CN108865913B - Method for constructing recombinant bacterium capable of efficiently secreting and expressing chondroitin sulfate hydrolase - Google Patents

Abstract

The invention discloses a method for constructing a recombinant bacterium for efficiently secreting and expressing chondroitin sulfate hydrolase, and belongs to the technical field of enzyme engineering. The present invention adopts the Pichia pastoris expression system to construct a method for expressing chondroitin sulfate hydrolase. The invention realizes the secretory expression of chondroitin sulfate hydrolase in the Pichia pastoris expression system, and plays an important role in promoting the industrial production of chondroitin sulfate hydrolase.

Description

A kind of method for constructing high-efficiency secreting and expressing chondroitin sulfate hydrolase recombinant bacteria

technical field

The invention relates to a method for constructing a recombinant bacteria for efficiently secreting and expressing chondroitin sulfate hydrolase, and belongs to the technical field of enzyme engineering.

Background technique

As a kind of high molecular carbohydrate, polysaccharide widely exists in animals, plants and microorganisms, and has various biological activities such as anti-tumor, anti-virus, anti-oxidation, and immune regulation. Polysaccharides are mainly composed of disaccharide units, which are divided into heparan sulfate, heparin, hyaluronic acid, chondroitin sulfate, and dermatan sulfate. The relative molecular weight of chondroitin sulfate (CS) has an important influence on its biological activity. Due to the large size of macromolecular polysaccharides, it is difficult to penetrate the cell membrane and thus cannot exert its various pharmacological functions in the body, while low molecular weight CS has the characteristics of good solubility, low viscosity, easy absorption and high bioavailability Therefore, in recent years, the preparation and application of low molecular weight CS has gradually become a hot spot.

At present, the methods to reduce the relative molecular weight of polysaccharides mainly include biodegradation, chemical degradation, physical degradation and oxidative degradation. Biodegradation is mainly enzymatic degradation, which is characterized by safety and high efficiency, but high cost; chemical degradation can be divided into inorganic acids and organic acids. It is safer, but the degradation efficiency of different organic acids on polysaccharides is quite different; the removal rate of low molecular sulfate groups obtained by acid degradation is high, and unsaturated structures such as unsaturated uronic acid are produced, and the color of the product is orange-red. Physical degradation is mainly ultrasonic degradation and irradiation degradation, which is characterized by easy control of the degradation process and no pollution; oxidative degradation is mainly hydrogen peroxide degradation, which is characterized by fewer by-products and relatively concentrated molecular mass distribution.

Compared with other degradation methods, enzymatic degradation has the advantages of mild reaction conditions, no pollution and simple process, and is suitable for the industrial production of CS oligosaccharides. However, the mechanisms of chondroitin sulfate degradation by enzymes from different sources may be different, resulting in different structures and functions of the CS oligosaccharides finally produced. Chondroitin sulfate lyase, the cleavage effect contains unsaturated double bonds, and the advantage of chondroitin sulfate hydrolase is that it has no unsaturated bonds.

There is no report on the heterologous expression of chondroitin hydrolase: the gene source for constructing the recombinant strain secreting chondroitin hydrolase is bovine, the host Pichia pastoris GS115, plasmid pPIC9K, and the defective fermentation period is long.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a recombinant bacterium for constructing and expressing chondroitin sulfate hydrolase with high efficiency.

In one embodiment of the present invention, the nucleotide sequence of the chondroitin sulfate hydrolase is shown in SEQ ID NO.1.

In an embodiment of the present invention, the amino acid sequence of the chondroitin sulfate hydrolase gene is shown in SEQ ID NO.2.

The second object of the present invention is to provide a method for constructing the recombinant bacteria: the chondroitin sulfate hydrolase gene whose nucleotide sequence is SEQ ID NO.1 is connected to the expression vector pPIC9K, and then transformed into the In Pichia pastoris GS115, the correct transformants were screened to obtain recombinant Pichia pastoris GS115/pPIC9K-CHASE.

In one embodiment of the present invention, the method for constructing the recombinant bacteria adds a histidine tag to the chondroitin sulfate hydrolase gene.

The third object of the present invention is to provide a method for producing chondroitin sulfate hydrolase by fermentation of the recombinant bacteria. The method adopts methanol induction strategy to ferment and produce chondroitin sulfate hydrolase in a fermenter.

In one embodiment of the present invention, the specific steps of the fermentation method are:

The YPD seed liquid cultured in shake flasks was inoculated into a 3L automatic fermenter (LiFlusGMBioTRON, Korea) with 10% inoculum, the temperature was controlled at 25-30°C, the pH was adjusted to 5-6 with 25% ammonia water, and the initial stirring speed was It is 500-1000r·min ^-1 , the ventilation volume is 2-4L·min ^-1 , and the dissolved oxygen is maintained above 30%-35%. Culture for about 20-40h, OD ₆₀₀ is about 70-80 until the glycerol is exhausted, add 30-50% (W·V ^-1 ) glycerol solution (containing 12mL·L ^-1 PTM1) in exponential flow, and the stirring speed is related to dissolved oxygen , control the speed at 500-1000r·min ^-1 for feeding for 12h, stop feeding until the glycerol is exhausted again, the OD ₆₀₀ is about 250 (dry weight is about 60.0g·L ^-1 ), and the speed is at 600-1000r·min ^-1 and 100% methanol (containing 12 mL·L ^-1 PTM1) was added in flow, the methanol concentration was controlled at 18.0 g·L ^-1 , and the induction temperature was 30°C, 25°C, 22°C and stage-controlled temperature induction strategy, respectively.

The fourth object of the present invention is to provide a method for separating and purifying the chondroitin sulfate hydrolase. The specific steps are as follows: centrifuging the fermentation broth, collecting the supernatant, and loading the sample; using a Ni column and balancing the column with a phosphate buffer 10 column volumes; use imidazole gradient elution, imidazole is prepared with the above-mentioned phosphate buffer, and eluted according to a gradient of 10 mmol·L ^-1 , 30 mmol·L ^-1 , and 200 mmol·L ^-1 .

Beneficial effects of the present invention:

(1) The genetically engineered bacteria constructed by the method of the present invention are expressed in Pichia pastoris, which is a method for rapidly producing chondroitin sulfate hydrolase. The purification by nickel column is simple and convenient.

(2) Pichia pastoris has a strong promoter AOX, high expression level, and high stability of recombinant strains. The heterologous protein gene and expression plasmid are integrated into the P. pastoris genome through homologous recombination, and they are inherited with chromosome replication and not independent. easy to lose. It has post-translational modification processing functions such as glycosylation, protein phosphorylation, and fat phthalation. Strong adaptability and low nutritional requirements. There are few types of proteins secreted to the outside of the cell, and the medium does not contain proteins, which is easy to separate and purify the downstream products. The high-density fermentation process is relatively mature, and it is easy to scale up the culture.

(3) The chondroitin sulfate hydrolase secreted and expressed by Pichia pastoris has high expression level, stable enzymatic properties, pH resistance and wide temperature range.

Description of drawings

Fig. 1 is the plasmid map of CHase constructed in Pichia pastoris;

Figure 2 shows that CHase has the activity of hydrolyzing heparin in Pichia pastoris: the control group A substrate heparin was added to the inactivated enzyme, and the experimental group B substrate was added CHase;

Figure 3 shows that CHase has the activity of hydrolyzing chondroitin sulfate A (CSA) in Pichia pastoris: the control group A substrate CSA was added to the inactivated enzyme, and the experimental group B substrate was added CHase;

Figure 4 shows that CHase has the activity of hydrolyzing chondroitin sulfate C (CSC) in Pichia pastoris: the control group A substrate CSC was added to the inactivated enzyme, and the experimental group B substrate was added CHase;

Fig. 5 is the SDS-PAGE electrophoresis result of constructing CHase chondroitin sulfate hydrolase in Pichia pastoris;

Detailed ways

BMGY medium: yeast powder 10g L ^-1 , peptone 20g L ^-1 , glycerol 10mL L ^-1 , 100mM potassium phosphate buffer, 0.34% (mass percentage) amino-free yeast nitrogen source (YNB), 1% ( _NH4 ) _2SO4 , _4x ^10-5 % biotin, pH=6.0.

Chondroitin sulfate hydrolase enzyme activity assay method: 300 μL of substrates heparin (HP)-fluorescein FITC, chondroitin sulfate A (CSA-fluorescein FITC), chondroitin sulfate C (CSC-fluorescein FITC) (2 mg·mL ^{- 1} ), add 100 μL of fermentation broth, hydrolyze at 37°C for 1 h, and detect the enzyme activity using a fluorescence detector of HPLC.

Example 1: Construction of Pichia pastoris recombinant bacteria containing chondroitin sulfate hydrolase gene

The construction of a genetically engineered bacteria producing chondroitin sulfate hydrolase is to take Pichia pastoris as a host and pPIC9K as a carrier to clone the chondroitin sulfate hydrolase gene (nucleotide sequence as shown in SEQ ID NO.1). ), a 6-his tag was added after the gene ATG. The upstream primer was introduced into the EcoR I restriction enzyme site; the downstream primer was introduced into the Not I restriction enzyme site; the gene CSHYDRO and the plasmid pPIC9K were digested and purified by the restriction enzymes EcoR I and Not I, and then ligated at 16°C overnight. Escherichia coli JM109 was used for amplification, and the plasmid with correct sequencing was selected and transferred to Pichia pastoris GS115 for expression, and the genetically engineered strain Pichia pastoris GS115/pPIC9K-CHASE producing chondroitin sulfate hydrolase was obtained. The upstream and downstream primers are:

F: CCG GAATTC ATGCATCACCATCACCA (SEQ ID NO: 3)

R: AAGGAAAAAA GCGGCCGC TTAAGGTGGTTTCAAGAA (SEQ ID NO: 4)

Using a similar method, a 6-his tag was added to the ATG of the gene whose nucleotide sequence is shown in SEQ ID NO. It was transformed into Pichia pastoris GS115 for expression, and the recombinant strain Pichia pastoris GS115/pPIC9K-CHASEQ was obtained.

Example 2: Shake flask horizontal fermentation to produce chondroitin sulfate hydrolase

The recombinant Pichia pastoris GS115/pPIC9K-CHASE containing the recombinant chondroitin sulfate hydrolase gene was used as the production strain, and a single colony was inoculated in YPD medium at 28°C and cultured at 200 rmp overnight to obtain a seed culture solution; the seeds were cultured The solution was inoculated into 25 mL of BMGY medium at an inoculum volume of 2% by mass, and 1% methanol was added to the medium for induction. The induction temperature was 28 °C, and the expression was induced for 70 h. After the fermentation, as can be seen from Figure 2, A is the control, B is the effect after treatment with the fermentation broth supernatant, the main peak is shifted backward, and it has a hydrolysis effect on heparin HP; as can be seen from Figure 3, A is the control, B is the effect. For the effect after processing with the fermentation broth supernatant, the main peak shifts back, and has a hydrolysis effect on CSA (chondroitin sulfate A); as can be seen from Figure 4, A is the control, and B is the effect after processing with the fermentation broth supernatant, The main peak shifts back, which has hydrolysis effect on CSC (chondroitin sulfate C). The enzyme activities of the substrates FITC-HP, CSA-FITC, and CSC-FITC were determined to be 300FI/mL, 500FI/mL, and 450FI/mL.

Control: recombinant strain Pichia pastoris GS115/pPIC9K-CHASEQ was fermented and cultured by the same method, and it had no activity on heparin and chondroitin sulfate.

Example 3: Production of Chondroitin Sulfate Hydrolase Enzyme by Fermentation Tank Horizontal Fermentation

The YPD seed liquid cultured in shake flasks was inoculated into a 3L automatic fermenter (LiFlus GMBioTRON, Korea) with 10% inoculum, the temperature was controlled at 30°C, the pH was adjusted to 5.5 with 25% ammonia water, and the initial stirring speed was 500r· min ^-1 , the ventilation volume was 2.5L·min ^-1 , and the dissolved oxygen was maintained above 30%. Culture for about 28h, OD ₆₀₀ is about 70-80 until the glycerol is exhausted, add 50% (W·V ^-1 ) glycerol solution (containing 12mL·L ^-1 PTM1) in exponential flow, and the stirring speed is related to dissolved oxygen. 500-1000r·min ^-1 for feeding for 12h, stop feeding until the glycerol is exhausted again, OD ₆₀₀ is about 250 (dry weight is about 60.0g·L ^{-1 )} , and 100% methanol ( Containing 12mL·L ^-1 PTM1), the methanol concentration was controlled at 18.0g·L ^-1 , and the induction temperature was 30℃, 25℃, 22℃ and the stage control temperature induction strategy respectively. The highest 96h fermentation enzyme activity was 5.5% of the shake flask level. times, the enzyme activities of the substrates FITC-HP, CSA-FITC, and CSC-FITC were determined to be 1650FI/mL, 2750FI/mL, and 2475FI/mL.

Example 4: Purification and preparation of recombinant chondroitin sulfate hydrolase

The specific steps for purifying recombinant chondroitin sulfate hydrolase: using a 5 mL Ni column, and equilibrating the column with phosphate buffer (50 mmol·L ^-1 pH 7.0) for 10 column volumes. The fermentation supernatant was collected by centrifugation at 4°C, and 20 mL was loaded. Gradient elution of imidazole was used, and imidazole was prepared with the above-mentioned phosphate buffer, and the gradient was eluted according to 10 mmol·L ^-1 , 30 mmol·L ^-1 , and 200 mmol·L ^-1 , and the target protein was finally eluted. The eluted target protein was desalted and deimidazole, and stored at 4°C. Enzyme activity after purification: 650FI/mL for HP enzyme activity. As shown in FIG. 5 , as shown in SDS-PAGE, lane 1 represents chondroitin sulfate hydrolase in the supernatant of the fermentation broth, and lane 2 represents the band of chondroitin sulfate hydrolase after purification.

Example 5: Determination of enzyme properties

A series of pH values (4.0-11.0) were used to determine the optimal pH for the reaction of chondroitin sulfate hydrolase, namely sodium acetate buffer (20mM, pH 4.0-5.0), phosphate buffer (20mM, pH 5.0-7.0) ), Tris-HCl buffer (20 mM, pH 7.0-9.0) and Gly-NaOH buffer (20 mM, pH 9.0-11.0), the optimum pH was determined to be 4.5. The influence of different temperatures on the activity of chondroitin sulfate hydrolase was mainly determined by using the temperature range of 20℃-70℃, and the optimum temperature was 37℃.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

SEQUENCE LISTING

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Claims (9)

1. The recombinant bacterium for secretory expression of chondroitin sulfate hydrolase is characterized in that the recombinant bacterium is pichia pastorisPichia pastoris GS115Expressing chondroitin sulfate hydrolase derived from cattle as a host, wherein an amino acid sequence coded by the chondroitin sulfate hydrolase gene is shown as SEQ ID NO. 2.

2. The recombinant bacterium according to claim 1, wherein the nucleotide sequence of the chondroitin sulfate hydrolase gene is represented by SEQ ID No. 1.

3. The recombinant bacterium according to claim 1, wherein the recombinant bacterium is constructed by a method comprising: the chondroitin sulfate hydrolase gene with the nucleotide sequence of SEQ ID NO.1 is connected to an expression vector pPIC9K and then is transformed into pichia pastorisPichia pastoris GS115Screening correct transformant to obtain recombinant Pichia pastorisPichia pastoris GS115/pPIC9K-CHASE。

4. The recombinant bacterium according to claim 3, wherein the recombinant bacterium is constructed by adding a nucleotide sequence encoding a histidine tag to the chondroitin sulfate hydrolase gene.

5. A method for producing a chondroitin sulfate hydrolase, which comprises using the recombinant bacterium according to any one of claims 1 to 4.

6. The method of claim 5, wherein the chondroitin sulfate hydrolase is produced fermentatively in a fermentor using a methanol induction strategy.

7. The method according to claim 5, characterized in that the method comprises the following specific steps:

inoculating YPD seed liquid cultured in shake flask into 3L full-automatic fermentation tank at 10 vol%, controlling temperature at 25-30 deg.C, adjusting pH to 5-6 with 25% ammonia water, and stirring at initial rotation speed of 500-1000 r.min ^-1 The ventilation rate is 2-4 L.min ^-1 The dissolved oxygen is maintained at more than 30 percent to 35 percent, the culture is carried out for 20 to 40 hours ₆₀₀ 70-80 until the glycerol is exhausted, the mass volume ratio of the exponential flow and the addition is 30-50%, and the glycerol content is 12 mL.L ^-1 Stirring speed of glycerol solution of PTM1 is related to dissolved oxygen, and the control speed is 500-1000 r.min ^-1 Feeding for 12h, stopping feeding until glycerol is exhausted again, and OD ₆₀₀ About 250 g.L dry weight of about 60.0 g.L ^-1 The rotating speed is 600-1000 r.min ^-1 And 100% of the mixture is fed-batch and contains 12 mL.L ^-1 The methanol concentration of PTM1 is controlled at 18.0 g.L ^-1 The induction temperature adopts 30 ℃,25 ℃ and 22 ℃ and a stage control temperature induction strategy respectively.

8. The method for separating and purifying chondroitin sulfate hydrolase according to claim 1.

9. The separation and purification method according to claim 8, comprising the following steps: centrifuging the fermentation liquor, collecting supernatant, and sampling; using a Ni column, and using a phosphate buffer solution to balance the volume of the column by 10 columns; gradient eluting with imidazole prepared from the above phosphate buffer solution at a concentration of 10 mmol.L ^-1 ，30 mmol·L ^-1 ，200 mmol·L ^-1 Gradient elution.

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