WO2022088662A1 - Mccj25 mutant, and preparation method therefor and application thereof - Google Patents

Abstract

The present invention relates to an MccJ25 mutant, and a preparation method therefor and an application thereof. The amino acid sequence of the MccJ25 mutant is as shown in SEQ ID NO. 1. A plasmid pUC5725 is taken as a template and substances having sequences shown as SEQ ID NO.3 and SEQ ID NO.4 are taken as primers to obtain an amplification product; an incision enzyme is added into the amplification product, to remove the pUC5725 template to obtain the MccJ25 mutant. The preparation method for the plasmid pUC5725 comprises synthesizing an MccJ25 coding gene sequence in an optimized mode according to the GenBank accession number AF061787, and then inserting the MccJ25 coding gene sequence into an EcoRI-SalI site of pUC57. The stability and bactericidal activity of the MccJ25 mutant having high water solubility are superior to or equal to those of a wild type, and the sensitivity to artificial gastrointestinal fluid is improved.

Description

MccJ25 mutant and preparation method and application thereof technical field

The invention belongs to the field of bioengineering, and in particular relates to a MccJ25 mutant and a preparation method and application thereof.

Background technique

MccJ25 is a bacteriocin with a length of 21AA produced by Escherichia coli AY25, isolated from human feces (Blond, A., J. Péduzzi, C. Goulard, et al. 1999. The cyclic structure of microcin j25, a 21-residue peptide antibiotic from escherichia coli. Febs Journal, 259(3), 747). The MccJ25 encoding gene is located in a 4.8kb fragment on the low-copy plasmid pTUC100, and contains 4 genes, namely mcjA, mcjB, mcjC and mcjD (Solbiati, J.O., M.Ciaccio, R.N.Farías, et al.1996. Genetic analysis of plasmid determinants for microcin j25 production and immunity. Journal of Bacteriology, 178(12), 3661-3). The mcjA gene is 174 bp long and is the precursor of MccJ25. During the process of maturation, the N-terminal 37aa is excised to form MccJ25.

Solution NMR (solution NMR) structural analysis shows that it forms a lariat-like structure, and the main interaction for stable folding is the van der Waals force between amino acid side chains, which is essentially the relationship between hydrophobic groups and polar groups. , two major hydrophobic regions are located on the surface: the first region involves the methylene side chains of Tyr20, Val6, and Glu8, the second region includes Pro7, Phe10, Pro16, and Phe19, and between His5 and Gly21 Forms a salt bridge due to electrostatic action, which helps to stabilize the C-terminus (Rosengren, K.J., R.J. Clark, N.L. Daly, et al. 2003. Microcin j25 has a threaded sidechain-to-backbone ring structure and not a head-to-tail cyclized backbone. Journal of the American Chemical Society, 125(41), 12464-74). Therefore, residue changes can directly affect the structural stability of MccJ25, and change its heat-resistant, acid-alkali-resistant, and enzyme-resistant properties. MccJ25 must be recognized, bound, and taken up by receptors before it can act on the target site. Residues Ala3 and His5 can bind to the outer membrane iron receptor protein FhuA, and residues Gly4, Pro7, Tyr9, Phe10, Phe19 and Gly21 inhibit RNA polymerase activity (Destoumieux-Garzón et al., 2005). Replacing the G12, I13 or T15 residues of MccJ25 will enhance its antibacterial activity (Pan et al., 2011). Therefore, residue changes can directly affect the recognition, uptake, and binding activity of MccJ25 to target sites, thereby affecting its bactericidal activity.

Two hydrophobic regions on the surface of MccJ25 (Rosengren, K.J., R.J.Clark, N.L.Daly, et al. 2003. Microcin j25 has a threaded sidechain-to-backbone ring structure and not a head-to-tail cyclized backbone. Journal of the American Chemical Society, 125 (41), 12464-74), so that its solubility in water is not high, and the recovery rate and concentration ratio will be reduced in the production process, thereby increasing the production cost.

As an antimicrobial peptide, MccJ25 can be used in many products for bactericidal or antiseptic effects. However, the unmodified MccJ25 has poor water solubility, which will limit the combined use of some products that require high water solubility. For example, MccJ25 can be used as an antimicrobial peptide to prepare veterinary drugs, and the biological veterinary drugs prepared by MccJ25 can kill Escherichia coli and Salmonella, reduce bacterial diarrhea in piglets and chicks, such as yellow pullorum in piglets, Salmonella pullorum in chickens, and reduce the number of Salmonella in laying hens. Reduce the incidence of Salmonella infection in eggs, thereby increasing the hatchability of breeders. After the veterinary drug can be dissolved in the water, it can be fed through the water line, and the effect is better. However, many veterinary drugs require good water solubility and no insoluble solids, which will block the water line and affect the animals' drinking water.

MccJ25 is antibacterial and is a good preservative for cosmetics, but unmodified MccJ25 is insoluble in water, and it is difficult to make cosmetic paste or milk. After the transformation, the water solubility is improved, and it can be dissolved in a large amount into cosmetics as a preservative to prolong the shelf life of cosmetics. There is currently no relevant literature reporting MccJ25 as a cosmetic preservative.

SUMMARY OF THE INVENTION

In view of this, the present invention aims to provide a method for improving the water solubility of wild-type MccJ25.

The residues I13 and T15 in the second hydrophobic region on the surface of MccJ25 were mutated to small side chain amino acids such as P and G, respectively.

Further, the method is specifically as follows: using plasmid pUC5725 as a template, and using the sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4 as primers to carry out PCR amplification to obtain an amplification product; adding an endonuclease to the amplification product to remove pUC5725 Template to obtain the MccJ25 highly water-soluble mutant; the plasmid pUC5725 is obtained by inserting the MccJ25 coding gene sequence into the EcoRI-SalI site of pUC57 after optimized synthesis according to AF061787.

Further, the PCR amplification program is: 98°C for 3 min; 98°C×10s, 55°C×15s, 72°C×2min for 15 cycles; 72°C×5min.

Further, the endonuclease is DpnI endonuclease.

Specifically, in some embodiments of the present invention, the amplification conditions of the PCR amplification are: 10 μl of 5× buffer reagent; 10-15 pmol of the primer of SEQ ID NO. 2; 10-15 pmol of the primer shown in SEQ ID NO. 3; template pUC5725<500ng; DNA buffer 0.5 μl; 1.25u/μl polymerase 0.5 μl, then add water to 50 μl.

Alternatively, MccJ25 mutants can be purified using liquid chromatography methods. The purification by the liquid chromatography method is as follows: mobile phase: A is 0.1% trifluoroacetic acid aqueous solution, B is 0.1% trifluoroacetic acid 90% acetonitrile; column temperature: 25° C.; injection volume: 100 μL; flow rate: 1.5 mL /min; detection wavelength: 280nm.

In addition, when designing primers, different primers are designed for different mutants, and different vectors are selected for PCR amplification.

In addition, in addition to the high water solubility mutant of MccJ25, other mutants with higher water solubility obtained by mutating different mutation sites on the basis of wild-type MccJ25, or mutants with other properties, are in the present invention. In certain embodiments, MccJ25 mutants also include improved acid resistance, alkali resistance, and resistance to gastrointestinal enzymes compared to the wild type. In certain embodiments of the present invention, the I13P, T15G residues are replaced in the beta hairpin region to obtain a MccJ25 mutant.

The present invention also aims to provide a MccJ25 mutant prepared by the aforementioned method.

Further, the amino acid sequence of the MccJ25 mutant is shown in SEQ ID NO. 1 and its identity is greater than or equal to 90%.

The present invention also aims to provide a nucleotide sequence encoding the aforementioned MccJ25 mutant, the nucleotide sequence is shown in SEQ ID NO. 2.

The present invention also aims to provide a recombinant plasmid comprising the aforementioned nucleotide sequence.

Further, the recombinant plasmid is constructed from the nucleotide sequence and the expression vector.

The present invention also aims to provide an Escherichia coli comprising the aforementioned recombinant plasmid.

Further, the Escherichia coli uses Escherichia coli DH5α as a host and pUC57 as an expression vector.

In addition, different mutants can be recombined with different E. coli hosts and different expression vectors to obtain different E. coli.

Specifically, in some embodiments of the present invention, the strains were streaked on LB plates containing 50 μg/ml ampicillin, and cultured at 37°C overnight. Pick a single clone in 3ml of liquid LB medium, and cultivate it at 37℃×220rpm for 12-16h as a seed solution. Take 40 μl of the seed liquid, inoculate it into a shake flask (250 ml capacity) containing 40 ml of fermentation medium, and cultivate at 37° C. × 220 rpm for 22 hours to obtain a fermentation broth containing bacterial cells and secreted mutants.

The fermentation broth containing cells and secreted mutants was centrifuged at 8000 rpm for 15 min, and the supernatant was taken, pre-frozen at -80°C for 3 h, and then lyophilized and concentrated. The lyophilized solid was weighed, washed three times with 80% acetonitrile (0.2 g/mL), centrifuged at 8000 rpm for 5 min, and the supernatant was saved. After the second and third supernatants were concentrated by rotary evaporation, purified mutants were obtained by filtration through a 0.22 μm organic filter membrane.

The purpose of the present invention is to also provide the application of the aforementioned MccJ25 mutant in the preparation of the aqueous phase in the liquid preparation.

The liquid preparation refers to a preparation containing water or a solid preparation containing water in the use state, and not only refers to a liquid preparation with relatively large fluidity in the traditional sense. The preparation containing water here, such as a dressing, is a paste-like preparation. , milky preparations or gel preparations; or use solid preparations containing water in the use state, such as oral granules or powder preparations, etc. When these granules or powders are used, after dissolving in water, the MccJ25 mutant solution is the water phase.

Preferably, the liquid preparation includes water-soluble veterinary drugs, cream or cream cosmetics.

Specifically, water-soluble biological veterinary drugs are prepared to prevent and treat animal bacterial diseases, such as killing Escherichia coli and Salmonella, reducing bacterial diarrhea in piglets and chicks, such as piglet yellow pullorum, chicken Salmonella pullorum, and reducing the body of laying hens Salmonella counts. As a cosmetic preservative to extend the shelf life of cosmetics, etc.

Specifically, usually cosmetic preparations including water phase, oil phase, emulsion and other excipients are made into paste or milky formulations for use, and MccJ25 mutant is dissolved in water and mixed with others as water phase to make paste or milky form and other preparations, that is, liquid preparations.

The purpose of the present invention is to also provide an aqueous phase with adjustable concentration of bacteriostatic components.

In the aqueous phase, the bacteriostatic component contains the aforementioned MccJ25 mutant, and the concentration of the MccJ25 mutant is 0.01-35.83 mg/ml.

Preferably, the concentration of the MccJ25 mutant is 33.90-35.83 mg/ml.

Specifically, the bacteriostatic component may also include other bacteriostatic agents. The bacteriostatic ability of the MccJ25 mutant and the wild-type mutant is almost the same. After the water solubility of the wild-type is improved, the MccJ25 mutant dissolves in water as the water phase to widen the wild-type. The use of MccJ25 as a bacteriostatic agent is not only the use of MccJ25 wild-type as an antimicrobial peptide, but many other biological antimicrobial peptides are relatively insoluble in water, and the highly water-soluble MccJ25 can meet the requirements of many liquid preparations.

The beneficial effects of the present invention are:

The water solubility of the MccJ25 highly water-soluble mutant provided by the present invention is about 35 g/L, which is 4 times higher than that of the wild type.

The antibacterial spectrum of the MccJ25 highly water-soluble mutant provided by the invention is the same as that of the wild type, the MIC is 2-128 μg/ml, and the bactericidal ability is equivalent to that of the wild type.

The MccJ25 highly water-soluble mutant provided by the present invention can withstand heat resistance up to 100° C., strong acid environment with pH as low as 2, and strong alkali environment with pH as high as 11.

The MccJ25 highly water-soluble mutant provided by the present invention has a degradation rate of 1.7% by artificial gastric juice and 9.3% by artificial intestinal juice, which is slightly higher than that of the wild type, but still belongs to the tolerance range.

The stability and bactericidal activity of the MccJ25 highly water-soluble mutant provided by the present invention are better than or equal to the wild type.

Description of drawings

Figure 1 is a mass spectrum of MccJ25 mutants.

Figure 2a is the purification chromatogram of MccJ25 mutant detected at 230 nm

Figure 2b is the purified chromatogram of MccJ25 mutant detected at 280 nm.

Figure 3 is a chromatogram for the detection of MccJ25 mutants.

Detailed ways

The examples are given to better illustrate the present invention, but the content of the present invention is not limited to the examples. Therefore, those skilled in the art make non-essential improvements and adjustments to the embodiments according to the above-mentioned contents of the invention, which still belong to the protection scope of the present invention.

In the examples of the present invention, Escherichia coli host strain DH5α was purchased from Beijing Tianjian Biochemical Technology Co., Ltd., and Escherichia coli M4j65-11 was preserved in the applicant's laboratory.

The indicator bacteria CVCC1497, CVCC1569, CVCC1499, CVCC1555 and CVCC1506 used in the examples of the present invention were purchased from China Veterinary Drug Administration.

In the examples of the present invention, ATCC6962 and ATCC25922 were purchased from ATCC, and E.coli O157, Salmonella typhimurium, Salmonella pullorum, Danai and K88 were stored in the applicant's laboratory.

In the example of the present invention, the formula of LB medium: 10 g/L tryptone, 5 g/L yeast extract and 10 g/L sodium chloride, the volume of purified water is adjusted to 1 L, and it is used after autoclaving at 121°C. Fermentation medium formula (g/L): 20g yeast powder, 6g KH2PO4, pH 6.2, dissolved in 1L deionized water, sterilized at high temperature and high pressure.

In the examples of the present invention, ampicillin, porcine pepsin, and bovine trypsin were purchased from Sigma Company in the United States.

Nucleic acid modifying enzymes such as Phusion DNA polymerase, T4 DNA ligase, and endonuclease in the examples of the present invention were purchased from Fermentas Company in the United States.

In the examples of the present invention, chromatographic grade trifluoroacetic acid (TFA) and chromatographic grade acetonitrile were purchased from Thermo.

In the examples of the present invention, the DNA purification kit Wizard DNA Clean-Up system (A7280) was purchased from Promega,

The plasmid mini-prep kit (DP103) in the examples of the present invention was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

In the examples of the present invention, MH solid medium, MH liquid medium, pepsin inhibitor and trypsin inhibitor were purchased from Beijing Soleibao Technology Co., Ltd.

Example 1

The MccJ25 coding gene sequence was optimized and synthesized according to AF061787, and inserted into the EcoRI-SalI site of pUC57, and the plasmid was named pUC5725.

Example 2 Preparation of mutants

SEQIDNO.3S2f (5'-AGTATTTTGTGGGGCCGGGTGGACCTATATCTT-3'), SEQIDNO.4S2r (5'-AAGATATAGGTCCACCCGGCCCCACAAAATACT-3') were amplified with plasmid pUC5725 as template, the amplification length was 3.6kb, PCR program: 98°C 3min; 98°C ×10s, 55°C×15s, 72°C×2min for 15 cycles; 72°C×5min, the amplification system is shown in Table 1.

Table 1 PCR system

reagent		Usage amount
5×Phusion buffer	10μl
S2f	10-15pmol
S2r	10-15pmol

pUC5725	<500ng
Phusion DNA Polymerase(1.25u/μl)	0.5μl
H2O	to 50μl

After the PCR reaction, 2 μl of DpnI endonuclease was added, and the pUC5725 template was removed in a water bath at 37° C. for 1 h.

After purification of the treated product, 10 μl was taken to transform E. coli DH5α, and the mutant was verified by sequencing.

Example 3 Preparation of mutant pure product

The wild-type and mutant clones (host M4j65-11) stored in glycerol tubes were streaked on LB plates containing 50 μg/ml ampicillin, and cultured at 37°C overnight. Pick a single clone in 3ml of liquid LB medium, and cultivate it at 37℃×220rpm for 12-16h as a seed solution. Take 40 μl of the seed liquid, inoculate it into a shake flask (250 ml capacity) containing 40 ml of fermentation medium, and cultivate at 37° C. × 220 rpm for 22 hours, which is called fermentation broth.

The fermentation broth was centrifuged at 8000 rpm for 15 min, and the supernatant was taken, pre-frozen at -80°C for 3 h, and then lyophilized and concentrated. The lyophilized solid was weighed, washed three times with 80% acetonitrile (0.2 g/mL), centrifuged at 8000 rpm for 5 min, and the supernatant was saved. After the second and third supernatants were concentrated by rotary evaporation, they were filtered through a 0.22 μm organic filter for use.

Purification chromatography conditions, mobile phase: A is 0.1% trifluoroacetic acid aqueous solution, B is 0.1% trifluoroacetic acid 90% acetonitrile; column temperature: 25 °C; injection volume: 100 μL; flow rate: 1.5 mL/min; detection wavelength: 230 , 280nm, see Figure 2. The gradient elution procedure is shown in Table 2 below.

Table 2 Gradient elution procedure

After purification, the collected fractions were lyophilized using a freeze dryer.

Example 4 Mutant detection

Accurately weigh 5-10 mg of wild-type and mutant pure products into a 2 mL centrifuge tube, add 1 mL of ultrapure water or phase B, shake well, and sonicate (35°C) for 15 min to dissolve them completely. Using the doubling (one-fold) dilution method, 10 samples with different concentrations were obtained by successive dilution.

Detection chromatographic conditions Mobile phase: A is 0.1% trifluoroacetic acid aqueous solution, B is 0.1% trifluoroacetic acid 90% acetonitrile; column temperature: 25 °C; injection volume: 10 μL; flow rate: 1.0 mL/min; detection wavelength: 214 nm, 230 nm, 254 nm, 280 nm; the gradient elution program is as follows in Table 3.

Table 3 Mutant detection elution procedure

Equilibrate the system to baseline levels. Starting from low-concentration samples, after filtration with a 0.22 μm filter, the samples were sequentially injected for analysis and detection, and the chromatogram was recorded, as shown in Figure 3. After 10 samples were detected, a new calibration table was established with the external standard quantification method, the method was renamed and saved, and the establishment of the standard curve was completed.

Example 5 Mutant mass spectrometry identification

(1) Identification step

Mass Spectrometry Conditions Triple Quadrupole Tandem Mass Spectrometer, Agilent 6460 Triple Quad LC/MS, Agilent Technologies, USA; Ion Source: Jet Stream Technology Ion Source (AJS); Analysis Software: Agilent MassHunter Workstation software(Version B.04.00); Electrospray ion source (ESI) positive ion scanning mode: capillary voltage is 3500V; nebulizer temperature is 350℃; nebulizer flow rate is 12L/min; sheath gas temperature is 400℃; sheath gas flow rate 10L/min.

Take a small amount of fractions, inject samples with constant flow of needle pump and scan in ESI+ mode and ESI- mode respectively, select the ion mode with better effect, and obtain the precursor ion as [M+H]+.

(2) Identification results

The amino acid sequence of the mutant is SEQ ID NO.1 (GGAGHVPEYFVGPGGPISFYG), and the theoretical molecular weight is 2047.3. In Figure 1: [M+2H]2+1024.2; [M+3H]3+683.2. It can be seen that the theoretical molecular weight of the mutant is consistent with the actual molecular weight.

Example 6 Determination of mutant solubility

(1) Measurement step

Determining the equilibrium solubility of substances often uses the classic shake flask method, which is widely used in pharmaceutical research and the preparation of high-purity materials, and is often used as a standard for calibrating new solubility determination methods. The specific method is as follows:

Take the excess mutant and put it in a 100 mL conical flask with a stopper, add 100 mL of ultrapure water, sonicate until the mutant no longer dissolves, place it in a 37°C water bath thermostatic oscillator, shake at 200 rpm, use a syringe to draw about 5 mL of the solution, and then pass through 0.22 Filter with a μm microporous membrane (preheated at the same temperature), discard the initial filtrate, draw 1 mL of the subsequent filtrate with a micropipette, quickly dilute it with HPLC mobile phase to a certain concentration and inject the sample. Generally, the sampling point is three or more times (12h, 24h and 36h) after shaking for 12h. The solubility of mutants in water was calculated from the standard curve. The RSD of the data before and after was less than 2%, and the average value was the equilibrium solubility.

(2) Measurement results

In the study, the equilibrium solubility of the mutant in water at 37 °C was measured to be 35 mg/ml, and its solubility was significantly higher than that of the wild-type MccJ25, mainly due to the relatively good water solubility of the residues at the mutant sites (I13P, T15G). According to the 2005 and 2010 editions of the Chinese Pharmacopoeia, "extremely soluble, easily soluble, soluble, slightly soluble, slightly soluble, very slightly soluble, almost insoluble or insoluble" is used to describe the solubility of drugs in different solvents, so the mutants are The solubility property in water is "dissolving". The solubility measurement results are shown in Table 4 below.

Table 4 Mutant solubility assay results

	MccJ25(mg/ml)	Mutant (mg/ml)
12h	8.59±0.10	34.55±0.65
24h	8.73±0.43	35.38±0.06
36h	8.77±0.16	35.16±0.67

Note: The unit of solubility is mg/ml, and MccJ25 is wild type.

Example 7 Determination of antibacterial spectrum and MIC

(1) Measurement step

(a) 12 strains of indicator bacteria stored in glycerol tubes were streaked on the MH plate and activated at 37°C for 20h; a single colony was picked in MH liquid medium, 37°C×220rpm, cultured for 12-16h, and the bacterial solution was diluted with physiological saline to OD600 It is 0.1 (about 108CFU/ml), for use (diluted 25-40 times with sterile normal saline).

(b) Pour 10ml of 1.5% water agar into the sterilized petri dish as a bottom layer, and place 8 Oxford cups evenly on each plate after cooling. When the MH solid medium is cooled to 50±5°C, add the diluted indicator bacteria liquid to the MH solid medium according to the ratio of 100 μL of indicator bacteria liquid to 100 ml of agar medium, shake evenly, and pour each plate. Add 25ml to ensure that there are more than 18 plates for each indicator bacteria. After cooling, take out the Oxford cup and place the punched plate in a 4°C refrigerator for later use. Take 1 plate of each indicator bacteria and use 25μg/ml chloramphenicol. Add samples with distilled water to check the paving effect.

(c) Hammami et al., in a study on the bacteriostatic effect of J25 mutants in 2015, the MIC of most mutants E. coli and Salmonella were measured in the range of 2-500 μg/ml, and the MIC of J25 wild-type was in the range of 0.2-13 μg/ml Within the range of ml, Hammami uses the 96-well plate method to measure the MIC, and the value measured by the punch plate method is 5-20 times higher (taking J25 wild-type versus K88 indicator bacteria as an example, the 96-well plate method determines the MIC value of 0.2 μg/ml , the MIC value of the punch plate method was 4 μg/ml), in view of the great difference in the bacteriostatic effect between the mutants made by Hammami and the wild type, the range of 1-512 μg/ml was selected for the test. Weigh 7.68mg of wild-type and mutant pure product, dissolve in 15ml of distilled water, shake and mix, and dilute with distilled water according to a 2-fold gradient to prepare 512, 256, 128, 64, 32, 16, 8, 4, 2, 1 μg/ml (both pH 7.5) sample solution, set aside. Add 200 μl of sample solution to each well of the perforated plate, and add 200 μl of distilled water as a blank control. After adding all the samples, place the perforated plate in a refrigerator at 4 °C for 1-2 hours, wait for the samples to diffuse, and incubate at 37 °C overnight. Antibacterial spectrum.

(2) Measurement results

MccJ25 and its mutants are taken up through the FhuA-Smba pathway and act on the target RNAP to exert bactericidal effects. Studies have shown that the β hairpin region Phe10-Ile17 in the structure of MccJ25 is related to uptake, and residue Ile13 is only recognized by the outer membrane protein FhuA . The mutants (I13P, T15G) in this example have mutation sites in the β hairpin region. Combined with the bacteriostatic spectrum and MIC results in Table 5, it can be seen that the bacteriostatic spectrum of the mutants has no change, and the bacteriostatic activity is equivalent to that of the wild type, but some The strains are less susceptible to mutants.

Table 5 MIC and antibacterial spectrum results of mutants

	MccJ25	mutant
Salmonella pullorum	2	4
CVCC1499	4	2
CVCC1506	4	4
ATCC25922	4	16
K88	4	4
CVCC1497	8	16
O157	128	128
CVCC1569	none	none
CVCC1555	none	none
ATCC6962	none	none
Salmonella typhimurium	none	none
Danai	none	none

Note: The unit of MIC is μg/ml, MccJ25 is wild type, “None” means that the indicator bacteria have no antibacterial activity when the concentration is 512 μg/ml.

Example 8 Heat resistance, acid resistance and alkali resistance test

(1) Test steps

(a) Wild-type and mutant were prepared with concentrations of 50 μg/ml and 120 μg/ml, respectively.

Take 400 μl of the solution in a 2.0 ml EP tube, put it in a metal bath at 60, 70, 80, and 100 °C for 20 min, and quickly cool the sample to room temperature with cold water.

(b) Take 400 μl of the solution into a 2.0 ml EP tube, adjust the pH to pH 2.0, 4.0, 6.0, 7.0, 8.0, 9.0 and 11.0 as needed, put the sample into a 37°C water bath for 2 hours, and the pH after treatment All were adjusted to 7.5, the content was determined by HPLC, and three parallel experiments were performed for each sample.

(2) Test results

The mutants are stable in the range of 50-100°C and pH 2-9. The results are shown in Table 6 and Table 7. Due to the evaporation of heating water, the data of some experimental groups in Table 5 are higher than those of the control group; The difference in volume is the main reason for the difference in the data in Table 7.

Table 6 Heat resistance test results

	MccJ25	mutant
temperature control	49.73±0.74	117.45±1.58
100℃	62.37±1.82	117.99±0.01

Note: The unit of data is μg/ml, and MccJ25 is wild type.

Table 7 Acid and alkali resistance test results

	MccJ25	mutant
pH control	49.73±0.74	117.45±1.58
pH2.0	49.66±3.19	110.96±0.83
pH11.0	50.31±2.70	106.40±2.03

Note: The unit of data is μg/ml, and MccJ25 is wild type.

Example 9 Pepsin resistance, trypsin test

(1) Test steps

(a) Prepare 50× artificial gastric juice mother liquor, weigh 0.1 g of sodium chloride and 0.175 g of pepsin, dissolve in 1 ml of ultrapure water, mix and filter for use.

(b) Prepare 10× artificial intestinal juice mother liquor, weigh 0.68 g of potassium dihydrogen phosphate and 0.1 g of trypsin, dissolve in 10 ml of ultrapure water, and adjust the pH to 7.8 with sodium hydroxide.

(c) Take 400 μl of solution (prepared from 3.7) and mix it with 8.17 μl of artificial gastric juice mother liquor, and make another set of 8.17 μl of 50× artificial gastric juice buffer (0.1 g/ml sodium chloride) mixed with 400 μl of the blank control, test The group and the control group were treated with the following treatments, adjusted pH to 2.5, water bath at 37 °C for 2 h, adjusted pH to 7.5, added 2.9 μl pepstatin at a concentration of 10 mg/ml (working concentration 0.7 μg/ml), water bath at 37 °C for 30 min , HPLC determination of the content, each sample to do three parallel experiments. The theoretical final concentration of pepsin is 3.5 mg/ml, and the theoretical final concentration of pepstatin is 0.7 μg/ml.

(d) Mix 400 μl of the solution (prepared from 3.7) with 44.45 μl of artificial intestinal fluid stock solution, and make another set of 44.45 μl of 10× artificial intestinal fluid buffer (0.02g/ml sodium chloride, 0.11g/ml sodium bicarbonate, 0.18g/ml ml bile salts) and 400 μl of solution mixed with blank control, the experimental group and the control group were treated as follows, adjusted pH to 8.0, 37 ° C water bath for 2h, adjusted pH to 7.5, added 0.85 μl of trypsin inhibitor with a concentration of 5 mg/ml (working concentration 0.1 μg/ml), 30min in water bath at 37°C, content was determined by HPLC, and three parallel experiments were performed for each sample. The theoretical final concentration of trypsin is 1 mg/ml, and the theoretical final concentration of pepstatin is 0.1 μg/ml.

(2) Test results

The degradation rate of mutants by artificial gastric juice was 1.7%, and the degradation rate of mutants by artificial intestinal juice was 9.3%, slightly higher than the wild type (Table 8), but still within the tolerance range.

Table 8 Test results of pepsin resistance and trypsin resistance

mutant	MccJ25	mutant
Control 1	50.99±1.47	111.96±1.65
artificial gastric juice	50.63±0.24	110.09±1.66
Degradation rate of artificial gastric juice (%)	0.7	1.7
Control 2	46.77±0.96	107.57±1.00
Artificial intestinal juice	42.99±0.61	97.51±1.40
Degradation rate of artificial intestinal juice (%)	8.1	9.3

Note: The unit of data is μg/ml, "Control 1" means artificial gastric juice control, "Control 2" means artificial intestinal juice control, MccJ25 is wild type.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be Modifications or equivalent replacements without departing from the spirit and scope of the technical solutions of the present invention should be included in the scope of the claims of the present invention.

Claims (10)

1. The method for improving the water solubility of MccJ25 is characterized in that the residues I13 and T15 in the second hydrophobic region on the surface of MccJ25 are mutated into P and G small side chain amino acids, respectively.
2. The MccJ25 mutant prepared by the method of claim 1, wherein the amino acid sequence of the MccJ25 mutant is as shown in SEQ ID NO. 1 and a sequence whose identity is greater than 90%.
3. The nucleotide sequence encoding the MccJ25 mutant of claim 2, wherein the nucleotide sequence is shown in SEQ ID NO. 2.
4. A recombinant plasmid comprising the nucleotide sequence of claim 3.
5. Escherichia coli comprising the recombinant plasmid of claim 4.
6. The Escherichia coli according to claim 5, characterized in that, Escherichia coli DH5α is used as a host, and pUC57 is used as an expression vector.
7. The application of the MccJ25 mutant of claim 2 in the preparation of the aqueous phase of the liquid preparation.
8. The application according to claim 7, wherein the liquid preparation includes water-soluble veterinary drugs, cream or milk cosmetics.
9. An aqueous phase with adjustable bacteriostatic component concentration, characterized in that the bacteriostatic component contains the MccJ25 mutant according to claim 2, and the concentration of the MccJ25 mutant is 0.01-35.83 mg/ml.
10. The aqueous phase according to claim 9, wherein the concentration of the MccJ25 mutant is 33.90-35.83 mg/ml.

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