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Strain for production of cyclodextrin glucosyltransferase and application of strain

A technology of bacterial strains and genetically engineered bacteria, applied in the fields of genetic engineering and enzyme engineering, enzyme engineering and genetic engineering, can solve problems such as distant kinship and difficult protein expression

Inactive Publication Date: 2015-05-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Like Escherichia coli, due to the distant relationship with many Gram-positive bacteria, it may be difficult to correctly express proteins from some positive bacteria

Method used

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  • Strain for production of cyclodextrin glucosyltransferase and application of strain
  • Strain for production of cyclodextrin glucosyltransferase and application of strain
  • Strain for production of cyclodextrin glucosyltransferase and application of strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: Cloning of Bacillus stearothermophilus α / β-CGTase coding gene and construction of expression vector

[0026] Bacillus stearothermophilus (preservation number CCTCC M 2013413) was cultured in LB liquid medium for 2 days, and the thalline was collected by centrifugation at 10,000 rpm, washed with sterile water, the collected precipitate was suspended in 500uL Tris-EDTA buffer, and 15uL lysozyme was added. Incubate at 37°C for 30min, then add 5uL RNase, incubate at 37°C for 30min, add 30µL 10% sodium dodecyl sulfate and 15µL proteinase K, incubate at 37°C for 60min, add 100uL of 5M NaCl and cetyltri 80 μL of methyl ammonium bromide, incubated at 65°C for 20 minutes, extracted with 700 μL of a mixed solvent with a volume ratio of phenol:chloroform:isoamyl alcohol of 25:24:1, centrifuged at 10,000 rpm, and the supernatant was mixed with 700 μL of chloroform:isoamyl alcohol Extract with a mixed solvent with an alcohol volume ratio of 24:1, centrifuge at 10,000 r...

Embodiment 2

[0031] Example 2: Transformation of Bacillus pumilus

[0032] 1) Fresh TM plate (TM medium: polypeptone 1%, beef extract 0.5%, yeast powder 0.2%, glucose 1%) pick Bacillus pumilus as Brevibacillus brevis (CCTCC AB 94025) and inoculate a single colony in 10ml TM culture cultured overnight.

[0033]2) Measure the OD in the shake flask, control the inoculum size, and make the OD of the medium after the inoculation complete. 600 Between 0.19-0.2. Medium is 50mL GM (peptone 0.5%, soybean peptone 0.5%, beef extract 0.5%, yeast extract 0.25%, glucose 0.5%, α-sodium glycerophosphate 1.9%, vitamin C 0.05%, MgSO 4 0.012%). 37°C, 200rpm culture to OD 600 =1.0 (about 3-4 hours).

[0034] 3) Take all the bacterial solution and bathe in ice water for 10 minutes, then centrifuge at 5000 rpm for 8 minutes at 4°C to collect the bacterial cells.

[0035] 4) Wash the cells with 40ml pre-cooled electroporation buffer ETM (9% sorbitol, 9% mannitol, 10% glycerol), centrifuge at 5000rpm, 8min,...

Embodiment 3

[0039] Embodiment 3: Shake flask fermentation produces enzyme

[0040] 1) Fermentation culture

[0041] The cyclodextrin glucosyltransferase-producing strain screened in Example 1 was inoculated in TM medium, and after culturing at 30°C for 12 hours, it was transferred to TM fermentation medium with a 5% inoculum size, and cultured at a constant temperature of 30°C for 44 hours. ~48h to produce enzyme. After the fermentation, the supernatant collected by centrifugation is the crude enzyme liquid.

[0042] TM medium (g / L): polypeptone 10, beef powder 5, yeast powder 2, glucose 10, pH7.0

[0043] 2) Determination of enzyme activity

[0044] The activity of the enzyme was determined by methyl orange method with soluble starch as substrate. Enzyme activity assay system: 2.5mL (containing soluble starch with a final concentration of 2%, 50mM KH 2 PO 4 -Na 2 HPO 4 Buffer, pH 6.0), add 100μL of appropriately diluted enzyme solution at 50°C and react for 10min, then add 200μl ...

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Abstract

The invention discloses a gene engineering strain for recombinant expression of Bacillus stearothermophilus alpha / beta-CGT enzyme and application of the gene engineering strain, belonging to the fields of gene engineering and enzyme engineering. According to the invention, the recombinant plasmid p SVEB-alpha / beta-CGTase is constructed, and Bacillus pumilus Brevibacillus brevis (CCTCC AB 94025) is transformed, so that recombinant Bacillus pumilus is obtained; based on the recombinant Bacillus pumilus as the strain, fermentation is carried to produce an alpha / beta-CGT enzyme which is applied to preparation of beta-cylodextrin and has good effect. The gene engineering strain for recombinant expression of Bacillus stearothermophilus alpha / beta-CGT enzyme disclosed by the invention has the advantages that the food-safe Bacillus pumilus is adopted as an expression host to recombine and produce alpha / beta-CGT enzyme, the recombinant strain is high in enzyme production, the ferment materials are wide in resources, and the production cost is relatively low.

Description

technical field [0001] The invention discloses a gene engineering strain for recombinantly expressing Bacillus stearothermophilus α / β-CGT enzyme and application thereof, belonging to the field of genetic engineering and enzyme engineering. The CGTase gene was linked into the Bacillus pumilus expression vector pSVEB, and then Brevibacillus brevis was transformed with the recombinant vector to realize the high expression of cyclodextrin glucosyltransferase (α / β-CGTase) in Bacillus pumilus (Brevibacillus brevis), It belongs to the fields of enzyme engineering and genetic engineering. Background technique [0002] CGTase is a multifunctional enzyme that catalyzes four different reactions: three transglycosylation reactions (disproportionation, cyclization, and coupling) and hydrolysis. CGTase can utilize starch to produce cyclodextrin through cyclization reaction, which is the basis of its industrial application. In addition to the production of cyclodextrins by cyclization re...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12N9/10C12P19/18C12P19/04C12R1/07
CPCC12N9/1074C12P19/18C12Y204/01019
Inventor 吴敬段绪果范博望
Owner JIANGNAN UNIV
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