CN105219748A - A kind of fermentation process of Escherichia coli fermentation Restruction arylsulfatase - Google Patents

Abstract

The invention discloses a fermentation method for Escherichia coli fermentation to produce recombinant aryl sulfatase. Firstly, the recombinant E. coli strain obtained by constructing pET-28a(+) and aryl sulfatase gene atsA gene includes: Activation and preparation, shake flask fermentation, tank fermentation and pilot-scale scale-up fermentation; on the basis of shake flask fermentation, the present invention carried out fermentation and cultivation of the screened recombinant Escherichia coli in a fermentation tank, and studied its production in the fermentation tank. Enzyme regulation, effectively improving the yield of recombinant arylsulfatase. Finally, the pilot-scale fermentation of arylsulfatase produced by recombinant Escherichia coli was carried out, which provided important technical parameter guidance for future large-scale production.

Description

A kind of fermentation method of Escherichia coli fermentation production recombinant arylsulfatase

technical field

The invention relates to the field of biotechnology, in particular to a fermentation method for Escherichia coli fermentation to produce recombinant arylsulfatase.

Background technique

Since the emergence of gene recombination technology, various prokaryotic and eukaryotic expression systems have been developed to produce recombinant foreign proteins to obtain proteins that are difficult to obtain or unstable under natural conditions to meet the needs of basic research and clinical application research. The Escherichia coli expression system has become one of the most commonly used exogenous protein gene expression systems after decades of development. Short and other advantages become people's preferred strains for cloning and expressing foreign genes.

IPTG is a very efficient lactose promoter inducer, which induces the production of sulfatase in genetically engineered bacteria. It is potentially toxic and has a certain inhibitory effect on bacterial growth, and is relatively expensive. At the same time, as a cheap disaccharide, lactose is not only non-toxic, but also has the effect of inducing the lactose operon, so it may become an inducer to replace IPTG. Compared with IPTG, lactose is cheap and harmless to bacteria, and lactose can also be used as a carbon source for E. coli fermentation to promote the growth of E. coli, so lactose makes it more valuable in large-scale industrial production.

At present, the main method for removing sulfate groups from agar is the alkali method, which reacts violently with strong alkali and causes huge environmental problems. The enzymatic modification of agar has the advantages of mild reaction conditions and no pollution. Therefore, the use of arylsulfatase-assisted extraction of agar will gradually replace the traditional alkaline method as the best method for agar preparation. However, the current research work on the effect of arylsulfatase on agar is still insufficient and there are problems such as less enzyme solution and low enzyme activity, which restricts the use of arylsulfatase to remove sulfate on agar to improve gel strength. In view of this, the inventors researched and designed a fermentation method for the production of recombinant arylsulfatase by Escherichia coli fermentation, and this case arose from this.

Contents of the invention

The object of the present invention is to provide a kind of fermentation method of Escherichia coli fermentation production recombinant aryl sulfatase, the present invention first transforms the host of the recombinant vector plasmid that utilizes pET-28a (+) and aryl sulfatase gene atsA gene to construct After optimizing the fermentation induction conditions, the unit yield of Escherichia coli fermentation to produce recombinant arylsulfatase can be effectively improved.

Another object of the present invention is to provide a method for carrying out Escherichia coli fermentation recombinant arylsulfatase using the above-mentioned optimized conditions, through the step-by-step amplification culture steps of seed activation, shake flask fermentation, tank fermentation and pilot scale-up fermentation, determine the optimum Optimal fermentation conditions, and then improve the unit yield of recombinant arylsulfatase in the final fermentation liquid.

In order to achieve the above object, the technical solution of the present invention to solve its technical problems is:

Firstly, the host bacteria of the recombinant vector plasmid obtained by constructing pET-28a(+) and the arylsulfatase gene atsA gene, and using LB medium to cultivate the host bacteria.

The LB medium is: bacteriological peptone 10g/l, yeast powder 5g/l, NaCl 10g/l, adjust the pH of the medium to 7.4, and sterilize at 121°C for 20min.

The present invention also relates to a fermentation method for producing recombinant arylsulfatase by said recombinant Escherichia coli using said LB medium, comprising the following steps:

Activation and preparation of seeds: Inoculate the above-mentioned recombinant E. coli strains into LB medium, culture at 37°C for 12 hours, and obtain shake flask seed liquid;

Shake flask fermentation: Inoculate the shake flask seed liquid into the shake flask containing LB medium, culture at 37°C until the OD ₆₀₀ grows to 0.6, add the inducer lactose to make the lactose concentration reach 4mg/ml, and then induce culture at 21°C 24-36h, obtain the fermentation broth containing recombinant arylsulfatase;

Fermentation on tanks: Inoculate the shake flask seed liquid into a 5L fermenter with LB medium, culture at 37°C until the OD ₆₀₀ grows to 0.6, add the inducer lactose to make the lactose concentration reach 4mg/ml, and then induce culture at 21°C 24-36h, obtain the fermentation broth containing recombinant arylsulfatase;

Pilot-scale scale-up fermentation: Inoculate the shake flask seed solution into 20L and 200L fermenters with LB medium, culture at 37°C until the OD ₆₀₀ grows to 0.6, add the inducer lactose to make the lactose concentration reach 4mg/ml, and then in 21 Induction culture at ℃ for 12-36 hours to obtain a fermentation broth containing recombinant arylsulfatase;

As a preferred mode of the embodiment, in the activation and preparation steps of the seeds, the glycerol tube strains stored at -20°C were thawed and placed on a plate containing the strain LB medium, and cultured at 37°C for 24 hours to obtain activated recombinant large intestine Bacillus species; then pick positive single colonies from the plate and place them in a 250mL shake flask filled with 50mL liquid LB medium, ferment at 37°C and 180r/min for 12-36h to obtain shake flask seed liquid.

As a preferred mode of the embodiment, the shake flask is fermented, and the cultured shake flask seed solution is inserted into a 250mL shake flask equipped with 50mL of LB medium at an inoculation amount of 1%, cultivated at 37°C until the _OD600 grows to 0.6, The inducer lactose was added to make the lactose concentration reach 4mg/ml, and then induced and cultivated at 21°C and 180r/min for 30h.

As a preferred mode of the embodiment, for fermentation on the tank, the cultivated shake flask seed liquid is inserted into a 5L fermenter equipped with 3.5LLB medium with an inoculation amount of 1%, and cultivated to OD at 37°C and 300r/min ₆₀₀ grows to 0.6, adding inducer lactose to make the lactose concentration reach 4mg/ml, and then inducing fermentation at 21°C for 24 hours to obtain a fermentation broth containing recombinant arylsulfatase.

As a preferred mode of the embodiment, in the scaled-up fermentation of the pilot scale, the cultured shake flask seed solution is inserted into a 20L fermenter equipped with 12LLB medium with an inoculation amount of 1%, and cultivated to OD at 37°C and 300r/min ₆₀₀ grows to 0.6, add inducer lactose to make the lactose concentration reach 4mg/ml, and then induce fermentation at 21°C for 12h to obtain a fermentation broth containing recombinant arylsulfatase.

As a preferred mode of the embodiment, in the scaled-up fermentation of the pilot scale, the shake flask seed solution was inoculated into 20L and 200L fermenters equipped with LB medium, cultivated at 37°C until the _OD600 grew to 0.6, and the inducer lactose was added to make The lactose concentration reaches 4 mg/ml, and then cultured at 21° C. and 200 r/min for 18 hours to obtain a fermentation broth containing recombinant arylsulfatase.

After the present invention adopts the above technical scheme, it has the following beneficial effects: on the basis of shake flask fermentation, the present invention carries out fermentation and cultivation of the screened recombinant Escherichia coli in a fermenter, and studies its enzyme production law in the fermenter, effectively Increased production of recombinant arylsulfatase. Finally, a pilot scale-up fermentation of arylsulfatase produced by recombinant Escherichia coli was carried out, which provided important technical parameter guidance for future large-scale production

Description of drawings

Fig. 1 Escherichia coli produces the shaking flask fermentation curve of recombinant arylsulfatase;

Fig. 2 Escherichia coli produces the tank fermentation curve of recombinant arylsulfatase;

Fig. 3 Escherichia coli produces the fermentation curve on the 20L tank of recombinant arylsulfatase;

Fig. 4 Escherichia coli produces the fermentation curve on the 200L tank of recombinant arylsulfatase.

detailed description

The detection method adopted in the following examples:

Determination of biomass: Evenly draw 3.0ml of fermentation broth, draw 700μL and add it to a cuvette, use LB medium as a blank, and select OD ₆₀₀ to measure the absorbance value.

Determination of recombinant arylsulfatase activity: the reaction system included 80 μL 20 mmol/L p-nitrophenyl potassium sulfate (p-NPS, prepared with 50 mmol/LTris-HCl (pH7.0) buffer), 20 μL enzyme solution (concentration of 6 μg /mL), after incubating at 50°C for 10 min, 25 μL of 1 mol/L NaOH was added to terminate the reaction, and the volume was made up to 1 mL with pre-cooled distilled water, and the absorbance was measured at 410 nm. The activity (U) of arylsulfatase is defined as the amount of enzyme required to catalyze the production of 1 μmol of p-nitrophenol (p-NP) per minute under the above conditions.

Induction conditions for Escherichia coli fermentation to produce recombinant arylsulfatase, including induction period, type and amount of inducer, temperature and induction time during induction;

The induction period of the strain is: when the OD ₆₀₀ of the bacteria grows to 0.6, it is the optimal condition, and the induction is carried out under this condition;

The type and amount of the inducer are as follows: use lactose instead of IPTG for induction, so that the final concentration of lactose reaches 4 mg/ml;

The temperature at the time of induction is: induction is carried out at 21°C, the enzyme activity is the largest, and the recombinant Escherichia coli is relatively stable;

All induction times are: the maximum enzyme activity can be obtained after induction for 24 hours, and the enzyme activity will decrease as time increases.

Embodiment 1: Recombinant Escherichia coli induces the optimization of producing recombinant sulfatase inducing conditions

(1) After thawing the glycerol tube strains stored at -20°C, they were placed on a plate containing strain LB medium, and cultured in a 37°C incubator for 12 hours to obtain activated recombinant Escherichia coli strains. The LB medium is: bacteriological peptone 10g/l, yeast powder 5g/l, NaCl 10g/l, pH adjusted to 7.4, sterilized at 121°C for 20min;

(2) Pick the cultured strains from the plate and place them in a 250 mL shake flask filled with 50 mL of LB medium, and ferment at 37° C. at 180 r/min for 12 hours to obtain shake flask seed liquid. ;

(3) Insert the cultured shake flask seed liquid into a 250mL shake flask equipped with 50mL LB medium with a 1% inoculum size. The LB medium contains 50 μg/mL of kanamycin, and the culture condition is a temperature of 37 °C, culture on a shaker until OD ₆₀₀ = 0.2-1.0, then add lactose to a final concentration of 0.05-6 mg/mL, induce at 15-28 °C for 12-28 h, and obtain a fermentation broth.

(4) Evenly take 3.0 mL of fermented liquid containing recombinant arylsulfatase, centrifuge to collect the fermented E.coliBL21 (DE3) thalline, resuspend the precipitate in the dissolution buffer and use ultrasonic wave to break the wall; the dissolution buffer The formula can be: 50mmol/LTris-HCl, pH7.0.

(5) The lysed suspension in (4) was refrigerated and centrifuged at 15,000×g at 4° C. for 20 min, and the supernatant was collected to obtain the crude recombinant arylsulfatase enzyme solution.

Embodiment 2: Shake flask fermentation of recombinant escherichia coli produces recombinant arylsulfatase

(1) After thawing the glycerol tube strains stored at -20°C, they were placed on a plate containing strain LB medium, and cultured in a 37°C incubator for 12 hours to obtain activated recombinant Escherichia coli strains. The LB medium is: bacteriological peptone 10g/l, yeast powder 5g/l, NaCl 10g/l, pH adjusted to 7.4, sterilized at 121°C for 20min;

(2) Pick the cultured strains from the plate in a 250mL shake flask equipped with 50mLLB medium, ferment at 37°C and 180r/min for 12h, and obtain the shake flask seed liquid;

(3) Insert the cultured shake flask seed liquid into a 250mL shake flask equipped with 50mL LB medium with a 1% inoculum size. The LB medium contains 50 μg/mL of kanamycin, and the culture condition is a temperature of 37 °C, cultured on a shaker until OD ₆₀₀ =0.6, then added lactose to a final concentration of 4 mg/mL, induced at 21 °C for 24 hours, and obtained a fermentation broth.

(4) Evenly take 3.0 mL of fermented liquid containing recombinant arylsulfatase, centrifuge to collect the fermented E.coliBL21 (DE3) thalline, resuspend the precipitate in the dissolution buffer and use ultrasonic wave to break the wall; the dissolution buffer The formula can be: 50mmol/LTris-HCl, pH7.0.

(5) The lysed suspension in (4) was refrigerated and centrifuged at 15,000×g at 4° C. for 20 min, and the supernatant was collected to obtain the crude recombinant arylsulfatase enzyme solution.

(6) Samples were taken to detect changes in the biomass and activity of recombinant arylsulfatase during the fermentation process, and the results are shown in Figure 1. The enzyme activity reached the maximum value of 165.41U/mL when fermented to 30h.

Embodiment 3: Fermentation production recombinant arylsulfatase on the tank of Escherichia coli

(1) After thawing the glycerol tube strains stored at -20°C, they were placed on a plate containing strain LB medium, and cultured in a 37°C incubator for 12 hours to obtain activated recombinant Escherichia coli strains. The LB medium is: bacteriological peptone 10g/l, yeast powder 5g/l, NaCl 10g/l, pH adjusted to 7.4, sterilized at 121°C for 20min;

(2) Pick the cultured strains from the plate in a 250mL shake flask equipped with 50mLLB medium, ferment at 37°C and 180r/min for 12h, and obtain the shake flask seed liquid;

(3) Insert the cultured shake flask seed solution with 1% inoculum size into a 5L fermentor equipped with 3.5LLB medium, the LB medium contains 50 μg/mL of kanamycin, and the culture conditions are temperature At 37°C, culture with shaking until OD ₆₀₀ =0.6, then add lactose to a final concentration of 4 mg/mL, induce at 21°C for 36 hours, and obtain a fermentation broth.

(4) Evenly take 3.0 mL of fermented liquid containing recombinant arylsulfatase, centrifuge to collect the fermented E.coliBL21 (DE3) thalline, resuspend the precipitate in the dissolution buffer and use ultrasonic wave to break the wall; the dissolution buffer The formula can be: 50mmol/LTris-HCl, pH7.0.

(5) The lysed suspension in (4) was refrigerated and centrifuged at 15,000×g at 4° C. for 20 min, and the supernatant was collected to obtain the crude recombinant arylsulfatase enzyme solution.

(6) Samples were taken to detect changes in the biomass and activity of recombinant arylsulfatase during the fermentation process, and the results are shown in Figure 2. The enzyme activity reached the maximum value of 162.12U/mL when fermented to 24h

Embodiment 4: Escherichia coli fermentation produces recombinant arylsulfatase pilot-scale scale-up fermentation

(1) After thawing the glycerol tube strains stored at -20°C, they were placed on a plate containing strain LB medium, and cultured in a 37°C incubator for 12 hours to obtain activated recombinant Escherichia coli strains. The LB medium is: bacteriological peptone 10g/l, yeast powder 5g/l, NaCl 10g/l, pH adjusted to 7.4, sterilized at 121°C for 20min;

(2) Pick the cultured strains from the plate in a 250mL shake flask equipped with 50mLLB medium, ferment at 37°C and 180r/min for 12h, and obtain the shake flask seed liquid;

(3) Insert the cultured shake flask seed solution with 1% inoculum size into a 20L fermenter equipped with 12LLB medium, the LB medium contains 50 μg/mL of kanamycin, and the culture condition is a temperature of 37 ℃, shaking culture to OD ₆₀₀ =0.6, then adding lactose to a final concentration of 4 mg/mL, and inducing at 21 ℃ for 24 hours to obtain a fermentation broth.

(4) Evenly take 3.0 mL of fermented liquid containing recombinant arylsulfatase, centrifuge to collect the fermented E.coliBL21 (DE3) thalline, resuspend the precipitate in the dissolution buffer and use ultrasonic wave to break the wall; the dissolution buffer The formula can be: 50mmol/LTris-HCl, pH7.0.

(5) The lysed suspension in (4) was refrigerated and centrifuged at 15,000×g at 4° C. for 20 min, and the supernatant was collected to obtain the crude recombinant arylsulfatase enzyme solution.

(6) Samples were taken to detect changes in the biomass and activity of the recombinant arylsulfatase during the fermentation process, and the results are shown in FIG. 3 . The enzyme activity reached the maximum value of 98.71U/mL after 12 hours of fermentation.

Example 5: Pilot scale-up fermentation of recombinant arylsulfatase produced by Escherichia coli fermentation

(1) After thawing the glycerol tube strains stored at -20°C, they were placed on a plate containing strain LB medium, and cultured in a 37°C incubator for 12 hours to obtain activated recombinant Escherichia coli strains. The LB medium is: bacteriological peptone 10g/l, yeast powder 5g/l, NaCl 10g/l, pH adjusted to 7.4, sterilized at 121°C for 20min;

(2) Pick the cultured strains from the plate and place them in a 250 mL shake flask filled with 50 mL of LB medium, and ferment at 37° C. at 180 r/min for 12 hours to obtain shake flask seed liquid.

(3) Insert the cultivated shake flask seed liquid with 1% inoculum size into a 200L fermenter with 12LLB medium, cultivate for 12h and then transfer to the 200L fermenter with 120LLB medium for LB cultivation The base contains 50 μg/mL of kanamycin, the culture condition is 37°C, shake culture to OD ₆₀₀ =0.6, then add lactose to a final concentration of 4 mg/mL, induce at 21°C for 24 hours, and obtain a fermentation broth.

(4) Evenly take 3.0 mL of fermented liquid containing recombinant arylsulfatase, centrifuge to collect the fermented E.coliBL21 (DE3) thalline, resuspend the precipitate in the dissolution buffer and use ultrasonic wave to break the wall; the dissolution buffer The formula can be: 50mmol/LTris-HCl, pH7.0.

(5) The lysed suspension in (4) was refrigerated and centrifuged at 15,000×g at 4° C. for 20 min, and the supernatant was collected to obtain the crude recombinant arylsulfatase enzyme solution.

(6) Sampling was performed to detect the changes in biomass and activity of recombinant arylsulfatase in the fermentation process, and the results are shown in Figure 4. The enzyme activity reached the maximum value of 69.19U/mL after 18 hours of fermentation.

On the basis of shake flask fermentation, the present invention carries out fermentation and cultivation of recombinant escherichia coli in a fermenter, studies the law of its enzyme production in the fermenter, and effectively improves the yield of recombinant aryl sulfate. Finally, a pilot scale-up test was carried out on the recombinant arylsulfatase produced by Escherichia coli, which provided important technical parameter guidance for future large-scale production.

All deformations that can be derived or associated directly from the disclosure content of the present invention by those skilled in the art should be considered as the protection scope of the present invention.

Claims (6)

1. a kind of fermentation method that Escherichia coli fermentation produces recombinant arylsulfatase, at first utilize the recombinant Escherichia coli strain that pET-28a (+) and arylsulfatase gene atsA gene construction obtains, and adopt LB culture medium to The host bacterium is cultivated; it is characterized in that: it also includes the following steps: Step 1. Activation and preparation of seeds: Inoculate the above-mentioned recombinant E. coli strains into the LB medium, culture at 37°C for 12 hours, and obtain shake flask seed liquid; Step 2. Shake flask fermentation: Inoculate the shake flask seed liquid into the shake flask equipped with LB medium, culture at 37°C until the OD ₆₀₀ grows to 0.6, add the inducer lactose to make the lactose concentration reach 4mg/ml, and then incubate at 21 Induction culture at ℃ for 24-36 hours to obtain a fermentation broth containing recombinant arylsulfatase; Step 3. Fermentation on the tank: Inoculate the shake flask seed liquid into a 5L fermenter equipped with LB medium, cultivate at 37°C until the OD ₆₀₀ grows to 0.6, add the inducer lactose to make the lactose concentration reach 4mg/ml, and then incubate at 21 Induction culture at ℃ for 24-36 hours to obtain a fermentation broth containing recombinant arylsulfatase; Step 4. Pilot scale-up fermentation: inoculate the shake flask seed solution into 20L and 200L fermenters equipped with LB medium, cultivate at 37°C until the OD ₆₀₀ grows to 0.6, add the inducer lactose to make the lactose concentration reach 4mg/ml, Then induce culture at 21° C. for 12-36 hours to obtain a fermentation broth containing the recombinant arylsulfatase. 2. a kind of Escherichia coli fermentation as claimed in claim 1 produces the fermentation method of recombinant arylsulfatase, it is characterized in that: the activation of described seed and preparation step, after the glycerol tube bacterial classification that -20 ℃ preserves is thawed Inoculate on a plate containing strain LB medium, culture at 37°C for 24 hours to obtain activated recombinant Escherichia coli strains; then pick positive single colonies from the plate and place them in a 250mL shake flask containing 50mL liquid LB medium, 37 °C, 180r/min fermentation for 12-36h, to obtain shake flask seed liquid. 3. a kind of Escherichia coli fermentation as claimed in claim 1 produces the fermentation method of recombinant arylsulfatase, it is characterized in that: described shake flask fermentation, the shake flask seed liquid that cultivates is received with 1% inoculum size Put them into a 250mL shake flask filled with 50mL of LB medium, culture at 37°C until the OD ₆₀₀ grows to 0.6, add the inducer lactose to make the lactose concentration reach 4mg/ml, and then induce culture at 21°C and 180r/min for 30h. 4. a kind of Escherichia coli fermentation as claimed in claim 1 produces the fermentative method of recombinant arylsulfatase, it is characterized in that: ferment on described tank, the shake flask seed liquid that cultivates is connected with 1% inoculum size Put it into a 5L fermenter equipped with 3.5LLB medium, cultivate at 37°C and 300r/min until the _OD600 grows to 0.6, add the inducer lactose to make the lactose concentration reach 4mg/ml, and then induce fermentation at 21°C for 24h to obtain Fermentation broth of recombinant arylsulfatase. 5. a kind of Escherichia coli fermentation as claimed in claim 1 produces the fermentative method of recombinant arylsulfatase, it is characterized in that: described pilot plant scale-up fermentation, with the inoculum size of 1% with the well-cultivated shake flask seed liquid Insert it into a 20L fermenter equipped with 12LLB medium, cultivate at 37°C and 300r/min until the OD ₆₀₀ grows to 0.6, add the inducer lactose to make the lactose concentration reach 4mg/ml, and then induce fermentation at 21°C for 12h to obtain Fermentation broth of recombinant arylsulfatase. 6. a kind of escherichia coli fermentation as claimed in claim 1 produces the fermentative method of recombinant arylsulfatase, it is characterized in that: described pilot plant enlarges fermentation, shake flask seed liquid is inoculated to the 20L that LB substratum is housed and in a 200L fermenter, cultivated at 37°C until the OD ₆₀₀ grows to 0.6, added the inducer lactose to make the lactose concentration reach 4mg/ml, and then induced and cultured at 21°C and 200r/min for 18h to obtain recombinant arylsulfatase fermentation broth.