Cloning And Expression Of A Bacillus Subtilis α-Amylase Gene In Pichia Pastoris

Screeningα-amylase-producing strains or constructing gene engineering strains may produce abundantα-amylase thus settle for the demand in the industry. In this paper, anα-amylase-producing strain was isolated from soil samples by initial screening under the low temperature and flasks fermentation, and theα-amylase activity measured was 30.0 U/ml. Based on partial 16S rDNA gene sequence aligned with the GenBank database and its morphological characteristics, the strain was identified as Bacillus subtilis XL-15. The basic properties of theα-amylase produced by the strain were examined after ammonium sulfate fraction and dialysis. The results showed that the optimum pH and temperature of the enzyme were 6.5 and 50℃respectively; the enzyme activity was obviously inhibited by Cu²⁺, Zn²⁺and EDTA, while stimulated by Ca²⁺ and Mn²⁺, and its measured Km was 1.726mg/mL.Subsequently, theα-amylase gene was amplified by PCR method by using B. subtilis XL-15 genomic DNA as template. The amplified DNA includedα-amylase gene signal sequences, and the Open Reading Frame (ORF) was composed of 1980bp, encoding 659 amino acid. The coding gene was cloned into the vectors of pET-28a(+) and pPIC9K respectively, then transformed into Escherichia coli BL21(DE3) and Pichia pastoris GS115 respectively. The results showed thatα-amylase activity was not tested in the supernatant of E.coli derupted by sonication, and the expressed protein were all present with inclusion body. But theα-amylase could secreted from the recombinant strain of Pichia pastoris GS115 withα-Factor singal peptide, promoter of AOX1 gene and termination signal of yeast genomic, and the activity reached 4.3U/mL, when the P. pastoris GS115 was fermented with high density liquid media and induced by 1.0%methanl, which brings a strong foundation for further expression in large scale.