CN102220360A - Lactobacillus plantarum display linoleic acid isomerase, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a Lactobacillus plantarum displaying linoleic acid isomerase and its preparation method and application. The method comprises a Usp45 signal peptide gene, a linoleic acid isomerase gene, an M6 anchor protein gene and a carrier pMG36e. Transform the recombinant plasmid into Lactobacillus plantarum CGMCC NO.3782, inoculate it in the MRS medium containing erythromycin, collect the bacteria by centrifugation after 5-12 hours of cultivation, wash to obtain Lactobacillus plantarum displaying linoleic acid isomerase, and then inoculate The collected Lactobacillus displaying linoleic acid isomerase was added to the buffer solution containing linoleic acid, shaken and cultured to catalyze the synthesis of conjugated linoleic acid. The present invention genetically engineered the Lactobacillus plantarum cells, so that the linoleic acid isomerase was expressed and secreted extracellularly, and at the same time, the linoleic acid isomerase was immobilized on the cell surface by M6 anchor cells, and the linoleic acid isomerase was used to Constructase catalyzes the conversion of conjugated linoleic acid and increases the yield of conjugated linoleic acid.

Description

Lactobacillus plantarum displaying linoleic acid isomerase and its preparation method and application

technical field

The invention relates to the technical field of bioengineering, in particular to a plant lactobacillus displaying linoleic acid isomerase and a preparation method and application thereof.

Background technique

Conjugated Linoleic Acid (CLA) has important physiological functions, such as anti-cancer (colon cancer, gastric cancer, breast cancer, prostate cancer), improving cellular immunity, reducing body fat content, preventing the occurrence of diabetes and inhibiting Atherosclerosis, etc., so CLA has attracted widespread attention. Many microorganisms can use their own linoleic acid isomerase to convert linoleic acid (LA) into conjugated linoleic acid. However, the currently used linoleic acid isomerase mainly has the following problems: the recombinant expression of the enzyme is generally intracellular expression and secretory expression. During intracellular expression, the enzyme cannot contact the extracellular substrate, which greatly affects the catalytic efficiency of the enzyme, and the accumulation of enzyme molecules in the cell forms feedback inhibition, which also affects the expression efficiency. The main disadvantage of secretory expression is that the expression efficiency is not high, and complete secretory expression cannot be achieved, and a considerable part of the expression product is always retained in the cell, so that the complete contact between the enzyme and the extracellular substrate cannot be realized. Both modes of expression lead to inefficient catalytic enzymes.

Contents of the invention

The invention provides a plant lactobacillus displaying linoleic acid isomerase, which realizes the complete contact between the enzyme and the extracellular substrate, and significantly increases the yield of conjugated linoleic acid.

A method for preparing Lactobacillus plantarum to display linoleic acid isomerase, comprising:

The recombinant plasmid was transformed into Lactobacillus plantarum CGMCC NO.3782, inoculated in MRS medium containing erythromycin, cultured for 24 to 48 hours, collected by centrifugation, and washed.

The recombinant plasmid consists of the original vector pMG36e and the Usp45 signal peptide gene, linoleic acid isomerase gene and M6 anchor protein gene which are sequentially inserted into the downstream of the original vector pMG36e promoter.

The linoleic acid isomerase gene is derived from Lactobacillus plantarum lp15-2-1, and the preservation number is CGMCC NO.3782. The isolation, purification and identification methods of this strain have been disclosed in Chinese patent application 201010251108.X Publicly, the Genbank number is HQ831447, and the base sequence is shown in SEQ ID NO: 1; the signal peptide gene can be selected from the Usp45 signal peptide gene amplified from Lactococcus lactis, and the Genbank number is LACUSP45, the base The base sequence is shown in SEQ ID NO: 2; the M6 anchor protein gene is derived from Streptococcus pyogenes (Streptococcus pyogenes), the Genbank number is STRM6, and the base sequence is shown in SEQ ID NO: 3.

The P32 constitutive promoter exists in the expression vector pMG36e.

The present invention also provides a linoleic acid isomerase displayed by Lactobacillus plantarum prepared by the method.

The present invention also provides an application of Lactobacillus plantarum prepared by the above method to display linoleic acid isomerase in catalyzing the synthesis of conjugated linoleic acid, including:

Add the linoleic acid isomerase displayed by Lactobacillus into the buffer solution containing linoleic acid, shake and cultivate for 5-12 hours.

The concentration of linoleic acid in the buffer solution is 3-5 mg/ml.

The pH value of the buffer solution is 6.0-7.0.

The present invention genetically engineered the Lactobacillus plantarum cells, so that the linoleic acid isomerase was expressed and secreted extracellularly, and at the same time, the linoleic acid isomerase was immobilized on the cell surface by M6 anchor cells, and the linoleic acid isomerase was used to The constitutive enzyme catalyzes the conversion of conjugated linoleic acid, increases the yield of conjugated linoleic acid, and reduces the production cost of conjugated linoleic acid.

Description of drawings

Figure 1 is a diagram of PCR and enzyme digestion identification results of cloning vectors pMDS-lai, pMDS-usp45, and pMDS-m6;

A: The results of PCR amplification of genes usp4 and m6; B the results of enzyme digestion and identification of cloning vectors pMDS-usp45 and pMDS-m6; C the results of PCR amplification and enzyme digestion of gene lai

Fig. 2 is a structural representation of lactic acid bacteria display expression vector pMG36e-usp45-lai-m6;

Fig. 3 is the PCR (A) of lactic acid bacterium display expression vector pMULM and enzyme cut (B) identify figure; A: to the PCR amplification of gene usp4, m6 and lai in the vector pMULM; B different enzyme (Cla I and Sal I; Sal I and Pst I; Pst I and Sph I) enzyme digestion identification of vector pMULM.

Fig. 4 is the PCR identification diagram of Lactobacillus plantarum engineering bacteria strain;

Fig. 5 is a diagram showing the enzyme activity measurement of Lactobacillus plantarum showing linoleic acid isomerase under different reaction times.

Detailed ways

Lactobacillus plantarum (Lactobacillus plantarum) lp15-2-1 has been preserved in the General Microorganism Center (CGMCC) of the China Microbiological Culture Collection Management Committee (CGMCC). The preservation date is April 27, 2010, and the preservation number is CGMCC NO.3782. The isolation, purification and identification methods of the strains have been disclosed in Chinese patent application 201010251108.X.

Example 1 Fusion of linoleic acid isomerase gene, signal peptide gene and anchor gene

The primers in Table 1 were used to amplify the lai gene (Genbank No.: HQ831447) from Lactobacillus plantarum lp15-2-1, and the signal peptide gene usp45 (Genbank No.: LACUSP45) from Lactococcus lactis (Lactococcus lactis) and The M6 anchor protein gene m6 (Genbank No: STRM6) was amplified from Streptococcus pyogenes; the PCR product of the gene sequence was connected to the pMD18-T simple (pMDS) vector, and the cloning vector pMDS- lai, pMDS-usp45 and pMDS-m6, and carry out PCR and enzyme digestion identification on the cloning vector, the reaction system and reaction procedure of PCR are as follows:

PCR reaction system (50μL):

The reaction program was: 94°C for 5min; 94°C for 30s, 48°C for 30s (primers Usp45-5' and Usp45-3')/50°C for 30s (primers M6-5' and M6-3')/50°C for 40s (primers LAI -5' and LAI-3'), 72°C for 30s, 30 cycles; 72°C for 5min.

The results are shown in Figure 1: the results of electrophoresis detection of the PCR products amplified by the usp45 and m6 genes are shown in Figure 1A, and fragments of 214bp (usp45) and 423bp (m6) were amplified respectively, which fully met the expected values. Use primers LAI-5' and LAI-3' to perform PCR amplification to obtain the lai gene containing restriction sites Sal I and Pst I, as shown in Figure 1C, about 1700bp.

_The results of enzyme digestion and identification of the cloning vectors _pMDS -usp45, pMDS-m ₆ and pMDS-lai are shown in Figure 1B and C, respectively. band, indicating that the cloning vector was constructed successfully.

Table 1 Primers for amplification

Example 2 Construction of lactic acid bacteria display expression vector

Lactococcus lactis universal expression plasmid pMG36e is a classic artificially constructed constitutive expression vector, which is constructed based on the transcription and translation signals of a Lactococcus lactis subsp. cremoris protease gene. It contains a strong promoter capable of expressing foreign proteins in a variety of bacteria. This expression vector was used in this example. First, the plasmid pMDS-usp45 and the empty vector pMG36e were digested with Cla I and Sal I respectively. After electrophoresis detection, the DNA fragment usp45 and the vector pMG36e were recovered and ligated, and the ligated product was transformed into Escherichia coli DH5α Competent cells were screened on LB medium containing erythromycin to obtain the plasmid pMG36e-usp45 (pMU); then the plasmids pMDS-lai and pMU were subjected to Sal I and Pst I double enzyme digestion, respectively, after electrophoresis detection, they were recovered and Ligate the DNA fragment lai and the vector pMU to construct pMG36e-usp45-lai (pMUL); finally, carry out Pst I and SphI double enzyme digestion on the plasmid pMDS-m6 and pMUL, recover and connect the DNA fragment m6 and the vector pMUL respectively, and construct the lactic acid bacteria display expression Carrier pMG36e-usp45-lai-m6 (pMULM), the structural schematic diagram of lactic acid bacteria display expression vector pMG36e-usp45-lai-m6 is shown in Figure 2; PCR and enzyme digestion identification of lactic acid bacteria display expression vector, PCR reaction system and reaction The procedure is as follows:

PCR reaction system (50μL):

The reaction program was: 94°C for 5min; 94°C for 30s, 48°C for 30s (primers Usp45-5' and Usp45-5')/50°C for 30s (primers M6-5' and M6-3')/50°C for 40s (primers LAI -5' and LAI-3'), 72°C for 30s, 30 cycles; 72°C for 5min.

The results are shown in Figure 3, where Figure A shows the PCR amplification results of genes lai, usp45, and m6, and bands with a size of 1700bp, 214bp, and 423bp were amplified respectively, while Figure B is the result of enzyme digestion identification, which was also cut The bands with the sizes of 1700bp, 214bp and 423bp showed that the display expression vector of lactic acid bacteria had been constructed successfully.

Example 3 Display and expression of linoleic acid isomerase on the cell surface of Lactobacillus plantarum

The lactic acid bacteria display expression vector pMG36e-usp45-lai-m6 was transformed into Lactobacillus plantarum CGMCC NO.3782 cells by electroporation method, and the transformants were screened on the MRS plate containing erythromycin, and further obtained by using primers M6-5' and M6 -3' for PCR verification, the PCR reaction system and reaction procedures are as follows:

PCR reaction system (50μL):

The reaction program was: 94°C for 5min; 94°C for 30s, 48°C for 30s (primers Usp45-5' and Usp45-3')/50°C for 30s (primers M6-5' and M6-3')/50°C for 40s (primers LAI -5' and LAI-3'), 72°C for 30s, 30 cycles; 72°C for 5min.

The results are shown in Figure 4. Compared with the control negative strains, the Lactobacillus plantarum engineered strains can amplify a band of about 500bp, indicating that the plasmid pYMLS has been successfully transferred into the Lactobacillus plantarum cells, thus obtaining the pMG36e- The Lactobacillus plantarum engineering strain usp45-lai-m6 realizes the display and expression of linoleic acid isomerase on the outer surface of the cell wall of Lactobacillus plantarum, that is, obtains Lactobacillus plantarum displaying linoleic acid isomerase.

The electric shock transformation of Lactobacillus plantarum includes the preparation of competent cells and electric shock transformation:

(1) Preparation of Lactobacillus plantarum Competent Cells

① Take 10 mL of the overnight culture of Lactobacillus plantarum lp15-2-1 and transfer it to 100 mL of MRS medium;

②Incubate at 30°C for 3 to 4 hours (OD600≈0.85;), then place in an ice bath for 30 minutes;

③ 4°C, 8000g centrifugation to collect bacteria;

④Resuspend the cells in 10mL of 10mM MgCl ₂ (pre-frozen at -20°C for 30 minutes), wash the cells twice, and collect the cells by centrifugation at 4°C and 8000g;

⑤ Resuspend the bacteria in 10 mL of 10% (w/v) glycerol solution containing 0.5M sucrose (pre-frozen in a refrigerator at -20°C for 30 minutes), and collect the bacteria by centrifugation at 4°C and 8000g;

⑥ The bacteria were resuspended with 3-4 mL of 10% (w/v) glycerol solution (pre-cooled) containing 0.5M sucrose;

⑦ Place the prepared Lactobacillus plantarum competent cells on ice for later use (it is better to use them up within 4 hours).

(2) Transformation of Lactobacillus plantarum by electric shock method

① Take 40 μL of the above-mentioned Lactobacillus plantarum competent cells, add 5 μL (containing 1~3ng DNA) of the plasmid DNA (pMULM and empty vector pMG36e) to be transformed, mix well, transfer to a pre-cooled electric shock cup, and place on ice for 5 minutes ;

②Electric shock transformation uses Bio-Rad's gene electric shock transformation instrument (BTX); transfer the electric shock cup into the electric shock seat for electric shock transformation (conditions: voltage 1.3KV; capacitance 25μF; resistance 200Ω; electric shock time 2-4s) ;

③After the electric shock is over, take out the electric shock cup immediately, add 1mL of fresh MRS liquid medium, mix well and transfer to a 1.5mL centrifuge tube;

④ 30°C, 180rpm shaking culture for 2h;

⑤ At room temperature, 3000rpm, centrifuge for 4min, and resuspend the bacteria with 200μL sterile ddH2O;

⑥Spread the bacterial liquid on the MRS plate containing 200 μg/mL erythromycin (Emr+), incubate upside down at 30°C for 48 hours, and identify the transformants after they grow into plaques of sufficient size.

Example 4 Collection of Lactobacillus plantarum displaying linoleic acid isomerase

The above Lactobacillus plantarum engineered bacteria strain was inoculated into the MRS culture concentration containing erythromycin (200 μg/ml) according to the inoculum amount of 1%, and cultured with shaking at 30° C. and 180 rpm for 24 hours. 3000rpm, centrifuge at 4°C for 10min, collect the bacterial cells, wash twice with physiological saline (0.9% NaCl solution, v/v), resuspend with phosphate buffer (0.1M, pH7.0), adjust OD (optical density) )600=2. That is, the Lactobacillus plantarum displaying linoleic acid isomerase is obtained.

Example 5 Lactobacillus plantarum displays linoleic acid isomerase enzyme activity and determination of conversion rate

Take 5ml of the collected Lactobacillus plantarum displaying linoleic acid isomerase enzyme solution, add 3mg/ml linoleic acid, and react 1, 2, 3, 4, 5, 6, 7, 8, 9 respectively at 30°C , 10, 11, and 12 hours, extract CLA with twice the volume of n-hexane, and use ultraviolet spectrophotometry to measure the content of CLA and calculate the enzyme activity. That is, the amount of enzyme required to produce 1 μg CLA per unit time (h).

Detection of linoleic acid isomerase activity of Lactobacillus plantarum by ultraviolet spectrophotometry:

Using n-hexane as a solvent, prepare CLA standard samples into solutions with different concentrations; use n-hexane as a reference, measure the absorbance value of CLA standard sample solution at 233nm, adjust the concentration of CLA so that the absorbance value ranges from 0.2 to 3.0 , with the CLA concentration (g/mL) as the abscissa and the absorbance as the ordinate, draw the UV absorbance standard curve of CLA, the linear regression equation is A=0.1058C+0.1452 (R ² =0.9996), A is the absorbance, C is the concentration of CLA.

Then detect the absorbance value at 233nm of CLA extracted from n-hexane in the conversion product of Lactobacillus plantarum linoleic acid isomerase, and calculate the enzymatic activity of Lactobacillus plantarum linoleic acid isomerase according to the formula obtained from the above CLA standard curve.

The results are shown in Figure 5, the enzyme activity reached the highest at 417U/ml at 5 hours after transformation. And at this time, the CLA content also reaches the maximum, which is 2.09 mg/ml, and the conversion rate reaches 69.7%.

Conversion rate = CLA production per unit volume of fermentation broth/addition of unit volume of LA

The control strain and Lactobacillus plantarum engineered strain were cultured in the MRS medium containing erythromycin for 48 hours, and then the bacterial cells were collected, and then cultured with shaking in phosphate buffer (0.1M, pH7.0) containing 3 mg/mL LA for 5 hours Finally, gas-phase detection was carried out on the synthesized CLA, and the results are shown in Table 2. Among them, the control strain, that is, Lactobacillus plantarum lp15-2-1 containing the empty plasmid pMG36e, was cultured for 48 hours, and when LA was added to the bacterial cells to transform and synthesize for 5 hours, there was a certain amount. The synthesis of CLA, and it contains c9t11-CLA and t10c12-CLA; compared with the control strain, the ability of Lactobacillus plantarum engineering strain to transform and synthesize CLA has been greatly improved, and the influence of endogenous LAI transformation product CLA has been removed , Lactobacillus plantarum engineering strains are mainly transformed to synthesize c9t11-CLA, that is, LAI displayed on the cell surface plays an important role. When cultured for 48 hours, among the 6 engineering strains, No. 1 Lactobacillus plantarum engineering strain had the highest CLA yield, reaching 2.084 mg/ml, and the conversion rate of LA was as high as 69.5%.

Table 2 Analysis of CLA products transformed and synthesized by Lactobacillus plantarum engineered strains after 48 hours of culture

Claims (9)

1. one kind prepares the method that plant lactobacillus is showed linoleate isomerase, comprising:

Change recombinant plasmid over to plant lactobacillus CGMCC NO.3782, be inoculated in the MRS substratum that contains erythromycin, cultivate centrifugal collection thalline after 24～48 hours, flushing;

Described recombinant plasmid is by initial carrier pMG36e and the Usp45 signal peptide gene, linoleate isomerase gene and the genomic constitution of M6 anchorin that are inserted into initial carrier pMG36e promotor downstream successively.

2. method according to claim 1 is characterized in that: the base sequence of described linoleate isomerase gene is shown in SEQ ID NO:1.

3. method according to claim 1 is characterized in that: the base sequence of described Usp45 signal peptide gene is shown in SEQ ID NO:2.

4. method according to claim 1 is characterized in that: the base sequence of the M6 anchorin gene of being told is shown in SEQ ID NO:3.

5. show linoleate isomerase according to the plant lactobacillus of the described method preparation of claim 1.

6. plant lactobacillus according to claim 5 is showed the application of linoleate isomerase in the catalysis synthesis of conjugated linoleic acid.

7. application according to claim 6 is characterized in that, comprising:

Plant lactobacillus is showed that the linoleate isomerase adding contains in the linoleic damping fluid shaking culture 5～12h.

8. application according to claim 6 is characterized in that: linoleic concentration is 3～5mg/ml in the described damping fluid.

9. application according to claim 6 is characterized in that: the pH value of described damping fluid is 6.0-7.0.

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