阅读说明：本技术 一种发酵生产辛伐他汀合成用酰化酶的方法 (Method for producing acylase for simvastatin synthesis by fermentation ) 是由 史磊 申洋 陈东 朱维忠 于 2019-11-26 设计创作，主要内容包括：本发明提供了一种发酵生产辛伐他汀酰化酶的方法,本发明属于发酵工程技术领域,所述方法,包括以下步骤：1)将重组大肠杆菌CH39807种子液接种至发酵培养基中36～38℃进行菌体发酵；2)当发酵液的OD<Sub>600</Sub>值为20～35时,开始对发酵液进行降温,降温至25℃,当所述发酵液的温度≤30℃时,加入IPTG诱导表达10～12h后,收集菌体；3)重悬收集到的菌体后,进行均质、絮凝、固液分离,收集上清酶液。所述方法发酵周期短、发酵获得的酶活力单位高、产量稳定、提取简单。(The invention provides a method for producing simvastatin acylase by fermentation, belonging to the technical field of fermentation engineering, and the method comprises the following steps: 1) inoculating the recombinant Escherichia coli CH39807 seed solution into a fermentation culture medium, and fermenting the thallus at 36-38 ℃; 2) when OD of fermentation broth 600 When the value is 20-35 ℃, beginning to cool the fermentation liquor to 25 ℃, when the temperature of the fermentation liquor is less than or equal to 30 ℃, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression for 10-12 h, and collecting thalli; 3) after the collected cells were resuspended, homogenization, flocculation, and solid-liquid separation were carried out, and the supernatant enzyme solution was collected. The method has the advantages of short fermentation period, high unit of enzyme activity obtained by fermentation, stable yield and simple extraction.)

1. A method for producing simvastatin synthetase by fermentation, comprising the following steps:

1) inoculating the recombinant Escherichia coli CH39807 seed solution into a fermentation culture medium, and fermenting the thallus at 36-38 ℃;

2) when OD of fermentation broth₆₀₀When the value is 20-35 ℃, beginning to cool the fermentation liquor to 25 ℃, when the temperature of the fermentation liquor is less than or equal to 30 ℃, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression for 10-12 h, and collecting thalli;

3) resuspending the collected thallus, homogenizing, flocculating, separating solid from liquid, and collecting supernatant enzyme solution.

2. The method according to claim 1, wherein the fermentation medium in step 1) comprises the following components in mass per 10L by taking water as a solvent: 160-190 g of disodium hydrogen phosphate dodecahydrate, 60-80 g of potassium dihydrogen phosphate, 45-60 g of ammonium chloride, 10-20 g of sodium sulfate, 8-15 g of magnesium sulfate heptahydrate, 90-110 g of peptone, 40-60 g of yeast powder, 90-110 g of glycerol, 3-8 mL of defoaming agent and 0.1-0.6 g of ferric chloride.

3. The method as claimed in claim 1 or 2, wherein the inoculation amount of the recombinant Escherichia coli CH39807 seed solution in step 1) is 4-6%, and the OD of the recombinant Escherichia coli CH39807 seed solution₆₀₀1.0 to 1.4.

4. The method according to claim 1, wherein the dissolved oxygen content in the fermentation of the bacterial cells in step 1) is controlled to 20% or more.

5. The method according to claim 1, wherein the pH value of the bacterial fermentation process in the step 1) is controlled to be 5.9-6.5.

6. The method according to claim 1, wherein the glucose solution is supplemented during the fermentation of the biomass in step 1).

7. The method of claim 1, wherein the method is performed in a batch modeCharacterized in that the OD of the fermentation broth in the step 1) is₆₀₀And when the value is 30-32, cooling the fermentation liquor.

8. The method of claim 1, wherein the final concentration of IPTG in step 2) in the fermentation system is 0.08-0.12 mmol/L.

9. The method according to claim 1 or 5, wherein the pH value during the induced expression in step 2) is 6.9-7.2.

10. The method according to claim 1, wherein the pressure for homogenization in step 3) is 700-800 bar, the temperature for homogenization is 4-6 ℃, and the number of times of homogenization is 1-3.

Technical Field

The invention belongs to the technical field of fermentation engineering, and particularly relates to a method for producing simvastatin acylase by fermentation.

Background

The method for producing acylase by fermentation of recombinant engineering bacteria is different from the conventional method. As for the selected biological materials, the former contains a recombinant vector with an exogenous gene, and the latter is a single microbial cell; in view of fermentation technology, the aim of fermentation production of recombinant bacteria is to obtain a large amount of exogenous gene products and reduce the pollution of host cells with proteins as much as possible. The high-level expression of the exogenous gene not only relates to the mutual relation among a host, a vector and a cloned gene, but also is closely related to the environmental conditions of the host, the vector and the cloned gene, so that the production of the acylase by the conventional fermentation process has long period, and the activity of the produced acylase is low.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for producing simvastatin acylase by fermentation, wherein the method has the advantages of short fermentation period, high enzyme activity unit obtained by fermentation, stable yield and simple extraction.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a method for producing simvastatin synthetic acylase by fermentation, which comprises the following steps:

1) inoculating the recombinant Escherichia coli CH39807 seed solution into a fermentation culture medium, and fermenting the thallus at 36-38 ℃;

2) when OD of fermentation broth₆₀₀When the value is 20-35 ℃, beginning to cool the fermentation liquor to 25 ℃, when the temperature of the fermentation liquor is less than or equal to 30 ℃, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression for 10-12 h, and collecting thalli;

3) after the collected thalli are resuspended, homogenizing, flocculating and separating solid from liquid are carried out, and supernatant is collected to be enzyme liquid.

Preferably, the fermentation medium in step 1) takes water as a solvent, and each 10L of the fermentation medium comprises the following components by mass: 160-190 g of disodium hydrogen phosphate dodecahydrate, 60-80 g of potassium dihydrogen phosphate, 45-60 g of ammonium chloride, 10-20 g of sodium sulfate, 8-15 g of magnesium sulfate heptahydrate, 90-110 g of peptone, 40-60 g of yeast powder, 90-110 g of glycerol, 3-8 mL of defoaming agent and 0.1-0.6 g of ferric chloride.

Preferably, the inoculation amount of the recombinant Escherichia coli CH39807 seed solution in the step 1) is 4% -6%, and the OD of the recombinant Escherichia coli CH39807 seed solution₆₀₀1.0 to 1.4.

Preferably, the dissolved oxygen content in the fermentation of the bacterial cells in step 1) is controlled to be 20% or more.

Preferably, the pH value of the thallus fermentation process in the step 1) is controlled to be 5.9-6.5.

Preferably, glucose solution is supplemented in the process of fermenting the thallus in the step 1).

Preferably, the OD of the fermentation broth in step 1)₆₀₀And when the value is 30-32, cooling the fermentation liquor.

Preferably, the final concentration of the IPTG in the fermentation system in the step 2) is 0.08-0.12 mmol/L.

Preferably, the pH value in the induced expression process in the step 2) is 6.9-7.2.

Preferably, the homogenizing pressure in the step 3) is 700-800 bar, the homogenizing temperature is 4-6 ℃, and the homogenizing times are 1-3 times.

The invention has the beneficial effects that: the method for producing simvastatin synthesis acylase by fermentation provided by the invention comprises the following steps of performing thallus fermentation on recombinant escherichia coli CH39807 and then inducing the expression of the acylase; the production of the acylase is realized by controlling the parameters of the fermentation stage and the time of induction expression; the method disclosed by the invention is short in production period, within 24h in fermentation period, high in fermentation enzyme activity unit, the enzyme activity unit reaches 110-130U/mL, the yield of the acylase obtained by the method is stable, the extraction method is simple, and the industrial production requirement can be met.

Detailed Description

The invention provides a method for producing simvastatin synthetic acylase by fermentation, which comprises the following steps: 1) inoculating the recombinant Escherichia coli CH39807 seed solution into a fermentation culture medium, and fermenting the thallus at 36-38 ℃; 2) when OD of fermentation broth₆₀₀When the value is 20-35 ℃, beginning to cool the fermentation liquor to 25 ℃, when the temperature of the fermentation liquor is less than or equal to 30 ℃, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression for 10-12 h, and collecting thalli; 3) after the collected thalli are resuspended, homogenizing, flocculating and separating solid from liquid, and collecting supernatant as simvastatin synthesis acylase.

In the invention, recombinant Escherichia coli CH39807 seed liquid is inoculated into a fermentation medium for thallus fermentation at 36-38 ℃.

In the invention, the Escherichia coli CH39807 is recombinant Escherichia coli, and the recombinant Escherichia coli CH39807 is recombinant into exogenous genes pET-25a-PHSP1 and pET-11bcrt EEscherichia coli. In the invention, the inoculation amount of the seed liquid is preferably 4-6% in terms of inoculation volume percentage, and more preferably 5%; in the invention, the OD of the recombinant Escherichia coli CH39807 seed liquid₆₀₀Preferably 1.0 to 1.4, and more preferably 1.1 to 1.3. In the present invention, the seed solution is preferably an activated seed solution; in the present invention, the activated culture medium is preferably LB culture medium containing ampicillin, and the amount of activated inoculum is preferably 0.5% to 1.5%, more preferably 1.0%, by volume percentage. The activating temperature is preferably 37 ℃, and the activating time is preferably 3-4 h; the activation process is preferably accompanied by oscillation, and the rotation speed of the oscillation is preferably 6000 rpm.

In the present invention, the fermentation medium, using water as a solvent, preferably comprises the following components in every 10L: 160-190 g of disodium hydrogen phosphate dodecahydrate, 60-80 g of potassium dihydrogen phosphate, 45-60 g of ammonium chloride, 10-20 g of sodium sulfate, 8-15 g of magnesium sulfate heptahydrate, 90-110 g of peptone, 40-60 g of yeast powder, 90-110 g of glycerol, 3-8 mL of defoaming agent and 0.1-0.6 g of ferric chloride, and the more preferable components include 179g of disodium hydrogen phosphate dodecahydrate, 68g of potassium dihydrogen phosphate, 53g of ammonium chloride, 14g of sodium sulfate, 10g of magnesium sulfate heptahydrate, 100g of peptone, 50g of yeast powder, 100g of glycerol, 5mL of defoaming agent and 0.3g of ferric chloride. In the present invention, the type of the defoaming agent is not particularly limited, and a commercially available defoaming agent for fermentation, which is conventional in the art, may be used, and a polyether defoaming agent is preferable.

In the present invention, the dissolved oxygen amount in the fermentation of the bacterial cells is preferably controlled to 20% or more; in the invention, the dissolved oxygen is controlled by aerating and supplementing glucose solution; in the present invention, the concentration (w/v) of the glucose solution is preferably 55% to 65%, more preferably 60%. In the invention, the pH value of the thallus fermentation process is controlled to be 5.9-6.5. In the present invention, the control of the pH is preferably achieved by adding ammonia. The adding amount and time of the ammonia water are not particularly limited, and the pH value of the fermentation system is preferably controlled to be 5.9-6.5.

In the present invention, the fermentation of the bacterial cells is preferably carried out in a fermentor; the volume of the fermentation tank is not particularly limited, and the specific volume of the fermentation tank is determined according to the fermentation requirement. When the fermentation tank is used for fermenting thalli, the elimination tank is preferably carried out before the thalli are fermented, the specific parameters of the elimination tank are not particularly limited, and the conventional elimination tank parameters are adopted; in the specific implementation process of the invention, the tank elimination preferably comprises the following steps: pouring the fermentation medium into a fermentation tank, and sterilizing at 121 ℃ for 30 min; after cooling, controlling the temperature to be 36-38 ℃, setting the air ventilation ratio to be 1:1, setting the rotating speed to be 180-220 rpm, and calibrating the dissolved oxygen to be 100% after stabilizing for 20 min; controlling the pH value of the fermentation medium to be 5.9-6.1. According to the invention, after the tank disinfection is finished, inoculation is carried out; in the invention, the inoculation is preferably flame inoculation, ampicillin is preferably added in the process of the inoculation, and the final concentration of ampicillin in the fermentation system is preferably 45-55 mg/L, and more preferably 50 mg/L.

In the present invention, when OD of fermentation broth is used₆₀₀And when the value is 20-35 ℃, cooling the fermentation liquor to 25 ℃, adding IPTG (isopropyl thiogalactoside) to induce expression for 10-12 h when the temperature of the fermentation liquor is less than or equal to 30 ℃, and collecting the thalli. In the present invention, it is preferable that OD of the fermentation broth is used₆₀₀And when the value is 30-32, cooling the fermentation liquor. In the invention, the final concentration of the IPTG in a fermentation system is preferably 0.08-0.12 mmol/L, and more preferably 0.1 mmol/L. In the invention, the pH value in the induction expression process is preferably 6.9-7.2, and more preferably 7.0. In the invention, after the induction expression, thalli are collected. In the invention, the method for collecting the thalli is preferably centrifugation, and the rotation speed of the centrifugation is preferably 5000-7000 rpm, more preferably 5500-6500 rpm, and most preferably 6000 rpm; the centrifugation time is preferably 10-20 min, and more preferably 15 min; the centrifugation temperature is preferably 3-5 ℃, and more preferably 4 ℃.

In the invention, after the thalli are collected, the collected thalli are resuspended, and then are homogenized, flocculated and separated from solid and liquid, and the supernatant is collected to be the enzyme liquid. In the present invention, the resuspension is preferably performed using a 4 ℃ pre-cooled PBNa buffer solution; the quality of the PBNa buffer solution is preferably 2.5-3.5 times of the wet weight of the collected thalli; the pH value of the PBNa buffer solution is preferably 8.5; the concentration of the PBNa buffer solution is preferably 40-60 mmol/L, and more preferably 50 mmol/L. Homogenizing after the heavy suspension, wherein the homogenizing pressure is preferably 700-800 bar, and more preferably 750 bar; the homogenizing temperature is preferably 4-6 ℃, and more preferably 5 ℃; the homogeneous temperature is preferably controlled by a cryogenic coolant circulating pump; the number of homogenization is preferably 1 to 3, and more preferably 2.

After homogenization, flocculation is carried out, a flocculating agent used for flocculation is preferably benzalkonium chloride, and the concentration of the benzalkonium chloride is preferably 30%; the addition amount of the benzalkonium chloride is preferably 50-60 ml/L, and more preferably 55 ml/L. In the invention, the flocculation temperature is preferably 36-38 ℃, and more preferably 37 ℃; the flocculation time is preferably 50-70 min, and more preferably 60 min; stirring is carried out in the flocculation process, and the rotating speed of the stirring is not particularly limited in the invention.

In the present invention, after the flocculation, solid-liquid separation is preferably performed. The solid-liquid separation method is preferably centrifugation, and the rotation speed of the centrifugation is preferably 5000-7000 rpm, more preferably 5500-6500 rpm, and most preferably 6000 rpm; the centrifugation time is preferably 10-20 min, and more preferably 15 min; the centrifugation temperature is preferably 3-5 ℃, and more preferably 4 ℃. In the invention, after solid-liquid separation, the collected liquid-phase component is the enzyme solution.

After the solid-liquid separation, the method preferably further comprises a precipitation purification step of the enzyme solution, wherein the reagent for precipitation purification is preferably ammonium sulfate; the amount of ammonium sulfate to be used is preferably calculated from the saturated ammonium sulfate amount obtained in the calculation table (0 ℃ C.) for the volume of the enzyme solution and the saturation of the ammonium sulfate solution. In the present invention, the temperature of the precipitation purification is preferably 0 ℃; the precipitation purification is preferably carried out on an ice bath; preferably, stirring is carried out along with the precipitation purification process, wherein the rotation speed of the stirring is preferably 100-200 rpm, and more preferably 150 rpm; the stirring time is preferably 1.5-2.5 h, and more preferably 2.0 h. The present invention preferably performs centrifugation after the purification of the precipitate, and collects the precipitate as a purified acylase. In the invention, the rotation speed of the centrifugation is preferably 11000-13000 rpm, and more preferably 12000 rpm; the centrifugation time is preferably 10-20 min, and more preferably 15 min; the centrifugation temperature is preferably 3-5 ℃, and more preferably 4 ℃.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.