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Method for high-efficiency expression of maltogenic amylase in bacillus subtilis

A technology of Bacillus subtilis and maltogenic amylase, which is applied in the fields of genetic engineering and fermentation engineering, can solve problems such as low enzyme activity, and achieve the effects of wide sources, good expression and low production cost

Active Publication Date: 2018-06-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the activity of maltogenic amylase expressed in Bacillus subtilis is generally low

Method used

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  • Method for high-efficiency expression of maltogenic amylase in bacillus subtilis
  • Method for high-efficiency expression of maltogenic amylase in bacillus subtilis
  • Method for high-efficiency expression of maltogenic amylase in bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Recombinant vector construction

[0021] Recombinant construction of signal peptide YvcE, LipA, YncM and expression vector linked with maltose amylase

[0022] Primers YvcE-F and YvcE-R (SEQ ID NO.5, SEQ ID NO.6) containing homology arms were designed to amplify the YvcE signal peptide using the Bacillussubtilis168 genome as a template, and the underlined part is the region of the homology arms. The recombinant construction of LipA and YncM signal peptide is the same as above, and the primers are LipA-F, LipA-R (SEQ ID NO.7, SEQ ID NO.8) and YncM-F, YncM-R (SEQ ID NO.9, SEQ ID NO. NO.10).

[0023] Primers P-F, P-R (SEQ ID NO.11, SEQ ID NO.12) were designed to amplify the expression vector sequence using the amyM-pHY300PLK vector plasmid as a template.

[0024] YvcE-F: 5'- TAAGGAGTGTCAAGA ATGAGAAAGAGTTTAATTACACTTGGT-3'

[0025] YvcE-R: 5'- GCTTGCAGAAGAAGA CGCCGATGCAGTTTTACTTGTAAA-3'

[0026] LipA-F: 5'- TAAGGAGTGTCAAGA ATGAAATTTGTAAAAAGAAGGATCAT-3...

Embodiment 2

[0037] Embodiment 2: Transformation of recombinant plasmid

[0038] 1) Fresh LB plate (LB solid medium: peptone 10g / L, yeast extract 5, NaCl 10g / L, 0.2g / L agar powder) pick a single colony of Bacillus subtilis (CCTCC M2016536) and inoculate it in 5ml LB liquid culture medium, 37°C, 200rpm for 10.5h.

[0039] 2) Take 2.5 mL and transfer it into 40 mL of LB medium with 0.5 M sorbitol, culture at 37° C. with shaking at 200 rpm for 4.5 hours.

[0040] 3) Take all the bacteria liquid and bathe in ice water for 10 minutes, then centrifuge at 5000 rpm and 4°C for 5 minutes to collect the bacteria.

[0041] 4) Wash the cells with 40ml of pre-cooled electroporation buffer (sorbitol 0.5M, mannitol 0.5M, glucose 10%), centrifuge at 5000rpm, 4°C for 5min to remove the supernatant, and rinse 4 times in this way.

[0042] 5) Resuspend the washed bacteria in 1mL electroporation medium, aliquot them into 1.5mL EP tubes, and fill each tube with 300ul competent cells.

[0043]6) Add 10 μL of...

Embodiment 3

[0045] Embodiment 3: Shake flask fermentation produces enzyme and the mensuration of maltose amylase enzyme activity

[0046] The recombinant Bacillus subtilis strain obtained in Example 2 was inoculated in LB medium, and after culturing at 37°C for 8-10 hours, it was transferred to TB fermentation medium with a 5% inoculation amount, and cultivated at 37°C and 200rpm for 2h, Then move to 33 ℃ constant temperature culture for 48h to produce enzyme. After the fermentation, the supernatant collected by centrifugation is the crude enzyme liquid.

[0047] Mix 1mL of 1% soluble starch solution and 0.9mL of 50mM, pH5.5 phosphate buffer well, preheat at 60°C for 10min, add 0.1mL of enzyme solution, oscillate and mix well, add 3mL of DNS after 10min of reaction, oscillate , boiled for 7 minutes, cooled rapidly, added distilled water to make up to 15ml, and measured the absorbance at 540nm (using the inactivated enzyme solution as a catalyst, the same operation was used as a blank). ...

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Abstract

The invention discloses a method for high-efficiency expression of maltogenic amylase in bacillus subtilis, and belongs to the fields of genetic engineering and fermentation engineering. According tothe method provided by the invention, by searching a signal peptide sequence, various signal peptide can be obtained from a bacillus subtilis 168 genome via PCR, recombinant construction is implemented together with target gene maltogenic amylase and a vector pHY300PLK and conversion into bacillus subtilis CCTCC M2016536 is promoted, so that three recombinant bacillus subtilis can be obtained. With the recombinant bacillus subtilis as a strain, the maltogenic amylase is obtained through fermenting production. In addition, the enzyme activity of the recombinant enzyme is improved by confirmingsuch optimized optimum shaker conditions as a nitrogen source, a carbon source, fermentation conditions and the like. The method provided by the invention has the advantages that it conforms that thehigh-efficiency expression of the signal peptide of the maltogenic amylase can be achieved in the bacillus subtilis, and the recombinant expression of the maltogenic amylase is implemented with the food-safety bacillus subtilis as an expression host; enzyme production of the recombinant bacterium undergoes fermentation optimization; a relatively good expression amount is achieved; the enzyme activity of obtained fermentation supernatant is 396U / mL; and the method is broad in source of fermentation raw materials and relatively low in production cost.

Description

technical field [0001] The invention relates to a method for efficiently expressing maltogenic amylase in Bacillus subtilis, belonging to the fields of genetic engineering and fermentation engineering. Background technique [0002] Maltogenic amylase or maltogenase, EC 3.2.1.133, can catalyze the hydrolysis of (1→4)-β-D-glucosidic bonds in maltotriose, starch and dextrin, remove maltose residues, usually in external random Hydrolyzed, but it can also undergo internal hydrolysis. In addition to hydrolysis, maltogenic amylase also catalyzes transglycosylation, which belongs to the glycoside hydrolase 13 family and has many sources, including Bacillus licheniformis, Bacillus stearothermophilus, thermophilic Actinomycete (Thermus vulgaris), etc., maltose amylase is widely used in starch saccharification industry, food baking, and flour industry. Maltogenic amylase can hydrolyze starch to produce maltose and some dextrins, prolong the shelf life of baked goods and reduce food w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/56C12N15/75C12N9/24C12R1/125
CPCC12N9/2402C12N15/75C12Y302/01133
Inventor 吴敬宿玲恰李雨桐
Owner JIANGNAN UNIV
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