, and plasmid (pHMSXD1) were provided by CSIRO and cultured in Luria–Bertani (LB) medium. Genomic DNA from B. subtilis was purified, and amylase gene from B. subtilis was amplified by polymerase chain reaction using three pairs of primers. The pHMSXD1 and amylase gene were partially digested with nucleases (Hind III and XbaI) and ligated with T4 DNA ligase. Ligation solution and E. coli cells were put into a pre–chilled cuvette (0.1 cm electrode gap); the electroporation apparatus was set to 1.7 kV. The transformed E. coli was aliquotted on to agar plates with starch (1%) and ampicillin (50 μg/ml); after overnight culture, the plates were stained by I2 and KI and the diameters of the haloes were measured as a semi–quantitive measure of amylase activity. For quantitative amylase measurement (Li et al. 1987), amylase in the culture supernatant was precipitated and incubated with a starch solution at 37oC for 7.5 min. Iodine was added and absorbance(A) was measured at 660 nm, whence: