阅读说明：本技术 青霉素发酵液的处理方法 (Method for treating penicillin fermentation liquor ) 是由 苗瑞春 刁夏 幸华龙 葛小波 彭亮亮 蒋远顺 刘国 于 2020-06-29 设计创作，主要内容包括：本发明涉及青霉素发酵液的处理方法,该方法包括：(1)对青霉素发酵液进行一级超滤；(2)对第一超滤稀液进行二级超滤；(3)对第二超滤稀液进行三级超滤；(4)对第三超滤稀液进行纳滤浓缩。本发明采用膜法处理青霉素发酵液,通过三步超滤可以取代实际生产中应用的破乳剂破乳、活性炭过滤、醋酸丁酯萃取等步骤,在工艺成本、环保上得到了突破性发展；在纳滤浓缩后得到的纳滤浓缩液,可以直接进行酶解反应,来制备6-氨基青霉烷酸,所得到的6-氨基青霉烷酸成品质量优异；所采用的膜为聚醚砜膜,其孔结构大小一致性好,且耐冲击性、耐腐蚀好,表面性能好,清洗通道宽,抗污染性很强,使用清洗剂正向循环清洗即可,清洗简单。(The invention relates to a method for treating penicillin fermentation liquor, which comprises the following steps: (1) performing primary ultrafiltration on the penicillin fermentation liquor; (2) performing secondary ultrafiltration on the first ultrafiltration dilute solution; (3) carrying out three-stage ultrafiltration on the second ultrafiltration dilute solution; (4) and carrying out nanofiltration concentration on the third ultrafiltration dilute solution. The invention adopts a membrane method to process the penicillin fermentation liquor, and can replace the steps of demulsification by a demulsifier, filtration by active carbon, extraction by butyl acetate and the like applied in actual production through three-step ultrafiltration, thereby achieving breakthrough development in process cost and environmental protection; the nanofiltration concentrated solution obtained after nanofiltration concentration can be directly subjected to enzymolysis reaction to prepare 6-aminopenicillanic acid, and the obtained 6-aminopenicillanic acid finished product has excellent quality; the adopted membrane is a polyether sulfone membrane, the pore structure of the polyether sulfone membrane has good size consistency, impact resistance and corrosion resistance, good surface performance, wide cleaning channel and strong pollution resistance, and the polyether sulfone membrane is cleaned by a cleaning agent in a forward circulating manner and is simple to clean.)

1. a method for processing penicillin fermentation liquor comprises the following steps:

(1) primary ultrafiltration of penicillin fermentation liquor

Keeping the temperature below 10 ℃, performing primary ultrafiltration on the penicillin fermentation liquor by using a high-molecular ultrafiltration membrane with the molecular weight cutoff of 30000-100000 daltons, and keeping the pressure difference value between the membrane inlet pressure and the membrane outlet pressure to be less than 0.02MPa to obtain a first ultrafiltration concentrated solution and a first ultrafiltration dilute solution;

(2) performing two-stage ultrafiltration on the first ultrafiltration dilute solution

Keeping the temperature below 10 ℃, performing secondary ultrafiltration on the first ultrafiltration dilute solution by using a macromolecular ultrafiltration membrane with the molecular weight cutoff of 10000-30000 daltons, and keeping the pressure difference value between the membrane inlet pressure and the membrane outlet pressure to be less than 0.02MPa to obtain a second ultrafiltration concentrated solution and a second ultrafiltration dilute solution;

(3) performing three-stage ultrafiltration on the second ultrafiltration dilute solution

Keeping the temperature below 10 ℃, performing three-stage ultrafiltration on the second ultrafiltration dilute solution by using a macromolecular ultrafiltration membrane with the molecular weight cutoff of 500-10000 Dalton, and keeping the pressure difference value between the membrane inlet pressure and the membrane outlet pressure less than 0.02MPa to obtain a third ultrafiltration concentrated solution and a third ultrafiltration dilute solution;

(4) performing nanofiltration concentration on the third ultrafiltration dilute solution

And (3) keeping the temperature below 10 ℃, performing nanofiltration concentration by adopting a high-molecular nanofiltration membrane with the molecular weight cutoff of 150-300 daltons, and keeping the pressure difference value between the membrane inlet pressure and the membrane outlet pressure to be less than 0.02MPa to obtain a nanofiltration concentrated solution.

2. The method for treating penicillin fermentation broth as claimed in claim 1, wherein said polymeric ultrafiltration membrane and said polymeric nanofiltration membrane are polyethersulfone membranes.

3. The method for treating penicillin fermentation broth as claimed in claim 2, wherein said polyethersulfone membrane is a polyethersulfone membrane prepared by the following method: and casting the casting solution containing the polyether sulfone on a substrate, solidifying and aging in an aqueous solution containing a surfactant, and drying.

4. The method for treating penicillin fermentation broth as claimed in claim 1, wherein the transmittance of said penicillin fermentation broth is controlled to 15% -20% for light with a wavelength of 430 nm; for light with the wavelength of 625nm, the light transmittance is 70-80%; the titer of the fermentation liquor is 45000-55000 u/ml.

5. The method for treating the penicillin fermentation broth as claimed in claim 1, wherein in the step (1) of primary ultrafiltration, the membrane inlet pressure is selected to be 0.30-0.50 MPa, the membrane inlet pressure is higher than the membrane outlet pressure, the pressure difference between the membrane inlet pressure and the membrane outlet pressure is less than 0.02MPa, and the light transmittance of the obtained first ultrafiltration dilute solution for light with a wavelength of 430nm is 25-30%; for light with a wavelength of 625nm, the light transmittance is 85% -90%, and the titer is 45000-55000 u/ml.

6. The method for treating penicillin fermentation broth as claimed in claim 1, wherein in said step (1) of primary ultrafiltration of penicillin fermentation broth, a polymeric ultrafiltration membrane with a molecular weight cutoff of 30000-50000 dalton, more preferably 30000-35000 dalton is used to perform primary ultrafiltration of penicillin fermentation broth.

7. The penicillin fermentation broth processing method as claimed in claim 1, wherein in said step (2) of performing secondary ultrafiltration on the first ultrafiltration dilute solution, a polymeric ultrafiltration membrane with cut-off molecular weight of 10000-20000 daltons, more preferably 10000-15000 daltons is used for performing secondary ultrafiltration on the first ultrafiltration dilute solution;

when secondary ultrafiltration is carried out, the membrane inlet pressure is selected to be 0.30-0.50 MPa, the membrane inlet pressure is greater than the membrane outlet pressure, the pressure difference value between the membrane inlet pressure and the membrane outlet pressure is less than 0.02MPa, and the light transmittance of the obtained second ultrafiltration dilute solution for light with the wavelength of 430nm is ensured to be 60-70%; for light with the wavelength of 625nm, the light transmittance is 90-95%, and the titer is 45000-55000 u/ml.

8. The method for treating penicillin fermentation broth as claimed in claim 1, wherein in the step (3) of performing tertiary ultrafiltration on the second ultrafiltration dilute solution, a polymeric ultrafiltration membrane with a cut-off molecular weight of 1000-5000 daltons, more preferably 2000-3000 daltons is used for performing tertiary ultrafiltration on the second ultrafiltration dilute solution.

9. The method for treating the penicillin fermentation broth as claimed in claim 1, wherein in the step (3), the second ultrafiltration dilute solution is subjected to three-stage ultrafiltration, wherein during the three-stage ultrafiltration, the membrane inlet pressure is selected to be 0.30-0.50 MPa, the membrane inlet pressure is greater than the membrane outlet pressure, the difference value between the membrane inlet pressure and the membrane outlet pressure is less than 0.02MPa, and the light transmittance of the obtained third ultrafiltration dilute solution for light with a wavelength of 430nm is ensured to be 65-75%; for light with the wavelength of 625nm, the light transmittance is 95-98%, and the titer is 45000-55000 u/ml.

10. The method for treating the penicillin fermentation liquor according to claim 1, wherein a polymeric nanofiltration membrane with the molecular weight cutoff of 180-250 daltons is adopted to carry out nanofiltration concentration on the third ultrafiltration dilute solution; when nanofiltration concentration is carried out, the pressure of a film inlet is selected to be 0.20-0.40 MPa, the pressure of the film inlet is greater than the pressure of a film outlet, and the pressure difference value of the film inlet and the film outlet is less than 0.02 MPa; the titer of the final nanofiltration concentrate is 250000-270000 u/ml.

Technical Field

The invention belongs to the technical field of biological pharmacy, and relates to a method for treating penicillin fermentation liquor.

Background

Penicillin is an important antibiotic with high efficiency, low toxicity and wide clinical application, and the first generation of penicillin refers to natural penicillin such as penicillin G (benzylpenicillin); the second generation penicillin is semi-synthetic penicillin obtained by changing side chain with penicillin mother nucleus-6-aminopenicillanic acid (6-APA), such as methicillin, amoxicillin, ampicillin, etc.

The industrial production of penicillin is to obtain penicillin G by fermentation, filtration, extraction, back extraction, crystallization and the like of strains. The industrial production of 6-amino penicillanic acid mainly includes microbial enzyme catalytic cracking process, i.e. the penicillin side chain enzyme is cracked into 6-APA. The penicillin fermentation broth has complex components, and contains a certain amount of hyphae, unused oil culture medium, protein, pigment, amino acid, penicillin homolog degradation product, etc. in addition to penicillin. In fact, the treatment process of the fermentation liquor is an impurity removal process, but more technical problems need to be broken through in the impurity removal process, and the impurity removal effect directly determines the quality and yield of the 6-APA finished product.

Therefore, the treatment technology of the penicillin fermentation broth is very critical in the production of penicillin G and 6-aminopenicillanic acid, determines the production benefits, the environmental protection problems and the product quality, and has important significance for the improvement and optimization of the treatment technology of the penicillin fermentation broth.

The method for processing penicillin fermentation liquor in the actual production at present is a solvent extraction method, and the method comprises the following specific steps: (1) treating hypha with flocculant, demulsifying agent and solvent butyl acetate to perform demulsification deposition under acidic condition to separate protein and solid macromolecular particles to obtain solvent phase liquid BA, and discarding water phase to remove protein impurities; (2) carrying out back extraction on the BA solution through a metal salt solution according to different concentrations to obtain a target titer aqueous phase liquid RB; (3) removing solvent from the film to achieve the enzymolysis condition to achieve the target residue; (4) and carrying out enzymolysis, separation, crystallization, filtration and drying on the RB solution to prepare the 6-APA. Although the method is the existing method for preparing 6-APA by stably and mature treating enzymolysis liquid, the method has more defects: (1) the use of the solvent not only has serious problems in cost and environment, but also has inevitable influence on the yield and quality in the extraction and back extraction processes; (2) the steps are too complex, the required precision is very high in operation, and the inconvenience is caused on the handling control of personnel and technology due to the difficulty in realizing automation; (3) in the process, auxiliary materials such as acid, alkali, demulsifier and the like are used to generate inevitable influence on the quality of the product.

Therefore, further improvements are needed for the processing of penicillin fermentation broths.

Disclosure of Invention

The invention aims to provide an effective penicillin fermentation broth treatment method for facilitating the subsequent preparation of penicillin G salt or 6-aminopenicillanic acid, and the treatment method has the advantages of simple and convenient process design, strong operability, high automation degree, good treatment effect, good product quality, low cost, economy, environmental protection and suitability for industrial popularization.

According to the invention, the method for treating the penicillin fermentation liquor comprises the following steps:

(1) primary ultrafiltration of penicillin fermentation liquor

Keeping the temperature below 10 ℃, performing primary ultrafiltration on the penicillin fermentation liquor by using a high-molecular ultrafiltration membrane with the molecular weight cutoff of 30000-100000 daltons, and keeping the pressure difference value between the membrane inlet pressure and the membrane outlet pressure to be less than 0.02MPa to obtain a first ultrafiltration concentrated solution and a first ultrafiltration dilute solution;

(2) performing two-stage ultrafiltration on the first ultrafiltration dilute solution

Keeping the temperature below 10 ℃, performing secondary ultrafiltration on the first ultrafiltration dilute solution by using a macromolecular ultrafiltration membrane with the molecular weight cutoff of 10000-30000 daltons, and keeping the pressure difference value between the membrane inlet pressure and the membrane outlet pressure to be less than 0.02MPa to obtain a second ultrafiltration concentrated solution and a second ultrafiltration dilute solution;

(3) Performing three-stage ultrafiltration on the second ultrafiltration dilute solution

Keeping the temperature below 10 ℃, performing three-stage ultrafiltration on the second ultrafiltration dilute solution by using a macromolecular ultrafiltration membrane with the molecular weight cutoff of 500-10000 Dalton, and keeping the pressure difference value between the membrane inlet pressure and the membrane outlet pressure less than 0.02MPa to obtain a third ultrafiltration concentrated solution and a third ultrafiltration dilute solution;

(4) performing nanofiltration concentration on the third ultrafiltration dilute solution

And (3) keeping the temperature below 10 ℃, performing nanofiltration concentration by adopting a high-molecular nanofiltration membrane with the molecular weight cutoff of 150-300 daltons, and keeping the pressure difference value between the membrane inlet pressure and the membrane outlet pressure to be less than 0.02MPa to obtain a nanofiltration concentrated solution.

The method for treating a penicillin fermentation broth according to the present invention will be described in more detail below.

In the step (1), during primary ultrafiltration of the penicillin fermentation liquor, a polymeric ultrafiltration membrane with the molecular weight cutoff of 30000-100000 daltons is adopted to perform primary ultrafiltration on the penicillin fermentation liquor at the temperature of below 10 ℃, and the difference value between the membrane inlet pressure and the membrane outlet pressure is kept to be less than 0.02MPa, so that a first ultrafiltration concentrated liquor and a first ultrafiltration dilute liquor are obtained.

Wherein, the components of the penicillin fermentation liquor are relatively complex, the penicillin fermentation liquor contains about 20 to 25 weight percent of penicillin after plate-frame filtration, and also contains a certain amount of hypha, unused oil culture medium, protein, pigment, amino acid, penicillin homolog degradation products and the like, and the general wet-solid content is about 2 to 3 weight percent. As the quality of the penicillin fermentation liquor directly influences the quality of a subsequently produced product, the requirement is that the higher the light transmission is, the better the light transmission is, the light transmission is preferably selected in the aspects of light transmittance and titer, the light transmittance is controlled to be light with the wavelength of 430nm, and the light transmittance is 15-20%; for light with the wavelength of 625nm, the light transmittance is 70-80%; the titer of the fermentation liquor is 45000-55000 u/ml.

The first stage ultrafiltration is mainly used for removing macromolecular proteins and amino acid polymerization saccharides in the penicillin fermentation liquor.

And (2) keeping the temperature below 10 ℃, particularly at 5 +/-2 ℃, and performing primary ultrafiltration on the penicillin fermentation liquor by using a high-molecular ultrafiltration membrane with the molecular weight cutoff of 30000-100000 daltons, preferably 30000-50000 daltons, and more preferably 30000-35000 daltons. When primary ultrafiltration is carried out, the front and back pressure of a membrane is ensured to be stable, the pressure of the membrane is selected to be 0.30-0.50 MPa, the pressure of the membrane is greater than the pressure of the membrane, the pressure difference value of the membrane is less than 0.02MPa, and the light transmittance of the obtained first ultrafiltration dilute solution to light with the wavelength of 430nm is ensured to be 25-30%; for light with a wavelength of 625nm, the light transmittance is 85% -90%, and the titer is 45000-55000 u/ml. The yield can be close to 99% by the following calculation:

yield ═ yield (volume of first ultrafiltration broth collected x its titer)/(volume of penicillin fermentation broth feed x its titer)

The obtained first ultrafiltration concentrated solution is mainly macromolecular protein and amino acid carbohydrate substances, and because the impurities are concentrated, the first ultrafiltration concentrated solution is subjected to biodegradation inactivation treatment after simple MVR treatment to achieve pollution-free discharge.

In the invention, the used high molecular ultrafiltration membrane and high molecular nanofiltration membrane are roll type polyethersulfone membranes. Since bioseparation requires a more precise pore size and more stable performance, a higher standard is put forward on the requirements of the membrane, the pore size is required to be highly uniform, the strength and the corrosion resistance are more stable, and the recovery of the membrane flux after cleaning is good, so that the durability is good. In the invention, the polyethersulfone membrane prepared by the following method is selected: the casting solution containing the polyether sulfone is subjected to membrane casting on a substrate, then is solidified and aged in an aqueous solution containing a surfactant, such as an organosilicon surfactant, a polyvinyl alcohol surfactant and the like, and then is dried. The polyether sulfone membrane obtained in the way has low condensation speed and long membrane forming time, and has small shrinkage stress in the membrane forming process, so that the pore structure has good size consistency; the impact resistance and the corrosion resistance are good; the surface performance is good, the cleaning channel is wide, the pollution resistance is strong, the cleaning is simple because the cleaning agent is used for forward circulating cleaning.

And (3) in the step (2), in the second-stage ultrafiltration of the first ultrafiltration dilute solution, keeping the temperature below 10 ℃, performing second-stage ultrafiltration on the first ultrafiltration dilute solution by using a macromolecular ultrafiltration membrane with the molecular weight cutoff of 10000-30000 daltons, and keeping the pressure difference value between the membrane inlet pressure and the membrane outlet pressure to be less than 0.02MPa to obtain a second ultrafiltration concentrated solution and a second ultrafiltration dilute solution.

The second-stage ultrafiltration is mainly used for removing organic matters such as pigment substances, residual proteins, small-molecular sugar oils and the like in the first ultrafiltration dilute solution.

And (2) keeping the temperature below 10 ℃, particularly 5 +/-2 ℃, and performing secondary ultrafiltration on the first ultrafiltration dilute solution by using a high-molecular ultrafiltration membrane with the molecular weight cutoff of 10000-30000 daltons, preferably 10000-20000 daltons, more preferably 10000-15000 daltons. During secondary ultrafiltration, the front and back pressure of the membrane is ensured to be stable, the membrane inlet pressure is selected to be 0.30-0.50 MPa, the membrane inlet pressure is greater than the membrane outlet pressure, the pressure difference value between the membrane inlet and the membrane outlet is less than 0.02MPa, and the light transmittance of the obtained second ultrafiltration dilute solution to light with the wavelength of 430nm is ensured to be 60-70%; for light with the wavelength of 625nm, the light transmittance is 90-95%, and the titer is 45000-55000 u/ml. The yield can be close to 99% by the following calculation:

yield ═ yield (volume of second ultrafiltration stream collected x its titer)/(volume of first ultrafiltration stream feed x its titer)

And (3) in the step (3), in the third-stage ultrafiltration of the second ultrafiltration dilute solution, the temperature is kept below 10 ℃, a high-molecular ultrafiltration membrane with the molecular weight cutoff of 500 to less than 10000 daltons is adopted to carry out the third-stage ultrafiltration of the second ultrafiltration dilute solution, and the pressure difference value between the membrane inlet pressure and the membrane outlet pressure is kept to be less than 0.02MPa, so that a third ultrafiltration concentrated solution and a third ultrafiltration dilute solution are obtained.

The three-stage ultrafiltration is mainly used for removing impurities with the molecular weight of more than 6-APA, and residual trace grease substances and salt substances in the second ultrafiltration dilute solution.

And (3) keeping the temperature below 10 ℃, particularly 5 +/-2 ℃, and performing three-stage ultrafiltration on the second ultrafiltration dilute solution by using a high-molecular ultrafiltration membrane with the molecular weight cutoff of 500-10000 Dalton, preferably 1000-5000 Dalton cut-off amount, and more preferably 2000-3000 Dalton cut-off amount. When three-stage ultrafiltration is carried out, the front and back pressure of the membrane is ensured to be stable, the membrane inlet pressure is selected to be 0.30-0.50 MPa, the membrane inlet pressure is greater than the membrane outlet pressure, the pressure difference value between the membrane inlet and the membrane outlet is less than 0.02MPa, and the light transmittance of the obtained third ultrafiltration dilute solution to light with the wavelength of 430nm is ensured to be 65-75%; for light with the wavelength of 625nm, the light transmittance is 95-98%, and the titer is 45000-55000 u/ml. The yield can be close to 99% by the following calculation:

yield ═ volume of the third ultrafiltration stream collected x its titer)/(volume of the second ultrafiltration stream feed x its titer)

After the three-stage ultrafiltration in the step (3), the obtained third ultrafiltration dilute liquid has the quality index of the BA product in the actual production, and the three-stage ultrafiltration can replace the steps of demulsification by using a demulsifier, filtration by using activated carbon, extraction by using butyl acetate and the like in the actual production, thereby achieving breakthrough development in process cost and environmental protection.

And (4) during nanofiltration concentration of the third ultrafiltration dilute solution in the step (4), a high-molecular nanofiltration membrane with the molecular weight cutoff of 150-300 daltons is adopted for nanofiltration concentration at the temperature below 10 ℃, and the difference value between the membrane inlet pressure and the membrane outlet pressure is kept less than 0.02MPa, so that a nanofiltration concentrated solution is obtained.

The titer of the third ultrafiltration dilute solution is 45000-55000 u/ml, but the titer needs to be improved to about 260000u/ml when 6-aminopenicillanic acid is produced by enzymolysis. And (3) keeping the temperature below 10 ℃, particularly 5 +/-2 ℃, and performing nanofiltration concentration on the third ultrafiltration dilute solution by adopting a high-molecular nanofiltration membrane with the molecular weight cutoff of 150-300 daltons, preferably 180-250 daltons. When nanofiltration concentration is carried out, the front and back pressure of the membrane is ensured to be stable, the membrane inlet pressure is selected to be 0.20-0.40 MPa, the membrane inlet pressure is greater than the membrane outlet pressure, and the pressure difference value between the membrane inlet pressure and the membrane outlet pressure is less than 0.02 MPa. The titer of the finally obtained nanofiltration concentrate is 250000-270000u/ml, for example, about 260000 u/ml.

The nanofiltration concentrated solution obtained by the treatment method of the penicillin fermentation liquor can be directly subjected to enzymolysis reaction to prepare 6-aminopenicillanic acid. Specifically, after the concentrated solution with the titer of 250000-270000u/ml is prepared to about 140000u/ml by boric acid (the boric acid plays a role of neutralization reaction as a slow release solution and can reach the concentration of crystallization), the conversion reaction is carried out for 70-150 minutes by using ammonia water to adjust the pH value to 8 +/-0.5 at the temperature of 26 +/-3 ℃ in the presence of immobilized penicillin acylase; and after the conversion reaction is finished, filtering to remove the immobilized penicillin acylase, adjusting the pH of the filtrate to 4.0 +/-0.2 by using an acid at the temperature of 7 +/-2 ℃, crystallizing and separating 6-aminopenicillanic acid, growing crystals for 1-2 hours, filtering and drying to obtain the 6-aminopenicillanic acid.

Advantageous effects

According to the method for treating the penicillin fermentation liquor, the membrane method is adopted to treat the penicillin fermentation liquor, the automation degree is high, the personnel configuration is simple, the steps of demulsifying by using a demulsifier, filtering by using activated carbon, extracting by using butyl acetate and the like applied to actual production can be replaced by three-step ultrafiltration, and breakthrough development is achieved in the aspects of process cost and environmental protection. The concrete points are as follows:

(1) the existing process occupies about 8-10% of the cost in the use of the extracting agent, is a main source of the cost, has huge cost in the recovery of the solvent, and is a huge challenge to the environment, the process completely avoids the use of the solvent, and the cost, including equipment and labor cost, can be reduced by about 15-20 percentage points.

(2) The use of solvent is banned, the yield loss caused by residue in extraction is avoided in the yield, and the yield is improved by about 1 percent.

(3) The use of solvent is cancelled, other impurities are not introduced in quality, the product purity also meets the requirements of customers, and the competitiveness is improved.

(4) Almost no pollution in the aspect of environmental protection, conforms to the strategic policy of China, and reduces the treatment cost of waste water and waste gas of companies.

(5) The adopted membrane is a polyether sulfone membrane, the pore structure of the polyether sulfone membrane has good size consistency, impact resistance and corrosion resistance, good surface performance, wide cleaning channel and strong pollution resistance, and the polyether sulfone membrane is cleaned by a cleaning agent in a forward circulating manner and is simple to clean.

Detailed Description

The method of the present invention for the treatment of penicillin fermentation broth is described in more detail below by way of examples, but the scope of the present invention is not limited to the following examples.