Acidproof medium-temperature alpha-amylase and preparation method thereof
An amylase and mesophilic technology, which is applied in the field of acid-resistant and mesophilic alpha-amylase and its preparation, can solve the problems that alpha-amylase cannot take into account the acid resistance and temperature adaptability at the same time, application limitations, etc. Effect of pH applicable range, good acid stability
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Embodiment 1
[0046] Example 1: Amplification of the Precursor Alpha-Amylase Gene
[0047] The chromosomal DNA of Bacillus subtilis [purchased from China Industrial Microorganism Culture Collection Center (CICC 10028)] was extracted. According to the reported Bacillus subtilis amylase gene in Genbank, the following primers were designed (the primers were commissioned to be synthesized by Shanghai Bioengineering Co., Ltd.):
[0048] Upstream primer F1: 5'-GCGC GAATTC GGAAACTGCAAACAAATCGAATGAGGTG-3' (SEQ ID NO: 1)
[0049] Downstream primer R1: 5'-CGCG AAGCTT ATGCGGAAGATAACCATTCAAACCGG-3' (SEQ ID NO: 2)
[0050] The 5' end of the upstream primer F1 contains an EcoR I restriction site (underlined), and the 5' end of the downstream primer contains a Hind III restriction site (underlined). Perform PCR amplification using Bacillus subtilis chromosomal DNA as a template, and mix the components in a sterilized thin-walled centrifuge tube in the following order: use 50 μL amplification system:...
Embodiment 2
[0056] Example 2: Site-directed mutagenesis of precursor alpha-amylases
[0057] The mutation diagram of α-amylase is shown in figure 1 , design complementary primers containing mutated amino acid residues as follows:
[0058] Primer 1: 5'-TTGGTCGGACGGATGGGACATCACT-3' (SEQ ID NO: 4)
[0059] Primer 2: 5'-AGTGATGTCCCATCCGTCCGACCAA-3' (SEQ ID NO: 5)
[0060] Primer 3: 5'-GGGCCATAGAAGATACCGGATCATG-3' (SEQ ID NO: 6)
[0061] Primer 4: 5'-CATGATCCGGTATCTTCTATGGCCC-3' (SEQ ID NO: 7)
[0062] Primer 1 and Primer 2 are complementary, and Primer 3 and Primer 4 are complementary. Primers 1 and 2 contained a mutation at amino acid 223, while primers 3 and 4 contained a mutation at amino acid 558. Use the recombinant plasmid pET22-ACN7 (constructed in Example 1) as a template for PCR amplification, and mix the components in a thin-walled centrifuge tube in the following order: PCR1: use 50 μL system, ddH 2 O3 8.5 μL, 10× buffer 5 μL, dNTP (2.5 mmol / L) 2 μL, primer 1 (20 μmol / L) 2 μL...
Embodiment 3
[0066] Embodiment 3: Construction of mutant α-amylase expression vector
[0067] The Bacillus subtilis expression vector used pWB980 [purchased from American Biogenetic Sciences, Inc], and the following primers were designed:
[0068] Upstream primer F2: 5'-CGC GGATCC AGGAAACTGCAAAACAAATCGAATGAGGTG-3' (SEQ ID NO: 9)
[0069] Downstream primer R2: 5'-CGCG GCATGC ATGCGGAAGATAACCATTCAAACCGG-3' (SEQ ID NO: 10)
[0070] The 5' end of the upstream primer F2 contains a BamH I restriction site (underlined), and the 5' end of the downstream primer contains a Sph I restriction site (underlined). Use the recombinant expression vector pETCN7-2 as a template for PCR amplification, and mix the components in a sterilized thin-walled centrifuge tube in the following order: use 50 μL amplification system: ddH 2 O 41.5 μL, 10× buffer 5 μL, dNTP (2.5 mmol / L) 1 μL, upstream and downstream primers 2 (20 μmol / L) each 0.5 μL, DNA template 1 μL, Taq DNA polymerase 0.5 μL. The amplification cond...
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