Cloning And Expression Of Thermostable α-Amylase Gene From Bacillus Licheniformis B13

Thermostableα-amylase,catalyzing hydrolysis of starch at high temperatures,is one of the most important enzymes,widely applied in sugar and fermentation industry.Bacillus licheniformis is one of the stains used to produce thermostableα-amylase in industry.The aim of this research was focused on cloning and expression of the gene encoding a thermostableα-amylase from Bacillus licheniformis B13.A DNA fragment containingα-amylase gene,amy with its own promoter and signal peptide sequences was obtained by PCR from genomic DNA of B.licheniformis B13.The result of sequencing indicated thatα-amylase gene from B.licheniformis B13 had 99% similarity to that from B.licheniformis 548.The amy gene could be expressed in E.coli using its own promoter andα-amylase expressed could secreted into the culture medium.B.subtilis WB800 was widely used as host for the expression of heterologous gene, containingα-amylase.By knocking out its amylase gene B.subtilis WB800D was constructed in order to expression heterologous amy gene.The amy gene from B.licheniformis B13 was cloned into shuttle vector pUBC19 and expressed in B.subtilis WB800D.But expression level was very low.The amy gene and signal peptide sequences was integrated into shuttle vector pP43NMK and expressed in B.subtilis WB800D.The results showed that theα-amylase gene was highly expressed under the control of promoter P43.The amount ofα-amylase expressed under the control of promoter P43 was 8.9 more times of that expressed under the control of itself promoter ofα-amylase gene of B.lichenformis B13.It was very helpful to constructα-amylase gene engineering strain.