A recombinant plasmid, a pichia pastoris engineering strain and application thereof

Abstract

The application relates to a recombinant plasmid, a Pichia pastoris engineering strain and application. In order to solve the problems of high cost and poor scale expression effect in the industrial production of existing penicillin acylase, the application constructs a recombinant plasmid with an amino acid sequence as shown in SEQ ID NO. 1, and converts the recombinant plasmid into Pichia pastoris to obtain an engineering strain; the engineering strain is subjected to activation culture, shake flask seed culture, primary seed culture, secondary seed culture and fermentation culture, so that the scale fermentation production of the engineering strain and the high-efficiency expression of penicillin acylase are realized, and the enzyme activity is stable at 49900 U / L after 100h of fermentation. The engineering strain has excellent heterologous expression performance and scale fermentation adaptability, can significantly improve the production efficiency of penicillin acylase, reduce the production cost, provides a high-efficiency and stable biological catalysis tool for the industrial synthesis of beta-lactam antibiotics, and has important industrial application value.

Description

Technical Field

[0001] This application belongs to the field of biocatalysis, specifically a recombinant plasmid, an engineered strain of Pichia pastoris, and its application in the production of penicillin acylase. Background Technology

[0002] β-lactam antibiotics, including cephalosporins and penicillins, are currently the mainstream drugs used in clinical anti-infective treatment both domestically and internationally, occupying a core position in the pharmaceutical industry. However, Chinese antibiotic pharmaceutical companies lag significantly behind developed countries in the research and development of high-end β-lactam antibiotics and the innovation of cutting-edge preparation technologies. The industrialization process of core products such as enzymatic synthesis of ampicillin, amoxicillin, and cephalosporins urgently needs to be accelerated.

[0003] Currently, the traditional preparation method for β-lactam antibiotics is mainly chemical synthesis. This process has many drawbacks, including high production costs, harsh reaction conditions, high energy consumption, large amounts of organic solvents used, and serious environmental pollution. Enzymatic synthesis technology, as a green preparation process, can catalyze the synthesis of semi-synthetic penicillins and cephalosporins under mild conditions, demonstrating significant advantages in process operation, product yield, economic benefits, and environmental protection. The enzymatic synthesis process for β-lactam antibiotics such as amoxicillin and cephalexin is now increasingly mature, with product yields reaching or even exceeding those of traditional chemical synthesis methods. With increasingly stringent global environmental requirements, enzymatic technology replacing chemical synthesis in the preparation of β-lactam antibiotics has become an inevitable trend in the industry.

[0004] Penicillin acylase (PA), also known as penicillin aminohydrolase or penicillin amidase, is a core enzyme preparation for the enzymatic preparation of β-lactam antibiotics. It can forward catalyze the enzymatic synthesis reaction of D-amino acid analogs with β-lactam nuclei to prepare novel semi-synthetic β-lactam antibiotics. It can also reverse catalyze the hydrolysis reaction of penicillin to efficiently produce key intermediates for β-lactam antibiotics such as 7-amino-3-deacetoxycephalosporanicacid (7-ADCA) and 6-amino-cephalosporanic acid (6-ACA). At the same time, this enzyme also plays an indispensable role in many fields such as biomedicine and food analysis.

[0005] The microbial sources of penicillin acylase are very diverse, currently including Escherichia coli (Escherichia coli) Escherichia coli ), Achromobacterium spp. ( Achromobacter Providencia retaliculata ( ), Providencia rettgeri ), Bacillus oryzae (Bacillus badius The enzyme-encoding gene was obtained by screening strains such as [list of strains]. Furthermore, regardless of whether the enzyme is expressed by a natural wild-type strain or a genetically engineered recombinant strain, it consistently exhibits the inherent enzyme activity characteristics. However, existing penicillin acylase-producing strains generally suffer from low enzyme expression levels, insufficient enzyme activity, and poor thermal stability and environmental tolerance. Prokaryotic expression systems also have drawbacks such as easy inclusion body formation of the expression product and complex subsequent separation and purification processes, making them unsuitable for the high-efficiency, low-cost requirements of large-scale industrial production.

[0006] Pichia pastoris ( Pichia pastoris As a classic eukaryotic expression system, it has advantages such as high expression levels, correct folding and modification of expression products, simple culture process, and easy industrial scale-up. It is the preferred host for efficient expression of enzyme preparations. For example, CN 110423704 A and CN 112877227 A used ultraviolet-mutated mutant strains of Pichia pastoris to ferment and express ferulic acid esterase and rhamnosylase, respectively, giving ferulic acid esterase an activity of 352 U / mL and rhamnosylase an activity of 536 U / mL. However, research on the efficient expression of penicillin acylase in engineered Pichia pastoris strains still needs breakthroughs. For example, CN115838753A introduced genes such as pcbCsc into yeasts such as Saccharomyces cerevisiae BY4741 to obtain engineered strains for the production of 7-ADCA, with a yield of 2.28 mg / g. CN110846342A used a UV-mutated mutant strain of Pichia pastoris to express penicillin acylase, aiming to reduce production costs and promote its application in the synthesis of β-lactam antibiotics, but no reports were found on the improvement of enzyme activity and yield. Therefore, it is urgent to construct engineered Pichia pastoris strains with high penicillin acylase production to meet the industrial production needs of enzymatic preparation of β-lactam antibiotics and promote the green upgrading and technological breakthroughs of the antibiotic industry. Summary of the Invention

[0007] To address the aforementioned issues, this application first reconstructed the recombinant plasmid and constructed a Pichia pastoris engineered strain incorporating the recombinant plasmid. This strain can efficiently express stable and highly active penicillin acylase through full fermentation culture. The constructed penicillin acylase-expressing Pichia pastoris engineered strain can also be effectively applied to the synthesis of β-lactam antibiotics, achieving high-yield preparations of products such as cefadroxil and cefaclor.

[0008] Specifically, this application is implemented through the following scheme: The applicant's primary objective is to provide a recombinant plasmid carrying a penicillin acylase gene derived from Escherichia coli, wherein the amino acid sequence encoded by the penicillin acylase gene is shown in SEQ ID NO.1.

[0009] The applicant's second objective is to provide an engineered strain of Pichia pastoris, which is obtained by transforming the aforementioned recombinant plasmid into Pichia pastoris.

[0010] The applicant's third objective is to provide the application of the aforementioned Pichia pastoris engineered strain in the production of penicillin acylase.

[0011] The application process is as follows: Pichia pastoris engineered strains are sequentially activated, shake-flask seed culture, primary seed culture, secondary seed culture, and fermentation culture to express penicillin acylase.

[0012] As a specific example, the above process is described as follows: S1. Activation culture: Pichia pastoris engineered strains were streaked onto YPD plates containing 100 μg / L Zeocin and cultured at 30℃ for 2-3 days to obtain activated strains; S2, Shake flask seed culture: Select a single colony of the activated strain and inoculate it into a baffle shake flask containing 100 μg / L Zeocin liquid medium. Incubate at 30℃ and 220 rpm for 20-36 h to obtain shake flask seed culture. S3. Primary seed culture: Take the shake flask seed solution obtained in step S2 and inoculate it with a flame into a seed tank containing BSM medium. The culture temperature is 30℃, the initial stirring speed is 200 rpm, the dissolved oxygen value is controlled at 10-40% by stirring and ventilation, and the pH is controlled at 5.3-5.7 by adding ammonia water. Culture for 20-36 hours to obtain the primary seed solution. S4. Secondary seed culture: Take the primary seed culture obtained in step S3 and inoculate it into a seed tank containing BSM medium. The culture temperature is 30℃, the initial stirring speed is 50 rpm, the dissolved oxygen value is controlled at 10-40% by stirring and ventilation, and the pH is controlled at 5.3-5.7 by adding ammonia water. Culture for 10-36 hours to obtain the secondary seed culture. S5. Fermentation culture: Take the secondary seed liquid obtained in step S4 and inoculate it into a fermenter containing BSM medium. The culture temperature is 30℃, the initial stirring speed is 20 rpm, and the dissolved oxygen value is controlled at 10-40% throughout the fermentation process by stirring and ventilation. The pH is controlled at 5.3-5.7 by adding ammonia water.

[0013] During fermentation, when dissolved oxygen (DO) rises, 50% glycerol is added to increase the bacterial concentration. When the bacterial concentration reaches 200-300 g / kg, glycerol feeding is stopped, and starvation culture is carried out for 2-4 hours.

[0014] During fermentation (as described above after starvation culture), pure methanol can be added to induce the expression of penicillin acylase.

[0015] This application has significant industrial application value and beneficial effects, specifically reflected in: 1) High-yield, high-activity penicillin acylase heterologous expression. The Pichia pastoris engineered strain obtained by this application has excellent heterologous expression performance of penicillin acylase and adaptability to large-scale fermentation. Under industrial-scale fermentation conditions in a fermenter, after about 100 hours of fermentation, the activity of the penicillin acylase expressed by this strain can stably reach 49,000 U / L, demonstrating efficient target enzyme expression ability and good large-scale fermentation characteristics.

[0016] 2) Excellent adaptability to large-scale fermentation. The Pichia pastoris engineered strain of this application can be directly applied to the industrial production of penicillin acylase. Relying on its excellent large-scale fermentation expression performance, it can effectively improve the industrial production efficiency of penicillin acylase, significantly reduce the industrial production cost of penicillin acylase, and solve the pain points of high cost and poor large-scale expression effect in the existing industrial production of penicillin acylase.

[0017] 3) Facilitates the industrialization of β-lactam antibiotics. The successful construction and application of the aforementioned recombinant plasmids and engineered strains can further promote the widespread application and large-scale use of penicillin acylase in the industrial synthesis of β-lactam antibiotics, providing an efficient, economical, and stable biocatalytic tool for the industrial synthesis of β-lactam antibiotics. This has significant practical implications for improving the quality and efficiency of the β-lactam antibiotic industry. Attached Figure Description

[0018] Figure 1 The image shows the pPICZαA-EcPGA plasmid pattern from Example 1.

[0019] Figure 2 This is an SDS-PAGE protein electrophoresis image of the Pichia pastoris engineered strain in Example 2.

[0020] Figure 3 This is a diagram of the Pichia pastoris engineered strain in Example 3 fermenting in 50L.

[0021] Figure 4 This is a diagram of the Pichia pastoris engineered strain's 5t fermentation scale-up production process in Example 4. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this application clearer, this application will be further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0023] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0024] Unless otherwise specified, the experimental materials used in the following examples are all conventional biochemical reagents.

[0025] The room temperature is 20-30℃, such as the common 20℃ or 25℃.

[0026] LB liquid medium composition: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, solvent: deionized water. LB solid medium requires the addition of agar to a final concentration of 20 g / L to the LB liquid medium. YPD medium, BSM basal salt medium, and PTM1 trace element solution are all prepared using standard formulations provided by Invitrogen. YPD solid medium requires the addition of agar to a final concentration of 20 g / L to the YPD liquid medium. BSM medium must be autoclaved first, and after cooling, PTM1 solution (filtered and sterilized) should be added at a ratio of 4.0 mL / L.

[0027] The following examples illustrate the method for detecting penicillin acylase activity: Weigh 4.0 g of penicillin G potassium salt and place it in a 100 mL beaker. Add 80 mL of purified water and 0.5 mL of phosphate buffer. Maintain the temperature of the system at 28±1℃ and stir until the sample is completely dissolved. Turn on the potentiometric titrator and perform cleaning and degassing operations on the titration system 2-3 times. After the system temperature stabilizes at 28±1℃, add 0.2 mL of enzyme solution to the reaction solution and pre-titrate with 0.1 mol / L sodium hydroxide solution until the pH of the system reaches 8.0. Then, stop the pre-titration operation. Start the repeat titration program and continue titrating with 0.1 mol / L sodium hydroxide solution, maintaining the pH of the system at 8.0. Record the volume of 0.1 mol / L sodium hydroxide solution consumed within 10 minutes of titration and calculate using the following formula: .

[0028] In the formula: U / mL is the amount of H produced per mL of enzyme per minute. + The amount of mmol is μmol; V is the volume of 0.1 mol / L sodium hydroxide titrant consumed in 10 minutes of titration, mL; C is the molar concentration of 0.1 mol / L sodium hydroxide titrant, C = 0.1 mol / L; 1000 is the conversion factor from mmol to μmol; G is the enzyme volume, G = 0.2 mL; t is the titration time, t = 10 min.

[0029] The primer sequences involved in the following examples are shown in Table 1.

[0030] Table 1: Primer Sequence Listing .

[0031] Example 1 This embodiment describes the synthesis and amplification of the penicillin acylase gene.

[0032] Combination Figure 1 Based on the gene sequence of penicillin acylase EcPGA published by NCBI (GenBank: M17609.1), the pPICZαA-EcPGA plasmid was constructed. Using the pPICZαA plasmid as a template and pPICZαA-F / pPICZαA-R primers (Table 1), the DNA fragment was recovered using a Clean-up kit to obtain the linearized pPICZαA-Line plasmid. Using the codon-optimized heterologous EcPGA pTrc99a plasmid (purchased from Beijing Qingke Biotechnology) as a template and EcPGA-F / EcPGA-R primers (Table 1), the PCR product was amplified. The DNA fragment was recovered using a Clean-up kit to obtain the EcPGA fragment (amino acid sequence shown in SEQ ID NO.1). Following the instructions of the One-step clonekit (Vazyme Biotech, Nanjing, China), the linearized pPICZαA-Line plasmid and the EcPGA fragment were ligated together. The ligation product was then transformed into a chemical transformation product. E.coli In DH5α competent cells, the cells were cultured and screened at 37℃. Clones were then selected and sequenced using primers pPICZαA-EcPGA-VF / pPICZαA-EcPGA-VR to obtain the pPICZαA-EcPGA plasmid.

[0033] Example 2

[0034] This embodiment describes the construction and verification of Pichia pastoris engineered strains.

[0035] 1. Construction of Pichia pastoris engineered strains Using the recombinant yeast expression plasmid pPICZαA-EcPGA as a template, PCR amplification was performed using the primer pairs pPICZαA-EcPGA-XF / pPICZαA-EcPGA-XR listed in Table 1. The product was recovered and purified using a Clean Up kit to obtain the linearized pPICZαA-EcPGA plasmid. Transformation was performed using the Pichia pastoris competent cell preparation and transformation kit from Shanghai Beyotime Biotechnology Co., Ltd. The transformation solution was plated on a YPD selection plate containing 100 μg / L Zeocin, and positive single clones were picked for colony PCR verification. After confirming that the band size was consistent with expectations by agarose gel electrophoresis, further sequencing verification was performed. Correct sequencing results indicated successful construction of the Pichia pastoris engineered strain.

[0036] 2. Validation of Pichia pastoris engineered strains Single colonies were selected and inoculated into YPD medium, and cultured with shaking at 30℃ and 220 rpm for 24 h. The seed culture was then transferred to BSM medium and cultured with shaking at the same temperature and speed, with 0.5% methanol added every 24 h to induce protein expression. After 3 days of induction, the bacterial cells were collected by centrifugation, and the crude enzyme solution was extracted after disruption. SDS-PAGE protein electrophoresis and penicillin acylase activity assays were performed. Figure 2 The electrophoresis results show that the cell lysate of the recombinant Pichia pastoris engineered strain exhibits distinct target bands at both 25 kD and 65 kD, which perfectly match the theoretical molecular weights of the α and β subunits of penicillin acylase EcPGA. This confirms that the Pichia pastoris engineered strain constructed in this embodiment can efficiently express active penicillin acylase.

[0037] Example 3

[0038] This example demonstrates the fermentation of Pichia pastoris engineered strains in a 50L tank to express penicillin acylase. The process is as follows: 1. Activation culture: The Pichia pastoris engineered strain constructed in Example 2 was streaked onto a YPD plate containing 100 μg / L Zeocin and cultured at 30°C for about 2 days to obtain activated bacteria; 2. Seed culture: Pick a single colony and inoculate it into a shaker flask containing 1000 mL of YPD liquid medium containing 100 μg / L Zeocin. Incubate at 37℃ and 220 rpm for about 36 h to obtain seed culture. 3. Fermentation Culture: Take the seed culture obtained in step 2 and inoculate it with a flame into a 50L fermenter containing 20 L of BSM medium. The culture temperature is 30℃, and the initial stirring speed is 300 rpm. Throughout the fermentation process, the dissolved oxygen value is controlled at around 30% by stirring and ventilation, and the pH is controlled at approximately 5.6 by adding ammonia. When the dissolved oxygen (DO) rises, 50% glycerol is added to increase the bacterial concentration. When the bacterial concentration reaches around 300 g / Kg, glycerol feeding is stopped, and starvation culture is performed for about 3 hours. After the starvation culture ends, pure methanol is added to induce the expression of penicillin acylase. During the induction process, the penicillin acylase content is monitored.

[0039] The results are as follows Figure 3As shown: In a 50L fermenter, the Pichia pastoris engineered strain showed a steady and rapid increase in bacterial concentration from an initial 100 g / kg starting at 12 hours of fermentation. The rate of increase slowed down after about 62 hours, and the concentration reached a peak of 588 g / kg at about 100 hours. The activity of penicillin acylase began to increase linearly after about 53 hours of fermentation, reaching a plateau of about 32 U / mL at about 75 hours. After nearly 90 hours of fermentation, the activity increased more rapidly again, reaching over 50 U / mL at about 100 hours, with a yield of about 125.2 U / mL methanol.

[0040] Example 4

[0041] This example demonstrates the fermentation of Pichia pastoris engineered strains in a 5t tank to express penicillin acylase. The process is as follows: 1. Activation culture: The Pichia pastoris engineered strain constructed in Example 2 was streaked onto a YPD plate containing 100 μg / L Zeocin and cultured at 30 °C for about 2 days to obtain activated bacteria; 2. Shake flask seed culture: Pick a single colony and inoculate it into a baffle flask containing 1000 mL of YPD liquid medium containing 100 μg / L Zeocin. Incubate at 30℃ and 220 rpm for about 36 h to obtain the shake flask seed culture. 3. Primary seed culture: Take the seed culture obtained in step 2 and inoculate it with flame into a 30L seed tank containing 15 L of BSM medium. The culture temperature is 30 ℃, the initial stirring speed is 200 rpm, the dissolved oxygen value is controlled at around 30% by stirring and ventilation, and the pH is controlled at about 5.6 by adding ammonia water. After culturing for about 24 hours, the primary seed culture is obtained. 4. Secondary seed culture: Take the seed culture obtained in step 3 and inoculate it into a 200L seed tank containing 150 L of BSM medium. The culture temperature is 30 ℃, the initial stirring speed is 50 rpm, the dissolved oxygen value is controlled at around 30% by stirring and ventilation, and the pH is controlled at about 5.6 by adding ammonia water. The culture is carried out for about 12 hours to obtain the secondary seed culture. 5. Fermentation Culture: The secondary seed culture obtained in step 4 was inoculated into a 5t fermenter containing 2000 L of BSM medium. The culture temperature was 30 ℃, and the initial stirring speed was 20 rpm. Throughout the fermentation process, the dissolved oxygen value was controlled at around 30% by stirring and ventilation, and the pH was controlled at approximately 5.6 by adding ammonia. When the dissolved oxygen (DO) rose, 50% glycerol was added to increase the bacterial concentration. When the bacterial concentration reached approximately 300 g / Kg, glycerol feeding was stopped, and starvation culture was performed for about 3 hours. After the starvation culture ended, pure methanol was added to induce the expression of penicillin acylase. During the induction process, the penicillin acylase content was measured.

[0042] The results are as follows Figure 4As shown: The constructed Pichia pastoris engineered strain, in a 5t fermenter, showed a steady and rapid increase in bacterial concentration from an initial 80 g / kg starting at about 13 hours of fermentation. At about 20 hours, the bacterial concentration reached its first peak (about 360 g / kg) and began to decline. Then, after about 30 hours of fermentation, the bacterial concentration rose again, reaching 521 g / kg at about 100 hours. The activity of penicillin acylase began to increase rapidly and linearly after about 40 hours of fermentation. After about 50 hours of fermentation, the rate of increase slowed down. When fermentation reached about 100 hours, the activity of penicillin acylase reached 49.9 U / mL, with a yield of about 121.2 U / mL methanol.

[0043] The results of comparison with Examples 3 and 4 further confirm that the Pichia pastoris engineered strain constructed in this example has outstanding potential for industrial application.

[0044] Comparative Example 1

[0045] The setup for this comparative example is the same as that for Example 3, except that glycerol was not added in step 3 to regulate dissolved oxygen. The expression process of penicillin acylase is as follows: Activation culture: The Pichia pastoris engineered strain constructed in Example 2 was streaked onto a YPD plate containing 100 μg / L Zeocin and cultured at 30°C for about 2 days to obtain activated bacteria; 2. Seed culture: Pick a single colony and inoculate it into a shaker flask containing 1000 mL of YPD liquid medium containing 100 μg / L Zeocin. Incubate at 37℃ and 220 rpm for about 36 h to obtain seed culture. 3. Fermentation culture: Take the seed liquid obtained in step 2 and inoculate it with flame into a 50L fermenter containing 20 L of BSM medium. The culture temperature is 30℃ and the initial stirring speed is 300 rpm. After culturing for about 3 hours, pure methanol is added to induce the expression of penicillin acylase. The enzyme activity of penicillin acylase is about 39.2 U / mL and the yield is about 72.6 U / mL of methanol.

[0046] Comparative Example 2

[0047] The expression process of penicillin acylase in this comparative example is as follows: 1. Activation culture: The Pichia pastoris engineered strain constructed in Example 2 was streaked onto a YPD plate containing 100 μg / L Zeocin and cultured at 30 °C for about 2 days to obtain activated bacteria; 2. Shake flask seed culture: Pick a single colony and inoculate it into a baffle flask containing 1000 mL of YPD liquid medium containing 100 μg / L Zeocin. Incubate at 30℃ and 220 rpm for about 36 h to obtain the shake flask seed culture. 3. Primary seed culture: Take the seed culture obtained in step 2 and inoculate it with flame into a 30L seed tank containing 15 L of BSM medium. The culture temperature is 30 ℃, the initial stirring speed is 200 rpm, the dissolved oxygen value is controlled at around 30% by stirring and ventilation, and the pH is controlled at about 5.6 by adding ammonia water. After culturing for about 24 hours, the primary seed culture is obtained. 4. Secondary seed culture: Take the seed culture obtained in step 3 and inoculate it into a 200L seed tank containing 150 L of BSM medium. The culture temperature is 30 ℃, the initial stirring speed is 50 rpm, the dissolved oxygen value is controlled at around 30% by stirring and ventilation, and the pH is controlled at about 5.6 by adding ammonia water. The culture is carried out for about 12 hours to obtain the secondary seed culture. 5. Fermentation Culture: The secondary seed culture obtained in step 4 was inoculated into a 5t fermenter containing 2000 L of BSM medium. The culture temperature was 30 ℃, and the initial stirring speed was 20 rpm. After culturing for about 3 hours, pure methanol was added to induce the expression of penicillin acylase. During the induction process, the penicillin acylase content was detected. The enzyme activity of penicillin acylase was approximately 30.9 U / mL, and the yield was approximately 66.8 U / mL of methanol.

[0048] Comparative Example 3

[0049] CN110846342A was used as comparative example 3. Its penicillin acylase gene originated from the genus *Achromobacter* (…). Achromobacter sp (CCM 4824) The engineered strain needs to be screened for high-yielding mutant strains through ultraviolet mutagenesis. The expression process of its penicillin acylase is as follows: the penicillin acylase gene is synthesized and amplified to obtain a recombinant plasmid - the linearized recombinant plasmid is transformed into ultraviolet-mutated Pichia pastoris PGA-3 to obtain the Pichia pastoris engineered strain - the engineered strain is inoculated in a culture medium and cultured with shaking, and methanol is added to induce the expression of penicillin acylase. The enzyme activity of fermentation in a 50L shake flask is about 30,000 U / mL.

[0050] This application uses bacteria derived from Escherichia coli (Escherichia coli) Escherichia coli The penicillin acylase gene of this application was used to construct an engineered Pichia pastoris strain. When cultured at room temperature (e.g., 30°C as mentioned above) and in a slightly acidic neutral environment (pH 5.3–5.7, approximately 5.6 as mentioned above), the strain exhibited excellent heterologous expression performance and adaptability for large-scale fermentation of penicillin acylase. It can directly express penicillin acylase in high yield through full fermentation. On a scaled-up industrial scale (e.g., in a 5t fermenter in Example 4), after 100 hours of fermentation, the enzyme activity stably reached 49900 U / L, which is the highest fermentation level disclosed in relevant patents to date. The penicillin acylase of this application can be applied to the industrial synthesis of β-lactam antibiotics.

[0051] The embodiments described in this application are merely illustrative examples illustrating several feasible implementation methods. Although the related descriptions are detailed and specific, they do not constitute a limitation on the scope of patent protection of this application, nor are they intended to restrict the scope of protection of the claims of this application. For those skilled in the art, various modifications and improvements can be made to this application without departing from the concept of this application; all equivalent implementations or changes made without departing from the spirit of this application should be included in the technical protection scope of this application.

Claims

1. A recombinant plasmid, characterized in that: The recombinant plasmid carries a penicillin acylase gene from Escherichia coli, and the amino acid sequence encoded by the penicillin acylase gene is shown in SEQ ID NO.

1.

2. A Pichia pastoris engineered strain, characterized in that: The Pichia pastoris engineered strain was obtained by transforming the recombinant plasmid of claim 1 into a Pichia pastoris host.

3. The application of the Pichia pastoris engineered strain according to claim 2 in the generation of penicillin acylase.

4. The application according to claim 3, characterized in that: The engineered strain of Pichia pastoris was successively activated, seed cultured, and fermented to express penicillin acylase.

5. The application according to claim 4, characterized in that, The activation culture conditions are as follows: Pichia pastoris engineered strains are streaked onto YPD plates containing 100 μg / L Zeocin and cultured at 30℃ for 2–3 days to obtain activated strains.

6. The application according to claim 4, characterized in that, The seed culture refers to shake flask seed culture, and the culture conditions are as follows: pick a single colony and inoculate it into a baffle shake flask containing 100 μg / L Zeocin in YPD liquid medium, and culture at 30℃ and 220 rpm for 20-36 h. The resulting shake flask seed liquid is then fermented.

7. The application according to claim 4, characterized in that, The seed culture includes shake flask seed culture, primary seed culture, and secondary seed culture: a single colony is picked and inoculated into a baffled shake flask containing 100 μg / L Zeocin in YPD liquid medium, and cultured at 30℃ and 220 rpm for 20–36 h. The resulting shake flask seed solution is then added for fermentation culture. The product obtained from the shake flask seed culture is flame-inoculated into a seed tank containing BSM medium, and cultured at 30℃ with dissolved oxygen at 10–40% and pH at 5.3–5.7 for 20–36 h to obtain primary seed solution. The primary seed solution is then inoculated into a seed tank containing BSM medium for secondary seed culture, with the secondary seed culture temperature at 30℃, dissolved oxygen at 10–40%, and pH at 5.3–5.7 for 10–36 h. The resulting secondary seed solution is then used for fermentation culture.

8. The application according to claim 4, characterized in that, The fermentation conditions are as follows: the product obtained from seed culture is inoculated into a fermenter containing BSM medium for fermentation culture. The fermentation temperature is 30℃, and the dissolved oxygen value is controlled at 10-40% and the pH is controlled at 5.3-5.7 throughout the fermentation process.

9. The application according to claim 4, characterized in that: Methanol was added during fermentation to induce penicillin acylase expression.

10. The application according to any one of claims 4 to 9, characterized in that: During the fermentation process, 50% glycerol is added to increase the bacterial concentration to 200-300 g / kg, and the culture is starved for 2-4 hours to control the rise of dissolved oxygen.

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