CN117230046A - Alkali-resistant streptomyces fradiae trypsin mutant - Google Patents

Abstract

The invention discloses an alkali-resistant streptomyces fradiae trypsin mutant, and belongs to the fields of genetic engineering and enzyme engineering. The invention removes trypsin leader peptide sequence APAP from Streptomyces fradiae (Streptomyces fradiae), fuses N end with thioredoxin TrxA, partial bovine trypsin leader peptide sequence DDDDK and hydrophobic propeptide FVEF, and obtains trypsin recombinant strain aSFT with artificial self-activated leader peptide sequence. The recombinant enzyme shows better alkaline environment stability and catalytic activity, and is more beneficial to industrial application compared with wild enzyme.

Description

Alkali-resistant streptomyces fradiae trypsin mutant

Technical Field

The invention relates to an alkali-resistant streptomyces fradiae trypsin mutant, belonging to the technical fields of genetic engineering and enzyme engineering.

Background

Trypsin (EC 3.4.21.4) is a serine proteolytic enzyme of the PA family superfamily, extracted from mammalian pancreatic tissue. Trypsin is highly selective and specifically catalyzes the hydrolysis of peptide or amide bonds at the carboxy-terminus of arginine or lysine residues in the peptide chain of proteins. Therefore, the method has been widely applied to various industrial fields such as leather softening, food processing, pharmacy, detergents, clinical diagnosis and biochemical detection.

At present, commercial production of trypsin is mainly dependent on extraction from bovine pancreatic tissues, but the method is limited by raw materials, and has the disadvantages of high separation and purification cost and harm to mammal immunogenicity. The heterologous expressed trypsin can stably produce a large amount of enzymes in a short time, and the expression quantity and the catalytic property can be improved by means of transformation and fermentation optimization.

The common host bacteria of the prokaryotic expression system is escherichia coli, and has the advantages of short period, low cost, diversified expression vector selection and the like, but also has the defects of high inclusion body denaturation and protein renaturation difficulty, difficult purification, difficult mass and continuous expression of target proteins and the like. Animal derived trypsin is more suitable for preparation and production using eukaryotic expression systems, while pichia pastoris is the most commonly used and perfected host for eukaryotic expression systems today.

With the development of recombinant DNA technology, human, bovine, porcine and shrimp trypsinogens are expressed in pichia pastoris in soluble form. Streptomyces trypsin has a similar structure, amino acid sequence and substrate specificity to bovine trypsin and is therefore considered to be an important alternative enzyme to potential bovine trypsin. Compared with six disulfide bonds of bovine trypsin, SGT contains only three disulfide bonds, so that SGT is more flexible, so that heterologous expression of SGT is easier than bovine trypsin, but the expression level of trypsin is low, the bovine trypsin cannot be self-activated, activation of SGT depends on enterokinase or trypsin digestion, and trypsin zymogen has a certain harm to host bacteria.

Therefore, the self-activatable alkali-resistant streptomyces fradiae (Streptomyces fradiae) trypsin is obtained, and has important significance for industrial application of the streptomyces fradiae enzyme.

Disclosure of Invention

The invention finds and modifies a key area affecting self-activation in the leader peptide through structural analysis of trypsin derived from streptomyces freundii ATCC 14544. The modified mutant is successfully expressed, so that the modified mutant can obtain active trypsin without enterokinase activation.

The invention firstly provides a trypsin mutant aSFT, which has an amino acid sequence shown as SEQ ID NO. 1.

In one embodiment, the amino acid sequence shown in SEQ ID NO.1 contains an artificial self-activating leader peptide and mature trypsin SFT, the amino acid sequence of mature trypsin is shown in SEQ ID NO.3, and the gene sequence encoding the mature trypsin is shown in SEQ ID NO. 5; the amino acid sequence of the artificial self-activation leader peptide is shown as SEQ ID NO.4, the gene sequence of the coding artificial self-activation leader peptide is shown as SEQ ID NO.6, and the coding artificial self-activation leader peptide comprises thioredoxin TrxA, enterokinase activation site DDDDK and hydrophobic propeptide FVEF, and is positioned at the N end of mature trypsin.

The invention also provides a gene for encoding the trypsin mutant, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

The invention also provides an expression vector containing the gene.

In one embodiment, the expression vector comprises a pPIC9K plasmid, a pPIC9K-His plasmid, a pPIC9 plasmid, a ppiczαa plasmid, a pAO815 plasmid, or a pWB980 plasmid.

The invention also provides a recombinant microbial cell expressing the trypsin mutant.

In one embodiment, the host of the recombinant microbial cell includes, but is not limited to, E.coli, B.subtilis, and Pichia pastoris.

The invention also provides a recombinant pichia pastoris, which takes the pichia pastoris as a host, takes the pPIC9K plasmid as an expression vector, and expresses a gene of the coded trypsin mutant shown in SEQ ID NO. 2.

In one embodiment, the pichia comprises p.pastoris GS115, p.pastoris KM71, p.pastoris X-33, or p.pastoris SMD1168.

The invention also provides a method for improving the expression quantity of trypsin in yeast cells, which uses streptomyces fradiae trypsin as parent enzyme and replaces leader peptide APAP of the parent enzyme with artificial leader peptide; the artificial leader peptide includes TrxA, DDDDK, and fvaf.

In one embodiment, the streptomyces freundii trypsin contains 345 amino acids; wherein, amino acid 1-109 is fusion tag thioredoxin TrxA, amino acid 110-114 is bovine trypsin leader peptide DDDDK, amino acid 115-118 is hydrophobic propeptide FVEF, amino acid 119-339 is mature peptide of trypsin, and amino acid 340-345 is histidine tag.

In one embodiment, the artificial self-activating leader peptide with the amino acid sequence shown as SEQ ID NO.4 and mature trypsin with the amino acid sequence shown as SEQ ID NO.3 are fused and expressed; the artificial leader peptide sequence is fused to the N-terminus of trypsin.

In one embodiment, the thioredoxin is cleaved after secretion of the thioredoxin with trypsin outside the cell; the hydrophobic propeptide fvaf is cleaved off by endogenous proteins; the trypsin after cleavage is mature trypsin.

The invention also provides application of the artificial self-activation leader peptide in improving the expression quantity of trypsin, and the amino acid sequence of the artificial self-activation leader peptide is shown as SEQ ID NO. 4.

The invention also provides a method for synthesizing trypsin, which comprises the steps of inoculating the recombinant microbial cells or the recombinant pichia pastoris into a culture medium to be cultured to a logarithmic phase, and adding methanol or sorbitol to induce expression.

The invention also provides application of the trypsin mutant, gene, expression vector or recombinant microbial cell in preparing trypsin and trypsin-containing products.

The invention also provides application of the trypsin mutant, the gene, the expression vector or the recombinant microorganism cell in leather softening.

The beneficial effects are that:

the invention fuses the artificial self-activated leader peptide sequence at the N end of trypsin, and the mature trypsin is obtained by secretory expression of the self-catalyzed leader peptide sequence after trypsinogen in a zymogen form. The molecular weight of Streptomyces fradiae trypsin aSFT after modification of the N-terminal leader peptide obtained by the invention is about 27kDa, the expression quantity is improved by 9.6% compared with that before modification, the amidase activity is improved by 2.46 times, and the specific enzyme activity is improved by 67.89%.

The pancreatic protein aSFT enzyme has an optimal reaction temperature of 45deg.C, an optimal reaction pH of 9.0, and a pH of 8-11 at 25deg.C>80% of enzyme activity. From the kinetic parameters of the enzyme-catalyzed substrate BAPNA, the k of trypsin aSFT is seen _m The value was 0.0715mM, and its Vmax and k _cat /k _m The values respectively reach 12.93mM min ^-1 And 5.38X10 ⁵ min ^-1 ·mM ^-1 . The results show that the aSFT has better affinity with the substrate and has good application prospect in leather production.

Drawings

FIG. 1 SDS-PAGE electrophoresis of recombinant strain expressed trypsin; m: protein markers; s: trypsin supernatant concentrate before modification, aS: modified trypsin supernatant concentrate;

FIG. 2 shows the enzyme production curves of 0-120h before and after the reconstruction of the recombinant strain; SFT: trypsin before transformation; aSFT: trypsin after transformation;

FIG. 3 amidase activity of recombinase and comparison of specific enzyme activity; SFT: trypsin before transformation; aSFT: trypsin after transformation;

FIG. 4 is a graph comparing the optimum temperatures of recombinases; SFT: trypsin before transformation; aSFT: trypsin after transformation;

FIG. 5 is a graph comparing optimum pH of recombinant enzyme; SFT: trypsin before transformation; aSFT: trypsin after modification.

Detailed Description

The media and solutions involved in the following implementations are as follows:

LB medium: 1% tryptone, 0.5% yeast extract, 1% NaCl; 1.5% agar powder is added when preparing the solid culture medium; sterilization conditions: 121 ℃ for 20min.

YNB medium: 2% of tryptone, 1% of yeast extract and 2% of glucose; 1.5% agar powder is added when preparing the solid culture medium; sterilization conditions: 115 ℃ for 15min.

MD plates: 2% glucose, 1.5% agar powder; sterilizing at 115deg.C for 15min, taking out, cooling to 60-70deg.C, adding 10XYNB, 500 Xbiotin to 0.4mg/L.

BMGY medium (100 mL) 2g tryptone, 1g yeast extract, 70mL water; after sterilization at 121℃for 20min, 10mL of 10 XYNB (filter sterilization), 10mL 100mM pH 6.0 potassium phosphate buffer, 0.2mL of 500 Xbiotin (filter sterilization), 10mL of 10 Xglycerol were added.

The following trypsin enzyme activity determination method is as follows:

(1) Enzyme activity

The catalytic activity of trypsin was determined by Na-benzoyl-DL-arginine-p-nitroaniline hydrochloride (BAPNA). Unit enzyme activity definition: at 25℃and pH8.0, deltaA _410nm The increase of 0.001 per min is an amidase hydrolysis unit of trypsin. And (3) enzyme activity measurement: 43.5mg BAPNA was dissolved in 1mL dimethylformamide, after which the mixture was dissolved in a solution containing 10mM CaCl ₂ 50mM Tris-HCl buffer (pH 8.0). 200. Mu.L of crude enzyme solution and 3mL of BAPNA substrate solution were measured in a cuvette with an optical path of 1cm at 25℃and absorbance change at 410nm was measured within 10 min. The enzyme activity calculation formula is as follows: trypsin enzyme activity (U.mL) ^-1 )＝(△A _410nm /min x dilution)/(volume of enzyme solution x 0.001).

(2) Specific enzyme activity determination

Protein concentration determination:

the protein concentration was determined by Bradford method as follows: taking 200 mu L of Bradford working solution and 20 mu L of sample to be tested, and rapidly and uniformly mixing; the reaction was carried out at room temperature of 25-30℃for 10min, absorbance was measured at 595nm, three replicates for each sample, and a pH8.0 Tris-HCl buffer was used as the blank sample. And (3) bringing the obtained absorbance value into a standard curve to obtain the protein concentration of the sample (the result is shown in figure 2).

Calculating the specific enzyme activity: specific enzyme activity (U.mg) ^-1 ) =enzyme activity/protein concentration.

EXAMPLE 1 construction of recombinant plasmid containing Streptomyces fradiae trypsin Gene

1. Acquisition of Artificial leader Streptomyces fradiae trypsin Gene

The gene of trypsin zymogen (Trypsin) is derived from the coding sequence (GenBank Accession No. D16687.1) of the trypsin zymogen-containing gene in Streptomyces fradiae ATCC14544, wherein the 1 st to 639 th positions are the coding gene of the leader peptide APAP and the 640 th to 1419 th positions are the coding gene of the mature peptide of SFT. After codon preference optimization is carried out on the gene for encoding the SFT mature peptide, ecoRI and NotI enzyme cutting sites are respectively added at the 5 'end and the 3' end of the gene, so that the SFT gene with the nucleotide sequence shown as SEQ ID NO.5 is obtained, and the total synthesis is carried out by adopting a chemical method.

The nucleotide sequence of the artificial leader peptide (comprising thioredoxin TrxA, enterokinase activation site DDDDK and hydrophobic propeptide FVEF) is synthesized and shown as SEQ ID NO. 6.

The artificial leader peptide gene shown in SEQ ID NO.6 and the SFT mature peptide gene shown in SEQ ID NO.5 are connected to obtain an aSFT gene fragment (shown in SEQ ID NO. 2).

2. Construction of recombinant plasmid pPIC9k-aSFT

The in vitro amplified fragment of aSFT gene and the expression vector pPIC9k are respectively subjected to NotI and EcoRI double digestion, and the recovered fragment is used for T ₄ The ligase performs ligation. The ligation reaction was (10. Mu.L): 2. Mu.L of target gene fragment, 2. Mu.L of vector, 10 xT ₄ Ligase Buffer 1. Mu.L, T ₄ Ligase 1. Mu.L, ddH ₂ O 4μL。

The ligation product pPIC9k-aSFT plasmid was transformed into competent cells of E.coli JM109 by the following method:

(1) The competent cells were removed and placed in centrifuge tubes, and after thawing, 10. Mu.L of the pPIC9k-aSFT plasmid was added to 80. Mu.L of E.coli JM109 competent, and the tubes were ice-bathed for 30min.

(2) The centrifuge tube was removed from the ice bath and rapidly transferred to a 42℃water bath heat shock for 90s, followed by 5min of ice bath.

(3) 400-500. Mu.L of LB medium was added to each centrifuge tube, and the mixture was shaken at 200rpm at 37℃for 45min or more, and then a kana resistance plate (20 mg/mL) was applied, and the mixture was incubated overnight at 37℃and the presence or absence of transformants was observed the next day. Positive transformants were selected and sent to sequencing verification, and the results indicated successful construction.

The recombinant plasmid pPIC9k-SFT containing SFT coding gene was constructed by the same method as described above.

EXAMPLE 2 construction of genetically engineered bacterium producing Streptomyces fradiae trypsin

The expression vector pPIC9K-aSFT was linearized by SalI cleavage. The cleavage system was 50. Mu.L: recombinant plasmid 20. Mu.L, 10 XBuffer 5. Mu.L, salI 1. Mu.L, ddH ₂ O24. Mu.L. The linearized product is purified and recovered by a PCR product purification kit after metal bath for 1h at 37 ℃, and competent pichia pastoris GS115 is transformed by an electric shock method, and the specific method is as follows:

(1) Inoculating YPD plate activated P.pastoris GS115 into 25mL/250mL triangular flask, and culturing at 30deg.C overnight; 1% inoculating the culture solution into a 50mL/500mL triangular flask, and culturing until the concentration of the bacterial cells OD ₆₀₀ 1.3 to 1.5;

(2) Centrifuging at 4deg.C for 10min at 5000r/min to collect thallus, and suspending cells with 50mL and 25mL sterile water respectively;

(3) 5mL of 1M sorbitol was used to resuspend the cells, and the cells were collected by centrifugation at 5000r/min and 4℃for 10 min;

(4) The cells were resuspended in 500. Mu.L 1M sorbitol and dispensed into 80. Mu.L/1.5 mL EP tubes;

(5) Soaking the electric rotating cup in 20% ethanol, pouring the soaked electric rotating cup onto paper with sterilized forceps, sterilizing with ultraviolet for 5min, sterilizing with ultraviolet for 10min, and placing on ice for 5min;

(6) Mixing 30. Mu.L of linearization product with 50. Mu.L of competent cells, adding into an electrorotating cup, and placing on ice for 5min;

(7) Opening an electric rotating cup, performing electric shock once at 1500V, 25 mu F and 200Ω, rapidly adding 1mL of 1M precooled sorbitol after electric rotating, completely sucking out to a new 1.5mL EP tube, and oscillating for 2h at 30 ℃;

(8) The electrotransfer product was coated onto MD plates and water was used as a control. Inversion culture is carried out for 2 to 3 days at the temperature of 30 ℃;

(9) White colonies GS115-aSFT grown in the plates were picked, inoculated in 1, 2, 3, and 4mg/mL (geneticin) YPD plates, and single colonies in 4mg/mL geneticin plates were selected for shake flask fermentation.

The genetic engineering bacteria GS115-SFT containing SFT are constructed by the same method.

EXAMPLE 3 expression purification and enzymatic Property determination of Streptomyces fradiae trypsin

1. Expression of Streptomyces fradiae trypsin

Single colonies of the genetically engineered bacteria GS115-aSFT and GS115-SFT were inoculated into BMGY medium and cultured at 30℃for 400 Xg for about 24 hours until the OD was about 2.0-6.0. The bacterial liquid was added to a 50mL centrifuge tube and centrifuged at 2000 Xg for 5min. Removing the supernatant, re-suspending the thalli with 5mL of sterile physiological saline for 2 times, washing, centrifuging at 2000 Xg for 5min, and pouring out the supernatant to collect thalli; resuspending yeast in BMMY medium to OD ₆₀₀ 1 and cultured at 30 ℃ with 400 Xg shaking until bacteria are harvested; starting from the BMMY-transferred culture medium, methanol was added to a final concentration of 1% every 24 hours, and induction was performed for 120 hours. The supernatant was analysed by SDS-PAGE and the results are shown in FIG. 1, as there are significant bands around the theoretical molecular weight of both SFT and aSFT, indicating successful expression of both enzyme molecules.

The obtained fermentation broth was centrifuged at 8000rpm at 4℃for 20 minutes, and the supernatant was collected by removing the cells, and the cell amount and enzyme activity were measured. As shown in FIG. 3, the trypsin activity expressed by the strain GS115-aSFT is 33.9U/mL, which is improved by 2.46 times compared with 13.8U/mL of trypsin activity expressed by the strain GS 115-aSFT. The cell amounts (dry weight) obtained after induction of expression of the strains GS115-aSFT and GS115-SFT were 3.45g/L and 3.46g/L, respectively, without significant differences.

2. Purification of Streptomyces fradiae trypsin

Purifying the target protein by using a Ni-NTA affinity chromatographic column. The 1 Xbinding Buffer and imidazole solutions with different concentrations used in the protein purification process should be vacuum filtered through a 0.45 μm filter membrane and sonicated for 20min before use. The Ni-NTA affinity chromatography column is first equilibrated with 1 Xbinding Buffer, the supernatant obtained is loaded onto the column, and then eluted with 1 Xbinding Buffer until OD ₂₈₀ The target proteins were eluted smoothly with 50mM, 100mM and 250mM final concentration imidazole solutions, respectively, and the eluates were collected. The eluate containing the target protein was concentrated by ultrafiltration tube with a molecular weight cut-off of 3000Da, and then the target protein was replaced by 50mM Tris-HCl buffer (pH 8.0) by GEPD-10 desalting column to obtain purified mutant enzyme sample. The trypsin yield of the modified strain obtained by the same purification method is 47.02 mug/mL, and is improved by 9.6% compared with the expression quantity 42.90 mug/mL of the original strain.

3. Specific enzyme activity determination

The specific enzyme activity of the mutant enzyme and the wild enzyme is calculated by measuring the enzyme activity and the protein concentration of the mutant enzyme and the wild enzyme. As can be seen from FIG. 3, the specific enzyme activity of the trypsin mutant was 20.98U/mg, which was 67.89% higher than that of the wild-type enzyme.

4. Determination of optimum temperature

Taking 20, 30, 35, 40, 45, 50, 55, 60, 70 and 80 ℃ as reaction temperatures, preheating for 5min, measuring enzyme activity, calculating relative enzyme activity, wherein the highest enzyme activity is recorded as 100%, the rest enzyme activities are expressed as residual enzyme activity percentages, each point is repeated three times, and the temperature corresponding to the highest enzyme activity is the optimal temperature. As shown in FIG. 4, the optimum temperature of the modified trypsin aSFT was 45℃and the same as that of the original enzyme SFT.

5. Determination of optimum pH

The substrate solution was prepared with 50mM buffers of different pH and the enzyme activity was measured by preheating at 25℃for 5min. A B-R buffer system with pH of 4.0-5.5; phosphate buffer solution with pH value of 6-8; pH 8.5-10, B-R buffer. At the optimum temperature, the enzyme activity at each pH was measured. The relative enzyme activity percentage was calculated from the enzyme activities at other pH values, with the highest enzyme activity value being 100%. Each point was repeated three times. As shown in FIG. 5, the modified trypsin aSFT had an optimum pH of 9, which was the same as that of the original enzyme SFT.

Example 4: kinetic parameter analysis of Streptomyces fradiae trypsin

Under optimal conditions, the reaction rate of trypsin at various concentrations (0.01-0.1 mmol/L) of substrate BAPNA was measured by the Miq constant equation V=V _max ·[S]/(K _m +[S]) K is calculated by fitting Michaelis-Menten equation _m And V _max Values. V (V) _max Dividing the number of moles of the corresponding protein to obtain the conversion number k _cat Values. The experimental results are shown in Table 1. V of aSFT _max And k _cat /K _m The values respectively reach 12.93mM min ^-1 And 5.38X10 ⁵ min ^-1 ·mM ^-1 Has no obvious difference from SFT (p>0.05). K of aSFT compared with SFT _m A value of 0.0715mM, K compared to SFT _m The value was 10% lower. This indicates that the affinity between aSFT and substrate is higher than the affinity between SFT and substrate. The reason for this may be that the trypsin structure is more flexible due to the presence of the N-terminal hydrophobic pro-peptide FVEF.

TABLE 1 kinetic parameter comparison of the original enzyme and the modified leader peptide trypsin mutant

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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.

Claims (10)

1.A trypsin mutant is characterized by having an amino acid sequence shown in SEQ ID NO. 1.

2. A gene encoding the trypsin mutant of claim 1.

3. An expression vector comprising the gene of claim 2.

4. The expression vector of claim 3, including but not limited to a pPIC9K plasmid, a pPIC9K-His plasmid, a pPIC9 plasmid, a ppiczαa plasmid, a pAO815 plasmid, or a pWB980 plasmid.

5. A recombinant microbial cell expressing the trypsin mutant of claim 1.

6. The recombinant microbial cell of claim 5, wherein the host includes, but is not limited to, E.coli, B.subtilis, and Pichia pastoris.

7. The method for improving the expression quantity of the trypsin is characterized in that the artificial self-activation leader peptide with the amino acid sequence shown as SEQ ID NO.4 and the trypsin with the amino acid sequence shown as SEQ ID NO.3 are fused and expressed; the artificial self-activation leader peptide is fused at the N end of the amino acid sequence of trypsin; after secretion of the fusion protein to the outside of the cell in the form of a trypsin zymogen, the artificial self-activating leader peptide is cleaved off.

8. The method of claim 7, wherein the nucleotide sequence encoding the artificial self-activating leader peptide is shown in SEQ ID No.6 and the nucleotide sequence encoding the trypsin is shown in SEQ ID No. 5.

9. Use of a trypsin mutant according to claim 1, or a gene according to claim 2, or an expression vector according to claim 3 or 4, or a recombinant microbial cell according to claim 5 or 6 for the preparation of trypsin, trypsin-containing products.

10. Use of a trypsin mutant according to claim 1, or a gene according to claim 2, or an expression vector according to claim 3 or 4, or a recombinant microbial cell according to claim 5 or 6 for leather softening.