CN114181925B - Industrial purification and freeze-drying method for recombinant proteinase K - Google Patents

Abstract

The invention discloses a recombinant proteinase K industrialized purification and freeze-drying method. Clarifying and filtering recombinant proteinase K fermentation liquor expressed by pichia pastoris, adding diluent to dilute, concentrating and dialyzing by ultrafiltration step to obtain proteinase K dialysate containing trace metal ions and pigment, separating and purifying the dialysate by cation exchange chromatography to obtain eluent containing recombinant proteinase K, standing the eluent containing recombinant proteinase K at room temperature for crystallizing and precipitating for 4-6h, stirring and mixing uniformly, pouring into a positive pressure filter device, press-filtering to obtain proteinase K filter cake, re-dissolving the filter cake, adding freeze-drying excipient, freeze-drying by optimized freeze-drying process, and packaging to obtain proteinase K finished product. The method has the advantages of simple process steps, high yield, low cost and easy industrialized mass production. The obtained proteinase K target has high yield and good activity.

Description

Industrial purification and freeze-drying method for recombinant proteinase K

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for industrially purifying and freeze-drying recombinant proteinase K.

Background

Proteinase K was a serine proteinase, first obtained in 1974 by purification of an extract of Candida albicans (Tritirachium album limber) at Lin Bashi. The main cleavage site is a peptide bond at the carboxyl end of an aliphatic or aromatic hydrophobic amino acid. Research shows that the enzyme has activity under the conditions of pH 4-12 and 20-60 ℃ and has the capability of cutting protein in SDS, urea, chelating agents (such as EDTA) and sulfhydryl agents (such as trypsin inhibitor or chymotrypsin inhibitor). Thus, its application is very extensive, including the preparation of pulsed-electrophoresis chromosomal DNA, western blotting and the removal of nucleases in DNA and RNA preparation.

The application of proteinase K is not limited to the fields of medical diagnosis and scientific research, but also can replace the traditional chemical method for treating raw materials possibly existing pollution in the fields of industrial leather making, paper making, feed and the like. Therefore, the proteinase K with high activity and low cost has great social and economic values by using genetic engineering technology and protein engineering technology.

Currently, the recombinant proteinase K produced industrially is one with prokaryotic cell, such as colibacillus, and eukaryotic cell, such as yeast, as host. The method has the advantages of simplicity, convenience and economy by using the escherichia coli as a host. However, the proteinase K obtained by the system has low activity and low yield, and is difficult to meet the market demand. The yeast is used as host, hexahistidine tag is added at C end of recombinant proteinase K, fermentation broth is subjected to centrifugation, metal chelating column chromatography and weak cation exchange column chromatography separation, macroporous decolorizing gel decolorizing treatment, and the like, and the process steps are more complicated. In view of the application value of proteinase K, it is necessary to develop a separation and purification technology which is rapid and economical and suitable for industrial scale and continuous production.

Disclosure of Invention

The invention aims to provide a recombinant proteinase K industrialized purification and freeze-drying method.

In order to achieve the above purpose, the present invention provides the following technical solutions:

A chromatographic method for separating and purifying recombinant proteinase K from Pichia pastoris sequentially comprises the following steps:

1) Clarifying supernatant of recombinant proteinase K fermentation liquid expressed by pichia pastoris, and then adding an equal volume of diluent for dilution and uniformly mixing. Concentrating the diluted fermentation supernatant by adopting an ultrafiltration membrane bag, adding an equal volume of dialysate to carry out repeated dialysis after concentrating to an end point, and obtaining proteinase K dialysate containing trace metal ions and pigments. Wherein the ultrafiltration membrane can be selected from Millipore 5kD, 10kD or Sartorius 5kD, 10kD. The preferred membrane package cutoff pore size is 5kD.

2) Regulating pH and electric conductivity of the dialyzate containing a small amount of metal ions and pigment of recombinant proteinase K, and performing cation exchange chromatography to obtain proteinase K eluent with higher concentration. Wherein the filler for cation exchange chromatography is selected from NanoGel 30/50SP、UniGel 30/80SP、SP Bestarose FF、SP Bestarose HP、Bestarose Diomond MMC、Uniphere S、MacroPrep S、POROS XS、SP-6FF、SP-6HP、SP Sepharose^TM Fast Flow. in one embodiment, the filler for cation exchange chromatography is preferably SP Bestarose FF. Cation exchange chromatography may be performed by selecting a pH or salinity gradient elution, preferably a sodium chloride gradient elution.

3) And (3) crystallizing and precipitating the obtained proteinase K eluent at room temperature, wherein the proteinase K eluent is preferably regulated to have the protein concentration within the range of 40-50 mg/ml before crystallizing and precipitating. And after the precipitation is finished, obtaining a proteinase K precipitation filter cake by adopting a positive pressure filter device. Wherein the filter paper board is Shenyang great wall filter paper board, the aperture of the filter board is 0.2-5 μm, preferably 2.5-5 μm.

4) And adding the obtained proteinase K precipitate filter cake into a redissolution buffer solution for redissolution, wherein in one implementation mode, the redissolution buffer solution is 10-50 mM Tris, 0-5 mM CaCl ₂, pH 7.5-9.0 or 10-50 mM NaAc, 0-5 mM CaCl ₂ and pH 4.5-5.5. After being uniformly mixed by adopting a preferred freeze-drying formula, the mixture is freeze-dried according to a preferred freeze-drying process. And packaging the obtained freeze-dried powder, and finally obtaining a proteinase K freeze-dried powder finished product.

Wherein the proteinase K activity of the supernatant of the recombinant proteinase K fermentation broth is 30-40U/mgP.

The invention also discloses a specific implementation mode, the implementation mode is amplified by a process, the protein purification yield of the obtained proteinase K freeze-dried powder is not less than 70%, and the activity is 30-40U/mgP. The specific implementation steps are as follows:

1) Clarifying the supernatant of the recombinant proteinase K fermentation broth expressed by pichia pastoris, measuring the pH and the conductivity of the clarified supernatant, and then adding an equal volume of diluent for dilution and uniformly mixing, wherein the diluent is 10-50 mM Tris, 0-5 mM CaCl ₂ and the pH is 7.5-9.0;

And installing an ultrafiltration membrane package on an ultrafiltration system, controlling inlet pressure and reflux pressure, regulating transmembrane pressure, measuring water flux, and performing ultrafiltration concentration after installing the ultrafiltration membrane package, wherein the interception pore diameter of the ultrafiltration membrane package is 5-10 kD, and the transmembrane pressure is 1-1.25 bar, 14.5-18 psi, or 0.1-0.125 MPa.

And adding the concentrated solution concentrated to the end point into an equal volume of dialysis solution to carry out dialysis for a plurality of times until the end point of dialysis, wherein the dialysis solution is 10-50 mM Tris, 0-5 mM CaCl ₂ and pH 7.5-9.0, the color of the permeate solution at the end point of dialysis is colorless, the electric conductivity is 0.5-2 mS/cm, and the pH is 7.5-9.0.

2) The method adopts a cation exchange chromatography medium as a Bognoton SP Bestarose FF cation exchange filler, and comprises the following chromatography steps:

Adopting a balancing buffer solution with the pH of 7.5-9.0 and the volume of 5-15 times of column volume of which is composed of 10-50 mM Tris, 0-5 mM CaCl ₂ and balancing the chromatographic column at the flow rate of 50-200 cm/hour;

taking the proteinase K dialysate obtained in the step 1) as a sample loading sample, and loading the sample at a flow rate of 50-200 cm/hour, wherein the conductance of the sample loading sample is 0.5-2 mS/cm, the pH value is 7.5-9.0, and the loading capacity is 56-90 mg protein/ml filler;

10-50 mM Tris, 0-5 mM CaCl ₂, and balancing buffer solution with pH of 7.5-9.0, and re-balancing the chromatographic column at a flow rate of 50-200 cm/hr;

Eluting the chromatographic column by adopting an elution buffer solution with the components of 10-50 mM Tris, 0-5 mM CaCl ₂ and 0~100mM NaCl,pH 7.5~9.0 at the flow rate of 50-200 cm/hour to finally obtain proteinase K eluent;

The protein recovery of the cationic chromatography step-by-step amplification chromatography is more than 70%.

3) Cutting a filter paper board with proper size, putting the filter paper board into a positive pressure filter, and adopting an elution buffer solution with the components of 10-50 mM Tris, 0-5 mM CaCl ₂ and 0~100mM NaCl,pH 7.5~9.0 to rinse the positive pressure filter device and the filter paper board;

regulating the pH value of the proteinase K eluent in the step 2) to 7.5-9.0, cond to 0-10 mS/cm, and the protein concentration to 40-50 mg/ml, pouring the proteinase K eluent into a stainless steel barrel, controlling the environmental temperature to 20-25 ℃, and standing for more than 4 hours until proteinase K crystallization and precipitation are complete;

Pouring the proteinase K completely precipitated into a positive pressure filter tank for multiple times, introducing compressed air into the filter tank, controlling the pressure gauge of the filter tank to be 0-0.15 MPa, and starting filter pressing to obtain a proteinase K filter cake;

the protein recovery in the proteinase K filter cake is greater than 90%.

4) Adding 10-50 mM Tris, 0-5 mM CaCl ₂, pH 7.5-9.0 or 10-50 mM NaAc and 0-5 mM CaCl ₂ into the filter cake containing recombinant proteinase K obtained in the step 3), re-dissolving the filter cake, adding mannitol according to the ratio of W/V=1:20, and uniformly mixing to obtain a mixed solution;

Pouring the mixed solution into a freeze-drying plate, setting the pre-freezing temperature to be-40 to-45 ℃, and maintaining the temperature reduction time to be 30 minutes for 3-6 hours;

setting the temperature of the first section of primary drying at-35 to-40 ℃, the heating time at 30min, maintaining for 1-2 h, setting the vacuum degree at 10-20 Pa, and setting the temperature of the second section at 0-10 ℃ and the heating time at 4-6 h, and maintaining for 40-45 h.

The invention adopts the industrial process steps of ultrafiltration, cation exchange, filter pressing and the like, shortens the batch production time to 1-2 days, and ensures that the total yield of proteinase K in the freeze-dried preparation prepared after purification is up to more than 70 percent, thereby realizing large-scale and continuous production.

Drawings

FIG. 1 color change of the solution before and after ultrafiltration of proteinase K fermentation broth.

FIG. 2 shows a GE DEAE anion exchange chromatography.

FIG. 3:SP Bestarose FF A linear elution chromatography of a cation exchange chromatography 1ml column.

FIG. 4:SP Bestarose FF A cation exchange chromatography 84ml column gradient elution chromatography.

FIG. 5 comparison of proteinase K crystallization precipitation with delamination.

FIG. 6 morphological changes after proteinase K lyophilization.

FIG. 7 dissolution of proteinase K in sodium acetate-acetic acid buffer at different pH values.

FIG. 8:L-tyrosine standard curve.

Detailed Description

The technical scheme of the present invention will be described in detail below by examples and drawings, so that the features and advantages of the present invention will be better illustrated. The examples provided should be construed as illustrative of the method of the present invention and not limiting the disclosed technology in any way.

The reagents and apparatus used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 proteinase K purification and lyophilization Process optimization

1.1 Experimental reagents and instruments

Proteinase K fermentation broth from Jinan Bai Jie bioengineering Co., ltd;

the ultrafiltration system is purchased from Merck Mibo and is of the model CUF 100;

Ultrafiltration membrane bags purchased from celecoxib;

Chromatography packing SP Bestarose FF, available from Shanghai Boguy, DEAE anion exchange packing, available from general electric company;

Other chemical reagents are all commercially available products.

1. Tangential flow ultrafiltration

1.1 Ultrafiltration Process fumbling

The yeast fermentation liquor is dark reddish brown and contains a large amount of pigment, and according to the molecular weight of proteinase K, the yeast fermentation liquor is subjected to pigment removal treatment by adopting a proper ultrafiltration membrane bag, and the color of the solution after ultrafiltration is lighter than that of the fermentation liquor and before ultrafiltration, so that the pigment is removed in a large amount. The color change of the fermentation broth before and after ultrafiltration is shown in figure 1.

The invention selects ultrafiltration membrane packages with different interception pore diameters and membrane areas for comparison experiments, the results are shown in table 1, the ultrafiltration membrane packages with different interception pore diameters and membrane areas have protein recovery of more than 90%, and both the two membrane packages can meet the requirement of amplified production. In addition, after the area of the comparative experiment membrane is amplified to 33 times, the protein recovery after ultrafiltration is more stable, which shows that the process can be stably amplified.

TABLE 1 ultrafiltration membrane packet ultrafiltration results for different rejection pore sizes and membrane areas

1.2 Ultrafiltration Process optimization

In order to adapt to the treatment capacity of fermentation liquor in a larger scale, the ultrafiltration process parameters are required to be optimized. 500ml of fermentation broth was taken and diluted with an equal volume of ultrapure water while stirring.

Ultrafiltration concentration was performed using a Sartorius 0.1m ² kD ultrafiltration membrane pack, the normal water flux of the membrane pack product was 42L/h/m ², pin (max) =4bar. After optimization, pin (inlet pressure) =2mar, pres (reflux pressure) =0.5 bar, pout (permeate pressure) =0 bar were finally controlled. Calculate TMP (transmembrane pressure) of the membrane packet= (pin+pret)/2-pout=1.25 bar=18 psi.

And optimizing the process parameters of ultrafiltration concentration and dialysis steps according to the transmembrane pressure. The results are shown in tables 2 and 3. And calculating the batch processing amount and the actual membrane package using area according to the average water flux of the optimized concentration and ultrafiltration steps. The final result is 250L/batch feeding, and the required membrane area is obtained after 8 hours ultrafiltration

= { (250L/17 LMH) + (250L x 7/28LMH }/8h=9.65 m ², number of required packets=9.65 m ²/0.66m² ≡15 blocks.

TABLE 2 control Table of Process parameters for concentration step

Concentration step average flux = 0.5L/(18/60) h/0.1m ² ≡17LMH

TABLE 3 dialysis step Process parameter control Table

Average flux of dialysis step = 3.5L/(74/60) h/0.1m ² ≡28LMH

2. Ion exchange chromatography

According to the isoelectric point of proteinase K, two purification modes of anion penetration mode or cation combination mode can be adopted for separating and purifying proteinase K.

2.1 Anion exchange chromatography

And (3) adopting a GE DEAE chromatographic column to search anion exchange chromatography conditions, taking the proteinase K fermentation broth subjected to ultrafiltration treatment, adjusting the pH to 7.6, and loading the proteinase K fermentation broth after the conductivity is 0.9 mS/cm. Chromatography was performed with 10mM Tris-HCl,1mM CaCl ₂, pH 7.5 as the equilibration solution, and 10mM Tris-HCl,1M NaCl,1mM CaCl ₂, pH 7.5 as the elution solution. The results showed that proteinase K penetrated only 69.3% and thus the anion penetration mode was abandoned for proteinase K purification. The chromatographic chart is shown in figure 2.

2.2 Cation exchange chromatography

2.2.1 Cationic chromatography conditions fumbling

And (3) adopting a Bognong SP Bestarose FF chromatographic column to search cation exchange chromatography conditions, taking the proteinase K fermentation broth subjected to ultrafiltration treatment, adjusting the pH value to 7.5, and loading the proteinase K fermentation broth after the conductivity is 0.8 mS/cm. Chromatography was performed with 10mM Tris-HCl,1mM CaCl ₂, pH 7.5 as the equilibration solution, and 10mM Tris-HCl,0~1M NaCl,1mM CaCl ₂, pH 7.5 as the elution solution. The result shows that proteinase K is mainly eluted under the gradient of 0-100 mM NaCl, the protein recovery is 92.7%, and the activity is more than 30U/mg. The chromatographic pattern is shown in figure 3.

2.2.2 Progressive amplification by cationic chromatography

On the basis of fumbling of cationic chromatographic conditions, various chromatographic parameters of SP Bestarose FF are optimized and confirmed. Wherein the loading conductivity is 0.5-2 mS/cm, the loading capacity is 56-90 mg protein/ml filler, the elution condition is 10mM Tris-HCl,0~100mM NaCl,1~5mM CaCl ₂, and the pH is 7.5-9.0. To verify the stability of the chromatography process, the filler volumes were sequentially scaled up from 1ml to 8.6ml, 28ml, 84ml in the fumbling stage under the optimized various chromatographic parameters. The results show that the protein recovery of the step-by-step amplification chromatography is more than 70%. The results of the stepwise amplification chromatography are shown in Table 4. The SP Bestarose FF chromatographic chart amplified to 84ml is shown in figure 4.

Table 4SP Bestarose FF results of step-by-step amplification chromatography

3. Proteinase K solid-liquid separation

Proteinase K is easy to crystallize out in a low-salt environment under the condition of high protein concentration. According to the invention, by utilizing the characteristic of proteinase K, the pH of SP Bestarose FF chromatography eluent is regulated to 7.5-9.0, cond is regulated to 0-10 mS/cm, the protein concentration is regulated to 40-50 mg/ml, and the mixture is stirred and mixed uniformly and then kept stand for 4-6 hours at 20-25 ℃. Under the above conditions, proteinase K crystallized to separate out a layer. The results of crystallization precipitation and delamination are shown in FIG. 5.

And (3) utilizing a solid-liquid separation technical platform of my department, selecting a filtering paperboard with proper size and a positive pressure filtering device to collect the precipitated sediment. Wherein the filter paper board is a Shenyang great wall filter paper board, the aperture of the filter board is 0.2-5 mu m, the filter paper board is adopted for filtering, proteinase K precipitate is trapped on the filter paper board to form a filter cake, and the filtrate is clear and transparent. The suspension after 30ml precipitation was press-filtered and three experiments were performed. And taking the pressed filtrate and the filter cake complex solution for protein content measurement. The result shows that the recovery of the protein after crystallization and precipitation is more than 90%, and the purpose of removing impurities and concentrating can be achieved by collecting the precipitate in the way. The measurement results are shown in Table 5.

TABLE 5 recovery of proteinase K crystalline precipitated protein

4. Proteinase K freeze-drying process

Adding 10-50 mM Tris, 0-5 mM CaCl ₂, pH 7.5-9.0 or 10-50 mM NaAc, 0-5 mM CaCl ₂ and pH 4.5-5.5 into the obtained filter cake containing the recombinant proteinase K for redissolution. Mannitol is added according to the ratio of W/V= (1:20) and evenly mixed. And pouring the mixed solution into a freeze-drying plate, setting the pre-freezing temperature to be-40 to-45 ℃, and maintaining the temperature reduction time to be 30 minutes for 3-6 hours. Setting the temperature of the first section of primary drying at-35 to-40 ℃, heating for 30min, maintaining for 1-2 h, and setting the vacuum degree at 10-20 Pa. The temperature of the second section is 0-10 ℃, the temperature rise time is 4-6 h, and the time is maintained for 40-45 h.

The preferred freeze-drying process is controlled to have freeze-drying parameters of-45 ℃, the cooling time of 30min and maintaining for 6h, the first section of primary drying temperature of-40 ℃, the heating time of 30min and maintaining for 1h, the vacuum degree of 20Pa, the second section of primary drying temperature of 0 ℃ and the heating time of 6h and maintaining for 45h. The freeze-dried proteinase K is shown in figure 6.

The freeze-dried powder obtained by the freeze-drying process is used for activity measurement (casein is taken as a substrate), and the result shows that the activity measurement value of the freeze-dried powder at different sampling points is larger than 30U/mg and is equivalent to the activity of Merck proteinase K. The results of the activity measurement are shown in Table 6.

TABLE 6 determination of the Activity of proteinase K lyophilized powder at different sampling points

5. Proteinase K solubility studies

5.1 Protease K lyophilized powder dissolution buffer solution fumbling

In order to explore the optimal dissolution condition of the proteinase K freeze-dried powder, the proteinase K freeze-dried powder is dissolved by adopting 20mM Tris-HCl and pH9.68-7.15;20mM PB,pH 8.02~5.60;20mM NaAc,pH 7.60~5.20 respectively, and the dissolution state is observed. The results showed that proteinase K dissolved well in the 20mM NaAc,pH 5.20~5.60 range.

To verify the results, 7 parts of proteinase K freeze-dried powder are accurately weighed, each 100mg of proteinase K freeze-dried powder is respectively prepared into 20mM NaAc,pH 5.58, 5.48, 5.40, 5.28 and 5.21 buffer solutions of 100ml in an EP tube of 10ml, then 5ml of buffer solutions with different pH values are respectively added into the 5 tube, and after shaking and mixing, the dissolution state is observed. As a control, 5ml of 50% glycerol and 5ml of ultrapure water were added to the two tubes. The results of the activity measurement are shown in Table 7, and the results of the dissolution are shown in FIG. 7.

TABLE 7 determination of the dissolution Activity of proteinase K in sodium acetate-acetic acid buffers at different pH values

EXAMPLE 2 Pronase K Process amplification

2.1 Production raw materials and instrumentation

Proteinase K fermentation broth from Jinan Bai Jie bioengineering Co., ltd;

Tris (hydroxymethyl) aminomethane, purchased from south Beijing chemical agents Co., ltd., analytically pure;

anhydrous calcium chloride, available from Hebei Huachen pharmaceutical industry Co., ltd, pharmaceutical grade;

Sodium chloride, available from Hebei Huachen pharmaceutical Co., ltd., pharmaceutical grade;

sodium hydroxide, available from Chengdu Hua Yi pharmaceutical excipients manufacturing Limited company, pharmaceutical grade;

ethanol, available from Nanjing chemical reagent Co., ltd., pharmaceutical grade;

Mannitol, purchased in the stoneware Hua Xu pharmaceutical industry, pharmaceutical grade;

sulfur Liu Gongna, available from Guogong chemical reagent Co., ltd., analytical grade;

the ultrafiltration system is purchased from Merck Mibo and is of the model CUF 100;

Ultrafiltration membrane package purchased from Sidoris, model number of which is HYDRO,5KD;

An industrial grade chromatographic column, available from general electric company, model Chromflow 800,800;

chromatography packing material purchased from Shanghai Boguo, model SP Bestarose FF;

freeze dryer, available from Beijing tachygen midday science and technology Co., ltd., model number YLDZ-0.5

2.2 Production of stock solution

And (1) pretreating the raw materials.

Sterilizing 10 barrels, 25L/barrel fermentation broth with 10 inch, 0.22 μm sterilizing filter, and packaging into two barrels of about 125L each;

And (2) preliminary concentration.

Wrapping each barrel of fermentation liquor in the step (1) by using a 5KD membrane, carrying out isovolumetric dialysis on the dialysate 1 (the formula is 0.111g/L CaCl ₂ water solution) for 1-2 times, concentrating each barrel of fermentation liquor to 75L, combining two barrels into one barrel of 150L, continuing to carry out isovolumetric dialysis on the fermented liquid for 3-4 times by using the dialyzed liquid 1, and carrying out isovolumetric dialysis on the fermented liquid for 3-5 times by using the dialysate 2 (the formula is 1.21g/L Tris,0.111g/L CaCl ₂, pH 7.5+/-0.1 and Cond 0.8-1 mS/cm);

And (3) preparing a sample liquid.

Regulating the pH value of the concentrated feed liquid in the step (2) to 7.5+/-0.1 by using Tris, regulating the electric conduction to 0.8-1.2mS/cm, and diluting the protein content of the feed liquid to 10-15mg/ml by using a dialysate 2 (the formula is 1.21g/L Tris,0.111g/L CaCl ₂, pH value of 7.5+/-0.1 and Cond of 0.8-1 mS/cm) to form a chromatographic loading liquid;

And (4) chromatographic purification.

The chromatographic purification is carried out according to the following steps 1) to 7), and the chromatographic eluent is collected as a target component.

1) Filling the column, namely adopting GE Chromflow chromatographic columns with SP Bestarose FF filling materials, wherein the filling height of the column is 16-18cm, the filling volume of the filling chromatographic filling materials is 80-86L, and the detection column efficiency is more than 3000.

2) And (3) washing the packing with purified water, wherein the flow rate of the chromatographic column is about 145cm/h, and the washing time is longer than 30min.

3) Balancing with equilibration buffer (1.21 g/L Tris,0.111g/L CaCl ₂, pH 7.5.+ -. 0.1, cond)

0.8-1 MS/cm) balance chromatographic column 600-700L, flow rate of about 145cm/h, balance until pH and conductivity baseline of chromatographic column outlet permeate are stable.

4) Loading, namely loading the chromatographic loading liquid in the step (3), wherein the loading capacity is less than 67.5mg total protein/ml filler. The loading flow rate is about 145cm/h, and the inlet pressure of the loading chromatographic column is less than 2.0bar.

5) Re-equilibration 550-650L of column was re-equilibrated with the above equilibration buffer at a flow rate of about 145cm/h. And balancing until the ultraviolet absorption value of the chromatographic outlet permeate is less than 20 mv.

6) Eluting and collecting target components by eluting the chromatographic column with eluting buffer (1.21 g/L Tris,0.555g/L CaCl _2, 5.844.844 g/L NaCl, pH 7.5+ -0.1, cond 12+ -1 mS/cm) at a flow rate of 145cm/h, collecting when the peak is chromatographed and the ultraviolet absorption value is greater than 30mV, stirring uniformly while collecting, and stopping collecting when the ultraviolet absorption value is less than 30mV, thus obtaining the target components.

7) And after the elution is finished, the chromatographic column is regenerated by using 2M NaCl and 0.5M NaOH respectively, the regeneration flow rate is 145cm/h, and the regeneration volume is 300-420L. After the end of the regeneration process,

The column was kept with 20% ethanol.

And (5) separating the sediment from the filter pressing.

Purifying the collected target component by chromatography in the step (4), standing and precipitating for more than 5 hours, starting to carry out positive pressure filtration separation by using a 75 μm filter paper board, collecting precipitate (filter cake), and collecting clear supernatant. The inlet pressure during the press filtration process does not exceed 4bar.

And (6) preparing a stock solution.

Adding 20L of clear supernatant obtained in the step (5) into a filter cake for dissolution, adding 10L of stock solution to prepare mother solution (2.5 kg of mannitol and 5g of merthiolate for dissolution by using the clear supernatant), adding into the filter cake, and finally fixing the volume of the clear supernatant to 50L to obtain proteinase k stock solution.

2.3 Lyophilization

Step (1) pre-freezing, namely after shaking up the feed liquid, pouring the solution into a stainless steel freeze-drying plate, and pre-freezing for 4 hours at the temperature of-45 ℃;

cooling the rear box in the step (2), namely after the pre-freezing beam, starting cooling the rear box, keeping the temperature of the product at-45 ℃, and cooling the rear box for 30min;

Step (3) vacuumizing, namely vacuumizing the box body after the cooling of the rear box is finished, wherein the vacuum is controlled below 25 pa;

Heating, namely heating the plate layer for 6 hours to 0 ℃ and controlling the vacuum degree to be 20-30pa;

Step (5) of primary sublimation, namely maintaining the temperature of the plate layer to be 0 ℃, controlling the vacuum degree to be 15-30pa, and entering the next section after the temperature of the product reaches more than-1.5 ℃;

heating in the step (6), namely heating the plate layer to 10 ℃ for 30min, and controlling the vacuum degree to 10-15pa;

step (7) of secondary sublimation, namely maintaining the temperature of the plate layer to be 10 ℃ and controlling the vacuum degree to be 5-10pa;

And (8) judging the end point, namely closing the front and rear box valves after the vacuum degree is less than 5pa, and ending freeze-drying after the front box vacuum degree is less than 5pa for 3 min.

2.4, Packaging the finished product:

Crushing the freeze-dried powder in the step (1), namely crushing proteinase K after freeze-drying is finished, and collecting the crushed proteinase K into double-layer sterile bags to obtain freeze-dried fine powder.

And (2) packaging, namely weighing and packaging the proteinase K freeze-dried fine powder in a clean room isolator to obtain a recombinant proteinase K finished product.

The process scale-up was performed according to the above examples, the process was stable and reliable, and the results of the batch production of the prepared products are shown in table 8:

TABLE 8 different preparation batch proteinase K yields and purification yields

Batch of	Yield (%)
		Preparation batch 1	70.5%
Preparation batch 2	73.3%
		Preparation batch 3	70.5%
Preparation batch 4	70.0%
		Preparation batch 5	70.5%
Preparation batch 6	70.5%

EXAMPLE 3 proteinase K Activity assay

3.1 Principle of experiment the method uses bovine hemoglobin as a substrate for detecting proteinase K activity. The bovine hemoglobin solution is hydrolyzed into peptides and amino acids which are soluble in trichloroacetic acid under the action of proteinase K, and the peptides and amino acids reduce phenolic reagents under alkaline conditions to generate blue compounds, and the color depth of the compounds at the wavelength of 750nm is in direct proportion to the concentration of enzyme hydrolysate.

The definition of the activity units is that 1 unit of proteinase K per minute of hydrolysis of urea-denatured bovine hemoglobin produced a color with the Fulin phenol reagent at 37℃CpH7.5, equivalent to 1.0. Mu. Mol of tyrosine produced a color with the Fulin phenol reagent.

3.2 Major instrumentation

PH meter (Sartorius PB-10)

Enzyme mark instrument (Molecular Device Versamax)

3.3 Preparation of reagents and solutions

Potassium dihydrogen phosphate, bovine hemoglobin, sodium hydroxide, urea, calcium chloride, trichloroacetic acid, phenol reagent (Fu Lin Fen), L-tyrosine, and hydrochloric acid

3.3.1 Preparation of reagents

The reagent is prepared with high purity water (more than or equal to 18M omega xcm RESISTIVITY AT ℃ C.)

(1) 1M Potassium dihydrogen phosphate solution 13.61g of potassium dihydrogen phosphate was weighed and dissolved in 50ml of water, the pH was adjusted to 7.5 with 1M KOH at 37℃and the volume was fixed to 100ml.

(2) The substrate (100 mM potassium dihydrogen phosphate-2.0% (W/V) bovine hemoglobin-6M urea) was prepared by weighing 2.0g bovine hemoglobin, dissolving and stirring with 40ml of water at 37℃for 30min, adding 8.0ml of 1M NaOH, stirring with 37℃for 20min, adding 36.0g of urea, stirring with 37℃for 60min, adding 10ml of 1M potassium dihydrogen phosphate, adjusting pH to 7.5 with 5M HCl, and fixing volume to 100ml.

(3) A20 mM CaCl ₂ solution was weighed out and 0.295g of calcium chloride was dissolved in water and taken up to a volume of 100ml.

(4) 305MM TCA solution (trichloroacetic acid solution) 24.9g trichloroacetic acid were weighed out and dissolved in water and the volume was set to 500ml.

(5) NaOH solution:

a.1M NaOH solution, 20g of sodium hydroxide are weighed, dissolved in water and brought to a volume of 500ml.

0.5M NaOH solution, namely diluting the 1M NaOH solution with water in an equal volume.

(6) Hydrochloric acid solution:

1M hydrochloric acid 8.33ml of hydrochloric acid are taken, diluted with water and brought to a volume of 100ml.

0.5M hydrochloric acid 1M hydrochloric acid was taken and diluted with water in equal volumes.

3.3.2 Standard Curve preparation

20Mg of L-tyrosine was weighed, heated to 70-80℃with 80ml of water to dissolve completely, cooled to room temperature and taken up to 100ml with water. Can be stored in a refrigerator at 2-8deg.C for 6 months.

L-tyrosine standard curve preparation is carried out according to the preparation steps of Table 9, and the content of each standard substance in the final reaction system is 0.0088, 0.0264, 0.044, 0.0616, 0.0792, 0.0968, 0.1144 and 0 mu mol.

TABLE 9L-tyrosine standard curve formulation table

3.3.3 Preparation of proteinase K sample

A, weighing 0.5g of solid powder, dissolving the solid powder with high-purity water, fixing the volume to 25ml, and diluting the solid powder with CaCl ₂ solution to an activity range of 0.075-0.175U/ml for measurement.

Liquid sample, protein concentration is diluted to 20mg/ml by high purity water, and then CaCl ₂ solution is diluted to the activity range of 0.075-0.175U/ml for measurement.

3.4 Detection step

3.4.1 Measurement of protein concentration of proteinase K and Merck proteinase K (BCA method)

200Mg of proteinase K freeze-dried powder is accurately weighed, added into 10ml of high-purity water and vibrated to be fully dissolved. Then, the Merck proteinase K was diluted 40-fold with high purity water to a standard curve range.

25 Μl of the diluted sample and BSA standard were added to the microplate, 200 μl of BCA working solution was added, incubated at 37deg.C for 30min, and the microplate reader wavelength was set at 562nm for reading. The concentration measurement was carried out by referring to protocols in the kit "REFERENCE BCA PLUS ^TM Protein ASSAY REAGENT protol for measurement, prod #1856210, thermo".

Merck proteinase K concentration was determined in the same way.

3.4.2 Protease K Activity assay

3.4.2.1 According to the following table, 0.50ml of substrate was added per tube.

3.4.2.2 After equilibration for 10min at 37 ℃, a further 0.10ml of diluted enzyme solution was added, and the blank control was added with 0.10ml of 20mM CaCl ₂ solution.

3.4.2.3 Vortex mixing, 37 ℃ incubation for 10min, and then adding 1.00ml 305mM TCA solution.

3.4.2.4 Vortex shaking and mixing uniformly, and standing at room temperature for 20min.

3.4.2.5 Centrifuging at 13000rpm for 1min, taking out the whole supernatant, and shaking and mixing.

After 3.4.2.6 centrifugation, 0.20ml of the supernatant was added to each tube, followed by capping and vortexing.

3.4.2.7 Vortex mix L-tyrosine standard with standard blank for each concentration.

3.4.2.8 Taking 0.6mL of L-tyrosine standard substance prepared by 3.3.2 into a 2mL EP tube, then taking 0.12mL of Fu Lin Fen reagent, adding the reagent into the EP tube, shaking and mixing uniformly, synchronously adding 0.12mL of Fu Lin Fen reagent into a 3.4.2.6 treated sample to be tested, shaking and mixing uniformly, and reacting for 30min at room temperature.

3.4.2.9 Taking 300 mu l of each of the standard product and the sample to be tested after the reaction, adding the 300 mu l of each of the standard product and the sample to be tested into each hole of the ELISA plate, and setting the wavelength of the ELISA plate to 750nm for reading.

Note that if the solution is cloudy, it is filtered with a 0.45 μm filter head and the reading is taken.

3.4.3 Data processing

3.4.3.1 Standard Curve

Δa ₇₅₀ standard = a ₇₅₀ standard-a ₇₅₀ blank

The tyrosine content (mu mol) is taken as an X axis, the average OD value is taken as a Y axis for linear fitting, the formula is as follows, Y=A+BX, R ² is more than or equal to 0.99, and a fitted standard curve is shown in figure 8.

3.4.4 Analysis of results

3.4.4.1 The results of the multiple runs are shown in Table 10 below:

TABLE 10 homemade batch and commercially available control proteinase K Activity assay

3.4.4.2 Sample calculation formula:

Sample ΔA ₇₅₀＝A₇₅₀ sample-A ₇₅₀ blank

Tyrosine content (sample) = (sample Δa ₇₅₀ -a)/B

Sample enzyme activity = tyrosine content (sample) ×1.6×dilution/(0.1×10×0.2)

1.6 Volume of complete stop reaction (ml)

0.2 Volume of stop reaction with F & C reagent (ml)

Df=dilution factor

0.10 =Add enzyme solution (mL)

10 Time of incubation for=experiment (min)

M=weight of enzyme to be tested (mg)

V = dissolution volume of enzyme

Total activity = sample enzyme activity/(m/V)

Specific activity = sample enzyme activity/BCA method to determine protein content

Therefore, by adopting the method to detect, the specific activities of the two batches of homemade proteinase K are respectively 40U/mgP and 43U/mgP, which are equivalent to the specific activities (41U/mgP) of the proteinase K products of commercial Merck, and the detection method is reliable and stable.

Claims (3)

1. An industrialized purification and freeze-drying method of recombinant proteinase K sequentially comprises the following steps:

1) Clarifying the supernatant of the recombinant proteinase K fermentation broth expressed by yeast, adding an equal volume of diluent, diluting and uniformly mixing, concentrating the diluted fermentation supernatant by adopting an ultrafiltration membrane bag with the interception pore diameter of 5-10 kD, and adding an equal volume of dialysate for multiple dialysis to obtain proteinase K dialysate;

2) Regulating pH and conductivity of the proteinase K dialysate, and performing cation exchange chromatography by adopting pH or salinity gradient elution to obtain proteinase K eluent with high concentration, wherein the filler of the cation exchange chromatography is selected from SP Bestarose FF;

In the step 2), the chromatography step is as follows:

2a) Adopting a balancing buffer solution with the pH of 7.5-9.0 and the volume of 5-15 times of column volume of which is composed of 10-50 mM Tris, 0-5 mM CaCl ₂ and balancing the chromatographic column at the flow rate of 50-200 cm/hour;

2b) Taking the proteinase K dialysate obtained in the step 1) as a sample loading sample, and loading the sample at a flow rate of 50-200 cm/hour, wherein the conductance of the sample loading sample is 0.5-2 mS/cm, the pH value is 7.5-9.0, and the loading capacity is 56-90 mg protein/ml filler;

2c) 10-50 mM Tris, 0-5 mM CaCl ₂, and balancing buffer solution with pH of 7.5-9.0, and re-balancing the chromatographic column at a flow rate of 50-200 cm/hr;

2d) Eluting the chromatographic column by adopting an elution buffer solution with the components of 10-50 mM Tris, 0-5 mM CaCl ₂ and 0~100mM NaCl,pH 7.5~9.0 at the flow rate of 50-200 cm/hour to finally obtain proteinase K eluent;

the protein recovery of the cationic chromatography step-by-step amplification chromatography is more than 70%;

3) Crystallizing and precipitating the proteinase K eluent at room temperature, and obtaining a proteinase K precipitation filter cake by adopting a positive pressure filter device after the precipitation is finished;

The step 3) is as follows:

3a) Cutting a filter paper board with the aperture of 2.5-5 mu m, loading the filter paper board into a positive pressure filter, and adopting an elution buffer solution with the components of 10-50 mM Tris, 0-5 mM CaCl ₂ and 0~100mM NaCl,pH 7.5~9.0 to rinse the positive pressure filter device and the filter paper board;

3b) Regulating the pH value of the proteinase K eluent in the step 2) to 7.5-9.0, the conductivity to 0-10 mS/cm and the protein concentration to 40-50 mg/ml, pouring the proteinase K eluent into a stainless steel barrel, controlling the environmental temperature to 20-25 ℃, and standing for more than 4 hours until proteinase K crystallization and precipitation are complete;

3c) Pouring the proteinase K completely precipitated into a positive pressure filter tank for multiple times, introducing compressed air into the filter tank, controlling the pressure gauge of the filter tank to be 0-0.15 MPa, and starting filter pressing to obtain a proteinase K filter cake;

protein recovery in the proteinase K filter cake is greater than 90%;

4) Adding the proteinase K precipitate filter cake into a redissolution buffer solution for redissolution, and freeze-drying to finally obtain proteinase K freeze-dried powder finished products;

Wherein the ultrafiltration membrane package is selected from Millipore 5kD, 10kD,Sartorius 5kD and 10kD, the redissolution buffer in the step 4) is 10-50 mM Tris, 0-5 mM CaCl ₂, pH 7.5-9.0 or 10-50 mM NaAc, 0-5 mM CaCl ₂ and pH 4.5-5.5;

The specific steps of the step 1) are as follows:

5a) Clarifying the supernatant of the recombinant proteinase K fermentation broth, measuring the pH and the conductivity of the clarified supernatant, and then adding an equal volume of diluent for dilution and uniformly mixing, wherein the diluent is 10-50 mM Tris, 0-5 mM CaCl ₂ and the pH is 7.5-9.0;

5b) Installing an ultrafiltration membrane package on an ultrafiltration system, controlling inlet pressure and reflux pressure, regulating transmembrane pressure, measuring water flux, and performing ultrafiltration concentration after installing the ultrafiltration membrane package, wherein the interception pore diameter of the ultrafiltration membrane package is 5-10 kD, and the transmembrane pressure is 1-1.25 bar or 14.5-18 psi or 0.1-0.125 MPa;

5c) And adding the concentrated solution concentrated to the end point into an equal volume of dialysis solution to carry out dialysis for a plurality of times until the end point of dialysis, wherein the dialysis solution is 10-50 mM Tris, 0-5 mM CaCl ₂ and pH 7.5-9.0, the color of the permeate solution at the end point of dialysis is colorless, the electric conductivity is 0.5-2 mS/cm, and the pH is 7.5-9.0.

2. The method according to claim 1, wherein the specific activity of the recombinant proteinase K obtained by the method is 30-40U/mgP.

3. The method according to claim 1, wherein the step 4) is:

6a) Adding 10-50 mM Tris, 0-5 mM CaCl ₂, pH 7.5-9.0 or 10-50 mM NaAc and 0-5 mM CaCl ₂ into the filter cake containing recombinant proteinase K obtained in the step 3), re-dissolving the filter cake, adding mannitol according to the ratio of W/V=1:20, and uniformly mixing to obtain a mixed solution;

6b) Pouring the mixed solution into a freeze-drying plate, setting the pre-freezing temperature to be-40 to-45 ℃, and maintaining the temperature reduction time to be 30 minutes for 3-6 hours;

6c) Setting the temperature of the first section of primary drying at-35 to-40 ℃, the heating time at 30min, maintaining for 1-2 h, setting the vacuum degree at 10-20 Pa, and setting the temperature of the second section at 0-10 ℃ and the heating time at 4-6 h, and maintaining for 40-45 h.