Colibacillus-lactic acid bacteria shuttle plasmid capable of expressing and excreting beta-galactosidase and its construction method and application

A technology of galactosidase and Escherichia coli, which is applied in the direction of botany equipment and methods, biochemical equipment and methods, applications, etc., can solve unfavorable industrial preparation of enzyme products, β-galactosidase does not have secretion, β -Galactosidase can not be secreted and other problems, to achieve the effect of wide application value and direct application value

Inactive Publication Date: 2007-09-12
SICHUAN UNIV
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  • Abstract
  • Description
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Problems solved by technology

However, these plasmids have the following disadvantages: ① The β-galactosidase produced by expression is a fusion protein, that is, the N-terminus of the expressed protein is fused with a polypeptide from the vector, which may change the three-dimensional structure of the protein and affect the active group of the protein The formation of β-galactosidase reduces the enzyme activity; ②The output of the expressed β-galactosidase is not high, which will directly affect its application; ③The expressed β-galactosidase is not secreted, which is not conducive to the industrial preparation of enzyme products. , the expression of β-galactosidase can not be secreted and will also affect the live bacteria to directly play the role of enzymatic hydrolysis; ④ cannot be expressed in lactic acid bacteria

Method used

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  • Colibacillus-lactic acid bacteria shuttle plasmid capable of expressing and excreting beta-galactosidase and its construction method and application
  • Colibacillus-lactic acid bacteria shuttle plasmid capable of expressing and excreting beta-galactosidase and its construction method and application
  • Colibacillus-lactic acid bacteria shuttle plasmid capable of expressing and excreting beta-galactosidase and its construction method and application

Examples

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Effect test

Embodiment 1

[0034] Example 1: Construction of Escherichia coli-lactic acid bacteria shuttle plasmid capable of expressing and secreting β-galactosidase

[0035] In this example and subsequent examples, the Escherichia coli-lactic acid bacteria shuttle plasmid capable of expressing and secreting β-galactosidase was named pMG36e-lacZ. The steps of its construction method are as follows:

[0036] (1) Amplification of Lactobacillus delbrueckii subsp. bulgaricus β-galactosidase gene fragment (SEQ ID NO.3)

[0037] Take Lactobacillus delbrueckii subsp. bulgaricus out of the refrigerator at -20°C, melt in a water bath at 37°C, streak the MRS plate, culture at 37°C for 48 hours, pick colonies and transfer to 5ml MRS broth, culture at 37°C for 24 hours, use a small amount of The bacterial genomic DNA extraction kit extracts genomic DNA as a template, and the extracted product is checked by 0.8% agarose gel electrophoresis, and there is an obvious band at >10kb in the positive result (see Figure 3...

Embodiment 2

[0066] Example 2: Electrotransformation of Lactococcus lactis with the recombinant plasmid pMG36e-lacZ

[0067] (1) Preparation of Lactococcus lactis cells for electroporation

[0068] Lactococcus lactis strains were taken out from the -20°C refrigerator, melted in a 37°C water bath, and cultured on a streaked MRS plate at 30°C for 48 hours. Pick colonies from the plate and inoculate 5ml of MRS broth, incubate at 30°C for 12-16h, pipette 5ml of bacterial liquid into 100ml of MRS broth, and incubate at 30°C until A 600 It is 0.5-0.8 (about 4h). 1 h before stopping the culture, ampicillin was added to make the final concentration 20 μg / ml. After stopping the culture, draw the bacterial solution into a clean, sterile, pre-cooled 50ml polypropylene centrifuge tube, and place it in an ice bath for 10 minutes. Centrifuge at 4000r / min for 7min at 4°C, discard the supernatant, invert the tube, and absorb the residual liquid with a sterile filter paper strip. Add 20ml of ice-cold g...

Embodiment 3

[0080] Example 3: Expression of recombinant plasmid pMG36e-lacZ in Escherichia coli DH5α

[0081] The positive bacteria E.coli DH5α / pMG36e-lacZ were obtained after the recombinant plasmid pMG36e-lacZ was transformed into Escherichia coli DH5α, and the β-galactosidase activity of the bacteria was determined to study the expression of the recombinant plasmid pMG36e-lacZ in Escherichia coli.

[0082] E. coli DH5α / pMG36e-lacZ streaked LB-Ery-X-gal plate, cultured at 37°C for 48 hours, picked blue colonies on the plate and inoculated 15ml of LB-Ery broth, cultured with shaking at 220r / min at 37°C for 24 hours; E. coli DH5α streaked LB plate, cultured at 37°C for 24 hours, picked a single colony on the plate to inoculate 15ml of LB broth, and cultured at 37°C with shaking at 220r / min for 24 hours. Take 10ml of the bacterial solution in a 50ml centrifuge tube (10ml bacterial solution / centrifuge tube), centrifuge at 15000r / min for 3min at 4°C, discard the supernatant, wash the bacteri...

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Abstract

Gene of an E.coli-Lactobacillus shuttle plasmid expressing and secreting beta-galactosidase is composed of the gene encoding beta-galactosidase and its upstream regulatory sequence containing SD sites. Experiments proves that the recombinant plasmid can be transformed into Lactococcus lactis, and highly express the non-fused beta-galactosidase in E.coli and Lactococcus lactis, which is applicable in the lactic acid bacteria strain expressing beta-galactosidaseion. The construction method includes the following steps: taking Lactobacillus Bulgaria subspecies genomic DNA as template to amplify and obtain the beta-galactosidase gene containing the upstream regulatory sequence, digesting the gene to conjugate with the dephosphorylated vector digested with the same two enzymes and transform E.coli, then screening the positive one to extract the recombinant for enzyme identification and sequencing the inserted sequence.

Description

technical field [0001] The invention relates to an Escherichia coli-lactic acid bacteria shuttle plasmid capable of expressing and secreting β-galactosidase, a construction method and application thereof. Background technique [0002] β-Galactosidase (lactase) is very important in industry and medicine because it can catalyze lactose into galactose and glucose. The purified β-galactosidase can be used in dairy processing to produce low-lactose milk in industrial production, and can be used in the medical field to treat lactose intolerance and the like. On the other hand, some bacteria such as Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus have specific practical value because they can produce β-galactosidase. For example, probiotics such as lactic acid bacteria can be used to prepare probiotics for alleviating lactose intolerance because they can produce β-galactosidase; Lactobacillus delbrueckii subsp. It will...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/56C12N1/21
Inventor 汪川张朝武刘衡川余倩许欣裴晓方王国庆林怡伶
Owner SICHUAN UNIV
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