CN112920280A

CN112920280A - Method for efficiently expressing acid protease and application thereof

Info

Publication number: CN112920280A
Application number: CN202110436308.0A
Authority: CN
Inventors: 吴丹; 郑璞; 陈鹏程; 魏梦园
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-04-22
Filing date: 2021-04-22
Publication date: 2021-06-08
Anticipated expiration: 2041-04-22
Also published as: CN112920280B

Abstract

The invention discloses a method for efficiently expressing acid protease and application thereof, belonging to the field of genetic engineering. The recombinant pichia pastoris GS115/pPIC 9K-en-alpha SP-acid protein has the expression enzyme activity reaching 5395U/mL, the enzyme activity is improved by 109% compared with the enzyme production enzyme activity of a control strain containing original alpha-signal peptide, the acid protein can maintain more than 60% of the enzyme activity within the pH range of 1-5, the residual enzyme activity after 30min treatment at 20-40 ℃ is more than 80%, and the recombinant pichia pastoris P.pastoris GS115/pPIC 9K-en-alpha SP-acid protein can be applied to the industries of food, feed or brewing and the like. According to the technical scheme of the invention, the acidic protease suitable for industrial application can be produced by utilizing a genetic engineering means.

Description

Method for efficiently expressing acid protease and application thereof

Technical Field

The invention relates to a method for efficiently expressing acid protease and application thereof, belonging to the field of genetic engineering.

Background

Proteases are a class of enzymes that catalyze proteolysis, which is widely found in plants, animals, and microorganisms. Compared with protease derived from animals and plants, the protease derived from microorganisms has the characteristics of convenient culture, simple operation and high enzyme yield, and is convenient for industrial mass production and large-scale production and application. Therefore, proteases of microbial origin are an important source of current proteases.

Proteases are classified in many ways, and are classified into acid proteases, alkaline proteases and neutral proteases according to the pH at which the protease acts. The acidic protease is generally stable at a pH of 2.0-6.0, and the optimum pH value is slightly different depending on the species, but is generally about pH 3.0, for example, the acidic protease produced by fungi has an optimum pH of 3.0, Penicillium griseovirens has a pH of 3.5, and the yeast has a pH of 3.0. The enzyme has high sequence similarity, and the three-dimensional structure of the enzyme is in a symmetrical double-leaf shape. Proteases, depending on the active center, are classified into: serine proteases, aspartic proteases, cysteine proteases, and metallo proteases.

The acidic protease is widely applied to the industries of food, brewing, fur and leather, medicine, feed and the like. During the fermentation process of brewing the white spirit, acidic protease is utilized to play a synergistic role, particles of fermentation raw materials are dissolved, the utilization rate of the raw materials is improved, the growth of microorganisms is promoted, proteins are decomposed to provide aroma precursor substances and flavor substances, and yeast mycoprotein is decomposed. In the manufacturing process of fur and leather, acid protease removes the fiber matrix, so that the leather is softer and plump. In the feed industry, the addition of the acidic protease can improve the digestibility of protein, so that high-molecular protein is degraded into low-molecular peptide and amino acid, the digestion and absorption of livestock and poultry are facilitated, the stimulation of the feed to the digestive tract of young animals can be reduced, the nutritional disorder is reduced, the utilization rate of the feed is improved, and the growth of the livestock and poultry is promoted.

In the prior art, wild acid protease has poor performance and low yield and can not meet the market demand far away, so that the wild acid protease is difficult to be really applied to industrial production. At present, the means of constructing genetically engineered bacteria is often adopted to enhance the transcription and translation of acid protease genes so as to improve the yield of acid protease.

Most of the acid protease currently used for industrial production is mould acid protease, the optimum action pH growth value of the enzyme is about 3.0, when the pH value is increased, the enzyme activity of the acid protease can be obviously reduced, and the enzyme is thermolabile and is unstable when the temperature reaches above 50 ℃. Therefore, the enzyme activity of the acid protease is not high, and the difference between the optimal action condition of the enzyme and the environment condition catalyzed by the enzyme in the aspects of pH, temperature and the like causes the reduction of the catalytic efficiency of the enzyme, thereby limiting the application of the acid protease in industry.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for efficiently expressing acid protease.

The first purpose of the invention is to provide a fusion protein, wherein the fusion protein is an acid protease with an enhanced alpha-signal peptide connected to the N end; the nucleotide sequence of the acid protease is shown as SEQ ID NO. 2; the nucleotide sequence of the enhanced alpha-signal peptide is shown in SEQ ID NO. 4.

In one embodiment, the amino acid sequence of the acidic protease is shown as SEQ ID NO. 1; the amino acid sequence of the enhanced alpha-signal peptide is shown in SEQ ID NO. 3.

The second purpose of the invention is to provide a vector for expressing the fusion protein.

It is a third object of the present invention to provide a recombinant strain containing the fusion protein or vector.

In one embodiment, the recombinant strain is host pichia pastoris.

In one embodiment, the recombinant strain is vectored with pPIC 9K.

In one embodiment, the host pichia is GS115, KM71, and X33.

The fourth purpose of the invention is to provide a method for constructing the recombinant strain, which comprises the following specific steps: (1) connecting and inserting the acid protease and the enhanced alpha-signal peptide into an expression vector pUC57 to obtain a recombinant vector pUC57-en alpha-SP-acidiprotea; (2) using a recombinant vector pUC57-en alpha-SP-acidiprotea as a template, carrying out PCR amplification on a target fragment containing acid protease and enhanced alpha-signal peptide, and inserting the target fragment between BamHI HI and Not I of a restriction enzyme site of an expression vector pPIC9K to obtain a recombinant vector pPIC 9K-en-alpha SP-acidiprotea; (3) the plasmid was linearized with Sac I and electrotransferred to Pichia pastoris to obtain the recombinant strain P.pastoris GS115/pPIC9K-en- α SP-acidprotea.

The fifth purpose of the invention is to provide a method for improving the expression level of acid protease, which comprises the following steps: connecting the gene sequence of the enhanced alpha-signal peptide to the N end of the acid protease gene sequence; the nucleotide sequence of the enhanced alpha-signal peptide is shown as SEQ ID NO. 4; the acid protease comprises acid protease of animal or fungus origin.

In one embodiment, the acid protease has the sequence shown in SEQ ID NO. 2.

The invention also provides application of the fusion protein or the recombinant strain in casein decomposition.

The invention also provides application of the fusion protein or the recombinant strain in the fields of feed, brewing or food.

Advantageous effects

(1) The recombinant pichia pastoris GS115/pPIC 9K-en-alpha SP-acidoteta expression enzyme activity reaches 5395U/mL, and the enzyme activity is improved by 109% compared with that of a control strain containing the original alpha-signal peptide;

(2) the recombinant acid protease provided by the invention has the most suitable pH of 2.0, and can maintain the enzyme activity of more than 60% in the pH range of 1-5; the optimal temperature of the protease is 50 ℃, the residual enzyme activity is more than 80 percent after 30min of treatment at 20-40 ℃, and the protease has better stability. The invention realizes the production of protease with excellent property by using genetic engineering means, and applies the protease to industries such as feed, food, medicine and the like.

Drawings

FIG. 1 recombinant expression plasmid map;

FIG. 2 shows the optimum pH of the fungal-derived acid protease;

FIG. 3 shows the pH stability of fungal-derived acid protease;

FIG. 4 shows the optimum reaction temperature for fungal-derived acid protease;

FIG. 5 shows the thermostability of fungal-derived acid protease.

Detailed Description

(1) Culture medium:

seed medium YPD: tryptone 20g/L, yeast powder 10g/L, glucose 20 g/L.

Induction medium YP: tryptone 20g/L, yeast powder 10g/L, every 12h adding 1% (v/v) methanol.

(2) Detection of acid protease activity

And (3) carrying out activity analysis on the acidic protease by adopting a forskolin phenol reagent color development method. The specific method comprises the following steps: under the conditions of pH 3.0 and 55 ℃, 1mL of reaction system comprises 500 mu L of appropriately diluted enzyme solution and 500 mu L of substrate, the reaction is carried out for 10min, and 1mL of trichloroacetic acid (0.4mol/L) is added to stop the reaction; the reaction system was centrifuged at 12000rpm for 3min, 500. mu.L of the supernatant was aspirated, 2.5mL of sodium carbonate (0.4mol/L) was added, 500. mu.L of Folin's phenol reagent was added, and after development at 40 ℃ for 20min and cooling, the OD value was measured at 680 nm.

Protease activity unit definition: under certain conditions, the amount of enzyme required to decompose the substrate casein to 1. mu.g tyrosine per minute per ml reaction system was 1 activity unit (U).

Example 1 construction of recombinant Pichia pastoris

(1) Construction of recombinant plasmid pPIC 9K-en-alpha SP-acid

An enhanced alpha-signal peptide (en-alpha SP) with a nucleotide sequence shown as SEQ ID No.4 is connected to the N end of the acid protease with a nucleotide sequence shown as SEQ ID No.2, and is artificially synthesized, Bam HI and Not I enzyme cutting sites are respectively added at the head and the tail of the enhanced alpha-signal peptide, and the sequence connected with the enzyme cutting sites is integrated on a cloning vector pUC57 to form a plasmid pUC57-en alpha-SP-a-acid protein. The plasmid pUC57-en alpha-SP-acidiprotea is subjected to double enzyme digestion by Bam HI and Not I to obtain a 1428bp sequence containing an enhanced alpha-signal peptide and acid protease and is recovered, the vector pPIC9K is subjected to double enzyme digestion by Bam HI and Not I to obtain a 8998bp vector sequence and is recovered, the sequence containing the enhanced alpha-signal peptide and acid protease and the vector sequence are connected by DNA ligase to transform escherichia coli and coat a G418 resistant plate, after overnight culture, a single colony is picked up and used for amplifying thalli by an LB liquid culture medium, the plasmid is extracted and is sent to a company for sequencing verification, and the recombinant expression plasmid with correct sequencing is pPIC9K-en alpha SP-acidiprotea. The plasmid map is shown in FIG. 1.

(2) Construction of recombinant Pichia pastoris GS115/pPIC 9K-en-alpha SP-acidotea

Linearizing the recombinant plasmid pPIC 9K-en-alpha SP-acidprotea in the step (1) by using Sac I, electrically transforming a linearized product into pichia pastoris competence, and coating the pichia pastoris competence on a YPD solid culture medium containing G418 with a certain concentration for about 24-48 h at 28-30 ℃. And then respectively selecting full colonies with better morphology and transferring the colonies to a YPD liquid culture medium for re-screening. The method comprises the following specific steps:

1) pichia pastoris GS115 competence was prepared for transformation: sucking 30 mu L of P.pastoris GS115 wild strain into 10mL YPD liquid culture medium, and carrying out shake-flask culture at 30 ℃ and 200rpm for 24h to obtain activated P.pastoris GS 115; taking 100 mu L of activated P.pastoris GS115 to 100mL YPD culture medium, shaking and culturing at 30 ℃ and 200rpm until the bacterial concentration OD600 is 1.3-1.5, centrifuging all bacterial liquid at 4 ℃ and 5000rpm for 5min, discarding supernatant, adding 10mL Solution I to resuspend the thallus, and culturing in a water bath shaking table at 30 ℃ and 50rpm for 45 min; adding sterile water precooled by a refrigerator at 4 ℃ into the cultured thalli to a final volume of 30mL, resuspending the thalli, centrifuging at 5000rpm at 4 ℃ for 5min, and discarding the supernatant; resuspending the cells in 25mL of 4 ℃ pre-cooled sterile water, centrifuging at 4 ℃ and 5000rpm for 5min, removing the supernatant, and repeating the operation once; resuspending the thallus with 25mL of 1mol/L sorbitol precooled at 4 ℃, centrifuging at 5000rpm at 4 ℃ for 5min, removing supernatant, and repeating the operation once; the cells were resuspended in 1mL, 1mol/L sorbitol and aliquoted into EP tubes, 80. mu.L each, for further experiments.

2) The plasmid pPIC 9K-en-alpha SP-acididiotea extracted in the step (1) is linearized by endonuclease Sac I, the linearized plasmid (5-10ug) is added into 80uL of yeast competent cells obtained in the step 1), and the cells are transferred into ice-precooled 0.2cm electric rotating cups and placed on ice for 5-10 min. 2000V; performing electric shock once within 5ms, immediately adding 0.5-2mL of 1mol/L ice-precooled sorbitol, uniformly mixing, transferring the mixture into a sterile centrifuge tube, standing for 1-2 h at 30 ℃, adding 0.5-1.5 mL of YPD medium, and culturing for 1-2 h at 30 ℃ and 20-100 rpm to obtain a mixed solution; centrifuging the mixed solution at 3000-6000 rpm for 1-5 min, removing part of supernatant, remaining 100. mu.L of resuspended thallus, coating YPD resistant plates containing a certain concentration of G418, and culturing at 30 ℃ for 1-4 days to pick out the monoclonal. And then, respectively selecting full colonies with better morphology, transferring the colonies to a YPD solid culture medium, and carrying out secondary screening.

The construction method of the recombinant pichia pastoris GS115/pPIC9K-acidprotea containing the original alpha-signal peptide is the same as that of the recombinant pichia pastoris GS115/pPIC 9K-en-alpha SP-acidprotea, and only the enhanced alpha-signal peptide with the nucleotide sequence shown as SEQ ID NO.4 is replaced by the original alpha-signal peptide with the nucleotide sequence shown as SEQ ID NO. 5.

Example 2 production of acid protease by recombinant Pichia pastoris shake flask fermentation

From the plate containing recombinant Pichia pastoris GS115/pPIC9K-en- α SP-acidprotea obtained in example 1, 10 single colonies were picked and inoculated into 25mL of liquid medium YPD, cultured at 30 ℃ and 220rpm for 24 hours, at 6000rpm at room temperature, centrifuged for 5min, the cells were collected, the supernatant was discarded, the cells were resuspended in 30mL of liquid medium YP, induction expression was performed at 28 ℃ and 220rpm, methanol was added every 24 hours to a final concentration of 1% to continue induction, and the flask was subjected to flask shaking induction for 72 hours to obtain a fermentation broth.

The recombinant strain P.pastoris GS115/pPIC9K-acidprotea containing the original α -signal peptide obtained in example 1 was used as a control strain, and induction culture was performed simultaneously.

And finally, when the bottle is shaken, 2580U/mL of enzyme activity of the control strain using the original alpha-signal peptide is adopted, 5395U/mL of the average value of the enzyme activity of the strain using the enhanced alpha-signal peptide is adopted, and the expression level is improved by 109%.

EXAMPLE 3 enzymatic Properties of recombinant acid proteases

(1) Purification of acid proteases

Centrifuging the fermentation liquid obtained in example 2 at 4 deg.C and 8000r/min for 10min, collecting the supernatant to obtain crude enzyme solution, and adding 20mmol/L NaH₂PO₄-Na₂HPO₄(p H6.5.5) buffer solution, 10kDa ultrafiltration membrane package were used to dialyze the crude enzyme solution. Collecting the crude enzyme solution after dialysis, centrifuging at 12000r/min at 4 deg.C for 30min, and collecting supernatant.

The protein was purified using a HitrapTM Q FP (5mL) anion exchange chromatography column. The protein of the crude enzyme solution is separated and purified by using an AKTA protein purifier, and the column purification conditions are as follows: the anion column was equilibrated with 10 column volumes of buffer A (20mmol/L NaH2PO4-Na2HPO4 buffer, pH 6.5) and injected at a flow rate of 1 mL/min; after sample injection is finished, flushing the column by using buffer solution A with 5 column volumes; and linear elution was performed with 0-100% buffer B (20mmol/L NaH2PO4-Na2HPO4+1mol/L NaCl buffer, pH 6.5) at a flow rate of 1 mL/min. And collecting samples in the elution peak, determining a collecting pipe where the target sample is located through enzyme activity determination, and obtaining the electrophoretically pure target protein.

(2) Optimum pH and pH stability of acid protease

Optimum pH: recombinant acid proteases containing the enhanced alpha-signal peptide were enzymatically reacted at a temperature of 50 ℃ at various pH's to determine their optimum pH. The buffer solution is glycine-hydrochloric acid buffer solution with pH value of 1.0-3.0, citric acid-disodium hydrogen phosphate buffer solution with pH value of 3.0-80, and Tris-HC1 buffer solution with pH value of 8.0-10.0. The optimum pH of the enzyme was determined with the highest enzyme activity detected in this example being 100%, as shown in FIG. 2, the optimum pH of the acid protease was 2.0.

pH stability: the recombinant acid protease containing the enhanced alpha-signal peptide is placed in buffers with different pH values and treated for 60min at 50 ℃, the enzyme activity is measured, the untreated enzyme activity is 100 percent, and the pH stability of the enzyme is researched. As shown in FIG. 3, the acid protease can maintain more than 60% of enzyme activity between pH 1.0-5.0, which indicates that the enzyme has excellent pH stability.

(3) Optimum temperature and thermal stability of acid protease reaction

Optimum temperature: under the condition of pH 2.0, the enzyme activity of the recombinant acid protease containing the enhanced alpha-signal peptide is measured at 20-90 ℃, and the optimum temperature of the recombinant acid protease is determined by taking the highest enzyme activity detected in the implementation process as 100%. As shown in FIG. 4, the recombinant acid protease containing the enhanced alpha-signal peptide of the present invention has an optimum reaction temperature of 50 ℃ and an enzyme activity of 60% or more at 40 ℃.

Thermal stability: under the condition of pH 2.0, the recombinant acid protease containing the enhanced alpha-signal peptide is treated for 30min at the temperature of 20-90 ℃, the enzyme activity is measured, and the thermal stability of the enzyme is researched by taking the untreated enzyme activity as 100%. As shown in figure 5, the recombinant acid protease containing the enhanced alpha-signal peptide can still keep more than 90% of enzyme activity after being treated at 30 ℃ for 30min, and the residual enzyme activity is more than 80% after being treated at 40 ℃ for 30 min; after the enzyme is treated at 50 ℃ for 30min, the residual enzyme activity is more than 40 percent, even though the enzyme has better stability at 20-40 ℃.

EXAMPLE 4 use of recombinant acid proteases

Freeze-drying the acid protease obtained in the step (1) of the embodiment 3 to prepare enzyme powder; is prepared from grape 800 parts, purified water 800 parts, white granulated sugar 200 parts, Saccharomyces cerevisiae 0.06 part, pectase 0.01 part, cellulase 0.01 part, acid protease 0.04 part, CaCl₂0.01 part of the fruit wine is prepared by fermentation.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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Claims

1. A fusion protein is characterized in that the fusion protein is an acid protease with an enhanced alpha-signal peptide connected to the N end; the nucleotide sequence of the acid protease is shown as SEQ ID NO. 2; the nucleotide sequence of the enhanced alpha-signal peptide is shown in SEQ ID NO. 4.

2. A vector expressing the fusion protein of claim 1.

3. A recombinant strain comprising the fusion protein of claim 1 or the vector of claim 2.

4. The recombinant strain of claim 3, wherein the recombinant strain is host Pichia pastoris and vector pPIC 9K.

5. The recombinant strain of claim 4, wherein the Pichia pastoris comprises Pichia pastoris GS115, KM71, and X33.

6. A method for constructing the recombinant strain of claim 3, which comprises the following specific steps: (1) connecting and inserting the acid protease and the enhanced alpha-signal peptide into an expression vector pUC57 to obtain a recombinant vector pUC57-en alpha-SP-acidiprotea; (2) using the recombinant vector pUC57-en alpha-SP-acidiprotea in the step (1) as a template, amplifying a target fragment containing acid protease and enhanced alpha-signal peptide, and inserting the target fragment into an expression vector pPIC9K to obtain a recombinant vector pPIC 9K-en-alpha SP-acidiprotea; (3) and (3) linearizing the recombinant vector in the step (2) and transforming the recombinant vector into pichia pastoris to obtain a recombinant strain.

7. A method for increasing the expression level of acid protease, which comprises: connecting the gene sequence of the enhanced alpha-signal peptide to the N end of the acid protease gene sequence; the nucleotide sequence of the enhanced alpha-signal peptide is shown as SEQ ID NO. 4; the acid protease comprises acid protease of animal or fungus origin.

8. The method of claim 7, wherein the acid protease has the sequence shown in SEQ ID No. 2.

9. Use of the fusion protein of claim 1 or the recombinant strain of claim 3 for casein degradation.

10. Use of the fusion protein according to claim 1 or the recombinant strain according to claim 3 in the field of feed, brewing or food.