CN106967704B - A kind of PA ase for being used to prepare 6 aminopenicillanic acids isolates and purifies and immobilization coupling process - Google Patents

Abstract

The present invention relates to bioengineering field, and in particular to a kind of PA ase for being used to prepare 6 aminopenicillanic acids isolates and purifies and immobilization coupling process.This method comprises the following steps：(1) the inorganic mixed salt solution containing PA ase crude enzyme liquid and inorganic salts is prepared, which is 0.05 0.35g/mL；(2) inorganic mixed salt solution obtained by step (1) is mixed with the tert-butyl alcohol, then carries out split-phase, obtain upper phase solution, middle phase solid and the three-phase distribution system of the lower phase solution composition rich in salt and PA ase rich in the tert-butyl alcohol；(3) immobilization that carrier carries out PA ase is added into lower phase solution obtained by step (2).Method of the invention is easy to operate, the activity of gained PA ase and yield are higher.

Description

A kind of separation and purification of penicillin acylase for preparing 6-aminopenicillanic acid immobilized coupling method

technical field

The invention relates to the field of bioengineering, in particular to a coupling method for separation, purification and immobilization of penicillin acylase for preparing 6-aminopenicillanic acid.

Background technique

Penicillin acylase is a key enzyme for the preparation of β-lactam antibiotics. In industrial production, this enzyme has been successfully applied to the preparation of 6-aminopenicillanic acid, mostly in the form of immobilized enzyme. In order to prepare the immobilized enzyme that meets the production requirements, the penicillin acylase is firstly separated and purified from the fermentation broth, and then the enzyme is immobilized. The expression vectors of penicillin acylase mainly include Escherichia coli, Bacillus subtilis, Bacillus megaterium and Pichia pastoris.

Although the expression vectors are different, the separation and purification of penicillin acylase from the fermentation broth all face some common problems: (1) removal of unconsumed medium components in the fermentation broth; (2) endogenous expression of penicillin acylase, which requires The cells are crushed to release the enzyme; (3) remove impurities such as grease, polysaccharides, pigments, amino acids and cell fragments in the feed liquid after cell crushing; (4) remove impurities in the feed liquid after cell crushing, and increase the penicillin Acylase purity.

In order to prepare high-purity penicillin acylase, multi-step operations such as solid-liquid separation, concentration, impurity removal and separation and purification are required. As an industrial enzyme, fine separation techniques such as chromatography cannot be used due to cost constraints. Separation methods such as plate and frame filtration, centrifugation, ammonium sulfate precipitation, isoelectric point precipitation and ultrafiltration are usually used, and combined on this basis to form corresponding process. The penicillin acylase separation, purification and immobilization processes known in the art are summarized as follows: (1) fermentation broth pretreatment: first use mechanical methods such as high-pressure homogenization or bead milling, chemical methods such as organic solvents or surfactants, bacteriolysis Cells are broken by biological methods such as enzymes, and then flocculants, filter aids, etc. are added, and solid-liquid separation is carried out by centrifugation or plate and frame filtration to obtain crude enzyme liquid without bacteria; (2) the initial production of penicillin acylase Separation: First, use acid or alkali to adjust to the isoelectric point precipitation method to remove part of the impurity protein, then use centrifugation or plate frame filtration to obtain the penicillin acylase crude enzyme solution, and finally use membrane filtration to concentrate the penicillin acylase crude enzyme solution (3) fine separation of penicillin acylase: first carry out ammonium sulfate salting out to the crude enzyme solution of penicillin acylase, centrifuge or plate frame filter to obtain precipitate; then dissolve precipitate with buffered salt solution, centrifuge or plate frame Remove insoluble solids by filtration; finally use membrane filtration to remove impurities such as small molecule miscellaneous proteins, amino acids, inorganic salts; (4) immobilization of penicillin acylase: first add buffer salt to the penicillin acylase solution obtained by fine separation Appropriate concentration, centrifugation or plate and frame filtration to remove solid impurities, and then add epoxy-based carrier or activated amino carrier for immobilization.

The main problems in the above-mentioned process are as follows: (1) the process steps are too complicated and the operation time is long, resulting in low production efficiency; (2) more steps and some enzymes will be lost due to the long operation time, resulting in low overall yield of enzyme; ( 3) The main impurity removal methods are flocculation precipitation, ammonium sulfate precipitation, ultrafiltration and plate-and-frame filtration (or centrifugation). The separation means are relatively traditional, and the effect of impurity removal is not significant; (4) Solid-liquid separation and concentration are not considered from the perspective of process integration The interconnection between separation, purification, impurity removal, and immobilization operations leads to too many steps.

Therefore, it is very necessary to provide a new separation and purification method for penicillin acylase that can effectively simplify the operation steps, shorten the operation time, and effectively improve the total yield of enzyme separation and purification.

Contents of the invention

The object of the present invention is to overcome the above-mentioned problems in the prior art, and provide a coupling method for separation, purification and immobilization of penicillin acylase for preparing 6-aminopenicillanic acid. The method of the invention is easy to operate, and the activity and yield of the obtained penicillin acylase are relatively high.

The inventors of the present invention have found that by adopting the system of organic solvent-inorganic salt, by strictly controlling the concentration of inorganic salt, a three-phase distribution system can be formed, and penicillin acylase can be enriched in the lower phase solution (if it enters the upper In the phase solution, on the one hand, it is easy to inactivate the enzyme, and on the other hand, it cannot be directly immobilized; if it is enriched in the middle phase (solid phase), it cannot be directly immobilized, and additional operating steps are required); thus, it can be used in this The immobilization of penicillin acylase is directly carried out in the lower phase solution; thus, the method of the present invention can complete each step of the traditional method in one unit operation, which greatly simplifies the process, thus becoming a very promising process integration technology. The inventors of the present invention also found that tert-butanol can maintain the activity of penicillin acylase to the greatest extent compared with other types of alcohols used in the prior art.

The present invention provides a coupling method for separation, purification and immobilization of penicillin acylase for preparing 6-aminopenicillanic acid, wherein the method comprises the following steps:

(1) preparing an inorganic salt mixed solution containing penicillin acylase crude enzyme liquid and inorganic salt, the concentration of the inorganic salt in the salt mixed solution is 0.05-0.35g/mL;

(2) Mix the inorganic salt mixed solution obtained in step (1) with tert-butanol, and then separate phases to obtain an upper phase solution rich in tert-butanol, a middle phase solid, and a lower phase rich in salt and penicillin acylase A three-phase distribution system composed of solutions;

(3) adding a carrier to the lower phase solution obtained in step (2) to immobilize the penicillin acylase.

Compared with the prior art, the method of the present invention has the advantages that at least:

(1) The method of the present invention completes multiple operating units in the solid-liquid separation, primary separation and fine separation involved in the separation and purification of penicillin acylase in one operation unit, and couples the separation and purification of the enzyme with immobilization, The operation steps are greatly simplified, the operation time is shortened, and the total yield of enzyme extraction is improved;

(2) The method of the present invention saves the operation of multi-step plate and frame filtration or centrifugal, 1-2 step membrane filtration, reduces sewage discharge, reduces production cost, improves production efficiency;

(3) The total yield of the method for separating and purifying penicillin acylase of the present invention is significantly higher than that of the prior art;

(4) The enzyme activity of the immobilized enzyme prepared by the method of the present invention is significantly improved;

(5) the tert-butanol used in the present invention can be recycled and reused, which further reduces the production cost;

(6) The amplification effect of the method of the present invention is small, and has great industrial application prospects.

Other features and advantages of the present invention will be described in detail in the detailed description that follows.

Detailed ways

Neither the endpoints nor any values of the ranges disclosed herein are limited to such precise ranges or values, and these ranges or values are understood to include values approaching these ranges or values. For numerical ranges, between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, these values Ranges should be considered as specifically disclosed herein.

The present invention provides a coupling method for separation, purification and immobilization of penicillin acylase for preparing 6-aminopenicillanic acid, wherein the method comprises the following steps:

(1) preparing an inorganic salt mixed solution containing penicillin acylase crude enzyme liquid and inorganic salt, the concentration of the inorganic salt in the salt mixed solution is 0.05-0.35g/mL;

(2) Mix the inorganic salt mixed solution obtained in step (1) with tert-butanol, and then separate phases to obtain an upper phase solution rich in tert-butanol, a middle phase solid, and a lower phase rich in salt and penicillin acylase A three-phase distribution system composed of solutions;

(3) adding a carrier to the lower phase solution obtained in step (2) to immobilize the penicillin acylase.

The object of separation, purification and immobilization in the present invention is penicillin acylase for the preparation of 6-aminopenicillanic acid, which belongs to a type of penicillin acylase and is specifically used for the preparation of 6-aminopenicillin Penicillin acylase of alkanoic acid, ie the term "penicillin acylase for the production of 6-aminopenicillanic acid" refers in the present invention to this specific penicillin acylase. In the present invention, for the convenience of description, "penicillin acylase for the preparation of 6-aminopenicillanic acid" is referred to as "penicillin acylase", that is, "penicillin acylase" described in this text refers to is "penicillin acylase for the production of 6-aminopenicillanic acid".

The inventors of the present invention have found that the concentration of inorganic salts in the mixed solution of inorganic salts obtained in step (1) can significantly affect whether a three-phase distribution system can be obtained in step (2). Generally speaking, the inorganic salts When the concentration of the inorganic salt in the salt mixed solution reaches the above-mentioned 0.05-0.35g/mL, a three-phase distribution system can be obtained in step (2). The concentration of the salt is 0.15-0.3 g/mL, more preferably 0.2-0.25 g/mL.

In step (1), the pH of the inorganic salt mixed solution is 5-9, which is beneficial to maintain the enzyme activity of penicillin acylase without loss. In order to better form a three-phase distribution system and make penicillin acylase enter the lower phase solution as much as possible, the pH of the mixed inorganic salt solution is preferably 5-8, most preferably 6-8, and more preferably 7-8.

In step (1), the inorganic salt may be one or more of water-soluble phosphates, sulfates, carbonates, citrates and halides. Wherein, the water-soluble phosphate is, for example, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate; the sulfate is, for example, ammonium sulfate, sodium sulfate, potassium sulfate The carbonate is, for example, sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate; the citrate is, for example, sodium citrate, potassium citrate; the halide is, for example, sodium chloride, potassium chloride , when the inorganic salt is a halide, it is preferably used in conjunction with a water-soluble phosphate.

In step (1), in preferred cases, the inorganic salt is water-soluble phosphate and/or sulfate, that is, preferably selected from dipotassium hydrogen phosphate, potassium dihydrogen phosphate, dibasic hydrogen phosphate, dihydrogen phosphate One or more of sodium, ammonium sulfate, sodium sulfate and potassium sulfate; more preferably, the inorganic salt is a water-soluble phosphate, that is, most preferably selected from dipotassium hydrogen phosphate, potassium dihydrogen phosphate, dihydrogen phosphate One or more of sodium and sodium dihydrogen phosphate.

In step (1), in order to control the pH of the inorganic salt mixed solution within the above range, the inorganic salts can be combined and gradually added to the mixed solution according to acidity and alkalinity. For example, according to a preferred aspect of the present invention, when the inorganic salt is a mixture of dipotassium hydrogen phosphate and potassium dihydrogen phosphate, in order to adjust the pH to 7.5-8.5, potassium dihydrogen phosphate can be added first to adjust the pH to 6 -7, add dipotassium hydrogen phosphate to adjust the pH to 8-9, and finally add potassium dihydrogen phosphate to adjust the pH to 7.5-8.5.

In step (1), the term "penicillin acylase crude enzyme solution" conforms to the conventional definition in the field. The penicillin acylase crude enzyme liquid can be obtained by using an expression vector to carry out fermentation and then pretreatment; the expression vector of the penicillin acylase can be Escherichia coli, Bacillus subtilis, Bacillus megaterium or Pichia pastoris; the fermentation The culture conditions can be carried out using fermentation culture conditions known in the art. The purpose of the pretreatment is to crush the fermented crude enzyme solution containing bacteria, or to remove solid impurities such as bacteria after the fermentation of the crude enzyme solution containing bacteria obtained by fermentation, so as to obtain the release of penicillin acylation Enzyme crude enzyme solution. The specific pretreatment method is a conventional method in the art, and will not be repeated here. In the process of pretreatment, inorganic salts such as phosphate may be used, and the amount of these inorganic salts is seldom, or can be reacted and removed in the process of pretreatment, so it does not affect the salt content described in step (1). The concentration of inorganic salts in the mixture had a significant effect.

In step (1), the penicillin acylase crude enzyme solution can be the untreated penicillin acylase crude enzyme solution after the cells are broken, or the penicillin acylase after the cells are broken and solid impurities such as bacteria are removed. Crude enzyme liquid; the method for removing solid impurities such as bacteria can be plate-and-frame filtration or centrifugation.

In step (1), the method for preparing an inorganic salt mixed solution containing a crude penicillin acylase enzyme solution and an inorganic salt may include: dissolving an inorganic salt in the crude penicillin acylase enzyme solution.

In step (1), the penicillin acylase enzyme activity in the penicillin acylase crude enzyme liquid is 50-300IU/mL, preferably 70-300IU/mL. Preferably, the crude enzyme solution is the crude enzyme solution of penicillin acylase after the cells are broken and solid impurities such as bacteria are removed. When using this crude enzyme solution, the penicillin acylase in the crude enzyme solution of penicillin The enzyme activity of Liase is preferably 150-300IU/mL.

In step (2), the volume ratio of the inorganic salt mixed solution to tert-butanol may be 1:0.1-2, preferably 1:0.2-1.5, more preferably 1:0.25-0.6.

In step (2), the tert-butanol may be a commercially available tert-butanol liquid, or may be mixed with the inorganic salt mixed solution in the form of a mixture of tert-butanol and water. In order to make the method of the present invention more economical, preferably, the process of step (2) also includes: recovering tert-butanol from the upper phase solution and recycling it for mixing with the inorganic salt mixed solution. The recovery method is, for example, distillation, and the obtained tert-butanol is usually a mixed solution of tert-butanol and water, and the content of tert-butanol in the mixed solution of tert-butanol and water can be 70-100% by weight, It is preferably 80-100% by weight, more preferably 85-95% by weight.

In step (2), the mixing method is not particularly limited, such as shaking or stirring.

In step (2), the phase separation method is not particularly limited, such as centrifugation or standing.

In step (2), when the phase separation method is centrifugal phase separation, the phase separation temperature can be 0-40°C, preferably 4-37°C, more preferably 15-30°C; the centrifugal force can be 500 -5000g, preferably 1000-4000g, more preferably 2000-3500g; time can be 2-60min, preferably 2-40min, more preferably 5-20min.

In step (2), when the phase separation method is static phase separation, the phase separation temperature can be 4-30°C, preferably 10-25°C, more preferably 12-17°C; the time is 0.1 -4h, preferably 0.3-3h, more preferably 0.5-2h.

Can obtain the three-phase distribution system that is rich in the upper phase solution of tert-butanol, middle phase solid and the lower phase solution that is rich in salt and penicillin acylase composition by the method of the present invention, in this three-phase distribution system, penicillin acylase The enzyme is enriched into the inorganic salt solution of the lower phase, the activity of the enzyme can be well maintained, and the lower phase solution can be directly used for the immobilization of penicillin acylase, which realizes integrated operation and effectively simplifies It simplifies the operation steps and shortens the operation time, thereby reducing the industrial cost. The upper phase solution mainly contains tert-butanol and water. There will be a solid layer tightly sandwiched between the upper phase solution and the lower phase solution, which is the middle phase solid, and the solids in the middle phase solid mainly include impurity proteins, polysaccharides, and possible bacterial fragments, etc. .

In step (3), adding a carrier to the lower phase solution obtained in step (2) to immobilize the enzyme, this process may include: the enzyme activity of the lower phase solution is 50-150IU/mL, preferably 100-150IU /mL, if the enzyme activity is higher than 150IU/mL, dilute with water to the above range, then add phosphate until the concentration of inorganic salts in the total system is 0.2-0.45g/mL (preferably 0.3-0.4g/mL), and then Add the carrier to immobilize the enzyme; the process of adding phosphate can be carried out with reference to step (1), and during the adding process, the pH value is preferably controlled within the pH range required by step (1). The amount of the carrier can be added according to the conventional amount in the art, for example, the ratio of the total enzyme activity to the weight of the carrier can be 500-1000 IU/g, preferably 700-900 IU/g.

In step (3), the carrier may be various commercial carriers known in the art, such as amino carrier, epoxy carrier and the like. When the carrier is an epoxy carrier, it can be used directly without activation. When the carrier is an amino carrier, it is preferable to activate it first, and the activation method of the amino carrier can be carried out according to a conventional method in the art, and will not be repeated here.

In step (3), the conditions for the immobilization of the enzyme can be conventional operating conditions in the art, for example, it can include: a temperature of 10-40°C, preferably 20-30°C; a pH of 5-9, preferably 7-9; the rotation speed is 50-300rpm, preferably 100-200rpm; the time is 4-48h, preferably 16-40h.

The immobilized method of the enzyme of step (3) of the present invention can also comprise the process of post-treatment of immobilized enzyme, can obtain the finished product of immobilized penicillin acylase that can be directly used in industry to prepare 6-aminopenicillanic acid through post-treatment , the post-treatment method can be a conventional post-treatment method in the art, such as comprising: after the enzyme immobilization operation is finished, the solution is drained, and then 2-6 times the volume (mL/g) of purification is added according to the weight of the immobilized enzyme Wash with water 2-4 times, drain the solution, collect the immobilized enzyme and store it in a warehouse at 2-6°C.

The specific method of applying the immobilized penicillin acylase prepared by the method of the present invention to the preparation of 6-aminopenicillanic acid can be carried out according to the conventional method in the art, and will not be repeated here.

The present invention will be described in detail below by way of examples. At first, the material, test method and calculation method used in the following examples and comparative examples are introduced as follows:

1. The carrier used for immobilization

The carrier used for immobilization is the epoxy-based carrier (L-11) provided by Cangzhou Yuancheng Chemical Co., Ltd.

2. Determination of enzyme activity

The enzyme activity was determined by alkali titration. Enzyme activity is defined as: under the conditions of 28°C and pH 8.0, the enzyme activity of a unit of penicillin acylase consuming 1 μmol of penicillin G potassium salt in 1 minute is one unit (IU). The principle is: at 28°C and pH 8.0, penicillin acylase hydrolyzes penicillin G potassium salt to generate equimolar 6-APA and phenylacetic acid, and penicillin G can be calculated by accurately titrating phenylacetic acid with 0.1mol/L NaOH calibration solution Potassium consumption.

The specific steps are as follows: 1) Preparation of buffer solution: Weigh 4.76 g of dipotassium hydrogen phosphate and 0.55 g of potassium dihydrogen phosphate and dissolve them in 900 mL of distilled water, adjust the pH to 7.8 with potassium dihydrogen phosphate, and then adjust the volume to 1 L. 2) Preparation of 0.1mol/L NaOH solution: first prepare a saturated NaOH solution, after clarification, take 5mL of the supernatant and add it to 1000mL of carbon dioxide-free water, and shake well. Then use potassium hydrogen phthalate as the standard substance to calibrate and calculate its concentration. 3) Preparation of 3mol/L NaOH solution: Quickly weigh 30g NaOH with a clean small beaker, dissolve it with purified water and pour it into a 250mL volumetric flask, rinse the beaker with purified water 3 times and pour it into the volumetric flask, and constant volume After shaking well, it becomes 3mol/L NaOH solution. 4) Preparation of the substrate: Weigh 2 g of penicillin G potassium salt, dissolve it in 40 mL of enzyme activity buffer, adjust the pH to 8.0 with 3 mol/L NaOH, and prepare it for immediate use. 5) Determination of enzyme activity: Accurately measure a certain volume of liquid enzyme or weigh a certain mass of immobilized enzyme into the 40mL substrate solution preheated to 28°C in advance, keep the temperature and stir rapidly, use 0.1 or 3mol/L NaOH Adjust the pH of the solution to 8.0 and start timing, intermittently add 0.1mol/L NaOH calibration solution dropwise to maintain the pH at 8.0, react for about 5 minutes, record the amount of NaOH titration solution and the reaction time. 6) Liquid enzyme activity calculation: Enzyme activity=v ₁ *c*1000/t*v ₂ . Among them, the enzyme activity unit is IU/mL; v ₁ represents the NaOH titration solution volume (mL) consumed within the measurement time; c represents the NaOH titration solution (0.1mol/L) concentration (mol/L); 1000 represents millimolar and micromole Mole conversion ratio; t represents the reaction time (min); v ₂ represents the liquid enzyme volume (mL). 7) Calculation of immobilized enzyme activity: enzyme activity=v ₁ *c*1000/t*w. Among them, the enzyme activity unit is IU/g; v ₁ represents the NaOH titration solution volume (mL) consumed within the measurement time; c represents the NaOH titration solution (0.1mol/L) concentration (mol/L); 1000 represents millimolar and micromole Molar conversion ratio; t represents the reaction time (min); w represents the weight of immobilized enzyme (g).

3. Immobilized penicillin acylase for the preparation of 6-aminopenicillanic acid

(1) Preparation method

Weigh 50 g of immobilized penicillin acylase and place it in a reactor, and prepare the concentrated penicillin G potassium salt solution with 0.25% boric acid solution to form a reaction solution with a potency of penicillin G potassium salt of 130000 U/mL, which is ready-to-use. During the reaction process, 3 mol of ammonia water was used to control the pH value at 8.0, the temperature was controlled at 30° C., and the reaction time was 40 min. Record the reaction time, temperature, pH value and ammonia water consumption every 5 minutes, take samples and measure the content of penicillin G potassium salt and 6-APA by liquid chromatography, and calculate the residual potency of penicillin G potassium salt and the conversion of 6-APA Rate.

(2) Determination of penicillin G potassium salt

Penicillin G potassium salt was detected by liquid chromatography. The details are as follows: 1) Mobile phase preparation: Weigh 14.85 g of potassium dihydrogen phosphate in a 2000 mL beaker, add 1400 mL of water, stir to dissolve, adjust the pH value to 4.5 with 1mol/L KOH solution, then mix it with 600 mL of chromatographically pure acetonitrile, pass through 0.45 After filtering with μm microporous membrane, ultrasonic degassing for 20min; 2) Reference substance configuration: Weigh 0.0315g of penicillin G potassium salt reference substance into a 50mL volumetric flask, add water to dilute to volume, take 5mL from the above solution to a 50mL volumetric flask 3) Sample treatment: measure 2mL of the reaction solution and dilute it to 50mL with water; 4) Liquid phase conditions: the column is C18 (150mm*4.6mm), the injection volume is 20μL, and the flow rate is 1mL/min. The detection wavelength is 215nm.

(3) Determination of 6-APA

6-APA was detected by liquid chromatography. The details are as follows: 1) Mobile phase preparation: Weigh 5.0 g of disodium hydrogen phosphate and 2.7 g of potassium dihydrogen phosphate, dissolve them in a 2000 mL beaker, add 1900 mL of water, stir and dissolve, adjust the pH value to 7.0 with 1mol/L KOH solution, and then Mix with 100mL of acetonitrile, filter through a 0.45μm filter membrane, and ultrasonically degas for 20min; 2) Reference substance configuration: Weigh 0.1000g of 6-APA reference substance and 0.0750g of sodium phenylacetate into the same 500mL volumetric flask, add pH 7.0 Dilute the buffer to the mark; 3) Sample treatment: Dilute the sample 250 times with the same buffer as the reference substance; 4) Liquid phase conditions: The column is C18 (150mm*4.6mm), the injection volume is 20μL, and the flow rate is 1mL /min, the detection wavelength is 220nm.

4. Calculation of specific activity of penicillin acylase

The formula for calculating the specific activity of penicillin acylase is: specific activity (IU/mg)=enzyme activity (IU/mL)/protein concentration (mg/mL), wherein the determination of protein concentration adopts the Coomassie brilliant blue method.

5. Calculation of enzyme yield

(1) The formula for calculating the penicillin acylase yield (Y, %) in each step is: Y=c*v/(c ₀ *v ₀ )*100%, wherein, c represents the penicillin acylase in the feed liquid after treatment Concentration (IU/mL) of enzyme; v represents the feed liquid volume (mL) after processing; c ₀ represents the concentration (IU/mL) of penicillin acylase in the feed liquid before processing; v ₀ represents the feed liquid volume before processing ( mL);

(2) Enzyme extraction total yield (%)=fermentation broth pretreatment step yield (%)×enzyme separation and purification step yield (%).

Preparation example

(1) Fermentation culture of penicillin acylase

Using recombinantly expressed Pichia strains, adopt the method described in the existing literature (Luo Qian, high-density fermentation and immobilization of PGA produced by recombinant Pichia [D], master's degree thesis of Central South University of Forestry and Technology, 2013) Fermentation culture, test the enzyme activity of penicillin acylase in the obtained fermentation liquid is 83.2IU/mL.

(2) Fermentation broth pretreatment:

(i) After the fermentation is finished, add 0.01g/mL of ammonium sulfate according to the volume of the feed liquid, stir to dissolve, then add 0.008g/mL of cetyltrimethylammonium bromide according to the volume of the feed liquid, and stir for 25min; The temperature of the solution was raised to 45°C, and the pH was adjusted to 7.6 with 2mol/L NaOH solution at the same time; after the temperature and pH were adjusted in place, the temperature was maintained at 45°C, and 3mol/L NaOH solution was added intermittently to maintain the pH at 7.6±0.1, soaking for 80min, intervals Sampling was carried out to measure the enzyme activity until the cells were completely broken, and the penicillin acylase crude enzyme solution I was obtained. The enzyme activity of the penicillin acylase crude enzyme solution I was determined to be 79.8IU/mL, the protein concentration to be 11.9mg/mL, and the specific activity 6.7IU/mg;

(ii) After the cell disruption is completed, lower the temperature to below 40°C, add 0.06g/mL phosphate according to the volume of the feed solution (adjust the ratio of dipotassium hydrogen phosphate and dipotassium hydrogen phosphate according to the pH), adjust the pH to 8.0, and stir Dissolve the salt completely, then slowly add 0.4g/mL calcium chloride solution to adjust the pH to 5.1; add 0.09g/mL diatomaceous earth according to the volume of the feed solution, stir and mix evenly, then plate and frame filter, and collect the filtrate.

(iii) Use 2mol/L NaOH solution to adjust the pH of the filtrate to 8.0, add a small amount of diatomaceous earth, stir and mix evenly, filter the plate and frame, and collect the filtrate; then use a hollow fiber membrane to concentrate the filtrate and collect the concentrated solution, which is penicillin The crude enzyme liquid II of penicillin acylase has an enzyme activity of 210.2 IU/mL, a protein concentration of 12.0 mg/mL, and a specific activity of 17.5 IU/mg.

The penicillin acylase crude enzyme solution I or II obtained in the preparation example was subjected to separation, purification and immobilization of penicillin acylase according to the methods described in the following examples and comparative examples, respectively.

Example 1

(1) Dissolve phosphate (composed of K ₂ HPO ₄ .3H ₂ O and KH ₂ PO ₄ ) in the penicillinyl obtained in the preparation example within 90 minutes at a concentration of 0.2 g/mL under the condition of 14°C Add KH ₂ PO ₄ to adjust the pH to 6.5, then add K ₂ HPO ₄ .3H ₂ O to adjust the pH to 8.3, and finally add KH ₂ PO ₄ to adjust the pH to 8.0), to obtain a mixed solution of inorganic salts;

(2) Mix the inorganic salt mixed solution obtained in step (1) with the mixed solution of tert-butanol and water recovered in step (3) according to the volume ratio of 1:0.6, vortex for 2 minutes until fully mixed, and then centrifuge at 3500g for 10 minutes, Form a three-phase distribution system consisting of an upper phase solution rich in tert-butanol, a middle phase solid, and a lower phase solution rich in salt and penicillin acylase, and separate the three-phase solution;

(3) get the upper phase solution of step (2) gained, distill to obtain the concentration of tert-butanol and be the mixed solution of the tert-butanol and water of 85% by weight, and it is recycled and used in step (2);

(4) The lower phase solution obtained in step (2) was diluted with water until the enzyme activity was 100IU/mL, and phosphate was added until the phosphate concentration was 0.4g/mL, and during the addition process, K ₂ HPO ₄ . The ratio of 3H ₂ O and KH ₂ PO ₄ adjusted the pH value to 8.0, and then added epoxy-based carrier for immobilization according to the ratio of total enzyme activity to carrier weight of 900IU/g. The operating conditions were: temperature 27°C, The pH is 8, the rotation speed is 150rpm, and the time is 30h;

(5) Post-treatment of immobilized enzyme: after the enzyme immobilization operation, drain the solution, then add 4 times the volume (mL/g) of purified water according to the weight of the immobilized enzyme to wash three times, drain the solution, collect the immobilized enzyme and store it for 4 ℃ stored in the warehouse.

Calculate the fermentation broth pretreatment yield and record it in Table 1. Sampling was carried out to measure the enzyme activity and protein concentration of the lower phase solution obtained in the above step (2), and the specific activity and yield of the enzyme were calculated, and the results were recorded in Table 1. Sampling was taken to determine the enzyme activity and water content of the immobilized enzyme obtained in step (5), and the obtained results were recorded in Table 1.

Example 2

(1) Dissolve phosphate (composed of K ₂ HPO ₄ .3H ₂ O and KH ₂ PO ₄ ) in the penicillinyl obtained in the preparation example within 90 minutes at a concentration of 0.3 g/mL under the condition of 12°C Add enzyme crude enzyme solution I and adjust the pH value of the solution to 6 during the process of adding (add KH ₂ PO ₄ first to adjust the pH to 5, then add K ₂ HPO ₄ .3H ₂ O to adjust the pH to 7, and finally add KH ₂ PO ₄ adjust the pH to 6), add salt while stirring (stirring speed is 250rpm) to fully dissolve the salt, and obtain a mixed solution of inorganic salt;

(2) Mix the inorganic salt mixed solution obtained in step (1) with the mixed solution of tert-butanol and water recovered in step (3) according to the volume ratio of 1:0.25, vortex for 2 minutes until fully mixed, and then centrifuge at 2000g for 20 minutes, Form a three-phase distribution system consisting of an upper phase solution rich in tert-butanol, a middle phase solid, and a lower phase solution rich in salt and penicillin acylase, and separate the three-phase solution;

(3) get the upper phase solution of step (2) gained, distill to obtain the concentration of tert-butanol and be the mixed solution of the tert-butanol of 95% by weight and water, and it is recycled in the step (2);

(4) The lower phase solution obtained in step (2) is diluted with water until the enzyme activity is 50IU/mL, and the ratio of the total enzyme activity to the weight of the carrier is 700IU/g to add an epoxy-based carrier for immobilization. The operating conditions are: the temperature is 20°C, pH 9, rotation speed 100rpm, time 40h;

(5) Post-treatment of immobilized enzyme: after the enzyme immobilization operation, drain the solution, then add 4 times the volume (mL/g) of purified water according to the weight of the immobilized enzyme to wash three times, drain the solution, collect the immobilized enzyme and store it for 4 ℃ stored in the warehouse.

Calculate the fermentation broth pretreatment yield and record it in Table 1. Sampling was carried out to measure the enzyme activity and protein concentration of the lower phase solution obtained in the above step (2), and the specific activity and yield of the enzyme were calculated, and the results were recorded in Table 1. Sampling was taken to determine the enzyme activity and water content of the immobilized enzyme obtained in step (5), and the obtained results were recorded in Table 1.

Example 3

(1) Take the penicillin acylase crude enzyme solution II obtained in the preparation example, add it to the tank, control the temperature of the tank at 13°C, and stir at a speed of 270rpm, then add phosphate in an amount of 0.25g/mL within 90min and During the addition process, control the pH value of the solution to about 7.8 (add KH ₂ PO ₄ first to adjust the pH to 6.5, then add K ₂ HPO ₄ .3H ₂ O to adjust the pH to 8.0, and finally add KH ₂ PO ₄ to adjust the pH to 7.8) , to obtain a mixed solution of inorganic salts;

(2) the mixed solution of inorganic salts obtained in step (1) is mixed with the mixed solution of tert-butanol and water recovered in step (3) according to a volume ratio of 1:0.5, and stirred at 350rpm for 30min to fully mix, then control the temperature of the tank body at 15°C, stop stirring, stand still for phase separation for 1 hour, and form a three-phase distribution system consisting of an upper phase solution rich in tert-butanol, a middle phase solid, and a lower phase solution rich in salt and penicillin acylase. The solution is separated;

(3) get the upper phase solution of step (2) gained, distill to obtain the concentration of tert-butanol and be the mixed solution of the tert-butanol of 88% by weight and water, and it is recycled and used in step (2);

(4) The lower phase solution of step (2) gained is diluted with water to the concentration of penicillin acylase and is 100IU/mL, adds phosphate to phosphate concentration and is 0.35g/mL, and in the process of adding, by adjusting K ₂ HPO ₄ .3 The ratio of H ₂ O and KH ₂ PO ₄ adjusts the pH value to 7.8, and the epoxy-based carrier is added according to the ratio of the total enzyme activity to the carrier weight of 800IU/g for immobilization. The operating conditions are: the temperature is 26.5°C, pH 8, rotation speed 150rpm, time 32h, and finally stand for 8h;

(5) Post-treatment of immobilized enzyme: after the enzyme immobilization operation, drain the solution, then add 4 times the volume (mL/g) of purified water according to the weight of the immobilized enzyme to wash three times, drain the solution, collect the immobilized enzyme and place it for 4 ℃ stored in the warehouse.

Calculate the fermentation broth pretreatment yield and record it in Table 1. Sampling was carried out to measure the enzyme activity and protein concentration of the lower phase solution obtained in the above step (2), and the specific activity and yield of the enzyme were calculated, and the results were recorded in Table 1. Sampling was taken to determine the enzyme activity and water content of the immobilized enzyme obtained in step (5), and the obtained results were recorded in Table 1.

Table 1

Example 1 Example 2 Example 3 Fermentation broth pretreatment yield (%) 71 92 71 Separation and purification yield (%) 93 68 95 Enzyme extraction total yield (%) 66 63 67 Separate enzyme specific activity (IU/mg) 31.3 18.2 26.1 Immobilized enzyme activity (IU/g) 404 313 353 Cured enzyme water content (%) 55 55 55

Embodiment 4 and comparative example 1

Example 4 (including Examples 4-1 to 4-4) and Comparative Example 1 (including Comparative Examples 1-1 to 1-2) are used to illustrate the influence of different extraction systems on the separation and purification of penicillin acylase.

Example 4 was carried out according to the method of Example 1, except that the phosphate in step (1) was replaced with other inorganic salts of the same quality, as shown in Table 2.

Comparative Example 1 was carried out according to the method of Example 1, except that the tert-butanol in step (2) was replaced with other organic solvents of the same quality, as shown in Table 2.

Sampling and measuring the enzyme activity and protein concentration of the lower phase solution obtained in the above step (2) respectively, calculating the specific activity and yield of the enzyme, and recording the results in Table 2.

Table 2

As can be seen from Table 2, by using the extraction system of the present invention, the specific activity of the extracted enzyme can reach more than 22.8IU/mg, and the yield can reach more than 78%, especially when the tert-butanol-phosphate of Example 1 is used The specific activity of the enzyme extracted during the system can reach 31.3IU/mg, and the yield can reach 93%. However, when tert-butanol was replaced by ethanol or isopropanol in the comparative example, penicillin acylase was inactivated.

Embodiment 5 and comparative example 2

Embodiment 5 (comprising embodiment 5-1～5-3) and comparative example 2 (comprising comparative example 2-1～2-2) are used to illustrate the impact of the concentration of inorganic salt in the inorganic salt mixed solution in step (1) on penicillinyl The impact of enzyme separation and purification.

Example 5 is carried out according to the method of Example 1, the difference is that the weight of the phosphate is changed so that the concentration of the phosphate is changed, but the pH value of the solution is kept constant, as shown in Table 3.

Comparative Example 2 was carried out according to the method of Example 1, except that the concentration of phosphate was changed by changing the weight of phosphate, but the pH value of the solution was kept constant, as shown in Table 3.

Sampling and measuring the enzyme activity and protein concentration of the lower phase solution obtained in the above step (2) respectively, calculating the specific activity and yield of the enzyme, and recording the results in Table 3.

table 3

As can be seen from Table 3, when the concentration of inorganic salts is within the preferred range of the present invention, the specific activity of the enzyme obtained by extraction reaches more than 24.6IU/mg, and the yield can reach more than 81%; when the concentration of inorganic salts is within the present invention When within the preferred range of the invention, the specific activity of the extracted enzyme can reach more than 28.4 IU/mg, and the yield can reach more than 90%. However, when the concentration of the inorganic salt is too low (Comparative Example 2-1), the three-phase distribution system cannot be formed. When the concentration of the inorganic salt is too high (comparative example 2-2), most of the penicillin acylase is allocated to the middle phase, and the specific activity and yield of the enzyme obtained by the lower phase extraction are significantly decreased.

Example 6

Example 6 (including Examples 6-1 to 6-5) is used to illustrate the influence of the pH value of the inorganic salt mixture solution on the separation and purification of penicillin acylase in step (1).

Carry out according to the method for embodiment 1, difference is, on the basis of keeping phosphate weight constant, change the proportioning of dipotassium hydrogen phosphate and potassium dihydrogen phosphate so that the pH value of gained inorganic salt mixed solution reaches as table 4 As shown, it can be fine-tuned with phosphoric acid or 3mol/L NaOH if necessary.

Sampling and measuring the enzyme activity and protein concentration of the lower phase solution obtained in the above step (2) respectively, calculating the specific activity and yield of the enzyme, and recording the results in Table 4.

Table 4

As can be seen from Table 4, when the pH value of the inorganic salt mixed solution is within the preferred range of the present invention, the specific activity of the enzyme obtained by extraction can reach more than 27.0IU/mg, and the yield can reach more than 73%; in the present invention The specific activity of the extracted enzyme can reach more than 27.0 IU/mg when it is in the most preferable range, and the yield is significantly increased to more than 89%.

Example 7

Example 7 (including Examples 7-1 to 7-5) is used to illustrate the influence of the amount of tert-butanol used in step (2) on the separation and purification of penicillin acylase.

Carry out according to the method for embodiment 1, difference is, change the volume ratio of the mixed solution of inorganic salt mixed solution obtained in step (1) in step (2) and the mixed solution of tert-butanol and water that step (3) reclaims gained, specifically as table 5.

Sampling and measuring the enzyme activity and protein concentration of the lower phase solution obtained in the above step (2) respectively, calculating the specific activity and yield of the enzyme, and recording the results in Table 5.

table 5

As can be seen from Table 5, when step (1) gained inorganic salt mixed solution and step (3) reclaim the volume ratio of the mixed solution of tert-butanol and water gained in the preferred range of the present invention, the ratio of the enzyme obtained by extraction The activity reaches more than 22.4IU/mg, and the yield can reach more than 83%; when the specific activity of the extracted enzyme reaches more than 28.8IU/mg, the yield can reach more than 93% when it is within the most preferred range of the present invention.

Example 8

Example 8 (including Examples 8-1 to 8-2) is used to illustrate the influence of the resting time on the separation and purification of penicillin acylase when static phase separation is adopted in step (2).

Carry out according to the method for embodiment 3, difference is, in step (2) in static phase separation, different time points are sampled, measure the enzymatic activity and the protein concentration of the lower phase solution that above-mentioned step (2) gains, calculate enzyme The specific activity and yield, the results are recorded in Table 6, and the specific time points are set as shown in Table 6.

Sampling and measuring the enzyme activity and protein concentration of the lower phase solution obtained in the above step (2) respectively, calculating the specific activity and yield of the enzyme, and recording the results in Table 6.

Table 6

As can be seen from Table 6, with the growth of standing time, the specific activity and yield of the enzyme obtained by extraction all increased, but when the standing time exceeded 2 hours, the specific activity and yield of the enzyme obtained by extraction There was a slight decline, and the production efficiency was reduced.

Example 9

Example 9 (including Examples 9-1 to 9-4) is used to illustrate the influence of the enzyme concentration of penicillin acylase diluted in step (3) on the immobilization of penicillin acylase.

According to the method of Example 3, the difference is that in step (3), the lower phase solution obtained in step (2) was diluted to different enzyme activities, as shown in Table 7.

Sampling was taken to measure the enzyme activity and water content of the final immobilized enzyme, and the results were recorded in Table 7.

Table 7

As can be seen from Table 7, when the enzyme concentration of the diluted penicillin acylase in the step (3) is within a larger range of the present invention, the enzyme activity of the gained immobilized enzyme can reach more than 332IU/g, and the water content can be In the range of 53-56%; when in the most preferred range of the present invention, the enzyme activity of the obtained immobilized enzyme can reach more than 351U/mg, and the water content can be in the range of 53-55%.

Example 10

Example 10 (including Examples 10-1 to 10-4) is used to illustrate the effect of the inorganic salt concentration of the solution obtained after adding the inorganic salt in step (3) on the immobilization of penicillin acylase.

Carry out according to the method for embodiment 3, difference is, in step (3), add dipotassium hydrogen phosphate and potassium dihydrogen phosphate (control pH value constant) to the mass volume ratio that inorganic salt accounts for total system respectively as follows Table 8 shows.

Sampling was carried out to measure the enzyme activity and water content of the final immobilized enzyme, and the results were recorded in Table 8.

Table 8

As can be seen from Table 8, when the inorganic salt concentration of the solution obtained after adding inorganic salt in the step (3) is within a relatively wide range of the present invention, the enzyme activity of the gained immobilized enzyme can reach more than 302IU/g, and the water content It can be in the range of 54-56%; when it is in the most preferred range of the present invention, the enzyme activity of the obtained immobilized enzyme can reach more than 332IU/g, and the water content can be in the range of 54-56%.

Example 11

Example 11 (including Examples 11-1 to 11-4) is used to illustrate the effect of the dosage ratio of penicillin acylase to carrier in step (3) on the immobilization of penicillin acylase.

According to the method of Example 3, the difference is that in step (3), the amount of the carrier is changed so that the amount of penicillin acylase and the carrier is as shown in Table 9.

Samples were taken to measure the immobilization yield, the enzyme activity and water content of the final immobilized enzyme, and the results were recorded in Table 9.

Table 9

As can be seen from Table 9, when the consumption ratio of penicillin acylase and carrier in step (3) is within the larger range of the present invention, the immobilization yield can reach more than 39.3%, and the enzymatic activity of the gained immobilized enzyme can be Reach more than 311IU/g, the water content can be in the scope of 54-56%; When in the most preferred range of the present invention, the immobilization yield can reach more than 42.4%, and the enzymatic activity of the obtained immobilized enzyme can reach 352IU/g Above, the water content can be in the range of 55-56%.

Example 12

This Example 12 (including Examples 12-1 to 12-5) is used to illustrate the effect of multiple recycling of tert-butanol in step (2) on the separation, purification and immobilization of penicillin acylase.

Carry out multiple tests according to the method for embodiment 3, the difference is that the mixture of tert-butanol and water used in step (2) is the tert-butanol that the previous test reclaims, that is to say, every time a test is carried out The number of cycles of tert-butanol used was increased by one, as shown in Table 10.

Measure and calculate the fermented liquid pretreatment yield (%) of gained, the separation and purification yield (%) of enzyme, enzyme extraction total yield (%) (being the total yield of fermented liquid pretreatment and the separation and purification two steps of enzyme ), immobilized enzyme activity (IU/g) and immobilized enzyme water content (%) are recorded in Table 10.

Table 10

As can be seen from Table 10, the data reproducibility of the experiments using 5 times of recovered tert-butanol is good, the yield of fermentation broth pretreatment, the separation and purification yield of enzyme, the total yield of enzyme extraction, the activity of immobilized enzyme and The data of the water content of the immobilized enzyme is basically kept within the error range; the average yield of the separation and purification steps is 96±1%, the average total yield of the enzyme extraction is 68±1%, and the average enzyme activity of the immobilized enzyme product is 353±1%. 8IU/g, the average water content is 55±1%.

In addition, the immobilized enzyme finished products obtained in Example 12-1, Example 12-2 and Example 12-4 were respectively taken to prepare 6-aminopenicillanic acid and repeated reactions for 15 batches, and the average penicillin G potassium salt residual potency was respectively It is 2737, 2652 and 2807 U/mL, which proves that the catalytic performance of the immobilized enzyme obtained is good.

Comparative example 3

This comparative example is used to illustrate the situation of separation, purification and immobilization of penicillin acylase and immobilized enzyme to prepare 6-aminopenicillanic acid using the prior art in the art.

Use the penicillin acylase crude enzyme solution II obtained in the preparation example to separate, purify and immobilize penicillin acylase according to the following steps:

(1) Slowly add 36-40% ammonium sulfate to the penicillin acylase crude enzyme solution II, stir while adding salt, and stir for 30 minutes after adding salt, then add a small amount of diatomaceous earth plate frame to filter, and collect the precipitate;

(2) Dissolve the precipitate with a 0.05mol/L pH 8.0 phosphate buffer solution close to the volume of the crude enzyme solution II, then add a small amount of diatomaceous earth to filter, and collect the filtrate;

(3) The filtrate is repeatedly washed with a hollow cellulose membrane and purified water to remove pigments, inorganic salts and small molecular proteins, so that the conductance is less than 1000μS/mL, and finally the enzyme solution is concentrated until the enzyme activity of penicillin acylase is 150-250IU/mL mL, collect the concentrate;

(4) Add 0.4-0.45% benzoic acid, adjust the pH to 6.5-6.7, raise the temperature to 50-52°C, keep it for 10-15min, then quickly cool down to below 30°C, add a small amount of diatomaceous earth, filter the plate and frame, and collect the filtrate ;

(5) Enzyme immobilization and post-treatment: the operation mode of the immobilization and post-treatment in Example 1 is the same to obtain the immobilized enzyme.

test case

The immobilized enzyme products obtained in Example 3 and Comparative Example 3 were used to prepare 6-aminopenicillanic acid respectively, and the reaction was repeated for 15 batches, and the results were averaged, as shown in Table 11.

Table 11

Numbering Example 3 Comparative example 3 Fermentation broth pretreatment yield (%) 71 71 Enzyme separation and purification yield (%) 95 70 Enzyme extraction total yield (%) 67 50 Immobilized enzyme activity (IU/g) 353 305 Immobilized enzyme water content (%) 55 55 Response time (min) 35 39 Residual potency of penicillin G potassium salt (U/mL) 2805 3025

As can be seen from Table 11, compared with the method of the comparative example, the method of the present invention has a significant increase in the yield of separation and purification of the enzyme, thereby significantly increasing the total yield of the enzyme extraction. And the enzyme activity of the immobilized enzyme obtained in the present invention is obviously higher than that of the comparative example, so that the reaction time for synthesizing 6-aminopenicillanic acid is shortened compared with the comparative example. In addition, the penicillin G potassium salt residual potency of the present invention is significantly less than that of the comparative example, which shows that the immobilized enzyme obtained by the present invention has better catalytic performance than the comparative example.

The preferred embodiments of the present invention have been described in detail above, however, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the disclosed content of the present invention. All belong to the protection scope of the present invention.

Claims (13)

1. isolate and purify and the immobilization coupling process of a kind of PA ase for being used to prepare 6-amino-penicillanic acid, it is special Sign is that this method comprises the following steps：

(1) the inorganic mixed salt solution containing PA ase crude enzyme liquid and inorganic salts is prepared, this contains institute in mixed salt solution The concentration for stating inorganic salts is 0.15-0.3g/mL；

Wherein, the pH of the inorganic mixed salt solution is 5-8；

(2) inorganic mixed salt solution obtained by step (1) is mixed with the tert-butyl alcohol, then carries out split-phase, obtained rich in the tert-butyl alcohol The three-phase distribution system of upper phase solution, middle phase solid and the lower phase solution composition rich in salt and PA ase；

Wherein, the volume ratio of the inorganic mixed salt solution and the tert-butyl alcohol is 1：0.2-1.5；

(3) immobilization that carrier carries out PA ase is added into lower phase solution obtained by step (2)；

Wherein, the enzyme activity of the lower phase solution is 50-150IU/mL, if being diluted with water to above range higher than 150IU/mL.

2. according to the method described in claim 1, wherein, in step (1), inorganic salts is dense in the inorganic mixed salt solution Spend for 0.2-0.3g/mL.

3. method according to claim 1 or 2, wherein, in step (1), the inorganic salts are water-soluble phosphate, sulphur One or more in hydrochlorate, carbonate, citrate and halide.

4. according to the method described in claim 3, wherein, the inorganic salts are water-soluble phosphate and/or sulfate.

5. according to the method described in claim 4, wherein, the inorganic salts are water-soluble phosphate.

6. method according to claim 1 or 2, wherein, in step (1), the PA ase crude enzyme liquid is warp The untreated PA ase crude enzyme liquid for being used to prepare 6-amino-penicillanic acid after clasmatosis, or for through clasmatosis The PA ase crude enzyme liquid for being used to prepare 6-amino-penicillanic acid after the solid impurities such as thalline is removed afterwards.

7. method according to claim 1 or 2, wherein, the enzyme activity of the PA ase crude enzyme liquid is 50-300IU/ mL。

8. according to the method described in claim 1, wherein, in step (2), the mode of the split-phase is to centrifuge split-phase, this point Phase temperature is 0-40 DEG C, centrifugal force 500-5000g, time 2-60min.

9. according to the method described in claim 1, wherein, in step (2), the mode of the split-phase is to stand split-phase, this point Phase temperature is 4-30 DEG C, time 0.1-4h.

10. according to the method described in claim 1, wherein, the process of the step (2) further includes：From the upper phase solution The recycling tert-butyl alcohol is simultaneously circulated for during being mixed with inorganic mixed salt solution.

11. according to the method described in claim 1, wherein, in step (3), add phosphate to inorganic salts and account for total system Concentration is 0.2-0.45g/mL.

12. the method according to claim 1 or 11, wherein, in step (3), the addition of the carrier is total enzyme activity It is 500-1000IU/g with the ratio between vehicle weight.

13. according to the method for claim 12, wherein, the carrier is epoxy base carrier or the amino carrier of activation.