CN102747058A - Method for producing alpha amylase - Google Patents

Abstract

The invention discloses a method for producing alpha amylase. It belongs to the field of biotechnology. Firstly clone the α-amylase genes of barley, Bacillus licheniformis and Aspergillus respectively; construct the expression vectors of Pichia pastoris, Saccharomyces cerevisiae and Bacillus subtilis containing the above three α-amylase gene expression frameworks, and transform the vectors respectively Corresponding host bacteria; Recombinants highly expressing α-amylase were selected as engineering bacteria; Pichia pastoris engineering bacteria, Saccharomyces cerevisiae engineering bacteria and Bacillus subtilis engineering bacteria were fermented to produce recombinant mixed α-amylase. Different from the traditional α-amylase encoded by a single gene, the suitable reaction temperature and pH range of the recombinant mixed α-amylase produced by the present invention are wide, and are suitable for various purposes; The yield of α-amylase is significantly higher than that of the α-amylase expressed by engineering bacteria containing a single gene, which plays a role in reducing production costs.

Description

A kind of method of producing α-amylase

technical field

The invention belongs to the field of biotechnology and relates to the construction of microbial engineering bacteria to produce recombinant proteins. Specifically, the mixed α-amylase is produced by using microbial engineering bacteria.

Background technique

α-amylase (α-amylase, EC 3.2.1.1) widely exists in the biological world. Alpha-amylase catalyzes the random hydrolysis of the alpha-1,4-glucosidic linkages of starch to produce maltose, maltotriose and alpha-dextrin. α-amylase is widely used in industries such as maltose, beer, rice wine, glucose, alcohol, liquor, monosodium glutamate and medicine. The alpha amylase products currently on the market are derived from alpha amylase produced by natural strains. However, due to the slow growth of natural α-amylase-producing strains and higher nutritional requirements, the production cost of the enzyme is higher; in addition, the suitable reaction temperature range and suitable reaction pH range of the current α-amylase products are narrow, which cannot meet current market needs.

Since there are multiple sources of α-amylase, the gene sequences of α-amylases from different sources may be different, and the physicochemical properties of α-amylases from different sources may be different. Pichia pastoris, Saccharomyces cerevisiae and Bacillus subtilis are commonly used host bacteria for the production of recombinant proteins, but each has its own characteristics. The main feature of Pichia pastoris is that it secretes very little self-protein, which is beneficial to the purification of expression products. Bacillus subtilis and Saccharomyces cerevisiae have facultative aerobic characteristics, and are suitable for solid-state anaerobic fermentation to produce recombinant enzymes.

Contents of the invention

According to the fact that there are many sources of α-amylase, the gene sequence of α-amylase from different sources may be different, and the physical and chemical properties of α-amylase from different sources may be different. If some α-amylase genes with different physical and chemical properties are recombined into the same cell If the genome is expressed, the expressed mixed α-amylase will have a variety of physical and chemical properties, that is, multiple optimum temperatures and optimum pHs. This mixture of enzymes will be of higher value than any of the individual enzymes. α-amylase derived from barley (Hordeum vulgare) has an optimum pH of 5, an optimum temperature of 70°C, and >60% enzyme activity at pH 5-6 and temperature from 35 to 73°C; derived from Bacillus licheniformis ) α-amylase has an optimum pH of 8.5, an optimum temperature of 80°C, and has >60% enzyme activity at pH 5-9.5 and 55-90°C; Temperature is 70°C with >60% enzyme activity at pH 3-6 and 50-75°C. Since the functions of these three enzymes are the same, the three mixed α-amylases expressed in Pichia pastoris or Saccharomyces cerevisiae or Bacillus subtilis are suitable for use in acidic, neutral, alkaline, normal temperature, medium temperature and higher temperature. used under the conditions. That is, the use of this mixed enzyme will be wider than that of the individual enzymes. If different bacterial strains are used to express these three enzymes, the production cost will be higher than that of a mixed enzyme that produces these three enzymes at the same time with one bacterial strain. The invention constructs Pichia pastoris engineering bacteria, Saccharomyces cerevisiae engineering bacteria and Bacillus subtilis engineering bacteria containing barley α-amylase gene expression framework, Bacillus licheniformis α-amylase gene expression framework and Aspergillus α-amylase gene expression framework, Production of α-amylase encoded by the α-amylase gene of recombinant mixed barley, α-amylase encoded by the α-amylase gene of Bacillus licheniformis and α-amylase encoded by the α-amylase gene of Aspergillus.

The technical scheme adopted in the present invention is:

1. Cloning enzyme gene: Apply molecular biology techniques to amplify α-amylase gene from barley genome, Bacillus licheniformis genome and Aspergillus genome respectively.

2. Obtain the promoter, recombinant sequence, signal peptide, transcription terminator and resistance gene required for the construction of the expression vector.

3. Construction of the alpha amylase gene expression framework containing barley, the alpha amylase gene expression framework of Bacillus licheniformis and the Pichia expression of the alpha amylase gene expression framework of Aspergillus as accompanying drawing 1, accompanying drawing 2 and accompanying drawing 3 Vector, Saccharomyces cerevisiae expression vector and Bacillus subtilis expression vector.

4. Transform the Pichia pastoris expression vector containing the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene expression framework of Aspergillus into Pichia pastoris; Gene expression framework, α-amylase gene expression framework of Bacillus licheniformis and Saccharomyces cerevisiae expression vector of α-amylase gene expression framework of Aspergillus transform Saccharomyces cerevisiae; Bacillus subtilis was transformed with the Bacillus subtilis expression vector of the gene expression framework and the Aspergillus alpha-amylase gene expression framework. [Description. Expression frame: promoter-signal peptide-gene-transcription terminator]

5. Screen the α-amylase encoded by the α-amylase gene encoded by barley, the α-amylase encoded by the α-amylase gene of Bacillus licheniformis and the Pichia recombinants of the α-amylase encoded by the α-amylase gene encoded by Aspergillus with high expression The α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the Pichia pastoris engineering bacteria of the α-amylase gene expression framework of Aspergillus; Screen the α-amylase encoded by the α-amylase gene of high expression barley, Saccharomyces cerevisiae recombinants of α-amylase encoded by the α-amylase gene of Bacillus licheniformis and α-amylase encoded by the α-amylase gene of Aspergillus as expression frameworks for the α-amylase gene containing barley, α-amylase gene expression of Bacillus licheniformis Saccharomyces cerevisiae engineered framework and Aspergillus α-amylase gene expression framework; screening for high expression of α-amylase encoded by the α-amylase gene of barley, α-amylase encoded by the α-amylase gene of Bacillus licheniformis and α-amylase of Aspergillus The bacillus subtilis recombinant of gene-encoded α-amylase serves as a subtilis engineering bacterium containing the α-amylase gene expression framework of barley, the α-amylase gene expression framework of bacillus licheniformis and the α-amylase gene expression framework of aspergillus.

6. Production of Pichia pastoris, Saccharomyces cerevisiae and Bacillus subtilis engineering bacteria containing barley α-amylase gene expression framework, Bacillus licheniformis α-amylase gene expression framework and Aspergillus α-amylase gene expression framework Recombinant mixed alpha amylases.

The Pichia pastoris engineering bacteria, Saccharomyces cerevisiae engineering bacteria and Bacillus subtilis engineering bacteria containing barley alpha amylase gene expression framework, Bacillus licheniformis alpha amylase gene expression framework and Aspergillus alpha amylase gene expression framework constructed by the present invention The advantages of producing recombinant mixed alpha amylases are reflected in:

The present invention constructs Pichia pastoris engineering bacteria, Saccharomyces cerevisiae engineering bacteria and Bacillus subtilis engineering bacteria, and uses engineering bacteria to produce three kinds of recombinant α-amylase mixed enzyme products. On the one hand, it provides The mixed α-amylase new product used in different temperature environments, on the other hand, replaces the α-amylase encoded by the α-amylase gene encoded by barley and the α-amylase gene encoded by Bacillus licheniformis produced by fermenting strains containing these three enzyme genes respectively α-amylase and α-amylase encoded by the α-amylase gene of Aspergillus. That is, one fermentation process is used to produce three kinds of α-amylases instead of three processes to produce three kinds of α-amylases respectively. Achieve the effect of saving raw materials, energy consumption and human resources.

Description of drawings

Accompanying drawing 1. Pichia pastoris expression vector containing the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene expression framework of Aspergillus.

p. Glyceraldehyde triphosphate dehydrogenase promoter from Pichia pastoris; S. α factor signal peptide; H-amy. α-amylase gene from barley; B-amy. α-amylase gene from Bacillus licheniformis; A-amy . Aspergillus α-amylase gene; T. transcription terminator; G418.G418 resistance gene (applied to screening Pichia recombinants); ColE1. Escherichia coli replication origin; AMP. ampicillin resistance gene (applied to screening large intestine Bacillus transformant); p. pastoris rDNA. The rDNA sequence of Pichia pastoris.

Accompanying drawing 2. Saccharomyces cerevisiae expression vectors containing the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene expression framework of Aspergillus.

p. Glyceraldehyde triphosphate dehydrogenase promoter from Saccharomyces cerevisiae; S. α factor signal peptide; H-amy. α-amylase gene from barley; B-amy. α-amylase gene from Bacillus licheniformis; A-amy. Aspergillus α-amylase gene; T. transcription terminator; G418.G418 resistance gene (applied to screening Saccharomyces cerevisiae recombinants); ColE1. Escherichia coli replication origin; S. cerevisiae rDNA. The rDNA sequence of Saccharomyces cerevisiae.

Accompanying drawing 3. Bacillus subtilis expression vector containing the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene expression framework of Aspergillus.

p. Glyceraldehyde triphosphate dehydrogenase promoter from Bacillus megaterium; S. α factor signal peptide; H-amy. α-amylase gene from barley; B-amy. α-amylase gene from Bacillus licheniformis; A-amy . Aspergillus α-amylase gene; T. transcription terminator; G418.G418 resistance gene (applied to screening Bacillus subtilis recombinants); ColE1. Escherichia coli replication origin; AMP. ampicillin resistance gene (applied to screening large intestine Bacillus transformant); B.Subtilis rDNA. The rDNA sequence of Bacillus subtilis.

Detailed ways

The present invention will be further illustrated below with non-limiting examples.

Example 1:

1.1 Construct the Pichia pastoris expression vector containing the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene expression framework of Aspergillus

1.1.1 Construction of cloning vector

A professional DNA sequence synthesis company synthesizes two base complementary double strands containing ampicillin (AMP) gene sequence, polyclonal adapter and E. coli replication origin, and forms cohesive ends at both ends of each DNA strand sequence. It is circularized by the action of DNA ligase to form a DNA cloning vector. The cloning vector was named pPD.

1.1.2 Acquiring genes

①Amplification of barley α-amylase gene by reverse transcription PCR

Primer 1: 5'GGC GAATTC caagtcctctttcaggggtt3'3'[Description: The 8 bases at 5' are enzyme-cleaved protection bases (2 bases) and enzyme recognition site (6 bases underlined)]

gctccgttgtagtgttgccgcggcaccgt3’Primer 2: 5'CA GCGGCCGCGC CTA

gctccgttgtagtgttgccgcggcaccgt3'

[Explanation: The 10 bases of the 5' are enzyme-cleaved protection bases (2 bases) and enzyme recognition sites (8 bases), and the 18 bases in the box are encoding 6 histidines DNA sequence. ]

The total RNA of barley was extracted by plant RNA extraction kit, its cDNA was synthesized by cDNA synthesis kit, PCR amplification was carried out by primer 1 and primer 2, and the obtained PCR product was proved to be barley by sequence analysis and BLAST software analysis provided by NCBI. α-amylase gene sequence.

②Amplify the α-amylase gene (including signal peptide) of Bacillus Licheniformis by PCR

Primer 1: 5'GG TACGTA ATGAAACAACAAAAACGGCT3'

[Explanation: The 8 bases at the 5' end of the primer are enzyme-cleaved protection bases (2 bases) and DNA restriction endonuclease recognition sites (6 bases underlined)]

Primer 2:

TCTTTGAACATAAATTGAAACCGACCC3’[说明：5’的10个碱基是酶切保护碱基(2个碱基)和酶识别位点(8个碱基)，方框中的18个碱基是编码6个组氨酸的DNA序列。]5'AA -GCGGCCGC -CTA

TCTTTGAACATAAATTGAAACCGACCC3'[Description: 10 bases of 5' are restriction enzyme protection bases (2 bases) and enzyme recognition site (8 bases), and 18 bases in the box encode 6 histidines acidic DNA sequence. ]

Genomic DNA of Bacillus licheniformis was extracted, using the genomic DNA as a template, primer 1 and primer 2 were used for PCR amplification, and the PCR product was sequenced and analyzed by BLAST software provided by NCBI, which proved to be α-starch containing signal peptide of Bacillus licheniformis Enzyme gene sequence. [Description: Bacillus licheniformis genomic DNA extraction method: Add Bacillus licheniformis cells to 9mg/ml helicase solution (helicase is dissolved with 1mol/L sorbitol) and shake at 30°C for 30 minutes, then follow the conventional bacterial Genomic DNA extraction method extracts its genomic DNA. ]

③ Amplification of Aspergillus α-amylase gene by reverse transcription PCR

Primer 1: 5'gc gaattc gcaacgcctgcggactggcg3'[Explanation: The 8 bases at 5' are the enzyme protection bases (2 bases) and the enzyme recognition site (6 bases underlined)]

gccgtaacagatcttgctacctgccaac3’[说明：5’端的10个碱基是酶切保护碱基(2个碱基)和DNA限制性内切酶识别位点(有下划线的8个碱基)]Primer 2: 5'aa gcggccgc CTA

gccgtaacagatcttgctacctgccaac3'[Description: The 10 bases at the 5' end are enzyme-cleaved protection bases (2 bases) and DNA restriction endonuclease recognition sites (8 bases underlined)]

Use the fungal RNA extraction kit to extract the total RNA of Aspergillus, use the cDNA synthesis kit to synthesize its cDNA, and use primer 1 and primer 2 to carry out PCR amplification. The obtained PCR product is proved to be Aspergillus by sequence analysis and BLAST software analysis provided by NCBI. α-amylase gene sequence.

1.1.3 Construct the α-amylase gene expression framework containing barley, Bacillus licheniformis α-amylase gene expression framework and Aspergillus α-amylase gene expression framework respectively.

①Use PCR to amplify the promoter sequence of glyceraldehyde triphosphate dehydrogenase in Pichia pastoris.

The genomic DNA of Pichia pastoris was extracted, and the genomic DNA was used as a template, and the following primers (5'CCTACGTAGGATCCTTTTTTGTAGAAATGTC3', 5'GGGCATGCTGTGTTTTGATAGTTGTTCAA 3') were used for PCR amplification. The PCR product was sequenced and analyzed by BLAST software provided by NCBI. The promoter sequence of its glyceraldehyde triphosphate dehydrogenase gene [the 8 underlined bases at the 5' end and the 3' end of the following sequence are the enzyme cutting protection bases (2 bases) and the enzyme recognition site (6 bases), without underline is the glyceraldehyde triphosphate dehydrogenase promoter sequence]:

CCTACGTA GGATCCTTTTTTGTAGAAATGTCTTGGTGTCCTCGTCCAATCAGGTAGCCATCTCTGAAATATCTGGCTCCGTTGCAACTCCGAACGACCTGCTGGCAACGTAAAATTCTCCGGGGTAAAACTTAAATGTGGAGTAATGGAACCAGAAACGTCTCTTCCCTTCTCTCTCCTTCCACCGCCCGTTACCGTCCCTAGGAAATTTTACTCTGCTGAGAGCTTCTTCTACGGCCCCCTTGCAGCAATGCTCTTCCCAGCATTACGTTGCGGGTAAAACGGAGGTCGTGTACCCGACCTAGCAGCCCAGGGATGGAAAAGTCCCGGCCGTCGCTGGCAATAATAGCGGGCGGACGCATGTCATGAGATTATTGGAAACCACCAGAATCGAATATAAAAGGCGAACACCTTTCCCAATTTTGGTTTCTCCTGACCCAAAGACCTTAAATTTAATTTATTTGTCCCTATTTCAATCAATTGAACAACTATCAAAACACA GCATGCCC

[Description: Pichia pastoris genomic DNA extraction method: Add Pichia pastoris cells to 9mg/ml helicase solution (helicase is dissolved in 1mol/L sorbitol) and shake at 30°C for 30 minutes, then follow the conventional bacterial Genomic DNA extraction method extracts its genomic DNA. ]

②Synthesize the α-factor signal peptide by a professional DNA sequence synthesis company [the following sequences are underlined are enzyme protection bases (2 bases) and DNA restriction endonuclease recognition sites (6 bases), without underlines is the alpha factor signal peptide sequence]:

CCGCATGC ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCTTAC ACTAGTCC

③The transcription terminator sequence was synthesized by a professional DNA sequence synthesis company [the following sequences are underlined are the enzyme protection bases (2 bases) and DNA restriction endonuclease recognition sites (6 bases), without underlines is the transcription terminator sequence]:

GGACTAGT CCTTAGACATGACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCTGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGAAGTTCGTTTGTGCAAGCTT ATCGATCC

④: Synthesize the following G418 resistance gene sequence by nested PCR method [the underlined sequence in the following sequence is the restriction enzyme protection base (2 bases) and the DNA restriction endonuclease recognition site (6 bases), without Underlined is the G418 resistance gene sequence]

GGATCGAT GGTACCGG

⑤ PCR amplification of rDNA sequence from Pichia pastoris genome

PCR primers:

Primer 1: 5'CC GGTACC ggcttccaggacgtatcgcggatcgctgcgttcttcatc3'

Primer 2: 5'CC TTCGAA agccccgtggcacacgaccaatcttcccagccgaca 3'

Explanation: The 8 bases at the 5' end of the primer are restriction enzyme protection bases (2 bases) and enzyme recognition sites (6 bases underlined).

Genomic DNA of Pichia pastoris was extracted, using the genomic DNA as a template, primer 1 and primer 2 were used for PCR amplification, and the obtained PCR product was proved to be the rDNA sequence in the genome of Pichia pastoris by sequence analysis and BLAST software analysis provided by NCBI.

⑥Expression framework construction process:

A. Construction of the α-amylase gene expression framework of barley:

Through the action of DNA restriction endonuclease and T4 DNA ligase, the DNA sequence of the glyceraldehyde triphosphate dehydrogenase promoter of Pichia pastoris was recombined into the multiple cloning site of the pPD vector; the α factor signal peptide DNA sequence was recombined into pPD The multiple cloning site of the vector, and make it located downstream of the promoter sequence; recombine the α-amylase gene sequence of barley obtained by reverse transcription amplification into the multiple cloning site of the pPD vector, and make it located at the downstream of the signal peptide sequence Downstream: recombine the transcription terminator sequence into the multiple cloning site of the pPD vector and make it downstream of the gene sequence. The vector containing the barley α-amylase gene expression framework is called pPD1.

B. Construction of the α-amylase gene expression framework of Bacillus licheniformis:

Through the action of DNA restriction endonuclease and T4 DNA ligase, the DNA sequence of the glyceraldehyde triphosphate dehydrogenase promoter of Pichia pastoris was recombined into the multiple cloning site of the pPD vector; The α-amylase gene sequence containing the signal peptide is recombined in the multiple cloning site of the pPD vector, and it is located downstream of the promoter sequence; the transcription terminator sequence is recombined in the multiple cloning site of the pPD vector, and it is located in the gene sequence downstream. The vector containing the α-amylase gene expression framework of Bacillus licheniformis is called pPD2.

C. Construction of the α-amylase gene expression framework of Aspergillus:

Through the action of DNA restriction endonuclease and T4 DNA ligase, the DNA sequence of the glyceraldehyde triphosphate dehydrogenase promoter of Pichia pastoris was recombined into the multiple cloning site of the pPD vector; the α factor signal peptide DNA sequence was recombined into pPD The multiple cloning site of the vector, and make it located downstream of the promoter sequence; recombine the Aspergillus α-amylase gene sequence obtained by reverse transcription amplification into the multiple cloning site of the pPD vector, and make it located at the signal peptide sequence Downstream: recombine the transcription terminator sequence into the multiple cloning site of the pPD vector and make it downstream of the gene sequence. The vector containing the Aspergillus α-amylase gene expression framework is called pPD3

⑦ Complete the construction of Pichia pastoris expression vector

A. Three sets of expression frameworks were assembled in the same cloning vector.

Through the action of DNA restriction endonuclease and T4 DNA ligase, the expression frameworks of pPD2 and pPD3 vectors were excised respectively, and the two excised expression frameworks were recombined into the multiple cloning site of pPD1. This vector is called pPD123.

B. Through the action of DNA restriction enzyme and T4 DNA ligase, the rDNA sequence (DNA recombination sequence) and G418 gene of Pichia pastoris were recombined in the multiple cloning site of the pPD123 vector to form the gene expression of α-amylase containing barley Framework, Bacillus licheniformis α-amylase gene expression framework and Aspergillus α-amylase gene expression framework Pichia expression vector (accompanying drawing 1).

1.2 Construction of Pichia engineering bacteria containing barley α-amylase gene expression framework, Bacillus licheniformis α-amylase gene expression framework and Aspergillus α-amylase gene expression framework

The Pichia pastoris expression vector constructed above (see Figure 1) was transformed into Pichia pastoris by electroporation, and the electrotransformed Pichia pastoris cells were coated with a G418 resistance plate. Using recombinant (clones grown on G418 resistant plates) genomic DNA as a template, use specific primers for the α-amylase gene of barley, the α-amylase gene of Bacillus licheniformis and the α-amylase gene of Aspergillus Specific primers are used for PCR amplification, and the amplified PCR product conforming to the target molecular weight is subjected to DNA sequence determination to verify that the correct recombinant is obtained. The recombinants containing the above three α-amylase gene expression frameworks were inoculated in YPD [2% peptone, 1% yeast extract (yeast extract), 2% glucose] medium, and cultured on a shaker at 30°C for two days. The fermentation broth was centrifuged and the supernatant harvested. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was carried out using the fermentation broth, and the results showed that new protein bands appeared in accordance with the molecular weights of the enzyme proteins encoded by these three genes. Purify the target protein with the His6 fusion protein purification kit, and perform Western blot experiments on the purified three target proteins with the His-tag monoclonal antibody. The results show that the three target proteins can bind to the His-tag monoclonal antibody, proving that the α The amylase gene, the α-amylase gene of B. licheniformis and the α-amylase gene of Aspergillus can all be expressed in the framework containing the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene of Aspergillus The framework of Pichia pastoris is expressed and secreted extracellularly. Screening recombinants that highly express the three mixed α-amylases described above as Pichia pastoris containing the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene expression framework of Aspergillus Engineering bacteria.

1.3 Production of α-amylase encoded by the α-amylase gene of recombinant barley, α-amylase encoded by the α-amylase gene of Bacillus licheniformis and α-amylase encoded by the α-amylase gene of Aspergillus

Pichia pastoris engineering bacteria containing barley α-amylase gene expression framework, Bacillus licheniformis α-amylase gene expression framework and Aspergillus α-amylase gene expression framework, Pichia pastoris containing barley α-amylase gene expression framework Engineering bacteria, Pichia engineering bacteria containing the α-amylase gene expression framework of Bacillus licheniformis, Pichia engineering bacteria containing the α-amylase gene expression framework of Aspergillus, and Pichia pastoris without the α-amylase gene expression framework respectively Inoculate in YPD medium, and ferment on a shaker at 30°C for two days; inoculate the natural α-amylase-producing Bacillus licheniformis (the Bacillus licheniformis strain applied to the PCR amplification of α-amylase gene described in 1.1.2) in LB (1% tryptone, 1% sodium chloride, 0.5% yeast extract) culture medium, 37 ℃ of shaker fermentation two days; strains) were inoculated in a conventional fungal culture medium (potato juice 200g/L, glucose 20g/L), and fermented in a shaker at 30°C for 3 days. Fermentations for each of the strains described above were performed in triplicate. Collect the supernatant of the fermentation broth at 50°C, 70°C and 80°C to analyze the α-amylase in the supernatant of the fermentation broth in acidic (pH4 and pH5), neutral (pH7) and alkaline (pH8.5) environments active. The results showed that the fermentation broth supernatant (per liter) of Pichia engineering bacteria containing the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene expression framework of Aspergillus was above The average α-amylase activity at said temperature and pH are 50°C and pH 4 (1862109 units), 50°C and pH5 (2930226 units), 50°C and pH7 (3289090 units), 50°C and pH8.5 (1504093 units ), 70°C and pH4 (2418659 units), 70°C and pH5 (3761490 units), 70°C and pH7 (4518382 units), 70°C and pH8.5 (2151179 units), 80°C and pH4 (2432195 units), 80 ℃ and pH 5 (4346099 units), 80 ℃ and pH7 (3266072 units), 80 ℃ and pH8.5 (2729903 units); the fermentation broth supernatant of Pichia pastoris engineering bacteria containing barley α-amylase gene expression framework (per l) The average α-amylase activity at the most suitable temperature and pH (pH5 and 70°C) of barley α-amylase is 2,275,490 units; Fermentation of Pichia pastoris engineered bacteria containing the α-amylase gene expression framework of Bacillus licheniformis The average α-amylase activity of liquid supernatant (per liter) at the most suitable temperature and pH (pH8.5 and 80°C) of the α-amylase of Bacillus licheniformis is 1490156 units; The average α-amylase activity of the fermented liquid supernatant (per liter) of the α-amylase of Aspergillus at the most suitable temperature and pH (70°C and pH4) of Pichia pastoris engineering bacteria is 1,500,271 units; The average α-amylase activity of the fermentation broth supernatant (per liter) of Bacillus licheniformis at the optimum temperature and pH (pH8.5 and 80°C) of Bacillus licheniformis was 995486 units; The average α-amylase activity of clear (per liter) at the most suitable temperature and pH (pH4 and 70° C.) of the α-amylase of Aspergillus is 1,033,457 units; per liter) no alpha amylase activity was detected at the various temperatures and pHs described above. The results of enzyme activity analysis of the above fermentation expression products showed that: the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene expression framework of Aspergillus Pichia expressed α Amylase has good α-amylase activity in acidic, neutral and alkaline and different test temperatures, and its α-amylase activity is significantly higher (P<0.01) than that of Pichia pastoris engineering containing a single α-amylase gene Alpha-amylase activity of expression products of bacteria or natural strains producing alpha-amylase.

illustrate:

a. The difference between the method of constructing the Pichia engineering bacteria containing a single alpha amylase gene expression framework and the construction method of the Pichia engineering bacteria containing three alpha amylase gene expression frameworks is the construction of the expression vector; The difference between the construction method of the Pichia expression vector of a single α-amylase gene expression framework and the method of constructing the Pichia expression vector containing three α-amylase gene expression frameworks is that the former only contains one α-amylase gene expression framework , while the latter contains three α-amylase gene expression frameworks.

c. Determination method of α-amylase activity: use soluble starch plate assay method. Prepare an agar plate containing 2% soluble starch, place the Oxford cup on the plate, take 50 μl of α-amylase standard solution of different concentrations and the fermentation broth of different bacterial strains and add it to different Oxford cups, and in each cup Add 50 μl of phosphate buffered saline. Place at 50°C for 12 hours, then stain with iodine-potassium iodide (I ₂ -KI) solution, and calculate the diameter of the transparent circle. The above experiments were repeated three times, and the results were analyzed by taking the average value. Draw a standard curve according to the transparent circle of the α-amylase standard of different concentrations; calculate the α-amylase activity of the fermentation liquid of each test strain according to the transparent circle standard curve of the transparent circle diameter of the test sample and the α-amylase standard of different concentrations.

Example 2:

2.1 Construction of Saccharomyces cerevisiae expression vectors containing the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene expression framework of Aspergillus

2.1.1 Construction of cloning vector

A professional DNA sequence synthesis company synthesizes two base complementary double strands containing ampicillin (AMP) gene sequence, polyclonal adapter and E. coli replication origin, and forms cohesive ends at both ends of each DNA strand sequence. It is circularized by the action of DNA ligase to form a DNA cloning vector. The cloning vector was named pSD.

2.1.2 Acquiring genes

①Amplification of barley α-amylase gene by reverse transcription PCR

Primer 1: 5'GGC GAATTC caagtcctctttcaggggtt3'3'[Description: The 8 bases at 5' are enzyme-cleaved protection bases (2 bases) and enzyme recognition site (6 bases underlined)]

gctccgttgtagtgttgccgcggcaccgt3’Primer 2: 5'CA GCGGCCGCGC CTA

gctccgttgtagtgttgccgcggcaccgt3'

[Explanation: The 10 bases of the 5' are enzyme-cleaved protection bases (2 bases) and enzyme recognition sites (8 bases), and the 18 bases in the box are encoding 6 histidines DNA sequence. ]

The total RNA of barley was extracted by plant RNA extraction kit, its cDNA was synthesized by cDNA synthesis kit, PCR amplification was carried out by primer 1 and primer 2, and the obtained PCR product was proved to be barley by sequence analysis and BLAST software analysis provided by NCBI. α-amylase gene sequence.

②Amplify the α-amylase gene (including signal peptide) of Bacillus Licheniformis by PCR

Primer 1: 5'GG TACGTA ATGAAACAACAAAAACGGCT3'

[Explanation: The 8 bases at the 5' end of the primer are enzyme-cleaved protection bases (2 bases) and DNA restriction endonuclease recognition sites (6 bases underlined)]

Primer 2:

TCTTTGAACATAAATTGAAACCGACCC3’[说明：5’的10个碱基是酶切保护碱基(2个碱基)和酶识别位点(8个碱基)，方框中的18个碱基是编码6个组氨酸的DNA序列。]5'AA -GCGGCCGC -CTA

TCTTTGAACATAAATTGAAACCGACCC3'[Description: 10 bases of 5' are restriction enzyme protection bases (2 bases) and enzyme recognition site (8 bases), and 18 bases in the box encode 6 histidines acidic DNA sequence. ]

Genomic DNA of Bacillus licheniformis was extracted, using the genomic DNA as a template, primer 1 and primer 2 were used for PCR amplification, and the PCR product was sequenced and analyzed by BLAST software provided by NCBI, which proved to be α-starch containing signal peptide of Bacillus licheniformis Enzyme gene sequence. [Description: Bacillus licheniformis genomic DNA extraction method: Add Bacillus licheniformis cells to 9mg/ml helicase solution (helicase is dissolved with 1mol/L sorbitol) and shake at 30°C for 30 minutes, then follow the conventional bacterial Genomic DNA extraction method extracts its genomic DNA. ]

③ Amplification of Aspergillus α-amylase gene by reverse transcription PCR

Primer 1: 5'gc gaattc gcaacgcctgcggactggcg3'[Explanation: The 8 bases at 5' are the enzyme protection bases (2 bases) and the enzyme recognition site (6 bases underlined)]

gccgtaacagatcttgctacctgccaac3’[说明：5’端的10个碱基是酶切保护碱基(2个碱基)和DNA限制性内切酶识别位点(有下划线的8个碱基)]Primer 2: 5'aa gcggccgc CTA

gccgtaacagatcttgctacctgccaac3'[Description: The 10 bases at the 5' end are enzyme-cleaved protection bases (2 bases) and DNA restriction endonuclease recognition sites (8 bases underlined)]

Use the fungal RNA extraction kit to extract the total RNA of Aspergillus, use the cDNA synthesis kit to synthesize its cDNA, and use primer 1 and primer 2 to carry out PCR amplification. The obtained PCR product is proved to be Aspergillus by sequence analysis and BLAST software analysis provided by NCBI. α-amylase gene sequence.

2.1.3 Construct the α-amylase gene expression frameworks containing barley, Bacillus licheniformis α-amylase gene expression frameworks and Aspergillus α-amylase gene expression frameworks respectively.

①The promoter sequence of glyceraldehyde triphosphate dehydrogenase in Saccharomyces cerevisiae was amplified by PCR.

Primer 1:

5'CC TACGTA TCGAGTTTTATCATTATCAAT 3'

[Explanation: The 8 bases at the 5' end of the primer are enzyme-cleaved protection bases (2 bases) and DNA restriction endonuclease recognition sites (6 bases underlined)]

Primer 2:

5' GG GCATGC TCGAAACTAAGTTCTTGGTG 3'

[Explanation: The 8 bases at the 5' end of the primer are enzyme-cleaved protection bases (2 bases) and DNA restriction endonuclease recognition sites (6 bases underlined)]

Extract the genomic DNA of Saccharomyces cerevisiae, use the genomic DNA as a template, and use primer 1 and primer 2 to carry out PCR amplification. The PCR product is sequenced and analyzed with BLAST software provided by NCBI, and it is proved that it is the promoter of the glyceraldehyde triphosphate dehydrogenase gene sequence.

[Description: Saccharomyces cerevisiae genomic DNA extraction method: Add Saccharomyces cerevisiae cells to 9mg/ml helicase solution (helicase is dissolved in 1mol/L sorbitol) and shake at 30°C for 30 minutes, then follow the conventional bacterial genomic DNA extraction Methods to extract its genomic DNA. ]

②Synthesize the α-factor signal peptide by a professional DNA sequence synthesis company [the following sequences are underlined are enzyme protection bases (2 bases) and DNA restriction endonuclease recognition sites (6 bases), without underlines is the alpha factor signal peptide sequence]:

CCGCATGC ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCTTAC ACTAGTCC

③The transcription terminator sequence was synthesized by a professional DNA sequence synthesis company [the following sequences are underlined are the enzyme protection bases (2 bases) and DNA restriction endonuclease recognition sites (6 bases), without underlines is the transcription terminator sequence]:

GGACTAGT CCTTAGACATGACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCTGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGAAGTTCGTTTGTGCAAGCTT ATCGATCC

④: Synthesize the following G418 resistance gene sequence by nested PCR method [the underlined sequence in the following sequence is the restriction enzyme protection base (2 bases) and the DNA restriction endonuclease recognition site (6 bases), without Underlined is the G418 resistance gene sequence]

GGATCGAT GGTACCGG

⑤PCR amplification of rDNA sequence from Saccharomyces cerevisiae genome

Primer 1:

5'CC GGTACC TGAACTAACACCTTTTGTGG3'

Primer 2:

5'CC TTCGAA GCTAATACATGCTTAAAATC3'

[Explanation: The 8 bases at the 5' end of the primer are enzyme-cleaved protection bases (2 bases) and DNA restriction endonuclease recognition sites (6 bases underlined)]

Genomic DNA of Saccharomyces cerevisiae was extracted, using the genomic DNA as a template, primer 1 and primer 2 were used for PCR amplification, and the obtained PCR product was proved to be the rDNA sequence in the Saccharomyces cerevisiae genome by sequence analysis and BLAST software analysis provided by NCBI.

⑥Expression framework construction process:

A. Construction of barley-containing α-amylase gene expression framework:

Through the action of DNA restriction endonuclease and T4 DNA ligase, recombine the DNA sequence of glyceraldehyde triphosphate dehydrogenase promoter of Saccharomyces cerevisiae into the multiple cloning site of pSD vector; recombine the α factor signal peptide DNA sequence into pSD vector The multiple cloning site of the pSD vector, and make it located downstream of the promoter sequence; recombine the barley α-amylase gene sequence obtained by reverse transcription amplification into the multiple cloning site of the pSD vector, and make it located downstream of the signal peptide sequence ; Recombine the transcription terminator sequence into the multiple cloning site of the pSD vector, and make it located downstream of the gene sequence. The vector containing the barley α-amylase gene expression framework is called pSD1.

B. Construction of α-amylase gene expression framework containing Bacillus licheniformis:

Through the action of DNA restriction endonuclease and T4 DNA ligase, the glyceraldehyde triphosphate dehydrogenase promoter DNA sequence of Saccharomyces cerevisiae was recombined into the multiple cloning site of pSD vector; the inclusion of Bacillus licheniformis obtained by PCR amplification The α-amylase gene sequence of the signal peptide is recombined in the multiple cloning site of the pSD vector, and it is located downstream of the promoter sequence; the transcription terminator sequence is recombined in the multiple cloning site of the pSD vector, and it is located downstream of the gene sequence . The vector containing the expression framework of the α-amylase gene of Bacillus licheniformis is called pSD2.

C. Construction of Aspergillus-containing α-amylase gene expression framework:

Through the action of DNA restriction endonuclease and T4 DNA ligase, recombine the DNA sequence of the glyceraldehyde triphosphate dehydrogenase promoter of Saccharomyces cerevisiae into the multiple cloning site of the pSD vector; recombine the α factor signal peptide DNA sequence into the pSD vector Multiple cloning site, and make it be positioned at the downstream of promoter sequence; Recombine the α-amylase gene sequence of Aspergillus obtained by reverse transcription amplification into the multiple cloning site of pSD vector, and make it be positioned at the downstream of signal peptide sequence; The transcription terminator sequence was recombined into the multiple cloning site of the pSD vector and positioned downstream of the gene sequence. The vector containing the α-amylase gene expression framework of Aspergillus is called pSD3⑦Complete the construction of the expression vector of Saccharomyces cerevisiae

A. Three sets of expression frameworks were assembled in the same cloning vector.

Through the action of DNA restriction endonuclease and T4 DNA ligase, the expression frameworks of pSD2 and pSD3 vectors were respectively excised, and the two excised expression frameworks were recombined into the multiple cloning site of pSD1. This vector is called pSD123.

B. Recombine the rDNA sequence (DNA recombination sequence) and G418 gene of Saccharomyces cerevisiae into the multiple cloning site of pSD123 vector through the action of DNA restriction endonuclease and T4 DNA ligase to form the gene expression of α-amylase containing barley Framework, the α-amylase gene expression framework of Bacillus licheniformis and the Saccharomyces cerevisiae expression vector of the α-amylase gene expression framework of Aspergillus (accompanying drawing 2).

2.2 Construction of Saccharomyces cerevisiae engineering bacteria containing barley α-amylase gene expression framework, Bacillus licheniformis α-amylase gene expression framework and Aspergillus α-amylase gene expression framework

The Saccharomyces cerevisiae expression vector constructed above (see Figure 2) was transformed into Saccharomyces cerevisiae by electroporation, and the electrotransformed Saccharomyces cerevisiae cells were coated with G418 resistance plate. Using recombinant (clones grown on G418 resistant plates) genomic DNA as a template, use specific primers for the α-amylase gene of barley, the α-amylase gene of Bacillus licheniformis and the α-amylase gene of Aspergillus Specific primers are used for PCR amplification, and the amplified PCR product conforming to the target molecular weight is subjected to DNA sequence determination to verify that the correct recombinant is obtained. The recombinants containing the above three α-amylase gene expression frameworks were inoculated in YPD [2% peptone, 1% yeast extract (yeast extract), 2% glucose] medium, and cultured on a shaker at 30°C for two days. The fermentation broth was centrifuged and the supernatant harvested. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was carried out using the fermentation broth, and the results showed that new protein bands appeared in accordance with the molecular weights of the enzyme proteins encoded by these three genes. Purify the target protein with the His6 fusion protein purification kit, and perform western blot experiments on the purified three target proteins with the His tag monoclonal antibody. The results show that the three target proteins can bind to the His tag monoclonal antibody, proving the The α-amylase gene, the α-amylase gene of Bacillus licheniformis and the α-amylase gene of Aspergillus can be expressed in the gene expression framework containing barley α-amylase, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene of Aspergillus Expression framework in Saccharomyces cerevisiae and secreted extracellularly. Saccharomyces cerevisiae engineering for screening high-expression recombinants of the above three mixed α-amylases as gene expression frameworks for barley α-amylases, Bacillus licheniformis α-amylases and Aspergillus α-amylases bacteria.

2.3 Production of α-amylase encoded by the α-amylase gene of recombinant barley, α-amylase encoded by the α-amylase gene of Bacillus licheniformis and α-amylase encoded by the α-amylase gene of Aspergillus

Saccharomyces cerevisiae engineering bacteria containing barley α-amylase gene expression framework, Bacillus licheniformis α-amylase gene expression framework and Aspergillus cerevisiae gene expression framework, Saccharomyces cerevisiae engineering bacteria containing barley α-amylase gene expression framework , Saccharomyces cerevisiae engineering bacteria containing the α-amylase gene expression framework of Bacillus licheniformis, Saccharomyces cerevisiae engineering bacteria containing the α-amylase gene expression framework of Aspergillus, and Saccharomyces cerevisiae without the α-amylase gene expression framework were inoculated in YPD medium In 30 DEG C shaker fermentation for two days; the natural α-amylase-producing Bacillus licheniformis (applied to the Bacillus licheniformis strain of the PCR amplified α-amylase gene described in 1.1.2) was inoculated in LB (1% pancreatic Peptone, 1% sodium chloride, 0.5% yeast extract) culture medium, 37 ℃ shaker fermentation for two days; Aspergillus (applied to the Aspergillus strain of PCR amplification α-amylase gene described in 2.1.2) was inoculated in routine The fungal medium (potato juice 200g/L, glucose 20g/L) was fermented in a shaker at 30°C for 3 days. Fermentations for each of the strains described above were performed in triplicate. Collect the supernatant of the fermentation broth at 50°C, 70°C and 80°C to analyze the α-amylase in the supernatant of the fermentation broth in acidic (pH4 and pH5), neutral (pH7) and alkaline (pH8.5) environments active. The result showed that the fermented liquid supernatant (per liter) of the Saccharomyces cerevisiae engineered bacteria containing the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene expression framework of Aspergillus The average alpha amylase activity at the stated temperature and pH are 50°C and pH 4 (650760 units), 50°C and pH5 (976407 units), 50°C and pH7 (1096364 units), 50°C and pH8.5 (495433 units) , 70°C and pH4 (806217 units), 70°C and pH5 (1253830 units), 70°C and pH7 (1502794 units), 70°C and pH8.5 (717058 units), 80°C and pH4 (810731 units), 80°C and pH5 (1448699 units), 80°C and pH7 (1752024 units), 80°C and pH8.5 (906641 units); fermentation supernatant of engineered Saccharomyces cerevisiae containing barley α-amylase gene expression framework (per liter) The average α-amylase activity at the most suitable temperature and pH (pH5 and 70°C) of barley α-amylase is 758495 units; the fermentation supernatant of Saccharomyces cerevisiae engineered bacteria containing the α-amylase gene expression framework of Bacillus licheniformis (per liter) the average alpha amylase activity at the most suitable temperature and pH (pH8.5 and 80°C) of the alpha amylase of Bacillus licheniformis is 495685 units ± 62 units; The average α-amylase activity of the fermented liquid supernatant (per liter) of the α-amylase of Aspergillus at the most suitable temperature and pH (pH4 and 70°C) of Saccharomyces cerevisiae engineering bacteria is 499690 units; natural lichen producing α-amylase The average α-amylase activity of the fermentation broth supernatant (per liter) of Bacillus licheniformis at the optimum temperature and pH (pH8.5 and 80°C) of Bacillus licheniformis was 78065 units; the fermentation broth supernatant of Aspergillus (per liter) the average α-amylase activity at the most suitable temperature and pH (pH4 and 70° C.) of the α-amylase of Aspergillus is 87089 units; ) No alpha-amylase activity was detected at the various temperatures and pHs described above. The results of the enzyme activity analysis of the above fermentation expression products show that: the expression products of Saccharomyces cerevisiae engineering bacteria containing the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene expression framework of Aspergillus in acid , neutral and alkaline, and different test temperatures all show good α-amylase activity, and it is significantly higher (P<0.01) than the Saccharomyces cerevisiae engineering bacteria containing a single α-amylase gene or the natural strain producing α-amylase α-amylase activity of the expressed product.

illustrate:

a. The difference between the construction method of the Saccharomyces cerevisiae engineering bacteria containing a single α-amylase gene expression framework and the construction method of the Saccharomyces cerevisiae engineering bacteria containing three α-amylase gene expression frameworks lies in the construction of the expression vector; The difference between the construction method of the Saccharomyces cerevisiae expression vector of the α-amylase gene expression framework and the method for the construction of the Saccharomyces cerevisiae expression vector containing three α-amylase gene expression frameworks is that the former only contains a kind of α-amylase gene expression framework, while the latter contains Three α-amylase gene expression frameworks.

c. Determination method of α-amylase activity: use soluble starch plate assay method. Prepare an agar plate containing 2% soluble starch, place the Oxford cup on the plate, take 50 μl of α-amylase standard solution of different concentrations and the fermentation broth of different bacterial strains and add it to different Oxford cups, and in each cup Add 50 μl of phosphate buffered saline. Place at 50°C for 12 hours, then stain with iodine-potassium iodide (I ₂ -KI) solution, and calculate the diameter of the transparent circle. The above experiments were repeated three times, and the results were analyzed by taking the average value. Draw a standard curve according to the transparent circle of the α-amylase standard of different concentrations; calculate the α-amylase activity of the fermentation liquid of each test strain according to the transparent circle standard curve of the transparent circle diameter of the test sample and the α-amylase standard of different concentrations.

Example 3:

3.1 Construction of the Bacillus subtilis expression vector containing the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene expression framework of Aspergillus

3.1.1 Construction of cloning vector

A professional DNA sequence synthesis company synthesizes two base complementary double strands containing ampicillin (AMP) gene sequence, polyclonal adapter and E. coli replication origin, and forms cohesive ends at both ends of each DNA strand sequence. It is circularized by the action of DNA ligase to form a DNA cloning vector. The cloning vector was named pBD.

3.1.2 Acquiring genes

①Amplification of barley α-amylase gene by reverse transcription PCR

Primer 1: 5'GGC GAATTC caagtcctctttcaggggtt3'3'[Description: The 8 bases at 5' are enzyme-cleaved protection bases (2 bases) and enzyme recognition site (6 bases underlined)]

gctccgttgtagtgttgccgcggcaccgt3’Primer 2: 5'CA GCGGCCGCGC CTA

gctccgttgtagtgttgccgcggcaccgt3'

[Explanation: The 10 bases of the 5' are enzyme-cleaved protection bases (2 bases) and enzyme recognition sites (8 bases), and the 18 bases in the box are encoding 6 histidines DNA sequence. ]

The total RNA of barley was extracted by plant RNA extraction kit, its cDNA was synthesized by cDNA synthesis kit, PCR amplification was carried out by primer 1 and primer 2, and the obtained PCR product was proved to be barley by sequence analysis and BLAST software analysis provided by NCBI. α-amylase gene sequence.

②Amplify the α-amylase gene (including signal peptide) of Bacillus Licheniformis by PCR

Primer 1: 5'GG TACGTA ATGAAACAACAAAAACGGCT3'

[Explanation: The 8 bases at the 5' end of the primer are enzyme-cleaved protection bases (2 bases) and DNA restriction endonuclease recognition sites (6 bases underlined)]

Primer 2:

TCTTTGAACATAAATTGAAACCGACCC3’[说明：5’的10个碱基是酶切保护碱基(2个碱基)和酶识别位点(8个碱基)，方框中的18个碱基是编码6个组氨酸的DNA序列。]5'AA -GCGGCCGC -CTA

TCTTTGAACATAAATTGAAACCGACCC3'[Description: 10 bases of 5' are restriction enzyme protection bases (2 bases) and enzyme recognition site (8 bases), and 18 bases in the box encode 6 histidines acidic DNA sequence. ]

Genomic DNA of Bacillus licheniformis was extracted, using the genomic DNA as a template, primer 1 and primer 2 were used for PCR amplification, and the PCR product was sequenced and analyzed by BLAST software provided by NCBI, which proved to be α-starch containing signal peptide of Bacillus licheniformis Enzyme gene sequence. [Description: Bacillus licheniformis genomic DNA extraction method: Add Bacillus licheniformis cells to 9mg/ml helicase solution (helicase is dissolved with 1mol/L sorbitol) and shake at 30°C for 30 minutes, then follow the conventional bacterial Genomic DNA extraction method extracts its genomic DNA. ]

③ Amplification of Aspergillus α-amylase gene by reverse transcription PCR

Primer 1: 5'gc gaattc gcaacgcctgcggactggcg3'[Explanation: The 8 bases at 5' are the enzyme protection bases (2 bases) and the enzyme recognition site (6 bases underlined)]

gccgtaacagatcttgctacctgccaac3’[说明：5’端的10个碱基是酶切保护碱基(2个碱基)和DNA限制性内切酶识别位点(有下划线的8个碱基)]Primer 2: 5'aa gcggccgc CTA

gccgtaacagatcttgctacctgccaac3'[Description: The 10 bases at the 5' end are enzyme-cleaved protection bases (2 bases) and DNA restriction endonuclease recognition sites (8 bases underlined)]

Use the fungal RNA extraction kit to extract the total RNA of Aspergillus, use the cDNA synthesis kit to synthesize its cDNA, and use primer 1 and primer 2 to carry out PCR amplification. The obtained PCR product is proved to be Aspergillus by sequence analysis and BLAST software analysis provided by NCBI. α-amylase gene sequence.

3.1.3 Construct Bacillus subtilis expression vectors containing barley α-amylase gene expression framework, Bacillus licheniformis α-amylase gene expression framework and Aspergillus α-amylase gene expression framework respectively.

①Use PCR to amplify the glyceraldehyde triphosphate dehydrogenase promoter sequence primer 1 of Bacillus megaterium:

5'CCTACGTAGATCCATTATCGGTGAACCA 3'

Primer 2:

5' GG GCATGC GGGTATTTCCTCCTTGAATGT 3'

[Explanation: The 8 bases at the 5' end of the primer are enzyme-cleaved protection bases (2 bases) and DNA restriction endonuclease recognition sites (6 bases underlined)]

Extract the genomic DNA of Bacillus megaterium [Bacillus subtilis genomic DNA extraction method: add Bacillus subtilis cells to 9mg/ml helicase solution (helicase is dissolved with 1mol/L sorbitol) and shake at 30°C for 30 minutes , and then extract its genomic DNA according to the conventional bacterial genomic DNA extraction method. ], with the genomic DNA of Bacillus megaterium as a template, primer 1 and primer 2 were used to carry out PCR amplification, and the PCR product was sequenced and analyzed with BLAST software provided by NCBI to prove that it was the gene of Bacillus megaterium glyceraldehyde triphosphate dehydrogenase Promoter sequence [the underlined sequence is the enzyme protection base (2 bases) and the DNA restriction endonuclease recognition site (6 bases), and the ununderlined one is the glyceraldehyde triphosphate of Bacillus megaterium Dehydrogenase promoter sequence]:

CCTACGTA GATCCATTATCGGTGAACCATCTATTAAAGACATGCTTCATTTAATTAAGTCCGCTGGTATGGTTGTTCACGGAATAGGAGACGCTATGACAATGGCAGAACGCCGTAAAACACCACAAGCAGACTTAGAAAAAGTGAAAAATGGACATGCTGTAGGTGAGGCATTTGGATACTATTTTAATCATCAAGGCGAAGTTGTTCATAAAGTTAAAACAGTTGGCATACAACTCGATGATTTAAAGAACAATAAATGTGTTATTGCTGTTGCAGGAGGTTCATCAAAAGCAAAGGCAATTAAAGCGTTTATGCAACAAGCGCATGATTCGATTCTCATTACAGATGAAGGCGCCGCAAAAGAGTTAGTAAGGGATTTTAATTAATCCCTCATATAAAAAATACTTTTTACATTCAAGGAGGAAATACCC GCATGCCC

②Synthesize the α-factor signal peptide by a professional DNA sequence synthesis company [the following sequences are underlined are enzyme protection bases (2 bases) and DNA restriction endonuclease recognition sites (6 bases), without underlines is the alpha factor signal peptide sequence]:

CCGCATGC ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCTTAC ACTAGTCC

③The transcription terminator sequence was synthesized by a professional DNA sequence synthesis company [the following sequences are underlined are the enzyme protection bases (2 bases) and DNA restriction endonuclease recognition sites (6 bases), without underlines is the transcription terminator sequence]:

GGACTAGT CCTTAGACATGACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCTGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGAAGTTCGTTTGTGCAAGCTT ATCGATCC

④: Synthesize the following G418 resistance gene sequence by nested PCR method [the underlined sequence in the following sequence is the restriction enzyme protection base (2 bases) and the DNA restriction endonuclease recognition site (6 bases), without Underlined is the G418 resistance gene sequence]

GGATCGAT GGTACCGG

⑤Amplification of rDNA sequence from Bacillus subtilis genome by PCR

Primer 1.

5'CC GGTACC gacgaacgctggcggcgtgc3'

Primer 2.

5'CC TTCGAA gcaccttccgatacggctacct3'

[Explanation: The 8 bases at the 5' end of the primer are enzyme-cleaved protection bases (2 bases) and DNA restriction endonuclease recognition sites (6 bases underlined)].

Extract the Bacillus subtilis genomic DNA (the genomic DNA extraction method is the same as the above-mentioned Bacillus subtilis genomic DNA extraction method), use the genomic DNA as a template and apply primer 1 and primer 2 to carry out PCR amplification, and the PCR product obtained is analyzed by sequence And analysis with BLAST software provided by NCBI proves that it is the rDNA sequence in the Bacillus subtilis genome.

⑥Expression framework construction process:

A. Construction of barley-containing α-amylase gene expression framework:

Through the action of DNA restriction endonuclease and T4 DNA ligase, the DNA sequence of glyceraldehyde triphosphate dehydrogenase promoter of Bacillus megaterium was recombined in the multiple cloning site of pBD vector; the α factor signal peptide DNA sequence was recombined in The multiple cloning site of the pBD vector, and make it located downstream of the promoter sequence; recombine the barley α-amylase gene sequence obtained by reverse transcription amplification into the multiple cloning site of the pBD vector, and make it located at the signal peptide sequence Downstream of the gene sequence; the transcription terminator sequence was recombined into the multiple cloning site of the pBD vector and positioned downstream of the gene sequence. The vector containing the barley α-amylase gene expression framework is called pBD1.

B. Construction of α-amylase gene expression framework containing Bacillus licheniformis:

Through the action of DNA restriction endonuclease and T4 DNA ligase, the DNA sequence of the glyceraldehyde triphosphate dehydrogenase promoter of Bacillus megaterium was recombined into the multiple cloning site of the pBD vector; the Bacillus licheniformis obtained by PCR amplification The α-amylase gene sequence containing the signal peptide is recombined into the multiple cloning site of the pBD vector, and it is located downstream of the promoter sequence; the transcription terminator sequence is recombined into the multiple cloning site of the pBD vector, and it is located in the gene sequence downstream. The vector containing the expression framework of the α-amylase gene of Bacillus licheniformis is called pBD2.

C. Construction of Aspergillus-containing α-amylase gene expression framework:

Through the action of DNA restriction endonuclease and T4 DNA ligase, the DNA sequence of glyceraldehyde triphosphate dehydrogenase promoter of Bacillus megaterium was recombined in the multiple cloning site of pBD vector; the α factor signal peptide DNA sequence was recombined in The multiple cloning site of the pBD vector, and make it located downstream of the promoter sequence; recombine the Aspergillus α-amylase gene sequence obtained by reverse transcription amplification into the multiple cloning site of the pBD vector, and make it located at the signal peptide sequence Downstream of the gene sequence; the transcription terminator sequence was recombined into the multiple cloning site of the pBD vector and positioned downstream of the gene sequence. The vector containing Aspergillus α-amylase gene expression framework is called pBD3

⑦Complete the construction of Bacillus subtilis expression vector

A. Three sets of expression frameworks were assembled in the same cloning vector.

Through the action of DNA restriction endonuclease and T4 DNA ligase, the expression frameworks of pBD2 and pBD3 vectors were respectively excised, and the two excised expression frameworks were recombined into the multiple cloning site of pPD1. This vector is called pBD123.

B. Through the action of DNA restriction endonuclease and T4 DNA ligase, the rDNA sequence (DNA recombination sequence) and G418 gene of Bacillus subtilis were recombined in the multiple cloning site of pPD123 vector to form the α-amylase gene containing barley Expression framework, α-amylase gene expression framework of Bacillus licheniformis and Bacillus subtilis expression vector of α-amylase gene expression framework of Aspergillus (accompanying drawing 3).

3.2 Construction of Bacillus subtilis engineering bacteria containing the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene expression framework of Aspergillus

The Bacillus subtilis expression vector constructed above (see Figure 3) was transformed into Bacillus subtilis by electroporation, and the electrotransformed Bacillus subtilis cells were coated with G418 resistance plates. Using recombinant (clones grown on G418 resistant plates) genomic DNA as a template, use specific primers for the α-amylase gene of barley, the α-amylase gene of Bacillus licheniformis and the α-amylase gene of Aspergillus Specific primers are used for PCR amplification, and the amplified PCR product conforming to the target molecular weight is subjected to DNA sequence determination to verify that the correct recombinant is obtained. The recombinants containing the above three α-amylase gene expression frameworks were inoculated in YPD [2% peptone, 1% yeast extract (yeast extract), 2% glucose] medium, and cultured on a shaker at 30°C for two days. The fermentation broth was centrifuged and the supernatant harvested. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was carried out using the fermentation broth, and the results showed that new protein bands appeared in accordance with the molecular weights of the enzyme proteins encoded by these three genes. The target protein was purified with the His6 fusion protein purification kit, and the purified three target proteins were subjected to Western blot experiments with the His tag monoclonal antibody. The results showed that the three target proteins could bind to the His tag monoclonal antibody, proving that barley The α-amylase gene, the α-amylase gene of Bacillus licheniformis and the α-amylase gene of Aspergillus can be expressed in the gene expression framework containing barley α-amylase, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene of Aspergillus The Bacillus subtilis engineering strain expressing the framework is expressed and secreted to the extracellular space. Screening of recombinants that highly express the above-mentioned three mixed α-amylases as Bacillus subtilis containing the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene expression framework of Aspergillus Engineering bacteria.

3.3 Production of α-amylase encoded by the α-amylase gene of recombinant barley, α-amylase encoded by the α-amylase gene of Bacillus licheniformis and α-amylase encoded by the α-amylase gene of Aspergillus

The Bacillus subtilis engineering bacteria containing the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene expression framework of Aspergillus, and the Bacillus subtilis containing the α-amylase gene expression framework of barley Bacillus engineering bacteria, Bacillus subtilis engineering bacteria containing the α-amylase gene expression framework of Bacillus licheniformis, Bacillus subtilis engineering bacteria containing the α-amylase gene expression framework of Aspergillus, Bacillus subtilis without the α-amylase gene and natural The Bacillus licheniformis producing α-amylase (applied to the Bacillus licheniformis strain of the PCR amplification α-amylase gene described in 1.1.2) was inoculated in LB medium (1% tryptone, 1% sodium chloride, 0.5 % yeast extract), 30 DEG C of shaker fermentation for two days; Aspergillus (applied to the Aspergillus strain of the PCR amplification α-amylase gene described in 1.1.2) was inoculated in conventional fungal culture medium (potato juice 200g/L , glucose 20g/L), and fermented in a shaker at 30°C for 3 days. Fermentations for each of the strains described above were performed in triplicate. Collect the supernatant of the fermentation broth at 50°C, 70°C and 80°C to analyze the α-amylase in the supernatant of the fermentation broth in acidic (pH4 and pH5), neutral (pH7) and alkaline (pH8.5) environments active. The result showed that the fermented liquid supernatant (per liter) of the Bacillus subtilis engineering bacteria containing the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene expression framework of Aspergillus was above The average α-amylase activity at said temperature and pH were 50°C and pH 4 (175358 units), 50°C and pH5 (285105 units), 50°C and pH7 (318814 units), 50°C and pH8.5 (142762 units ), 70°C and pH4 (233669 units), 70°C and pH5 (367059 units), 70°C and pH7 (445030 units), 70°C and pH8.5 (210096 units), 80°C and pH4 (239046 units), 80 ℃ and pH5 (426087 units), 80 ℃ and pH7 (318690 units), 80 ℃ and pH8.5 (260819 units); the supernatant of the fermentation broth of Bacillus subtilis engineering bacteria containing the α-amylase gene expression framework of barley (per l) The average α-amylase activity at the most suitable temperature and pH (pH5 and 70°C) of barley α-amylase is 218095 units; fermentation of Bacillus subtilis engineering bacteria containing the α-amylase gene expression framework of Bacillus licheniformis The average α-amylase activity of liquid supernatant (per liter) at the most suitable temperature and pH (pH8.5 and 80°C) of α-amylase of Bacillus licheniformis is 138990 units; The most suitable temperature of the alpha amylase of aspergillus and the average alpha amylase activity of pH (70 ℃ and pH4) of the fermented liquid supernatant (per liter) of Bacillus subtilis engineered bacterium is 103027 units; The average α-amylase activity of the fermentation broth supernatant (per liter) of Bacillus licheniformis at the most suitable temperature and pH (pH8.5 and 80°C) of Bacillus licheniformis was 82917 units; Clear (per liter) the most suitable temperature of the α-amylase of Aspergillus and the average α-amylase activity of pH (pH4 and 70 ℃) is 7834 units; The fermentation broth supernatant ( per liter) no alpha amylase activity was detected at the various temperatures and pHs described above. The results of the enzyme activity analysis of the above fermentation expression products showed that: the α-amylase gene expression framework of barley, the α-amylase gene expression framework of Bacillus licheniformis and the α-amylase gene expression framework of Aspergillus subtilis engineering bacteria expressed α Amylase has good α-amylase activity in acidic, neutral and alkaline and different test temperatures, and its α-amylase activity is significantly higher (P<0.01) containing a single α-amylase gene engineering bacteria or production Alpha-amylase activity of expression products of native strains of alpha-amylase.

illustrate:

a. The difference between the construction method of the Bacillus subtilis engineering bacteria containing a single α-amylase gene expression framework and the construction method of the Bacillus subtilis engineering bacteria containing three α-amylase gene expression frameworks is the construction of the expression vector; The difference between the construction method of the Bacillus subtilis expression vector of a single α-amylase gene expression framework and the method of constructing the Bacillus subtilis expression vector containing three α-amylase gene expression frameworks is that the former only contains an α-amylase gene expression framework , while the latter contains three α-amylase gene expression frameworks.

c. Determination method of α-amylase activity: use soluble starch plate assay method. Prepare an agar plate containing 2% soluble starch, place the Oxford cup on the plate, take 50 μl of α-amylase standard solution of different concentrations and the fermentation broth of different bacterial strains and add it to different Oxford cups, and in each cup Add 50 μl of phosphate buffered saline. Place at 50°C for 12 hours, then stain with iodine-potassium iodide (I ₂ -KI) solution, and calculate the diameter of the transparent circle. The above experiments were repeated three times, and the results were analyzed by taking the average value. Draw a standard curve according to the transparent circle of the α-amylase standard of different concentrations; calculate the α-amylase activity of the fermentation liquid of each test strain according to the transparent circle standard curve of the transparent circle diameter of the test sample and the α-amylase standard of different concentrations.

Claims (2)

1. a method of producing αDian Fenmei is characterized in that, its concrete steps are following:

1), the αDian Fenmei gene of applied molecular biology technology clone barley, αDian Fenmei gene and the aspergillar αDian Fenmei gene of Bacillus licheniformis;

2), structure contains the αDian Fenmei gene expression construct of barley, the αDian Fenmei gene expression construct of Bacillus licheniformis and yeast expression vector, yeast saccharomyces cerevisiae expression vector and the subtilis expression vector of aspergillar αDian Fenmei gene expression construct respectively;

3) thereby, yeast expression vector transformed pichia spp obtain the pichia spp recon; Thereby yeast saccharomyces cerevisiae expression vector transformed saccharomyces cerevisiae is obtained the yeast saccharomyces cerevisiae recon, obtain the subtilis recon thereby the subtilis expression vector is transformed subtilis;

4), the pichia spp recon of the above-described three kinds of αDian Fenmeis of screening high expression level is as containing the αDian Fenmei gene expression construct of barley, the αDian Fenmei gene expression construct of Bacillus licheniformis and the Pichia yeast engineering of aspergillar αDian Fenmei gene expression construct; The yeast saccharomyces cerevisiae recon of the above-described three kinds of αDian Fenmeis of screening high expression level is as containing the αDian Fenmei gene expression construct of barley, the αDian Fenmei gene expression construct of Bacillus licheniformis and the saccharomyces cerevisiae engineered yeast of aspergillar αDian Fenmei gene expression construct, and the subtilis recon of the above-described three kinds of αDian Fenmeis of screening high expression level is as containing the αDian Fenmei gene expression construct of barley, the αDian Fenmei gene expression construct of Bacillus licheniformis and the subtilis engineering bacteria of aspergillar αDian Fenmei gene expression construct;

5), above-described Pichia yeast engineering, saccharomyces cerevisiae engineered yeast and the production of the subtilis engineering bacteria reorganization blend alpha glycase that ferments respectively.

2. according to the said a kind of method of producing αDian Fenmei of claim 1, it is characterized in that: utilize the method for the described production αDian Fenmei of claim 1 to be prepared into reorganization blend alpha glycase product.

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