1.1. Penicillin acylase and its characteristics
Penicillin acylase, also known as penicillin acylase aminohydrolase, or penicillin acylase aminohydrolase. It is mainly obtained from intracellular enzymes of Escherichia coli, extracellular enzymes of Bacillus megaceus, and intracellular enzymes of Pichia pastoris. It has been widely used in the production of important intermediates of β-lactam antibiotics and semi-synthetic β-lactam antibiotics. Penicillin acylase is an enzyme that specifically catalyzes penicillin reactions. Penicillin acylase is mainly used in the production process of 6-aminopenicillanic acid (6-APA). 6-APA is mainly obtained by deacylating penicillin G through penicillin acylase. In addition, due to the antibacterial, antigenic and allergenic properties of penicillin acylase, it is difficult to be decomposed by enzymes in bacterial cells; in addition, due to the easy formation of antibiotic β-lactam residues by penicillin acylase and the problem of carcinogenicity, the treatment and control of such substances has always been a difficult problem in the field of antibiotic research.
1.2. What is acylation?
Acylation is also called acylation. Acylation refers to the reaction of introducing aliphatic acyl RCO- or aromatic acyl ArCO- into nitrogen, oxygen, carbon or sulfur atoms in organic molecules. The products after acylation are compounds such as ketones (aldehydes), amides and esters. Acylation reactions can be divided into oxygen acylation, nitrogen acylation and carbon acylation according to the site of acyl introduction. Acylation agents are generally carboxylic acids or carboxylic acid derivatives. Acylation reactions can also be classified according to the type of acylating agents.
2 Extraction method of penicillin acylase
How to recycle and purify penicillin acylase, especially using Escherichia coli or Pichia pastoris for genetic engineering. This is a very important link, and this step has a great impact on the quality of penicillin acylase.
2.1. Ultrasonic disruption. Chromatography
Ultrasonic disruption, chromatography and extraction purification are commonly used bioseparation techniques that can be used to separate, purify and extract biomacromolecules such as proteins and nucleic acids. The whole process is usually carried out at low temperature (0~4℃) to avoid sample degradation and damage. In this study, Escherichia coli was used as the main genetically engineered bacteria for crude enzyme liquid extraction. Removing the cell wall, nucleic acid and lipid parts in the extract can make the target protein purer and more stable. In order to obtain better results, the study adopted a variety of efficient purification methods such as ammonium sulfate segmented sedimentation method, chromatographic column method, DAE-dietary fiber design engineering method-52 column chromatography, gel material-200-hydroxyapatite column chromatography, etc. Studies have shown that the specific activity of crude enzyme solution of Escherichia coli is poor, and generally four to five stages of purification are required to obtain a higher specific activity. Ultrasonic crushing, chromatography and extraction purification technology can be effectively used to purify crude enzyme solution of Escherichia coli, providing a reliable basis for subsequent functional research and application.
2.2. Impact osmotic pressure
High-pressure pulse technology is widely used in the extraction and purification of biomacromolecules. Penicillin acylase is an intracellular protease mainly distributed on the cell membrane and cell wall, and is often used in the preparation of antibiotics. In this study, penicillin acylase was extracted by high-pressure pulse technology, and the obtained crude enzyme solution had higher activity. Through precipitation with ammonium sulfate aqueous solution, a higher reaction rate can be obtained at one time, and it can be directly applied to immobilization. In order to effectively extract penicillin acylase, the study used the osmotic pressure shock method, which is a simple and easy extraction method that does not require too many instruments and has a wide range of applications. This method wets Escherichia coli in a hypertonic solution and then moves it to a hypotonic medium. Through the action of osmotic pressure, the cell wall ruptures and the enzyme is excreted from the body. This method can prevent the problem of excessive release of contained substances due to complete rupture, and effectively maintain the survival rate of the bacteria. After obtaining the crude enzyme, it can be purified by methods such as ammonium sulfate precipitation and DEAE-cellulose-52 column chromatography analysis to obtain a polyacrylamide gel electrophoresis enzyme with a single band structure. It is precisely because of the osmotic pressure impact method that the cell wall integrity and bacterial survival rate are maintained during the extraction process, thereby obtaining a crude enzyme solution with higher specific activity. In addition, this extraction method also helps to retain the enzyme band structure, thereby helping to obtain high-purity, high-specific activity penicillin acylase.
2.3. Adsorbent treatment
Penicillin acylase is an important enzyme and has important application value in the preparation of antibiotics. In this study, in order to separate penicillin acylase from the fermentation broth, the adsorption method was used for separation. Using ammonium hydroxide to treat diatomaceous earth can directly extract penicillin acylase, and operate at room temperature, with high enzyme activity yield and good industrial application prospects. For the collection of bacteria, the energy consumption of the centrifugal separation method is high due to the small size of the microorganisms and the difficulty in precipitation. In addition, the single flocculation method cannot completely collect the bacteria. Therefore, a combination of flocculants and centrifugation technology can be used to collect bacteria to improve collection efficiency and reduce centrifugal energy consumption. There are many factors that affect enzyme activity during the production and extraction of penicillin acylase, such as the type, concentration, temperature, etc. of solvents. The method of adjusting the dosage and pH value of the extracted enzyme is also very critical. Usually, the fermentation broth can be pretreated to maximize the enzyme activity, and then the adsorbate is added and the penicillin acylase is separated. This method can make the production and separation of penicillin acylase more efficient and stable.
3 Fermentation process of penicillin acylase
3.1. Preparation of spores
For the improved strains from sandy soil, genetic improvement can be carried out using culture media containing glycerol, glucose, protein, etc. Under the conditions of the optimal growth temperature of 25-26℃ and 6-8 days, single colonies can be obtained and slant propagation can be carried out, and slant spores can be obtained after 7 days. These spores can be transplanted into high-quality grain or rice solid culture medium, and the millet strain culture with 3 stages of growth for 7 days at 25℃ needs to add more carbon source and organic nitrogen source to obtain a large amount of hyphae. For the spores produced during the fermentation process, shake flask experiments must be carried out to check the efficacy and the condition of foreign bacteria. In addition, during the cultivation process of seed tanks and fermentation tanks, a large amount of hyphae needs to be obtained by adding carbon source and organic nitrogen source. Fermentation process for penicillin production During the fermentation of penicillin acylase, a high dissolved oxygen content and ventilation rate are required, and the air flow rate is usually 1:1~1:5. The stirring speed needs to be moderate, generally 150~200r/min. In the early stage of fermentation, the stirring power is large, which can promote the growth of the bacteria. In addition, the control of parameters such as time and temperature in each stage of fermentation also needs to be adjusted according to different strains and fermentation conditions. The fermentation tank is the main link in the large-scale production of penicillin acylase. In the fermentation tank, a variety of substances, such as peanut cake powder, bran powder, corn syrup, glucose, urea, etc., can be added to improve the nutritional status and growth rate of the bacteria. When inoculating, it is recommended to control the mycelium inoculation amount between 12% and 15% to avoid waste and reduce costs. In addition, when handling the waste liquid generated during the fermentation process, attention should be paid to environmental protection. The waste liquid may contain a variety of harmful substances and pollutants, which need to be strictly treated and discharged.
3.2. Penicillin acylase fermentation control
The preparation process of penicillin acylase obtained penicillin acylase under suitable culture medium, temperature, ventilation and stirring culture conditions. Before fermentation, the relevant equipment and media (mainly carbon source, nitrogen source, precursor and inorganic salt) are disinfected, and then the seed liquid is connected. During this period, in order to maintain a certain temperature and pressure, defoamers are often added. If the pH value of the fermentation liquid is controlled by acid and alkali, glucose and ammonium salts must be added intermittently or continuously to supplement the carbon source and nitrogen source, or other liquid precursors must be added to increase the yield of penicillin acylase.
3.3. Characteristics of penicillin acylase fermentation process
In the process of producing penicillin acylase, sugar solution is one of its main nutrients, and the content of sugar solution can directly affect the production and yield of penicillin acylase. If the sugar addition rate is too low, the growth rate of mycelium will decrease, thereby affecting the activity and production of the bacteria, while a high sugar ratio will cause the viscosity of the fermentation liquid to increase, the number of mycelium to increase, and the dissolved oxygen to decrease rapidly, affecting the normal progress of fermentation. Therefore, in order to produce efficient penicillin acylase, the content of sugar solution needs to be precisely controlled. At present, a certain sugar content curve is usually used, which is based on experimental data and has been optimized and improved. However, since the raw materials of each batch of production are different, and the variety characteristics of each batch are different due to different germplasms, if only the same sugar content curve is used for addition, some products will have insufficient yields. Therefore, how to accurately adjust the sugar content according to the metabolic status of the mycelium is one of the keys to controlling the production process of penicillin acylase. Different sugar content curves can be used for adjustment according to the metabolic status of the mycelium. When the number of mycelium is small in the early stage of fermentation, the sugar addition rate of the sugar solution can be gradually increased to accelerate the growth rate of the mycelium. At the same time, in the middle and late stages of fermentation, the content of the sugar solution should be controlled to avoid excessive sugar accumulation affecting the normal metabolism of the fermentation liquid. In addition, the sugar content curve can be dynamically adjusted according to the real-time detected sugar solution content, so as to more accurately control the sugar solution content.
4 Extraction process of penicillin acylase
4.1. Pretreatment
After the fermentation is completed, it exists in the Monascus fermentation broth in the form of a minimum intensity of 10~30kg/m3, which contains a large amount of inorganic ions with high oxygen content (Ca, Mg, Fe), mycelium, unused culture medium, easily contaminated cells, metabolites of pathogens, proteins, etc. Its main purpose is to enrich the target product and remove a large amount of impurities for further separation and purification.
4.2. Filtration
The fermentation broth needs to be pretreated before extraction. A small amount of flocculant (such as alum) can be added to the fermentation broth, or the pH value of the fermentation broth can be adjusted to the isoelectric point of the protein to precipitate the protein, and then the floating liquid is pumped into each sealed filter chamber of the filter through a vacuum drum (using negative pressure as the filtration power) or a flat filter cartridge. Under working pressure, solid-liquid separation is achieved through a filter membrane or other filter cakes. Since penicillin acylase is easily degraded at room temperature, the temperature of the fermentation broth and the filtrate is lower than 10°C, and its yield is above 90%.
4.3. Extraction
Penicillin acylase was isolated from penicillin acylase by solvent extraction. It is a method of concentrating and purifying antibiotics from one liquid phase (e.g. fermentation filtrate) to another liquid phase (e.g. organic solvent) in different pH conditions, in different chemical states (free acid or salts), in solvents that are insoluble in water. Since there is a carboxyl group in the structure of penicillin acylase molecules and its pKa is 2.75, free acid is formed when its aqueous solution is acidified to a pH value of about 2.0. Penicillin acylase acid has extremely low solubility and good solvent extraction properties in alcohols, ketones, ethers, esters, etc., so it can be separated and purified from the fermentation broth.
Penicillin acylase is transferred into an organic solvent under an acidic environment, the pH value is adjusted to about 2.0, and then it enters a neutral aqueous phase, extracted multiple times, and finally purified. Penicillin acylase with a higher partition coefficient is generally used. Butyl acetate and n-pentyl acetate are mainly used in industrial production. The pH of butyl acetate extracted from the fermentation broth is 1.8~2.0, and the pH value of butyl acetate back-extracted into the aqueous phase is 6.8~7.4. The experimental results show that the ratio of fermentation filtrate to butyl acetate solution is 1.5~2.1. Sulfate and carbonate buffer solutions are used for back extraction to prevent pH fluctuations. The ratio of fermentation broth to solution is 3~4. After multiple extractions and 10 concentrations, its concentration basically meets the crystallization conditions, and the total extraction yield reaches 85%. The obtained sieving solution is mostly secondary extraction, using 10% sulfuric acid solution with a pH of 2.0~3.0, and then butyl acetate is added, the amount of which is about 1/3 of the sieving solution, and the back extraction is usually carried out with sodium bicarbonate aqueous solution with a pH of 7.0~8.0. During the primary extraction process, due to the high protein concentration in the filtration, a demulsifier needs to be added. The first step is extraction, containing protein, and adding PPB 0.05%~0.1%.
4.4. Extraction
In order to reduce the degradation of penicillin acylase, it must be carried out at a low temperature below 10℃. The brine in the extraction tank is frozen. During extraction, temperature and acidity have a great influence on the degradation of penicillin acylase, which is an important factor affecting its degradation. Many scholars have conducted a lot of research on it, and have conducted a detailed discussion on the influence of sewage temperature and humidity of different pH values on its degradation half-life. The results show that the stability range of penicillin acylase is pH 5.0~8.0, and it shows strong stability at pH 6.0, and it can be rapidly degraded in both acid and alkaline conditions. Through curve fitting, the relationship between the degradation half-life of penicillin acylase at different temperatures and pH value is obtained.
4.5. Crystallization
Activated carbon is added to the extract to remove pigments, heat sources, filter, and remove activated carbon. The extract is usually refined by crystallization. In butyl acetate, since the solubility of penicillin acylase potassium salt is very low, penicillin acylase potassium salt will crystallize after adding potassium acetate-ethanol. On this basis, potassium is dissolved in KOH aqueous solution through recrystallization method, the pH value is adjusted to neutral, butanol is added, and finally the pure product is obtained through azeotropic distillation. (1) Direct crystallization method: The butyl acetate extract is reacted with sodium acetate-ethanol to obtain crystalline sodium salt. Penicillin acylase potassium salt is obtained by mixing potassium acetate-ethanol-potassium acetate. (2) Azeotropic distillation method: After extraction, it is extracted with 0.5 mol/L sodium hydroxide and the pH value is adjusted to 6.4~6.8 to obtain sodium-containing physiological saline. At 16~26℃ and 0.67~1.3 kPa, 2.5 times butanol is added. Water and n-butanol are azeotropically evaporated. Sodium ion crystals are precipitated. The crystals are washed and dried to obtain penicillin acylase products.
