A subtilisin protease mutant capable of catalyzing peptide bond synthesis in a pure water phase environment in one step and its application

Abstract

The present invention belongs to the field of protein engineering. The present invention provides a subtilisin mutant capable of catalyzing peptide bond synthesis in a pure water phase environment in one step and its application. The existing wild-type subtilisin structure is modified, and its catalytic performance is changed by semi-rational design of mutation points. The peptide bond synthetase after site-directed mutation finally realizes the application of catalyzing peptide bond synthesis in a pure water phase. The subtilisin mutant of the present invention can catalyze the synthesis of peptide bonds by natural amino acids in a pure water phase environment in one step, providing a new method for peptide bond synthesis, green synthesis, and meeting the industrial production needs of peptide synthesis.

Description

Technical Field

[0001] The invention belongs to the field of protein engineering, and in particular relates to a peptide bond synthetase for catalyzing peptide bond synthesis and an application thereof. Background Art

[0002] Peptide bonds are a special form of amide bonds (-CO-NH-) formed by the dehydration condensation reaction of the α-amino and α-carboxyl groups between two amino acid molecules. As the basis of the structure of polypeptides and proteins, peptide bond synthesis has received extensive attention. Due to the high reaction energy barrier of the carboxyl-amino dehydration condensation between amino acids and the kinetic inhibition of water, it is extremely difficult to synthesize peptide bonds in vitro, especially in pure water phase systems. Currently, there is no catalyst that can catalyze the "one-step dehydration condensation" between natural amino acids to form peptide bonds.

[0003] In chemical coupling, carboxyl preactivation is used to overcome high reaction energy barriers and kinetic inhibition of water, such as solid phase peptide synthesis (SPPS) and native chemical ligation (NCL). However, the multi-step reactions caused by carboxyl preactivation and amino acid side chain group protection and deprotection, as well as the use of hazardous chemical reagents, are major drawbacks of chemical coupling.

[0004] The ribosome is the only known mediator of the process of natural amino acid condensation peptide bonds. However, this reaction process is a synergistic process involving 60-80 proteins, which is complex and strictly controlled, and has almost no application scalability.

[0005] Discovering or designing a single enzyme that can realize the "one-step dehydration condensation" function between natural amino acids similar to ribosomes will bring great changes and endless application imagination to the life, medicine, chemical, material and other industries. This can not only completely change the current peptide and protein synthesis industry; it can also perform specific modifications or labeling such as biotinylation and PEG on the surface of living cells to reveal complex biological processes such as apoptosis, inflammation, bacterial and viral infections; it can also achieve targeted delivery of drugs by specifically identifying receptors on the surface of target cells, significantly improving drug targeting and biological effectiveness.

[0006] At present, Li et al. have found that an intracellular lipase can catalyze the dehydration condensation of carboxyl-amino groups in 20% water to synthesize amide small molecules, but its application scope is limited. Scientists have also rationally designed peptide ligases based on PatG, Butelase 1, Sortase A and Trypsin proteases. However, since it needs to recognize a specific substrate sequence to cut the peptide bond to expose the carboxyl group and then synthesize the peptide bond with the amino substrate, it is not a one-step synthesis reaction and its application scope is limited. Subtiligase is a peptide ligase modified by Wells et al. through kinetic control of subtilisin (a serine protease). It can catalyze the synthesis of peptide bonds between ester groups and amino groups after esterification, and is widely used in peptide connection, cyclization, peptide drug synthesis, protein synthesis, modular bioconjugation (Weeks AM, Wells JA. Subtiligase-Catalyzed Peptide Ligation. ChemRev. 2020Mar 25; 120(6): 3127-3160. doi: 10.1021 / acs.chemrev.9b00372. Epub 2019Oct30. PMID: 31663725.), protein N-terminal modification and cell N-terminal genomics identification (Schaefer K, Lui I, Byrnes JR, Kang E, Zhou J, Weeks AM, Wells JA. Direct Identification of Proteolytic Cleavages on Living Cells Using a Glycan-Tethered Peptide Ligase.ACS CentSci.2022Oct26;8(10):1447-1456.doi:10.1021 / acscentsci.2c00899.Epub 2022Oct11.PMID:36313159;PMCID:PMC9615116.) and other fields, showing great value and significance. Unfortunately, all of the above enzyme preparations require organic phase catalysis, substrate recognition or modification, which will limit their further application in living cell systems under physiological reaction conditions.

[0007] Therefore, further modifying Subtilisin and developing a peptide synthetase that catalyzes peptide bond synthesis in a pure water phase in one step is one of the research and development directions that technicians in this field are committed to. Summary of the invention

[0008] In order to solve the above-mentioned problems of the prior art, the present invention is based on the study of wild-type Bacillus subtilisin protease Subtilisin, and provides a Bacillus subtilisin protease mutant that can catalyze the synthesis of peptide bonds in a pure water phase environment in one step, which can realize the one-step catalysis of natural amino acid synthesis of peptide bonds in a pure water phase environment, green synthesis, and provide a new technical idea for peptide bond synthesis to meet the industrial production needs of peptide synthesis.

[0009] The object of the present invention is achieved in that:

[0010] The present invention provides a subtilisin mutant capable of catalyzing peptide bond synthesis in a pure water phase environment in one step, and its amino acid sequence is shown in one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.

[0011] The gene encoding the above-mentioned Bacillus subtilis protease mutant capable of catalyzing peptide bond synthesis in a pure water phase environment in one step.

[0012] The present invention also provides a recombinant vector comprising a nucleotide sequence as shown in one of SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12.

[0013] A host cell is also provided, which comprises the above recombinant vector.

[0014] Also provided is a use of the Bacillus subtilis protease mutant capable of catalyzing peptide bond synthesis in a pure water phase environment in one step in the preparation of polypeptide products.

[0015] The present invention obtains the peptide bond synthetase mutants M1, M2, M3, M4, M5 and 4M of the present invention by semi-rational design of mutation points, namely, the asparagine at position 62 of the wild-type subtilisin is changed to phenylalanine, the serine at position 63 is changed to valine, and the proline at position 225 is mutated to alanine; the asparagine at position 62 of the wild-type subtilisin is changed to phenylalanine, the serine at position 63 is changed to leucine, and the proline at position 225 is mutated to alanine; the asparagine at position 62 of the wild-type subtilisin is changed to valine, the serine at position 63 is changed to leucine, and the proline at position 225 is mutated to The wild-type subtilisin is mutated into alanine; the asparagine at position 62 is changed to phenylalanine, the serine at position 63 is changed to isoleucine, and the proline at position 225 is mutated into alanine; the tyrosine at position 217 is changed to leucine, the asparagine at position 218 is changed to phenylalanine, and the proline at position 225 is mutated into alanine; the asparagine at position 62 of the wild-type subtilisin is changed to leucine, the serine at position 63 is changed to leucine, the tyrosine at position 217 is changed to leucine, the asparagine at position 218 is changed to phenylalanine, and the proline at position 225 is mutated into alanine.

[0016] The inventors provide key mutation sites at positions 62, 63, 217, 218 and 225 by structural analysis of wild-type Subtilisin, and perform different combinations of the mutation sites to obtain the subtilisin mutants of the present invention that can catalyze peptide bond synthesis in a pure water phase environment in one step. The results show that the application of the subtilisin mutants of the present invention in peptide bond synthesis can achieve one-step catalysis of natural amino acid peptide bond synthesis in a pure water phase environment compared with the prior art, meet the industrial production needs of peptide synthesis, eliminate the need for organic phase catalysis or substrate recognition or modification steps in the prior art, solve the problems of environmental restrictions on existing peptide bond synthesis, and have better application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the nucleic acid electrophoresis diagram of the mutant plasmid in Example 1;

[0018] Figure 2 is the protein electrophoresis diagram of the purified mutant and wild-type Subtilisin (WT) in Example 2;

[0019] Figure 3 This is the UPLC-MS detection chart of the product Suc-Ala-Ala-Pro-Phe-Ala-Phe-NH2 generated in the pure water phase of the mutant in Example 3. DETAILED DESCRIPTION

[0020] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Biomaterials

[0022] The amino acid sequence of wild-type Bacillus subtilisin protease Subtilisin is shown in SEQ ID NO.13, and the encoding gene sequence thereof is shown in SEQ ID NO.14.

[0023] The promoter-signal peptide-leader peptide-gene-terminator sequence of wild-type Bacillus subtilisin Subtilisin was synthesized by a fully artificial synthesis method (codons were optimized according to the properties of the cloning vector), the nucleotide sequence is shown in SEQ ID NO.15, and a pP43NMK plasmid containing the Bacillus subtilisin Subtilisin gene, namely, a pP43NMK-Subtilisin plasmid, was constructed.

[0024] Example 1 Construction of plasmid containing mutation site gene

[0025] The M1 mutant is a mutant in which asparagine at position 62, serine at position 63, and proline at position 225 of the wild-type Bacillus subtilisin are replaced by phenylalanine, valine, and alanine, and its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.7; the M2 mutant is a mutant in which asparagine at position 62, serine at position 63, and proline at position 225 of the wild-type Bacillus subtilisin are replaced by phenylalanine, leucine, and alanine, and its amino acid sequence is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.8; the M3 mutant is a mutant in which asparagine at position 62, serine at position 63, and proline at position 225 of the wild-type Bacillus subtilisin are replaced by valine, leucine, and alanine, and its amino acid sequence is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.8; the M4 mutant is a mutant in which phenylalanine, isoleucine and alanine are substituted for asparagine at position 62, serine at position 63 and proline at position 225 in the wild-type Bacillus subtilisin protease Subtilisin, and its amino acid sequence is shown in SEQ ID NO.4, and its nucleotide sequence is shown in SEQ ID NO.10; the M5 mutant is a mutant in which leucine, phenylalanine and alanine are substituted for tyrosine at position 217, asparagine at position 218 and proline at position 225 in the wild-type Bacillus subtilisin protease Subtilisin, and its amino acid sequence is shown in SEQ ID NO.5, and its nucleotide sequence is shown in SEQ ID NO.11; the 4M mutant is a mutant in which leucine, leucine, leucine, phenylalanine and alanine are substituted for asparagine at position 62, serine at position 63, tyrosine at position 217, asparagine at position 218 and proline at position 225 in the wild-type Bacillus subtilisin protease Subtilisin, and its amino acid sequence is shown in SEQ ID No. 6, and the nucleotide sequence is shown in SEQ ID No. 12. A 6xHis tag was added to the C-terminus of the above mutants.

[0026] The mutant was constructed using the wild-type Bacillus subtilisin gene sequence as a template using the PCR method. The specific primer design is shown in the following table:

[0027] Primer name Primer sequences 62L / 63L-F 5'-AATCCTTCCAAGACAACTTACTACACGGAACT-3' 62F / 63V-F 5'-AATCCTTCCAAGACAACTTTGTACACGGAACT-3' 62F / 63L-F 5'-AATCCTTCCAAGACAACTTTCTACACGGAACT-3' 62V / 63L-F 5'-AATCCTTCCAAGACAACGTACTACACGGAACT-3' 62F / 63I-F 5'-AATCCTTCCAAGACAACTTTATACACGGAACT-3' 62 / 63-R 3'-GTCTTGGAAAGGATTTGTTTCAGA-5' 217 / 218-F 5'-GGAAACAAATACGGGGCCGCTATTCGGTACGTCA-3' 217 / 218-R 3'-CCCGTATTTGTTTCCAGGAAGCGT-5' 225-F 5'-TCAATGGCATCTGCACACGTTGCCGGA-3' 225-R 3'-AGATGCCATTGACGTACCATTGTACGC-5'

[0028] Using the wild-type Bacillus subtilisin gene sequence as a template, the Bacillus subtilisin gene sequence is subjected to site-directed mutagenesis by the above method to obtain a mutant target gene. The specific steps are as follows:

[0029] 1. Amplification of the original plasmid pP43NMK-Subtilisin

[0030] The glycerol bacteria containing the subtilisin gene plasmid was inoculated at 1% in 10 mL LB resistant liquid medium, cultured overnight at 37° C. for about 12 hours, and the plasmid was extracted the next day.

[0031] 1. Specific operation of plasmid DNA extraction

[0032] (1) Take 3 mL of overnight cultured bacterial solution and centrifuge at 8000 g for 2 min to collect the bacterial cells, remove the supernatant, and add 200 μL FAPD1 to resuspend the precipitated bacterial cells.

[0033] (2) After adding 200 μL FAPD2, slowly invert the tube 5-10 times to mix the liquid in the centrifuge tube and let it stand at room temperature for 2-4 minutes.

[0034] (3) After adding 350 μL FAPD3, slowly invert the tube 5-10 times to mix the liquid in the centrifuge tube.

[0035] (4) After centrifugation at 12000g for 5 min, aspirate the supernatant onto the adsorption column and centrifuge at 9000g for 30 s. Pour off the effluent and place the adsorption column into the same collection tube.

[0036] (5) Add 500 μL of W1 Buffer to the adsorption column and centrifuge at 9000 g for 30 seconds. Pour off the effluent and place the adsorption column in the same collection tube.

[0037] (6) Add 500 μL Wash Buffer to the adsorption column and centrifuge at 9000 g for 30 seconds. Pour off the effluent and place the adsorption column in the same collection tube.

[0038] (7) Place the empty adsorption column and collection tube into a centrifuge and centrifuge at 18,000 g for 1 min. Discard the effluent.

[0039] (8) Add 50 μL of Elution Buffer to the center of the adsorption membrane, let stand at room temperature for 2 min, and centrifuge at 18,000 g for 1 min. Determine the concentration of the obtained plasmid DNA solution using Nanodrop and store at -20°C or use in subsequent experiments.

[0040] 2. Obtaining the target gene containing the mutation site and amplifying the plasmid

[0041] 1. Construction of plasmid containing mutation site

[0042] Using the Subtilisin plasmid as a template, a 50 μL PCR system was constructed and PCR was performed according to the following system and conditions. The PCR product was subjected to nucleic acid electrophoresis at a voltage of 120 V for 30-50 min and observed under ultraviolet light. The nucleic acid electrophoresis results of the mutant plasmid are as follows: Figure 1 As shown, M: standard molecular weight marker; 1 is the M1 mutant plasmid; 2 is the M2 mutant plasmid; 3 is the M3 mutant plasmid; 4 is the M4 mutant plasmid; 5 is the M5 mutant plasmid; 6 is the 4M mutant plasmid. The length of the sequence fragment of subtilisin promoter-signal peptide-leader peptide-gene-terminator of Bacillus subtilisin is 1304bp, and the pP43NMK expression vector is 7412bp, so the length of the entire plasmid is 8716bp. Figure 1 The build was successful.

[0043] The PCR system is shown in the following table:

[0044]

[0045] The PCR conditions are shown in the following table:

[0046]

[0047]

[0048] The above PCR products were blunt-ended and phosphorylated using the Blunting and Kination Kit from Shanghai Bao Biotechnology Co., Ltd. The reaction system is as follows: Incubate at 37°C for 15 minutes and incubate at 70°C for 5 minutes to inactivate the enzyme. The blunt-ended and phosphorylated reaction system was stored at -20°C or used directly for ligation reactions. The PCR product blunt-ended and phosphorylated reaction system is shown in the following table:

[0049]

[0050] Take the reaction system for blunt-end and phosphorylation, add each component according to the following reaction system, mix well, incubate at 16°C for 1 hour, and then store the reaction system at -20°C or use it for the next transformation.

[0051] The reaction system is shown in the following table:

[0052] Components volume Blunting and Kination reaction system 2.5μL Ligation Solution 2.5μL Total volume 5μL

[0053] 2. E. coli cloning host transformation

[0054] The transformation steps of competent E.coli DH5α ligation system are as follows:

[0055] (1) Thaw the competent cells stored at -80°C on ice and dispense 100 μL of the cells into pre-cooled 1.5 mL sterile centrifuge tubes.

[0056] (2) Take 2 μL of the ligation system and add it to the dissolved competent cells. Flick twice to mix well and place on ice for 30 min.

[0057] (3) Heat shock at 42°C for 45-90 seconds and quickly place on ice for 2-5 minutes.

[0058] (4) Add 1 mL of LB medium and incubate at 37°C, 200 rpm, on a shaking platform for 1 h.

[0059] (5) Take 50-100 μL of transformed cells and spread them on a plate containing 50 μg / mL Amp resistance, and culture them upside down in a 37°C incubator for 12-16 hours.

[0060] 3. Screening of positive clones

[0061] In the clean bench, pick 3-5 single colonies from the plate, tap them onto the solid LB plate containing Amp resistance, mark them, invert and culture at 37℃ for 12-16h, rinse the corresponding gun tip in 1mL Amp resistance medium for a few times, and incubate at 37℃, 250rpm, shaker for 8-9h. Take 500μL of the incubated bacterial solution for sequencing, and add 500μL of 50% glycerol to the remaining 500μL bacterial solution for seed preservation, and store at -80℃. When the company returns the sequencing results, use SnapGene software to align the sequences. Pick the colony corresponding to the bacterial solution with the correct sequence alignment, inoculate it in 10mL Amp LB liquid culture medium, shake it at 37℃, 250rpm overnight, and extract the plasmid the next day. The specific steps are the same as the specific operations of plasmid DNA extraction in step 1 above. Continue to construct according to the same method based on the correct positive clone until all mutation sites are constructed.

[0062] Example 2 Obtaining the target protein of mutants M1, M2, M3, M4, M5, 4M and wild-type Subtilisin (WT)

[0063] 1. Transformation of mutants M1, M2, M3, M4, M5, and 4M into Bacillus subtilis expression hosts

[0064] 1. The types and amounts of reagents required for competent cell preparation are as follows:

[0065] SPI-I Salts Solution, its components and contents are shown in the following table:

[0066] SPI-I Salts Solution Components content <![CDATA[ddH2O]]> 100mL <![CDATA[(NH4)2SO4]]> 0.4g <![CDATA[K2HPO4·3H2O]]> 2.8g <![CDATA[KH2PO4]]> 1.2g Trisodium citrate dihydrate 0.2g

[0067] SPI-I Salts Solution was sterilized at 121°C for 20 min.

[0068] SPI-II Salts Solution, its components and contents are shown in the following table:

[0069] SPI-II Salts Solution Components content <![CDATA[ddH2O]]> 100mL <![CDATA[MgSO4·7H2O]]> 0.04g

[0070] SPI-II Salts Solution was sterilized at 121°C for 20 min.

[0071] 100×CAYE Solution, its components and contents are shown in the following table:

[0072]

[0073]

[0074] 100×CAYE Solution was sterilized at 121°C for 20 min.

[0075] SPI Medium, its components and contents are shown in the following table:

[0076] SPI Medium Components content <![CDATA[ddH2O]]> 20mL SPI-I Salts Solution 9.8mL SPI-II Salts Solution 9.8mL Glucose 200ul 100×CAYE 200ul

[0077] SPII Medium, its components and contents are shown in the following table:

[0078] SPII Medium Components content <![CDATA[ddH2O]]> 6mL SPI Medium 5.88mL <![CDATA[50mM CaCl2]]> 60ul 250mM MgCl2 60ul

[0079] Glucose (50% W, sterilized at 115℃ for 20 min)

[0080] 100×EGTA Solution: 10mM EGTA solution. When dissolving, add a small amount of NaOH to pH 8.0.

[0081] 2. Preparation of competent cells of WB600 Bacillus subtilis

[0082] (1) Activate the strain: Striking the frozen WB600 Bacillus subtilis host on an LB plate for 12 h.

[0083] (2) A single WB600 colony with good growth was selected and inoculated into 3 mL of LB medium and cultured in a shaking incubator at 37°C for 12 h.

[0084] (3) Take 100 μl of bacterial solution and inoculate it into 6 mL of SPI Medium. Incubate it at 37°C in a shaking incubator. When the OD600 of the culture reaches 0.85-0.95, take 300 μl and inoculate it into 3 mL of SPII Medium. Incubate it at 37°C in a shaking incubator at 100 rpm for 1.5 h.

[0085] (4) Add 20 μl of 100× EGTA solution, incubate at 37°C with a shaker at 100 rpm for 10 min, and dispense into 1.5 mL precooled centrifuge tubes, 500 μl per tube.

[0086] 3. Transformation of recombinant plasmid

[0087] (1) Add 10 μL of plasmid to the centrifuge tube containing the competent cells, mix gently, and incubate at 37°C at 100 rpm for 30 min.

[0088] (2) Transfer the centrifuge tube to a shaking incubator at 250 rpm and incubate at 37°C for 1.5 h.

[0089] (3) Collect the cells by centrifugation at 4000 rpm, discard part of the supernatant, keep 100 μL of the resuspended cells, spread on LB medium plates (containing 100 μg / mL kanamycin), and culture at 37°C for 12 h.

[0090] 2. Fermentation, expression and purification of mutant and wild-type subtilisin (WT)

[0091] (1) Pick a single plump colony after transformation and inoculate it into 6 mL 2×YT medium (containing 100 μg / mL Kanamycin) and culture it at 37°C and 200 rpm for 12 h.

[0092] (2) Inoculate the culture in (1) into 50 mL of 2×YT medium (containing 100 μg / mL Kanamycin, 5 mM CaCl2) to OD600 = 0.03, and culture in a baffled conical flask at 37°C and 200 rpm for 20-24 h.

[0093] (3) The culture in (2) was centrifuged and the supernatant was collected (4000 g, 15 min, 4°C).

[0094] (4) Slowly add 3 volumes of pre-cooled 95% ethanol to the supernatant and stir at 4°C for 1-2 hours. Centrifuge (18000g, 20 min, 4°C) and discard the supernatant. Invert the centrifuge cup on a paper towel and allow the precipitate to dry at room temperature for 15 minutes.

[0095] (5) Resuspend the precipitate with 10 mL of nickel column purification equilibrium solution, centrifuge (18000 g, 20 min, 4°C), retain the supernatant, and remove the insoluble matter.

[0096] (6) The supernatant was filtered through a 0.22 μm microporous filter membrane to remove insoluble impurities and particles, and then the protein was purified using a HisTrap HP1 mL column. The purified recombinant protein was eluted using a linear gradient imidazole elution buffer using a nickel column.

[0097] (7) The purity and molecular weight of the purified protein were analyzed by SDS-PAGE. The electrophoretic diagrams of the purified mutant and wild-type subtilisin (WT) proteins are shown in Figure 2. Figure 2 As shown, M is Protein Marker; 1 is wild-type Subtilisin (WT), 2 is M1 mutant, 3 is M2 mutant; 4 is M3 mutant, 5 is M4 mutant, 6 is M5 mutant, and 7 is 4M mutant.

[0098] Thus, in this example, mutants M1, M2, M3, M4, M5, 4M and wild-type Subtilisin (WT) were finally expressed and purified to obtain target proteins.

[0099] Example 3 Application of the hexapeptide Suc-Ala-Ala-Pro-Phe-Ala-Phe-NH2 in synthesis

[0100] In this example, the tetrapeptide Suc-Ala-Ala-Pro-Phe and the dipeptide NH2-Ala-Phe-NH2 were used as substrates, and the substrates were catalyzed by M1, M2, M3, M4, M5, 4M mutants and wild-type Subtilisin (WT) to generate the hexapeptide Suc-Ala-Ala-Pro-Phe-Ala-Phe-NH2, and the peptide bond synthesis ability of the two was investigated. The specific method is as follows:

[0101] The components and dosage of the reaction system are shown in the following table:

[0102]

[0103] Add the sample as above and react at 40°C, 700 rpm for 8 hours. After the reaction, UPLC-MS was performed to detect the product. The UPLC-MS detection chart of the product Suc-Ala-Ala-Pro-Phe-Ala-Phe-NH2 generated in the pure water phase of the mutant is shown in the figure below. Figure 3 As shown, a is the product standard, the enzyme used in group b is the M1 mutant, the enzyme used in group c is the M2 mutant, the enzyme used in group d is the M3 mutant, the enzyme used in group e is the M4 mutant, the enzyme used in group f is the M5 mutant, and the enzyme used in group g is the 4M mutant. The hexapeptide product Suc-Ala-Ala-Pro-Phe-Ala-Phe-NH2 was not detected in the WT sample, while the mutants M1, M2, M3, M4, M5, and 4M samples were successfully detected. The mutants M1, M2, M3, M4, M5, and 4M achieved the "one-step dehydration condensation" catalysis between natural amino acids in pure water phase to form peptide bonds.

[0104] Therefore, the Subtilisin mutant of the present invention can catalyze the synthesis of peptide bonds from natural amino acids in a pure water phase environment in one step, meeting the industrial production needs of peptide synthesis, and has important significance and value.

[0105] The above embodiments are only used to illustrate the present invention, rather than to limit the present invention. Those skilled in the relevant technical field may make various changes or modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions should also belong to the scope of the present invention and should be defined by the claims.

Claims

1. A subtilisin mutant capable of catalyzing peptide bond synthesis in a pure aqueous environment in one step, characterized in that: Its amino acid sequence is shown in one of SEQ ID NO.

6.

2. A gene encoding the subtilisin mutant of claim 1 that is capable of catalyzing peptide bond synthesis in a pure water phase environment in one step.

3. A recombinant vector, characterized in that: It comprises a nucleotide sequence as shown in one of SEQ ID NO.

12.

4. A host cell, characterized in that It comprises the recombinant vector according to claim 3.

5. Use of the Bacillus subtilisin mutant capable of catalyzing peptide bond synthesis in a pure water phase environment according to claim 1 in the preparation of polypeptide products.

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