阅读说明：本技术 一种头孢菌素c酰化酶的发酵方法 (Fermentation method of cephalosporin C acylase ) 是由 何锡林 张云辉 李琨 韩原亮 蒋成辉 马利萍 王文静 尹凡钦 原振刚 于 2020-06-23 设计创作，主要内容包括：本发提供了一种头孢菌素C酰化酶的发酵方法,它包括补加乳糖和甘油的工艺；所述补加乳糖的工艺是：培养8h且40≤OD-(600)≤50时一次性补入1％乳糖,在6-60h随补料(甘油)流加1％的乳糖；所述补加甘油的工艺是：6-8h,流加补入甘油100L/h；8-18h,流加补入甘油75L/h；18-30h,流加补入甘油85L/h；30-40h,流加补入甘油90L/h；40-50h,流加补入甘油95L/h；50-60h,流加补入甘油100L/h。本发明的发酵方法得到的发酵产物酶活超过170U/ml,应用前景良好。(The invention provides a fermentation method of cephalosporin C acylase, which comprises a process of supplementing lactose and glycerol; the lactose supplementing process comprises the following steps: culturing for 8h and OD not less than 40 600 1% lactose is added at one time when the temperature is less than or equal to 50, and 1% lactose is added along with the feeding (glycerol) in 6-60 h; the process for supplementing the glycerol comprises the following steps: adding 100L/h of supplementary glycerol in a flowing manner for 6-8 h; adding supplemental glycerol for 75L/h after 8-18 h; adding supplemental glycerol at a ratio of 85L/h for 18-30 h; adding 90L/h of supplementary glycerol in a flowing manner for 30-40 h; adding 95L/h of supplementary glycerol in a flowing way for 40-50 h; adding 100L/h of glycerol in the feed for 50-60 h. The fermentation product obtained by the fermentation method has the enzyme activity of over 170U/ml and has good application prospect.)

1. A method for fermenting cephalosporin C acylase, comprising:

(1) seed transferring: transferring the seed liquid into a fermentation culture medium;

(2) fermentation: fermenting for 60h, maintaining pH value at 6.8-7.2, controlling fermentation culture temperature at 37 + -1 deg.C for 0-7h, and fermenting at 25 + -1 deg.C for 8-60 h; feeding materials during the period, adjusting the stirring speed and controlling the dissolved oxygen amount to be 20-30%;

the fermentation medium in the step (1) contains: 43.0-48.0g/l of yeast extract, 3.0-8.0g/l of sodium chloride, 8.0-13.0g/l of disodium hydrogen phosphate, 4.17-4.22g/l of dipotassium hydrogen phosphate, 0.71-1.10g/l of sodium sulfate, 1.78-2.00g/l of magnesium sulfate, 2.68-2.98g/l of ammonium sulfate, 19.4-20.8g/l of glycerol, 0.015-0.020g/l of ferric chloride and 0.42-0.50g/l of defoaming agent;

supplementing lactose and glycerol for the feeding in the step (2);

the lactose supplementing process comprises the following steps: culturing for 8h and OD not less than 40₆₀₀When the volume is less than or equal to 50, lactose accounting for 1 percent +/-0.1 percent of the volume of the fermentation culture medium is added at one time, and lactose accounting for 1 percent +/-0.1 percent of the volume of the fermentation culture medium is fed along with glycerol in the 6 th to 60 th hours;

the process for supplementing the glycerol comprises the following steps: feeding 100 +/-1L/h of supplementary glycerol in the 6 th to 8 th hours; feeding glycerol at a ratio of 75 + -1L/h in 8-18 h; adding supplementary glycerol 85 + -1L/h in 18-30 h; feeding 90 +/-1L/h of supplementary glycerol in 30-40 h; feeding 95 +/-1L/h of glycerol at 40-50 h; adding 100 + -1L/h of glycerol into the feed stream at 50-60 h.

2. The method of claim 1, wherein: in the step (2), the fermentation culture temperature is controlled to be 37 ℃ in 0-7h, and the fermentation temperature is controlled to be 25 ℃ in 8-60 h.

3. The method of claim 1, wherein:

the lactose supplementing process comprises the following steps: culturing for 8h and OD not less than 40₆₀₀Adding lactose 1% of the fermentation medium at one time when the volume is less than or equal to 50, and adding glycerol in 6-60hLactose was fed in an amount of 1% by volume of the fermentation medium.

4. The method of claim 1, wherein:

the process for supplementing the glycerol comprises the following steps: feeding 100L/h of supplementary glycerol in the 6 th to 8 th hours; feeding glycerol for 75L/h in the 8 th-18 th hour; adding 85L/h of supplementary glycerol in the flow from 18h to 30 h; feeding 90L/h of supplementary glycerol in the 30 th-40 th hour; feeding 95L/h of supplementary glycerol in the 40-50h period; adding 100L/h of glycerol into the feed stream from 50h to 60 h.

5. The method according to claim 1, wherein the amount of the inoculated cells in step (1) is 1.25% (v/v).

6. The method according to any one of claims 1 to 5, wherein step (1) is preceded by a seed preparation step comprising:

a. preparing shake flask bacterium suspension: taking out the glycerol tube, naturally thawing, inoculating to a culture medium in a sterile shake flask, and shake culturing at 37 deg.C and pH of 7.0-7.2 for 15-17 hr;

b. preparing first-grade seeds: b, inoculating the shake flask bacterial suspension in the step a to a primary culture medium in a primary seeding tank, and culturing for 5-6h at the temperature of 37 ℃ and at the pH of 6.9-7.5;

c. preparing second-level seeds: b, inoculating the primary seeds in the step b into a secondary culture medium in a secondary seed tank, and culturing for 5-6h at the temperature of 37 ℃ and at the pH of 6.9-7.5;

the culture medium in the step a contains 10g/l of peptone, 5g/l of yeast extract and 10g/l of sodium chloride.

7. The method of claim 6, wherein:

the primary culture medium in the step b contains peptone 12g/l, yeast extract 6g/l and sodium chloride 10 g/l.

8. The method of claim 6, wherein:

the secondary culture medium in the step c contains peptone 12g/l, yeast extract 6g/l and sodium chloride 10 g/l.

9. The fermentation process of claim 1, wherein step (3) is followed by a step of detecting enzyme activity.

10. The fermentation method according to claim 7, wherein the enzyme activity detection step is: enriching fermentation thalli, adding water to a constant volume to an original volume, homogenizing to obtain a homogenized solution, and measuring enzyme activity by using a potentiometric titration method by using cephalosporin C as a substrate.

Technical Field

The invention relates to the field of fermentation engineering, in particular to a fermentation method of cephalosporin C acylase.

Background

7-aminocephalosporanic acid (7-ACA) is a key intermediate for synthesizing cephalosporin antibiotics, and cephalosporin antibiotics with different properties are formed by connecting different side chains on active groups of the cephalosporin antibiotics. The developed countries of the medicine industry all pay attention to the research and production of 7-ACA in the world.

The one-step enzyme method is a common technology for producing 7-ACA. Cephalosporin C acylase (CPC acylase, CCA) is a key enzyme for producing 7-ACA by a one-step enzyme method, and can directly catalyze raw material cephalosporin C (CPC) to remove a 7-position D-D aminoadipoyl side chain to generate the 7-ACA. The action molecular formula is as follows:

the cephalosporin C acylase is mainly obtained by fermentation culture of genetically engineered bacteria. In order to improve the expression amount and activity of the enzyme, researchers are continuously trying to screen strains with superior fermentation performance or construct genetically engineered strains (endogenous signal peptide DSE4 mediates the secretory expression of cephalosporin C acylase in pichia pastoris, proceedings of the university of eastern physics (nature science edition), 2015). However, there are few reports on improvement of fermentation processes at present, mainly because the fermentation process of cephalosporin C acylase is very complicated, many conditions such as culture medium, fermentation temperature regulation control (including temperature and corresponding time), feeding types, feeding time, feeding speed and the like are matched with each other, and it is difficult to optimize and obtain a better fermentation method.

Disclosure of Invention

The invention aims to provide a simple and efficient fermentation process of cephalosporin C acylase, and the specific technical scheme comprises the following steps:

a method of fermenting a cephalosporin C acylase comprising:

(1) seed transferring: transferring the seed liquid into a fermentation culture medium;

(2) fermentation: fermenting for 60h, maintaining pH value at 6.8-7.2, controlling fermentation culture temperature at 37 + -1 deg.C for 0-7h, and fermenting at 25 + -1 deg.C for 8-60 h; feeding materials during the period, adjusting the stirring speed and controlling the dissolved oxygen amount to be 20-30%;

the fermentation medium in the step (1) contains: 43.0-48.0g/1 of yeast extract, 3.0-8.0g/l of sodium chloride, 8.0-13.0g/l of disodium hydrogen phosphate, 4.17-4.22g/l of dipotassium hydrogen phosphate, 0.71-1.10g/l of sodium sulfate, 1.78-2.00g/l of magnesium sulfate, 2.68-2.98g/l of ammonium sulfate, 19.4-20.8g/l of glycerol, 0.015-0.020g/l of ferric chloride and 0.42-0.50g/l of defoaming agent;

supplementing lactose and glycerol for the feeding in the step (2);

the lactose supplementing process comprises the following steps: culturing for 8h and OD not less than 40₆₀₀When the volume is less than or equal to 50, lactose accounting for 1 percent +/-0.1 percent of the volume of the fermentation culture medium is added at one time, and lactose accounting for 1 percent +/-0.1 percent of the volume of the fermentation culture medium is fed along with glycerol in the 6 th to 60 th hours;

the process for supplementing the glycerol comprises the following steps: feeding 100 +/-1L/h of supplementary glycerol in the 6 th to 8 th hours; feeding glycerol at a ratio of 75 + -1L/h in 8-18 h; adding supplementary glycerol 85 + -1L/h in 18-30 h; feeding 90 +/-1L/h of supplementary glycerol in 30-40 h; feeding 95 +/-1L/h of glycerol at 40-50 h; adding 100 + -1L/h of glycerol into the feed stream at 50-60 h.

As the method, in the step (2), the fermentation culture temperature is controlled to be 37 ℃ in 0-7h, and the fermentation temperature is controlled to be 25 ℃ in 8-60 h.

The process for supplementing lactose as described above is: culturing for 8h and OD not less than 40₆₀₀When the volume of the fermentation medium is less than or equal to 50, lactose accounting for 1 percent of the volume of the fermentation medium is added at one time, and the lactose accounting for 1 percent of the volume of the fermentation medium is fed along with the glycerol in the 6 th to 60 th hours.

As in the previous method, the process of supplementing glycerin is as follows: feeding 100L/h of supplementary glycerol in the 6 th to 8 th hours; feeding glycerol for 75L/h in the 8 th-18 th hour; adding 85L/h of supplementary glycerol in the flow from 18h to 30 h; feeding 90L/h of supplementary glycerol in the 30 th-40 th hour; feeding 95L/h of supplementary glycerol in the 40-50h period; adding 100L/h of glycerol into the feed stream from 50h to 60 h.

As described above, the amount of the inoculated cells in step (1) was 1.25% (v/v).

As the method, step (1) is preceded by a seed preparation step, and the shake flask strain preparation step comprises:

a. preparing shake flask bacterium suspension: taking out the glycerol tube, naturally thawing, inoculating to a culture medium in a sterile shake flask, and shake culturing at 37 deg.C and pH of 7.0-7.2 for 15-17 hr;

b. preparing first-grade seeds: b, inoculating the shake flask bacterial suspension in the step a to a primary culture medium in a primary seeding tank, and culturing for 5-6h at the temperature of 37 ℃ and at the pH of 6.9-7.5;

c. preparing second-level seeds: b, inoculating the primary seeds in the step b into a secondary culture medium in a secondary seed tank, and culturing for 5-6h at the temperature of 37 ℃ and at the pH of 6.9-7.5;

the culture medium of the step a contains 10g/l of peptone, 5g/l of yeast extract and 10g/l of sodium chloride;

the primary culture medium in the step b contains peptone 12g/l, yeast extract 6g/l and sodium chloride 10 g/l;

the secondary culture medium in the step c contains peptone 12g/l, yeast extract 6g/l and sodium chloride 10 g/l.

As the method, the step (3) is followed by a step of enzyme activity detection.

As the method, the enzyme activity detection step comprises the following steps:

enriching fermentation thalli, adding water to a constant volume to an original volume, homogenizing to obtain a homogenized solution, and measuring enzyme activity by using a potentiometric titration method by using cephalosporin C as a substrate.

In the prior art (CN105112392A), the activity of the cephalosporin C acylase in the obtained fermentation product is 5000U/L, namely 5U/mL. The cephalosporin C acylase activity of the homogeneous liquid of the fermentation thallus obtained by the fermentation method is as high as more than 170U/mL.

In addition, the method of the invention does not need to refer to the OD value of the fermentation liquor corresponding to the material supplementing process, thereby saving the cost generated by the real-time monitoring of the OD value.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The foregoing aspects of the present invention are explained in further detail below with reference to specific embodiments. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Detailed Description

The production strain used in the examples is BL21(DE3) Escherichia coli expression strain, which is recombinant Escherichia coli with cephalosporin C acylase expressed by exogenous gene. The Escherichia coli expression strain is a protein expression host which takes T7RNA polymerase as a high-efficiency exogenous gene of an expression system, the T7RNA polymerase is expressed by a lacUV5 promoter controlled by a DE3 region, and the region is integrated on a chromosome of BL 21.

EXAMPLE 1 fermentation of CPC acylase

1. Method of producing a composite material

(1) Preparation of Shake flask bacterial suspension

Shake flask medium contains: 10g/l of peptone, 5g/l of yeast extract and 10g/l of sodium chloride, inoculating the production strain into a sterile shake flask filled with a shake flask culture medium through a glycerol pipe, and carrying out shake culture at 37 ℃ for 15-17h at pH 7.0-7.2.

(2) Preparation of first seed

The first-stage culture medium proportion is as follows: peptone 12g/l yeast extract 6g/l sodium chloride 10g/l, pure strain shake flask bacterial suspension inoculated to the first seed tank, cultured for 5-6h under the conditions of pH6.9-7.5 and 37 ℃.

(3) Preparation of Secondary seeds

And (3) proportioning a secondary culture medium: peptone 12g/l yeast extract 6g/l sodium chloride 10g/l, pure strain shake flask bacterial suspension inoculated to the secondary seed tank, cultured for 5-6h under the conditions of pH6.9-7.5 and 37 ℃.

(4) Fermentation of

Fermentation tank proportioning: 43.0-48.0g/l of yeast extract, 3.0-8.0g/l of sodium chloride, 8.0-13.0g/l of disodium hydrogen phosphate, 4.17-4.22g/l of dipotassium hydrogen phosphate, 0.71-1.10g/l of sodium sulfate, 1.78-2.00g/l of magnesium sulfate, 2.68-2.98g/l of ammonium sulfate, 19.4-20.8g/l of glycerol, 0.015-0.020g/l of ferric chloride and 0.42-0.50g/l of defoaming agent.

And (4) taking the secondary seeds obtained in the step (3), transplanting the seeds into a fermentation culture medium according to the seed transplanting amount of 1.25% (v/v), fermenting for 60h, wherein the pH value is 6.80-7.20 at the temperature of 8-60h25 ℃ and at the temperature of 0-7h37 ℃.

During the period, lactose and glycerol are added.

The lactose supplementing process comprises the following steps: culturing for 8h and OD not less than 40₆₀₀Less than or equal to 50 percent, 1 percent of lactose is added at one time, and 1 percent of lactose is added along with the feeding flow in 6 to 60 hours.

The glycerol supplementing process comprises the following steps: when the fermentation culture period is 6-8h, the dissolved oxygen can rise back (the dissolved oxygen rises from 20% to 100% within 1 min), and the dissolved oxygen rises back to 100%, namely, the glycerol is added in a flowing mode, and the flowing speed is 100L/h; starting from 8h, the glycerol feed rate was: 8-18h75L/h, 18-30h85L/h, 30-40h90L/h, 40-50h95L/h, 50-60h 100L/h. In the process of replenishing the glycerol, adjusting the stirring speed and controlling the dissolved oxygen to be 20-30 percent;

(5) and (3) taking 1.5L of fermentation liquor after fermentation, centrifuging at 6000rpm for 25min by using a centrifuge, collecting cells, carrying out volume constant on purified water to the original volume, and homogenizing for 2 times by using a homogenizer at 80MPa to obtain the cephalosporin C acylase homogeneous solution.

(6) Taking 1ml of homogeneous liquid, 10000rpm for 5min, centrifuging, taking appropriate supernatant, adding a CPC substrate with the substrate concentration of 5mmol/I, titrating by using a potentiometric titrator, titrating for 7min by using 0.1mol/L sodium hydroxide standard titration solution, keeping the pH of the solution all the time in the titration process, discarding the first 2min, and recording the consumption of the 5min sodium hydroxide standard titration solution; when 0.1mol/L sodium hydroxide is consumed, 0.1 mol/L7-ACA is equivalently generated, corresponding to 1U enzyme activity, and the enzyme activity of the homogeneous liquid is obtained through calculation.

2. Results

Table 1 shows the fermentation results of 2 groups of parallel experiments in the feeding mode of the invention, and the activity of the corresponding tank-placed homogenized liquid enzyme can reach more than 170U/ml.

Table 1 example 1 fermentation results

Lactose dosage (kg)	Glycerol addition (kg)	Tank OD600	Jar homogenized liquid enzyme activity (U/ml)
				240	2500	147±34.24	173±7.07
240	2500	153±34.24	187±107.07

EXAMPLE 2 fermentation of CPC acylase

1. Fermentation process

(1) Preparation of Shake flask bacterial suspension

Shake flask medium contains: 10g/l of peptone, 5g/l of yeast extract and 10g/l of sodium chloride, inoculating the production strain into a sterile shake flask filled with a shake flask culture medium through a glycerol pipe, and carrying out shake culture at the temperature of 7.0-7.237 ℃ for 15-17 h.

(2) Preparation of first seed

The first-stage culture medium proportion is as follows: peptone 12g/L yeast extract 6g/L sodium chloride 10g/L, and volume of 30L. Inoculating the pure shake flask bacterial suspension to a first-stage seeding tank, and performing shake culture at 37 ℃ for 5-6h under the conditions of pH 6.9-7.5.

(3) Preparation of Secondary seeds

And (3) proportioning a secondary culture medium: peptone 12g/L yeast extract 6g/L sodium chloride 10g/L, metering volume 1500L, inoculating the pure strain shake flask suspension to the first-stage seed tank, and shake culturing at 37 deg.C and pH6.9-7.5 for 5-6 h.

(4) Fermentation of

Fermentation tank proportioning: 43.0-48.0g/L of yeast extract, 3.0-8.0g/L of sodium chloride, 8.0-13.0g/L of disodium hydrogen phosphate, 4.17-4.22g/L of dipotassium hydrogen phosphate, 0.71-1.10g/L of sodium sulfate, 1.78-2.00g/L of magnesium sulfate, 2.68-2.98g/L of ammonium sulfate, 19.4-20.8g/L of glycerol, 0.015-0.020g/L of ferric chloride, 0.42-0.50g/L of defoaming agent and 12000L of metered volume.

And (4) taking the secondary seeds prepared in the step (3), transplanting the seeds into a fermentation culture medium according to the seed transplanting amount of 1.25% (v/v), fermenting for 60h, wherein the pH value is 6.80-7.20 at the temperature of 8-60h25 ℃ and at the temperature of 0-7h37 ℃.

During the period, lactose and glycerol are added.

The lactose supplementing process comprises the following steps: culturing for 8h and OD not less than 40₆₀₀When the volume is less than or equal to 50, 1 percent of lactose (accounting for 12000L of the volume of the fermentation liquor) is added at one time, and 1 percent of lactose (accounting for 12000L of the volume of the fermentation liquor) is fed along with the feeding (glycerol) in 6-60 h.

Glycerol supplementation process 1: when the fermentation culture period is 6-8h, the dissolved oxygen can rise back (the dissolved oxygen rises from 20% to 100% within 1 min), and the dissolved oxygen rises back to 100%, namely, the glycerol is added in a flowing mode, and the flowing speed is 100L/h; starting from 8h, the glycerol feed rate was: 8-18h75L/h, 18-30h85L/h, 30-40h90L/h, 40-50h95L/h, 50-60h 100L/h. In the process of replenishing the glycerol, the stirring speed is adjusted, and the dissolved oxygen is controlled to be 20-30 percent.

Glycerol supplementation process 2: when the fermentation culture period is 6-8h, the dissolved oxygen can rise back (the dissolved oxygen is increased from 20% to 100% within 1 min), and the glycerol is supplemented at the speed of 89L/h. In the process of replenishing the glycerol, adjusting the stirring speed and controlling the dissolved oxygen to be 20-30 percent;

glycerol supplementation process 3: the dissolved oxygen rises back when the fermentation culture period is 6-8h (the dissolved oxygen rises from 20% to 100% within 1 min), and the stirring speed is 85%And (4) rpm, adjusting the amount of the supplement, and controlling the dissolved oxygen to be 20% -30% until 18 h. During the period from 18h to the end of the feed supplement, the tank is taken out according to OD₆₀₀Adjusting the growth rate and feeding: if the increase per hour is 1.5-2.0, maintaining the feeding speed of the previous 1 hour; if OD₆₀₀When the growth rate is less than 1.5, the feeding is increased by 3-5L/h. Correspondingly adjusting the stirring speed and controlling the dissolved oxygen within the range of 20-30 percent. Stopping the machine and placing the tank after the material is supplemented.

(5) And (3) taking 1.5L of fermentation liquor after fermentation is finished, centrifuging at 6000rpm for 25min by using a centrifuge to collect cells, carrying out volume constant on purified water to the original volume, homogenizing at 80MPa by using a homogenizer for 2 times to obtain the CPC acylase homogeneous solution.

(6) And taking 10000rpm of the homogeneous liquid for 5min, taking the centrifuged supernatant, adding a CPC substrate, wherein the substrate concentration is 20mol/l, titrating for 5min by using a potentiometric titrator, reaching the titration end point, and obtaining the enzyme activity of the homogeneous liquid by calculating the enzyme activity of 1U/ml corresponding to the consumption of 0.1mol/l of sodium hydroxide.

2. Results

As shown in Table 2, the enzyme activity of the tank-placing homogenizing liquid can reach 202U/ml by using the third feeding process because feeding is strictly adjusted according to the OD value; the OD value is monitored at any time in the process, the supplement amount is required to be frequently adjusted, so that the dissolved oxygen is controlled by frequently adjusting the stirring rotating speed, the dissolved oxygen meets the technological requirements, the method has high requirements on detection data and fine control process, the process is complicated, the tank placing index is unstable, and the production cost is high.

The glycerol supplementing process 1 (the invention) can ensure that the enzymatic activity of the canning homogenized liquid reaches 176U/ml; the feeding mode is relatively determined, and the culture process is simple and easy to operate. In combination, the feeding process is more suitable for industrial production.

TABLE 2 comparative results of three feeding processes

In conclusion, in the fermentation method, the glycerol and the lactose are supplemented, and the supplement amount and the supplement mode are specially selected, so that the enzyme activity of the CPC acylase homogeneous liquid can be remarkably improved, the production cost is reduced, and the industrial application prospect is good.