A penicillinase mutant and its recombinant strain and its application
By modifying penicillinase through site-directed mutagenesis, the problem of penicillinase activity dropping sharply in highly alkaline environments was solved, achieving efficient purification treatment within the pH range of 5-10. This method is suitable for penicillin G production wastewater, reducing operating costs and improving treatment efficiency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HUNAN INSTITUTE OF ENGINEERING
- Filing Date
- 2026-05-07
- Publication Date
- 2026-06-02
AI Technical Summary
Existing penicillinases exhibit a sharp drop in activity under highly alkaline conditions, making them unsuitable for the wide pH fluctuations in penicillin production wastewater. This results in unstable enzymatic pretreatment effects, high operating costs, and an inability to meet industrial-scale requirements.
By performing site-directed mutagenesis on wild-type penicillinase, a penicillinase mutant that maintains high catalytic activity in the pH range of 5-10 was obtained, especially maintaining high enzyme activity under highly alkaline conditions of pH 9-10, which is suitable for the purification treatment of penicillin G production wastewater.
It maintains high catalytic activity over a wide pH range, solves the problem of sudden drop in enzyme activity, simplifies the process, reduces operating costs, and improves processing efficiency and stability, making it suitable for industrial applications.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of bioengineering, and more specifically to a penicillinase mutant and its recombinant strain and applications. Background Technology
[0002] Penicillin G (penicillin G sodium / potassium), as the earliest β-lactam antibiotic to be widely used, can cause multiple potential hazards to natural water bodies such as rivers, lakes, and groundwater if discharged into them without effective treatment. These hazards are also characterized by their insidious, cumulative, and transmissible nature.
[0003] Penicillin G is completely degraded into CO2, H2O, and NH4. + / NO3 — SO4 2- Inorganic substances are the core objective of antibiotic wastewater treatment. Single enzymatic methods or common treatment processes typically only achieve ring-opening inactivation of the β-lactam ring, failing to achieve complete mineralization. Currently, the industry's efficient and engineering-feasible integrated treatment pathway is: pretreatment to break down the ring—deep mineralization—stable compliance. Specifically: first, penicillinase (β-lactamase) rapidly hydrolyzes the β-lactam ring, generating penicillin thiazolic acid, which has no antibacterial activity, significantly reduced molecular stability, and is easily degraded by subsequent processes; then, through advanced oxidation, bioaugmentation, and physicochemical-biochemical coupling technologies, the thiazolic acid ring, benzene ring, and side-chain carbon chains are gradually broken down, ultimately mineralizing into CO2, H2O, and inorganic ions (NH4+). + SO4 2- (etc.) to achieve complete mineralization.
[0004] In the entire penicillin G degradation process, the rapid and complete hydrolysis of the β-lactam ring is the key to determining the overall treatment efficiency. However, commonly used penicillinases in industry generally have application defects: their optimal pH is 7.0-8.0, and their activity decreases rapidly in highly alkaline environments with pH>9, with even more significant activity loss at pH10. They cannot adapt to the actual working conditions of wide pH fluctuations in penicillin production wastewater, resulting in unstable enzymatic pretreatment effects, high operating costs, and difficulty in meeting the requirements for stable industrial treatment. Summary of the Invention
[0005] To address the technical problems of existing penicillinase, such as its narrow optimal pH range, sharp drop in activity under high alkalinity, inability to adapt to wide pH fluctuations in wastewater, frequent pH adjustments required for enzymatic pretreatment of penicillin G production wastewater, high operating costs, and poor process stability, this invention provides a penicillinase mutant with wide pH applicability and high alkalinity stability, along with its recombinant strain and applications. By performing site-directed mutagenesis on wild-type penicillinase, its catalytic activity and stability within the pH range of 5-10 are significantly improved, especially maintaining high enzyme activity under high alkalinity conditions of pH 9-10. It can be directly used for the efficient purification treatment of penicillin G production wastewater.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] A penicillinase mutant was obtained by using wild-type penicillinase with the amino acid sequence shown in SEQ ID NO:1 as the parent, and by mutating isoleucine at position 71 to threonine and lysine at position 180 to aspartic acid. The amino acid sequence of the penicillinase mutant is shown in SEQ ID NO:2.
[0008] Furthermore, the amino acid sequence of the wild-type penicillinase was obtained from the protein sequence library GenBank:CAA84711.1. Using the Lésign platform, the wild-type penicillinase sequence was designed to obtain the computationally optimal enzyme mutant.
[0009] Furthermore, the amino acid sequence of the penicillinase mutant, as shown in SEQ ID NO:3, is obtained by mutating phenylalanine at position 99 to valine and phenylalanine at position 283 to aspartic acid, based on SEQ ID NO:2.
[0010] Furthermore, the amino acid sequence of the penicillinase mutant, as shown in SEQ ID NO:4, is obtained by mutating phenylalanine at position 99 to leucine and phenylalanine at position 283 to asparagine, based on SEQ ID NO:2.
[0011] Furthermore, the amino acid sequence of the penicillinase mutant, as shown in SEQ ID NO:5, is obtained by mutating glycine at position 230 to arginine and isoleucine at position 244 to alanine, based on SEQ ID NO:4.
[0012] Furthermore, the amino acid sequence of the penicillinase mutant, as shown in SEQ ID NO:6, is obtained by mutating glutamine at position 55 to glutamic acid and alanine at position 232 to threonine, based on SEQ ID NO:4.
[0013] Furthermore, the amino acid sequence of the penicillinase mutant, as shown in SEQ ID NO:7, is obtained by mutating methionine at position 44 to lysine and glycine at position 230 to arginine, based on SEQ ID NO:4.
[0014] The present invention also provides a recombinant genetic material of a penicillinase mutant, wherein the recombinant genetic material is DNA or RNA capable of expressing the above-mentioned penicillinase mutant.
[0015] The present invention also provides a recombinant strain of penicillinase mutant, comprising the recombinant genetic material of the above-mentioned penicillinase mutant.
[0016] The present invention also provides applications of the above-mentioned penicillinase mutant, including its application in the preparation of penicillinase and its application in the purification of penicillin G-containing wastewater, which is a wide pH environment of pH 5 to 10, and is particularly suitable for highly alkaline penicillin G production wastewater of pH 9 to 10.
[0017] Terminology explanation or definition:
[0018] Recombinant gene: refers to DNA or RNA capable of expressing the penicillinase of the present invention. Typically, the recombinant gene is initially synthesized in vitro via solid-phase phosphoramidite synthesis, TdT biosynthesis, or other suitable techniques known in the art. Once a template sequence is available, it can be amplified by PCR or other suitable techniques known in the art. With a recombinant bacterial strain, further large-scale amplification can be achieved by culturing the strain. In some embodiments, the recombinant gene may also include residual restriction enzyme sites, other accessory elements such as control elements (e.g., promoters), labeling substances (e.g., fluorescent labels), and other sequences that do not affect the expression of the target gene.
[0019] Clonal scars refer to the promoter sequence of transcription, which is dependent on the initiator ribonucleotide (mRNA) for protein expression, followed by the ribosome-binding site (RBS) that attracts the translation machinery, and then the signal peptide sequence that facilitates protein transport to the periplasm. Mature proteins are typically cloned after the signal peptide, cleaved from it by a signal peptidase as they cross the membrane. However, when cloning constructs after the signal peptide, restriction endonucleases often require specific sequences to cut the DNA, leaving clonal scars after the signal peptide sequence.
[0020] Signal peptides are short peptides (typically 16-30 amino acids long) located at the N-terminus of most newly synthesized proteins, which are destined for the secretion pathway. They can also be called signal sequences, targeting signals, localization signals, localization sequences, transport peptides, leader sequences, or leader peptides. Signal peptides are usually cleaved from proteins by signal peptidases.
[0021] Signal peptide cleavage site: This refers to the dipeptide between which a signal peptidase cleaves the signal peptide from the mature protein. In most (but not all) cases, the dipeptide is Ala-Ala. Signal peptide cleavage sites can be calculated using algorithms such as SignalP 4.1, which are available online at http: / / www.cbs.dtu.dk / services / SignalP / (Center for Biological Sequence Analysis, Technical University of Denmark).
[0022] A promoter is a DNA region that initiates the transcription (writing to mRNA) of a specific gene. Promoters are typically located near the transcription start site of a gene, on the same strand of the DNA and upstream of the DNA strand (pointing to the 5' region of the sense strand). Promoters can be inducible, meaning that the expression of a gene operatively linked to the promoter can be activated in the presence of an inducing agent. Alternatively, promoters can be constitutive, meaning they are not regulated by any inducing agent.
[0023] RBS stands for ribosome-binding site, or ribosome binding site. This is the sequence of nucleotides upstream of the start codon in mRNA transcripts, responsible for recruiting ribosomes during the initiation of protein translation.
[0024] Expression: refers to the process of DNA being transcribed into messenger RNA (mRNA) and then translated into protein. To achieve successful expression and screening of penicillinase, the aforementioned signal peptide, promoter, and RBS may be introduced into the recombinant gene. Therefore, some corresponding peptide segments may remain on the expressed penicillinase protein. These peptide segments do not affect the function of penicillinase; therefore, even if the product contains additional peptide segments, as long as the amino acid sequence of the main component is identical to the sequence of this invention, the product is still an infringing product.
[0025] Expression vectors: These have the ability to incorporate and express heterologous polynucleotide fragments in host cells. Many prokaryotic and eukaryotic expression vectors are commercially available. Choosing a suitable expression vector is within the knowledge of a technical person.
[0026] Chassis cell: refers to a suitable host vector used to express DNA containing the present invention. The host can be any organism capable of containing and expressing the nucleic acids or genes disclosed herein, but is not limited thereto. The chassis cell can be a prokaryote or eukaryote, unicellular or multicellular, including mammalian cells, plant cells, fungi, etc. The chassis cell may be selected from at least one of the following: *Escherichia coli*, *Pichia pastoris*, *Saccharomyces cerevisiae*, *Hansenula polymorpha*, *Candida albicans*, *Rhodotorula rubrum*, *Bacillus*, *Escherichia coli*, *Salmonella*, *Clostridium*, *Streptomyces*, *Staphylococcus*, *Neisseria*, and *Shigella*. Chassis cells are preferably *Escherichia coli*, and suitable *E. coli* strains (including many others) include BL21(DE3), C600, DH5αF′, 1113101, JM83, JM101, JM103, JM105, JM107, JM109, JM110, MC1061, MC4100, MM294, NM522, NM554, TGI, χ1776, XL1-Blue, and Y1089. + The above E. coli strains are all commercially available strains.
[0027] Identity: This means that residues in two sequences are identical when aligned to the maximum correspondence, as measured using sequence comparison or analysis algorithms such as those described in this paper. For example, if corresponding fragments of two sequences have the same residues at 5 out of 10 positions when correctly aligned, the two sequences are said to have 50% identity. Most bioinformatics programs report the percentage identity of aligned sequence regions, which are typically not the entire molecule. If the alignment is long enough and contains enough identical residues, an expected value can be calculated, indicating that the level of identity in the alignment is unlikely to occur randomly.
[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0029] (1) The penicillinase mutant of this invention has a wide pH adaptability and can maintain stable enzyme activity under highly alkaline conditions. The optimal pH of wild-type penicillinase is around 6, and the highest enzyme activity is 2.78 × 10⁻⁶. 9 However, when the pH reaction environment changes to pH 9, the enzyme activity is only about 70% of the maximum enzyme activity, and it drops to below 50% at pH 10. The penicillinase mutant of this invention maintains high catalytic activity in the pH range of 5 to 10. In a highly alkaline environment of pH 9 to 10, the enzyme activity can still reach more than 90% of the maximum enzyme activity of the wild type, completely solving the problem of rapid enzyme activity drop under highly alkaline conditions.
[0030] (2) The penicillinase mutant of the present invention can be directly adapted to the highly alkaline environment of penicillin G production wastewater, without the need to frequently adjust the pH of the wastewater to adapt to enzyme activity, which significantly simplifies the process flow, improves the operational stability, and solves the industry pain point that the existing enzymatic pretreatment is difficult to adapt to the pH fluctuation of wastewater.
[0031] (3) The penicillinase mutant of the present invention can rapidly and efficiently hydrolyze the β-lactam ring of penicillin G under a wide pH range, ensuring the stability and efficiency of the wastewater pretreatment process and laying a good foundation for subsequent deep mineralization. The overall treatment efficiency is significantly better than that of wild-type penicillinase.
[0032] (4) This invention has low operating costs and is environmentally friendly, making it suitable for industrial application. Because it eliminates the need for a large amount of acid-base adjustment agents, the operating cost is significantly reduced. At the same time, it uses enzymatic biodegradation, which eliminates secondary pollution and meets the requirements of green environmental protection and sustainable production. It has important industrial application value in the field of penicillin antibiotic wastewater purification. Detailed Implementation
[0033] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited thereto.
[0034] Penicillinase (β-lactamase) mainly exerts its catalytic effect by hydrolyzing the β-lactam ring. Its mechanism of action is as follows:
[0035] Substrate hydrolysis: Penicillinase specifically recognizes and binds to penicillin G molecules, catalyzing the hydrolysis of the β-lactam cyclic amide bond, thus rapidly opening the ring and inactivating the antibiotic;
[0036] Product conversion: Penicillin G is converted into penicillin thiazolic acid. This product has no antibacterial activity, its stability is significantly reduced, and it is more easily degraded by subsequent advanced processing.
[0037] Environmental adaptability: The modified penicillinase mutant can maintain the stability of the enzyme protein spatial structure over a wide pH range (especially in highly alkaline environments), ensuring continuous and stable catalytic efficiency.
[0038] In the following examples, 0.1M PB buffer represents 0.1 mol / L phosphate buffer, 150mM NaCl represents 150 mmol / L NaCl solution, and M and mM in molar concentration refer to mol / L and mmol / L, respectively.
[0039] Example 1
[0040] The preparation of penicillinase mutants includes the following steps:
[0041] (1) Construction of recombinant strain: The nucleotide sequence of the target gene was synthesized by Beijing Qingke Biotechnology Co., Ltd., and the nucleotide sequence was inserted into the expression vector using molecular cloning technology. Specifically, it was inserted into plasmid pET28a(+) to construct the recombinant plasmid. Subsequently, the recombinant plasmid was transformed into Escherichia coli BL21(DE3 competent cells) by heat shock. After verification by resistance selection, colony PCR and sequencing, a recombinant strain containing the target penicillinase gene was obtained. Other suitable expression vectors and host strains can also be used in the prior art. This embodiment only provides a specific scheme that can stably achieve efficient expression.
[0042] (2) Induction of penicillinase expression: The above recombinant strain was inoculated into LB liquid seed medium at an inoculum rate of 1% (v / v) and cultured overnight at 37°C and 200 rpm until mid-log (OD) expression. 600 ≈1.0-1.2), to obtain seed culture. Then, at an inoculum rate of 1% (v / v), the seed culture was transferred to TB liquid fermentation medium and cultured at 37℃ with shaking at 200-220 rpm, with real-time monitoring of the bacterial OD. 600 Value, pending OD 600 When the concentration was approximately 6.0, IPTG was added to a final concentration of 100 μM, the temperature was lowered to 16℃, and the culture was induced at 180 rpm for 16-18 h to achieve soluble and efficient expression of penicillinase.
[0043] After induction culture, the fermentation broth was centrifuged at 4℃ and 4000g for 10 min, and the cell pellet was collected. The cell pellet was washed twice with pre-cooled 0.1M PB buffer (pH 7.0, containing 150mM NaCl to prevent protein aggregation). After each wash, the pellet was centrifuged at 4℃ and 4000g for 10 min, the supernatant was discarded, and the washed cell pellet was collected and placed on ice for later use.
[0044] Add an appropriate amount of pre-cooled 0.1M PB buffer (pH 7.0) to the bacterial pellet and resuspend thoroughly, controlling the bacterial concentration to 0.1-0.2 g / mL. Perform sonication on ice for disruption. Sonication parameters: power 200-300W, 3-second interval, 5-second pause, total disruption time 20-30 min. Maintain ice bath throughout to prevent enzyme inactivation due to high temperature. After disruption, centrifuge at 12000 rpm for 1 hour at 4℃. Collect the supernatant, which is the crude penicillinase solution, and store temporarily at 4℃ for later use.
[0045] (3) Purification of penicillinase: Penicillinase (adapted histidine-tagged recombinant protein) was purified by nickel affinity chromatography (Ni-NTA). The specific operation was as follows: The crude enzyme solution was filtered through a 0.22 μm filter membrane to remove impurities, and then slowly loaded onto a Ni-NTA column that had been fully equilibrated with 0.1 M PB buffer (pH 7.0). The flow rate was controlled at 1 mL / min to ensure that the target protein was fully bound to the nickel column. The nickel column was washed with equilibration buffer (0.1 M PB, pH 7.0, containing 150 mM NaCl) at a flow rate of 1.5 mL / min until the elution peak was stable (OD was monitored by a UV detector). 280 Value, when OD 280 When the value drops below 0.05, it indicates that the contaminating proteins have been washed away. Subsequently, the target protein is eluted using a gradient elution method with 0.1M PB buffer (pH 7.0, containing 150mM NaCl). Gradient elution is performed by gradually increasing the imidazole concentration (20mM, 50mM, 100mM, 200mM, 500mM) while controlling the elution flow rate at 1mL / min. 5mL of eluent is collected from each tube, and the eluted components are detected by SDS-PAGE electrophoresis. The eluted peak component containing the target penicillinase is collected.
[0046] The collected target protein fraction was dialyzed and desalted for 24 hours with 0.1M PB buffer (pH 7.0) (changing the buffer 2-3 times) to remove imidazole and other salts. After dialysis, purified penicillinase was obtained and could be stored at -80℃ for later use.
[0047] Example 2
[0048] The method for determining penicillinase activity includes the following steps:
[0049] (1) Solution preparation
[0050] Penicillin solution: Weigh an appropriate amount of sodium (potassium) penicillin and dissolve it in phosphate buffer (pH 7.0) to prepare a solution containing 10,000 units of penicillin per 1 ml.
[0051] Penicillinase dilution solution: Take penicillinase fermentation broth or purified enzyme solution, dilute 1000-5000 times, and preheat at 37℃ for later use.
[0052] Iodine titrant (0.005 mol / L): Accurately measure 10 mL of 0.05 mol / L iodine titrant and dilute to 100 mL with pH 4.5 sodium acetate buffer.
[0053] (2) Measurement method
[0054] Test sample determination: Accurately measure 50 ml of penicillin solution and place it in a 100 ml volumetric flask. Preheat to 37°C, accurately add 25 ml of preheated penicillinase dilution, mix quickly, and react accurately at 37°C for 1 hour. Accurately pipette 3 ml of the reaction solution and immediately add it to 25 ml of iodine titrant (0.005 mol / L). Place in the dark at room temperature for 15 min, and titrate with 0.01 mol / L sodium thiosulfate titrant. Near the endpoint, add starch indicator solution and continue titrating until the blue color disappears.
[0055] Blank test: Take 2 ml of preheated penicillin solution, place at 37°C for 1 hour, accurately add 25 ml of iodine titrant (0.005 mol / L), then add 1 mL of penicillinase diluent, place at room temperature in the dark for 15 minutes, and titrate with 0.01 mol / L sodium thiosulfate titrant.
[0056] (3) Enzyme activity calculation
[0057] E = (BA) × M × F × D × 100,
[0058] In the formula, E is the penicillinase activity, U / (L·h); B is the volume of sodium thiosulfate consumed in the blank group, ml; A is the volume of sodium thiosulfate consumed in the test group, ml; M is the concentration of sodium thiosulfate titrant, mol / L; F is the equivalent potency of penicillin for each 1 ml of the above iodine titrant (0.005 mol / L) under the same conditions, abbreviated as equivalent potency coefficient; D is the dilution factor of penicillinase solution.
[0059] The pH of the penicillin solution was adjusted to 5, 6, 7, 8, 9, and 10 using 0.1 mol / L HCl and 0.1 mol / L NaOH, respectively. The enzyme activity of wild-type and mutant penicillinase was measured under different pH conditions, and the test results are shown in Tables 1-7.
[0060] Table 1. Enzyme activity of wild-type penicillinase at different pH values
[0061]
[0062] Table 2. Enzyme activity of penicillinase mutant QMS-2 at different pH values.
[0063]
[0064] Table 3. Enzyme activity of penicillinase mutant QMS-3 at different pH values.
[0065]
[0066] Table 4. Enzyme activity of penicillinase mutant QMS-4 at different pH values.
[0067]
[0068] Table 5. Enzyme activity of penicillinase mutant QMS-5 at different reaction temperatures.
[0069]
[0070] Table 6. Enzyme activity of penicillinase mutant QMS-6 at different pH values.
[0071]
[0072] Table 7. Enzyme activity of penicillinase mutant QMS-7 at different pH values.
[0073]
[0074] As shown in Table 1, the optimal pH for wild-type penicillinase is around 6, and the highest enzyme activity is 2.78 × 10⁻⁶. 9 However, when the pH reaction environment changes to pH 9, the enzyme activity rapidly decreases, reaching only about 70% of its maximum activity. When the pH reaction environment changes to pH 10, the enzyme activity drops to below 50% of its maximum activity. Therefore, the activity of the wild-type penicillinase mutant drops sharply in the pH range of 9-10, making it unsuitable for adapting to wide pH fluctuations in wastewater. As shown in Tables 2-7, the penicillinase mutant can still achieve more than 90% of the maximum activity of the wild type in the pH range of 9-10. It can be directly used for enzymatic pretreatment of highly alkaline penicillin G production wastewater without significant pH adjustment. It has advantages such as high degradation efficiency, low operating cost, stable process, and environmental friendliness, and has important engineering application value in the purification of penicillin antibiotic wastewater.
Claims
1. A penicillinase mutant, characterized in that, The amino acid sequence of the penicillinase mutant is shown in SEQ ID NO:
2. The penicillinase mutant is obtained by mutating isoleucine at position 71 to threonine and lysine at position 180 to aspartic acid, based on the wild-type penicillinase shown in SEQ ID NO:
1.
2. The penicillinase mutant according to claim 1, characterized in that, The amino acid sequence of the penicillinase mutant is shown in SEQ ID NO:
3. The penicillinase mutant is obtained by further mutating phenylalanine at position 99 to valine and phenylalanine at position 283 to aspartic acid, based on SEQ ID NO:
2.
3. The penicillinase mutant according to claim 1, characterized in that, The amino acid sequence of the penicillinase mutant is shown in SEQ ID NO:
4. The penicillinase mutant is obtained by further mutating phenylalanine at position 99 to leucine and phenylalanine at position 283 to asparagine, based on SEQ ID NO:
2.
4. The penicillinase mutant according to claim 3, characterized in that, The amino acid sequence of the penicillinase mutant is shown in SEQ ID NO:
5. The penicillinase mutant is obtained by further mutating glycine at position 230 to arginine and isoleucine at position 244 to alanine, based on SEQ ID NO:
4.
5. The penicillinase mutant according to claim 3, characterized in that, The amino acid sequence of the penicillinase mutant is shown in SEQ ID NO:
6. The penicillinase mutant is obtained by further mutating glutamine at position 55 to glutamic acid and alanine at position 232 to threonine, based on SEQ ID NO:
4.
6. The penicillinase mutant according to claim 3, characterized in that, The amino acid sequence of the penicillinase mutant is shown in SEQ ID NO:
7. The penicillinase mutant is obtained by further mutating methionine at position 44 to lysine and glycine at position 230 to arginine, based on SEQ ID NO:
4.
7. A recombinant genetic material of a penicillinase mutant, characterized in that, The recombinant genetic material is DNA or RNA capable of expressing the penicillinase mutant of any one of claims 1 to 6.
8. A recombinant strain of penicillinase mutant, characterized in that, Recombinant genetic material including the penicillinase mutant as described in claim 7.
9. The application of the penicillinase mutant according to any one of claims 1 to 6, characterized in that, The penicillinase mutant was used to prepare penicillinase.
10. The application of the penicillinase mutant according to any one of claims 1 to 6, characterized in that, The penicillinase mutant is used to purify wastewater containing penicillin G.