Abstract:
21A Transformed Escherichia coli with amylase gene from Bacillus subtilis Qingqiang Yin1, Gangping Xue2, A. King3 and J.G. Dingle1 1 2 3 School of Animal Studies, The University of Queensland, Gatton Qld 4343 Molecular Biology Laborator y, Plant Industry, CSIRO, Indooroopily Qld 4067 Australasian Pig Institute, The University of Queensland, Gatton Qld 4343 yin@sas.uq.edu.au 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 dif ferent primers can produce dif ferent 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 �g/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: Amylase activity (U/L) = [(A blank The results showed that the three kinds of amylase genes had similar molecular weights (2.3 kb). After the amylase gene was connected with a plasmid (pHMSXD1) and inserted into E. coli, the transformed E. coli expressed and excreted amylase. The activities of that excreted amylase and that from the initial B. subtilis were 1.0376 and 0.0226 U/ml in LB medium, respectively. After transformation in E. coli, the amylase activity was increased 45 times. The reasons may be: (i) the plasmid can replicate itself abundantly in the host (Brown 1990) and produce more amylase than the initial bacteria; (ii) the different regulatory and metabolic systems in E.coli could increase the secretion of amylase; (iii) the high voltage used during transformation could increase the expression of the amylase gene. The transformed E.coli with high amylase activity may be suitable for use as a prebiotic/probiotic in animals. Brown, T.A. (1990). Gene Cloning. Second edition, pp. 84_102. Chapman and Hall, London. Li, Y.L., Lu, S.W. and Liu, Y.C. 1987. Examination Handbook of Clinical Medicine, pp. 378_379. Jilin Science and Technology Press. Lin, L.L. and Hsu, W.H. (1997). Lactose_induced expression of Bacillus sp. TS_23 amylase gene in Escherichia coli regulated by a T7 promoter. Letters in Applied Microbiology 24, 365_368. _ Areaction ) / Ablank] x 8000 Recent Advances in Animal Nutrition in Australia, Volume 13 (2001)