阅读说明：本技术 一种高比活力碱性磷酸酶工程菌、工程菌构建及碱性磷酸酶纯化方法 (High specific activity alkaline phosphatase engineering bacteria, engineering bacteria construction and alkaline phosphatase purification method ) 是由 高玉舟 李海超 何欣 乔善鹏 刘珍妮 于 2020-06-11 设计创作，主要内容包括：本发明公开了一种高比活力碱性磷酸酶工程菌、工程菌构建及碱性磷酸酶纯化方法,属于生物工程技术领域。本发明通过对Cobetia marina的碱性磷酸酶基因进行密码子优化,利用优化后的碱性磷酸酶基因构建工程菌,该工程菌的保藏编号为CGMCC NO.18926。用本发明所构建的基因工程菌表达,其发酵量可达10mg(1L发酵液)。本发明还对蛋白的纯化方法进行了创新,用本发明中的提纯方法,可以得到最终纯度大于95％的酶蛋白,比活力可达12000U/mg,相对现有国际上常见的碱性磷酸酶,是一种活力极高的碱性磷酸酶,为进一步实现工业化铺垫,也为工业大规模生产碱性磷酸酶提供了有效地方法。(The invention discloses an alkaline phosphatase engineering bacterium with high specific activity, an engineering bacterium construction method and an alkaline phosphatase purification method, and belongs to the technical field of biological engineering. The invention optimizes the codon of the alkaline phosphatase gene of Cobeta marina, and constructs engineering bacteria by using the optimized alkaline phosphatase gene, wherein the preservation number of the engineering bacteria is CGMCC NO. 18926. The gene engineering bacteria constructed by the invention can be used for expression, and the fermentation amount can reach 10mg (1L fermentation liquor). The invention also innovates a protein purification method, and by using the purification method, the enzyme protein with the final purity of more than 95 percent can be obtained, the specific activity can reach 12000U/mg, and compared with the existing internationally common alkaline phosphatase, the alkaline phosphatase with extremely high activity is an alkaline phosphatase, so that the method provides an effective method for further realizing industrialized bedding and industrial large-scale production of the alkaline phosphatase.)

1. A gene encoding CmAP, wherein the nucleotide sequence is as shown in SEQ ID No. 1.

2. The use of the gene of claim 1 in the construction of alkaline phosphatase engineering bacteria.

3. An alkaline phosphatase engineering bacterium Escherichia coli BL21(DE3)/pET16b-CmAP, the biological preservation number is: CGMCC NO. 18926.

4. The method of claim 3, comprising the steps of:

(1) using an alkaline phosphatase gene CmAP of Betebrata Hayata as a template, and performing codon optimization suitable for an escherichia coli expression system on the CmAP gene, wherein the nucleotide sequence of the optimized CmAP gene is shown as SEQ ID NO. 1;

(2) connecting the CmAP gene optimized in the step (1) with a pET16b expression vector after enzyme digestion to obtain a recombinant expression vector pET16 b-CmAP;

(3) transforming the recombinant expression vector pET16b-CmAP into an Escherichia coli expression host Escherichia coli BL21(DE3) to construct and obtain alkaline phosphatase engineering bacteria Escherichia coli BL21(DE3)/pET16 b-CmAP;

preferably, the recombinant expression vector pET16b-CmAP is obtained by inserting the alkaline phosphatase gene CmAP between Nde I and BamH I sites of the expression vector pET16 b.

5. Use of the alkaline phosphatase of claim 3 for producing alkaline phosphatase with high specific activity.

6. The use according to claim 5, wherein the amino acid sequence of the produced high specific activity alkaline phosphatase is as shown in SEQ ID No. 2.

7. A method for expressing and purifying high specific activity alkaline phosphatase is characterized by comprising the following steps:

inoculating the seed liquid of the alkaline phosphatase engineering bacteria of claim 3 into a fermentation culture medium containing ampicillin, wherein the inoculation amount is 2-5 per mill, and performing fermentation culture to obtain engineering bacteria fermentation liquid;

and centrifuging the engineering bacteria fermentation liquor, collecting thalli, carrying out cell ultrasonic disruption, and carrying out 40% and 70% ammonium sulfate fractional separation, Phenyl HP hydrophobic chromatography and High Q anion exchange chromatography for 2 times to obtain the purified High-specific activity alkaline phosphatase CmAP.

8. The expression purification method according to claim 7, wherein the composition of the fermentation medium containing ampicillin is: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of NaCl and 100 mu g/mL of ampicillin; the pH value of the culture medium is 7.0-7.2;

preferably, the conditions of the fermentation culture are as follows: shaking and culturing at 37 deg.C and 180r/min to OD₆₀₀When the concentration is 0.6-0.8, 0.5mM IPTG is added to induce, and the temperature is adjusted to 16 ℃ at the same time, and the fermentation is induced for 18-24 h.

9. The expression purification method according to claim 7, wherein the cell ultrasonication is specifically: dissolving the thallus in a buffer solution A, and placing the thallus on ice for ultrasonic crushing until the thallus is transparent from a viscous state;

preferably, the 40% and 70% ammonium sulfate fractionation is specifically: centrifuging the bacterial solution after cell ultrasonication, collecting supernatant protein solution, slowly adding ground ammonium sulfate powder into the supernatant protein solution at 4 ℃ and continuously stirring, standing at 4 ℃ for 2h, centrifuging at 12000r/min for 15min, collecting supernatant, continuously slowly adding the ground ammonium sulfate powder into the supernatant protein solution at 4 ℃ and continuously stirring, standing at 4 ℃ for 2h, centrifuging at 12000r/min for 15min, collecting precipitate, and redissolving the precipitate with 15-20mL of buffer solution A;

preferably, the Phenyl HP hydrophobic chromatography is specifically as follows: centrifuging the protein reconstituted in the step of fractionation by 40% and 70% ammonium sulfate at 12000r/min for 15min, collecting supernatant, performing Phenyl HP hydrophobic chromatography, performing linear elution with buffer solution A containing ammonium sulfate at concentration of 1-0M, detecting with reaction solution, collecting and combining target protein, dialyzing the collected protein solution with buffer solution B overnight, and desalting;

preferably, the 2 times of High Q anion exchange chromatography specifically comprises: carrying out High Q anion exchange chromatography on the protein solution dialyzed in the Phenyl HP hydrophobic chromatography step, linearly eluting by using a buffer solution B containing 0-0.5M NaCl, detecting by using a reaction solution, collecting and combining target proteins, and dialyzing the collected protein solution by using the buffer solution B overnight for desalting; and (3) performing High Q anion exchange chromatography on the dialyzed protein solution again, linearly eluting with a buffer solution B containing 0.1-1M NaCl, detecting with the reaction solution, collecting and combining the target protein, and dialyzing with the buffer solution B to remove salt.

10. The method for purifying expression according to claim 9, wherein the buffer a is Tris-HCl at pH 10.0; the buffer B is Tris-HCl with the pH value of 8.0;

the reaction solution is prepared by the following method:

taking 10.5g of diethanolamine, adding 50mL of deionized water for mixing, adjusting the pH to 10.3, fixing the volume of the solution to 100mL, and then adding 37.1mg of PNPP.6HH₂O。

Technical Field

The invention relates to the technical field of bioengineering, in particular to high specific activity alkaline phosphatase engineering bacteria, engineering bacteria construction and an alkaline phosphatase purification method.

Background

Alkaline phosphatase (Apase; EC 3.1.3.1) catalyzes the hydrolysis of phosphate compounds under alkaline conditions, and is widely used for immunolabeling and chemiluminescence. In addition, alkaline phosphatase has also found wide application in DNA and protein immunoassays. The specific activity of the enzyme directly affects the sensitivity of immunoassay and chemiluminescence, so that it is important to obtain a large amount of alkaline phosphatase with high specific activity.

Alkaline phosphatase is contained in most organism cells in nature from microbial cells to human cells. The enterobacter coli alkaline phosphatase is the best studied alkaline phosphatase at present, the enzyme is in a homodimer structure, each subunit consists of 449 amino acids, and the specific activity of the commercial escherichia coli alkaline phosphatase is 40-100U/mg, which is relatively low. The commercialized alkaline phosphatase with the highest specific activity is derived from the small intestine of a cow and can reach about 7000U/mg at most.

The alkaline phosphatase derived from deep-sea bacteria Cobeta marina is named as CmAP, the specific activity of the alkaline phosphatase can reach 15000U/mg and is far greater than that of calf intestinal alkaline phosphatase. See the references for details: a Highly Active alkalinecellphone from the Marine Bacterium Bacillus Cobeta, E.Yu Plusova et al, MarineBiotechology, Volume 7, 173-. However, Cobeta marina is industrially difficult to grow on a large scale, and the alkaline phosphatase production of this strain is low. Although the engineering bacteria constructed by the genetic engineering method can express alkaline phosphatase exogenously, the N end of CmAP is provided with a signal peptide consisting of 32 amino acids, the signal peptide plays an essential role in folding protein into a correct conformation, and after the protein is folded into an active conformation, the signal peptide is automatically cut off, so that the enzyme is not suitable for adding an affinity tag at the N end; however, the C-terminal affinity tag of the peptide chain seriously affects the expression and activity of the enzyme, which makes the purification of the exogenously expressed CmAP difficult.

Therefore, how to increase the expression level of CmAP and how to obtain high-purity CmAP is a problem that needs to be solved.

Disclosure of Invention

In view of the prior art, the invention aims to provide an alkaline phosphatase engineering bacterium with high specific activity and a method for expressing and purifying alkaline phosphatase CmAP by using the engineering bacterium. The fermentation amount of the alkaline phosphatase of the engineering bacteria constructed by the invention is greatly improved compared with that of a wild strain Cobeta marina, and the engineering bacteria are more suitable for the industrial production requirement; the invention also optimizes the protein purification process to finally obtain the protein with the purity not inferior to that of the protein purified by the affinity tag.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, there is provided a gene encoding CmAP, which has a nucleotide sequence as shown in seq id No. 1.

The invention takes the alkaline phosphatase gene derived from the Befibrate Cobeta marina as a template to carry out codon optimization aiming at an enterobacter coli expression system, so that the optimized gene for coding the alkaline phosphatase CmAP is more suitable for an escherichia coli expression system.

In a second aspect of the invention, the application of the gene in constructing alkaline phosphatase engineering bacteria is provided.

The third aspect of the invention provides an alkaline phosphatase engineering bacteria Escherichia coli BL21(DE3)/pET16b-CmAP, the classification name of the alkaline phosphatase engineering bacteria is Escherichia coli, the designated name of the alkaline phosphatase engineering bacteria is (E.coli) CmAP, the alkaline phosphatase engineering bacteria has been preserved in China general microbiological culture Collection center (CGMCC for short, the address is: China institute of sciences institute of microbiology No. 3, North Cheng Xilu 1, Kyoho, Beijing city) on 11-and-08 days 2019, and the biological preservation number is: CGMCC NO. 18926.

The alkaline phosphatase engineering bacteria can efficiently express the alkaline phosphatase CmAP with high specific activity, the specific activity of the enzyme can reach 12000U/mg, and a salt solution with a certain concentration also has an activating effect on the specific activity of the alkaline phosphatase; and the activity of the enzyme is stable, and the activity of the enzyme is not changed after being preserved by 50% (v/v) glycerol for one year at the temperature of-20 ℃.

The fourth aspect of the present invention provides a method for constructing the above alkaline phosphatase engineering bacteria, comprising the steps of:

(1) using an alkaline phosphatase gene CmAP of Betebrata Hayata as a template, and carrying out codon optimization suitable for an escherichia coli expression system on the CmAP gene, wherein the nucleotide sequence of the optimized CmAP gene is shown as SEQ ID NO. 1;

(2) connecting the CmAP gene optimized in the step (1) with a pET16b expression vector after enzyme digestion to obtain a recombinant expression vector pET16 b-CmAP;

(3) the recombinant expression vector pET16b-CmAP is transformed into an Escherichia coli expression host Escherichia coli BL21(DE3) to construct alkaline phosphatase engineering bacteria Escherichia coli BL21(DE3)/pET16 b-CmAP.

Preferably, the recombinant expression vector pET16b-CmAP is obtained by inserting the alkaline phosphatase gene CmAP between Nde I and BamH I sites of the expression vector pET16 b.

In a fifth aspect of the present invention, there is provided the use of the above alkaline phosphatase engineering bacteria for producing alkaline phosphatase with high specific activity.

In the application, the amino acid sequence of the produced high specific activity alkaline phosphatase is shown as SEQ ID NO. 2.

The sixth aspect of the invention provides a method for expressing and purifying high specific activity alkaline phosphatase, which comprises the following steps:

inoculating the seed liquid of the alkaline phosphatase engineering bacteria into a fermentation culture medium containing ampicillin, wherein the inoculation amount is 2-5 per mill (v/v), and performing fermentation culture to obtain engineering bacteria fermentation liquid;

and centrifuging the engineering bacteria fermentation liquor, collecting thalli, carrying out cell ultrasonic disruption, and then carrying out 40% and 70% ammonium sulfate fractionation, Phenyl HP hydrophobic chromatography and High Q anion exchange chromatography for 2 times to obtain the purified High specific activity alkaline phosphatase CmAP.

Preferably, the seed solution of the alkaline phosphatase engineering bacteria is prepared by the following method:

inoculating alkaline phosphatase engineering bacteria into a seed culture medium, wherein the inoculation amount is 2-5 permillage (v/v), and performing shaking culture at 37 ℃ and 180r/min until OD is reached₆₀₀The concentration is approximately equal to 6.0, and seed liquid is obtained;

the solvent of the seed culture medium is water, and the solute and the final concentration thereof in the seed culture medium are respectively as follows: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of NaCl; the pH value of the culture medium is 7.0-7.2.

Preferably, the composition of the fermentation medium containing ampicillin is: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of NaCl and 100 mu g/mL of ampicillin; the pH value of the culture medium is 7.0-7.2.

Preferably, the conditions of the fermentation culture are as follows: shaking and culturing at 37 deg.C and 180r/min to OD₆₀₀When the concentration is 0.6-0.8, 0.5mM IPTG is added to induce, and the temperature is adjusted to 16 ℃ at the same time, and the fermentation is induced for 18-24 h.

Preferably, the cell ultrasonication is specifically: and dissolving the thallus in the buffer solution A, and placing the thallus on ice for ultrasonic disruption until the thallus is transparent from a viscous state.

Preferably, the 40% and 70% ammonium sulfate fractionation is specifically: centrifuging the bacterial solution after cell ultrasonication, collecting supernatant protein solution, slowly adding ground ammonium sulfate powder into the supernatant protein solution at 4 ℃ and continuously stirring, standing at 4 ℃ for 2h, centrifuging at 12000r/min for 15min, collecting supernatant, continuously slowly adding the ground ammonium sulfate powder into the supernatant protein solution at 4 ℃ and continuously stirring, standing at 4 ℃ for 2h, centrifuging at 12000r/min for 15min, collecting precipitate, and re-dissolving the precipitate with 15-20mL of buffer solution A.

Preferably, the Phenyl HP hydrophobic chromatography is specifically as follows: centrifuging the protein reconstituted in the 40% and 70% ammonium sulfate fractionation step at 12000r/min for 15min, collecting supernatant, performing Phenyl HP hydrophobic chromatography, performing linear elution with buffer solution A containing ammonium sulfate at concentration of 1-0M, detecting with reaction solution, collecting and combining target proteins, dialyzing the collected protein solution with buffer solution B overnight, and desalting.

Preferably, the 2 times of High Q anion exchange chromatography specifically comprises: carrying out High Q anion exchange chromatography on the protein solution dialyzed in the Phenyl HP hydrophobic chromatography step, linearly eluting by using a buffer solution B containing 0-0.5M NaCl, detecting by using a reaction solution, collecting and combining target proteins, and dialyzing the collected protein solution by using the buffer solution B overnight for desalting; and (3) performing High Q anion exchange chromatography on the dialyzed protein solution again, linearly eluting with a buffer solution B containing 0.1-1M NaCl, detecting with the reaction solution, collecting and combining the target protein, and dialyzing with the buffer solution B to remove salt.

The buffer solution A is Tris-HCl with the pH value of 10.0; the buffer B is Tris-HCl with the pH value of 8.0; the reaction solution is prepared by the following method: taking 10.5g of diethanolamine, adding 50mL of deionized water for mixing, adjusting the pH to 10.3, fixing the volume of the solution to 100mL, adding 37.1mg of PNPP.6HH₂And storing at 4 ℃ under O.

According to the invention, the alkaline phosphatase expression and purification conditions are optimized, so that the fermentation amount of the alkaline phosphatase is greatly improved compared with that of a wild strain Cobeta marina, and the method is more suitable for the industrial production requirement; particularly, the purification process of the protein is optimized, an affinity tag is not required in the purification process, the method is particularly suitable for purifying the alkaline phosphatase CmAP, and the protein with the purity not lower than that of the protein purified by the affinity tag can be obtained.

The invention has the beneficial effects that:

(1) the invention carries out codon optimization on the alkaline phosphatase gene CmAP derived from the Befibrate Cobeta marina of the Hitaceae, replaces the original codon of gene mRNA with the codon commonly used by Escherichia coli, and increases the Codon Adaptation Index (CAI) from 0.57 to 0.97 after codon optimization (CAI is favorable for expression between 0.8 and 1.0).

(2) Based on CmAP gene after codon optimization, the invention constructs the engineering bacteria of alkaline phosphatase, the fermentation amount of the alkaline phosphatase in the engineering bacteria constructed by the invention is greatly improved compared with that of a wild strain Cobeta marina, the yield of the alkaline phosphatase prepared by the invention can reach 10mg (1L fermentation liquor), the specific activity of the enzyme can reach 12000U/mg, and compared with the existing internationally common alkaline phosphatase, the alkaline phosphatase is the alkaline phosphatase with extremely high activity, thereby providing an effective method for further realizing industrialized bedding and industrial large-scale production of the alkaline phosphatase.

(3) The invention optimizes the purification process of the alkaline phosphatase CmAP, does not need to adopt an affinity tag in the purification process, and can obtain the protein with the purity not inferior to that of the protein purified by the affinity tag.

Drawings

FIG. 1 is an SDS-PAGE protein electrophoresis of CmAP after final purification, in which lane 1 is Marker and lanes 2-5 (High Q1-High Q4) are each tube samples collected for the second High Q chromatography.

FIG. 2 is a pH optimum curve for the CmAP enzyme of the invention.

FIG. 3 is a thermal stability curve for the CmAP enzyme of the invention.

FIG. 4 is a KCl and NaCl effect curve for the CmAP enzyme of the invention.

FIG. 5 is a graph of the storage stability of the CmAP enzyme of the invention.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As described in the background section, the specific activity of the alkaline phosphatase CmAP derived from the deep-sea bacterium Cobeta marina can reach 15000U/mg. However, since Cobeta marina is industrially difficult to be cultured on a large scale and the alkaline phosphatase yield of the strain is low, it is difficult to produce the alkaline phosphatase CmAP on a large scale by directly fermenting and culturing Cobeta marina.

The engineering bacteria are constructed by means of genetic engineering, and the exogenous expression of the alkaline phosphatase CmAP can be realized theoretically. On one hand, however, because of the difference between the expression systems of the deep sea bacteria Cobeta marina and the existing genetic engineering host bacteria, the gene CmAP of the Cobeta marina coding alkaline phosphatase can not be stably expressed in the existing genetic engineering host bacteria; on the other hand, the CmAP has a signal peptide consisting of 32 amino acids at the N-terminus, which plays an essential role in the correct protein folding into conformation, and which is automatically cleaved after the protein is folded into active conformation, so that the enzyme is not suitable for N-terminal affinity-tagging; furthermore, it was verified that the C-terminal affinity tag of CmAP severely affected the expression and activity of the enzyme. Therefore, due to the specificity of the CmAP structure, exogenously expressed CmAP is difficult to purify by means of affinity tags.

In summary, there are two major technical difficulties in the large-scale production and application of CmAP for alkaline phosphatase: firstly, how to improve the CmAP expression quantity and secondly how to obtain the CmAP with high purity.

Based on the above, the invention aims to provide an alkaline phosphatase engineering bacterium with high specific activity, a construction method of a genetic engineering strain and a purification method of recombinant alkaline phosphatase.

The method comprises the steps of firstly, using an alkaline phosphatase gene CmAP derived from Betebrata marina of the family Craiferae as a template, carrying out codon optimization aiming at an escherichia coli expression system, replacing an original codon of gene mRNA with a common codon of escherichia coli, and increasing CAI (codon adaptation index) from 0.57 to 0.97(CAI is favorable for expression between 0.8 and 1.0) after adjusting the codon to be an escherichia coli preference codon; repetitive regions in the original sequence were removed to avoid stem-loop structures in the mRNA. The nucleotide sequence of the gene CmAP after codon optimization is shown as SEQ ID NO. 1.

On the basis of obtaining the codon optimized gene CmAP, the optimized alkaline phosphatase gene CmAP is connected to an expression vector pET16b to obtain a recombinant expression vector, and the recombinant expression vector is transformed into Escherichia coli E.coli BL21(DE3) to obtain recombinant expression alkaline phosphatase engineering bacteria. The fermentation amount of the alkaline phosphatase in the engineering bacteria is greatly improved compared with that of a wild strain Cobeta marina, the engineering bacteria are more suitable for industrial production requirements, the alkaline phosphatase CmAP (amino acid sequence is shown as SEQ ID NO. 2) prepared by fermenting the engineering bacteria has many characteristics, the specific activity of the enzyme can reach 12000U/mg, the optimum reaction pH of the enzyme is 10.3, the optimum reaction temperature is 45 ℃, and the activity of the enzyme is kept unchanged for one year by using 50% (v/v) glycerol at the temperature of minus 20 ℃. The activity of the product is reduced by 20 percent when the product is stored at the temperature of 4 ℃ for one year; a saline solution of a certain concentration may act as an activator of the enzyme. Therefore, the inventors have carried out biological preservation on the constructed genetically engineered bacteria.

The engineering bacteria expression product constructed by the invention is cell soluble protein, the invention also innovates the purification process of the protein, and the pure enzyme protein, namely CmAP, is obtained by separation and purification through cell ultrasonic disruption, ammonium sulfate fractional separation, hydrophobic chromatography and 2 times of ion exchange chromatography. The purification process does not need to adopt an affinity tag, and the protein with the purity not lower than that of the protein purified by the affinity tag can be obtained.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments. If the experimental conditions not specified in the examples are specified, the conditions are generally conventional or recommended by the reagent company; reagents, consumables, and the like used in the following examples are commercially available unless otherwise specified.

The protein content was determined in the following examples using a Nano Drop micro spectrophotometer to measure protein concentration.