High-thermal-stability lysostaphin mutant and application thereof

Abstract

The invention provides a series of lysostaphin mutants with high thermal stability as well as a preparation method and application thereof, and belongs to the technical fields of biology and enzyme engineering. On the basis of structural analysis, in combination with various thermal stability analysis software, by taking thermal stability as an evaluation index, wild-type lysostaphin is rationally designed and modified, and a series of mutants with obviously improved thermal stability are obtained. Wherein the enzyme activity loss of the beneficial mutant at 60 DEG C is reduced by 5.8-37.9%, the Tm value is increased by 0.3-7.0 DEG C, and the enzyme activity is equivalent to that of a wild type. Therefore, compared with a wild type, the lysostaphin mutant provided by the invention has better thermal stability and has a better industrial application scene.

Description

Technical Field

[0001] The invention belongs to the fields of biotechnology and enzyme engineering, and relates to a series of lysostaphin mutants with high thermal stability and preparation methods and applications thereof. Background Art

[0002] Lysostaphin (LST) is a zinc-dependent peptidase synthesized by Staphylococcus aureus. It can effectively cut the peptide bond formed by the second and third glycine in the glycine pentapeptide bridge of the peptidoglycan cross-linking structure of the bacterial cell wall, thereby playing a role in lysing and killing bacteria. Therefore, the protein is specific and can specifically kill Staphylococcus aureus, Staphylococcus epidermidis and hemolytic Staphylococcus. At present, domestic and foreign researchers have successfully expressed this enzyme in a variety of engineered bacteria using genetic recombination technology, and a batch of new drug clinical trials for lysostaphin have been approved in China. It can be used in medicine, beauty, food, mucosal disinfection, oral care, private care, veterinary drugs, pet care and other fields, and has strong market potential.

[0003] The thermal stability of enzymes characterizes the ability of enzyme molecules to maintain their own structure and activity at high temperatures, and is an important criterion for evaluating the application value of enzymes. Thermally stable enzymes have the advantages of increasing chemical reaction rates, simplifying processes, reducing costs, improving product quality, and maintaining stable activity and storage resistance. Therefore, improving the thermal stability of lysostaphin has always been a hot topic in scientific research and production, and is also an important factor limiting its expansion of application scenarios.

[0004] Based on the above background, the present invention obtains a series of highly thermally stable lysostaphins through rational design, laying a foundation for subsequent production and expansion of application scenarios. Summary of the invention

[0005] The purpose of the present invention is to provide a series of highly stable lysostaphin mutants, as well as preparation methods and applications thereof, so as to solve the existing thermal stability problem.

[0006] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0007] The invention provides a lysostaphin mutant with high thermal stability. The amino acid sequence of the wild-type lysostaphin is shown in SEQ ID NO.1. The mutant comprises one or more of the following mutation sites: K45Y, Y85F, N120G, M131L, S190A and 190W.

[0008] Furthermore, the lysostaphin includes but is not limited to the sequence shown in SEQ ID NO.1, and also includes a truncated form of the sequence.

[0009] Furthermore, the mutant comprising multiple mutation sites is preferably selected from a combination of the following sites, including but not limited to the following mutants: K45Y / Y85F; K45Y / N120G; K45Y / S190A; Y85F / S190A; Y85F / S190W; Y85F / M131L; Y85F / N120G; N120G / S190A; K45Y / Y85F / N120G; Y85F / N120G / S190A; Y85F / M131L / S190A; Y85F / N120G / M131L.

[0010] The present invention also provides an expression vector of the mutant, including but not limited to pET30a + .

[0011] The invention also provides a recombinant strain expressing the mutant.

[0012] Furthermore, the recombinant strain is preferably Escherichia coli, but is not limited to Escherichia coli, and may also be other prokaryotic cells or eukaryotic cells.

[0013] Furthermore, the eukaryotic cells include Pichia pastoris; the prokaryotic cells include Lactococcus lactis, Bacillus subtilis, Staphylococcus and Streptococcus.

[0014] The present invention also provides a method for preparing the mutant, comprising the following steps: constructing a mutant expression vector by site-directed mutagenesis, transferring the mutant expression vector into competent Escherichia coli BL21 (DE3) and inducing expression, collecting the bacteria by centrifugation and ultrasonically crushing them, taking the supernatant and purifying it by Ni column, and concentrating and desalting it by ultrafiltration tube.

[0015] The present invention also provides the use of the mutant, polynucleotide molecule, recombinant expression vector and host cell in the field of anti-staphylococcus.

[0016] Furthermore, the staphylococci include but are not limited to Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugtownensis, Staphylococcus saprophyticus and Staphylococcus aureus.

[0017] The beneficial effect is that the present invention is based on structural analysis, combined with systematic testing of various thermal stability design software, and uses the thermal stability of lysostaphin as an indicator to design mutants, and obtains a series of mutants with improved Tm values. Among them, the beneficial mutants screened out have a 5.8%-37.9% reduction in enzyme activity loss at 60°C, and a Tm value increase of 0.3-7.0°C. And compared with the wild-type lysostaphin, the enzyme activity of the mutant is basically the same. It provides convenience for subsequent storage, transportation and expansion of application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Single site mutant enzyme activity assay and thermal stability assay. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiment of the present application will be clearly and completely described below in combination with the embodiment of the present application. Obviously, the described embodiment is only a part of the embodiment of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present application.

[0020] In this embodiment, the various biological reaction reagents, reaction facilities, hardware and software facilities involved are only used to select reagents for a method for preparing lysostaphin in this application, and are displayed as the optimal implementation scheme of this technology. However, it does not mean that this application only uses the materials provided in the following embodiments to express the selection of reagents, reaction facilities, hardware and software facilities. As long as the various biological reaction reagents, reaction facilities, hardware and software facilities that can be replaced by those skilled in the art can achieve the technical purpose of this application, they can all be used in the application of this research and should be protected.

[0021] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail with reference to the embodiments below.

[0022] The implementation method of each preparation step will be described in detail below.

[0023] The present invention predicts the flexible region, ΔG value, mutation site and conservation of lysostaphin by using software such as RESQ, Rosetta ddg, Fireprot and ConSurf, respectively, and screens mutants by multi-data cross-analysis. Finally, 30 mutants are selected for experimental verification, and the mutant sites are summarized as shown in Table 1.

[0024] Table 1: Summary of mutation sites

[0025]

[0026]

[0027] Note: The larger the B-factor value, the higher the flexibility; the higher the color value, the more conservative;

[0028] Example 1: Construction of mutant expression strains

[0029] The lysostaphin gene (amino acid sequence shown in SEQ ID NO.1, nucleotide sequence shown in SEQ ID NO.2) was used as a template to design primers for mutation sites, and the primer sequences are shown in Table 2. K45Y Taking the mutant expression strain as an example, the construction process of the mutant expression strain is briefly described. + As template, PCR amplification was performed using primer pair 45Y-F / 45Y-R to obtain a band of 5994 bp in size. After gel excision and recovery, the band was amplified according to C112-01. II One Step Cloning Kit instructions for fragment recombination, then transformed into E. coli competent BL21 (DE3) competent cells and coated on LB plates containing kan resistance. Correct transformants were screened by colony PCR and sequencing reactions.

[0030] Table 2: Summary of primer sequences

[0031]

[0032]

[0033] Note: 39-r is the downstream universal primer of N39F-f and N39P-f; 67-r is the downstream universal primer of G67M-f and G67Q-f; 74-r is the downstream universal primer of V74K-f and V74T-f; 190-r is the downstream universal primer of S190A-f and S190W-f.

[0034] Example 2: Expression and purification of lysostaphin

[0035] The mutant expression strain with correct sequencing was inoculated into 5 ml LB liquid medium (containing 50ug / mL kan) and cultured overnight at 37°C and 220rpm. The strain was transferred into 100 ml LB liquid medium at an inoculation ratio of 2% and cultured until OD 600Add IPTG at a final concentration of 0.5mM to induce expression at 0.6-0.8, and culture at 16°C, 220rpm for 16h. After centrifugation, take 20ml A phase buffer (20mM Tris-HCl, 150mM NaCl, pH7.5) for suspension, mix well and then perform ultrasonic disruption. Ultrasonic conditions: power 250W, working time 2s, interval time 4s, time lasting about 10-15min, until the bacterial solution is completely clear. Centrifuge the bacterial solution after ultrasonication (12000rpm, 30min) and separate the supernatant and precipitate. The supernatant was filtered through a 0.22 μm microporous membrane to remove insoluble impurities and particles, and then the protein was purified using a HisTrap HP 1 mL column, and the purified lysostaphin was eluted with a linear gradient imidazole using a B phase buffer (20 mMTris-HCl, 150 mM NaCl, 500 mM imidazole, pH 7.5, filtered through a 0.22 μm membrane, and ultrasonicated for 15 min). The eluted protein was desalted and concentrated using a 10 kDa ultrafiltration tube, and the protein concentration was determined using a micro-spectrophotometer K5600.

[0036] Example 3: Enzyme activity assay

[0037] Strain culture: Activate the Staphylococcus aureus stored at -80℃ on an LB plate, pick a single colony and inoculate it into 5ml LB liquid medium, and culture it at 220℃ for 16h. Collect the bacteria by centrifugation at 6000rpm for 5min, wash the bacteria once with 5ml A phase buffer, and finally set the OD value of the bacteria to 1.0±0.1 for use;

[0038] Protein treatment: The mutant protein concentration was adjusted to 1 mg / ml using phase A buffer, and 100 μL was heat treated at 25°C, 37°C and 60°C for 30 min. The treated protein was centrifuged (12000 rpm, 10 min), and the supernatant was used for enzyme activity determination;

[0039] Enzyme activity determination: Use a pipette to take 190μL of diluted bacterial solution into a 96-well plate, then use a pipette to take 10μL of treated protein solution and add it to the bacterial solution, and measure the change in absorbance at 450nm. When the protein is added to the bacterial solution, it is recorded as 0, and data is recorded every 15s. The total reaction time is 10min. The control group is wild-type LST, the blank control is to replace the protein with A phase buffer, and the experimental group is LST mutant protein. Because the initial OD value is unstable, the difference between OD at 1min and 10min is taken as the basis for judging the enzyme activity, that is, the △OD value. Enzyme activity loss ratio: (△OD 37℃ -△OD 60℃ ) / △OD 37℃ *100%, including OD60℃ Indicates the △OD value of the protein treated at 60℃, △OD 37℃ Indicates the △OD value of the protein treated at 37℃. △Enzyme activity loss: wild type (WT) enzyme activity loss ratio minus mutant enzyme activity loss ratio. The enzyme activity determination results of 30 mutants are shown in Table 3 (Note: The OD values ​​in the table are the difference between the control group or experimental group and the blank control group).

[0040] Table 3: Enzyme activity assay of single-site mutants

[0041]

[0042]

[0043] Results: The thermostability of 30 mutants was characterized by enzyme activity assay using wild-type LST as control. The data showed that the enzyme activity or thermostability of most mutants was inferior to that of wild-type LST, but the enzyme activities of K45Y, Y85F, N120G, M131L, S190W and S190A were comparable to those of the wild-type, and the thermostability was improved to varying degrees, with enzyme activity loss ratios reduced by 2.8%, 15.5%, 17.3%, 12%, 23.8% and 21.8%, respectively.

[0044] Example 4: Construction of double-site superposition mutant strain and enzyme activity determination

[0045] In order to obtain a mutant with better thermal stability, the effective mutation sites in Example 3 were superimposed. K45Y ,Y85F Take LST as an example, the specific operation is as follows: K45Y -pET30a + As a template, PCR amplification was performed using primer pair Y85F-f / Y85F-r, followed by recombination according to Example 1, and finally a mutant expression vector with correct sequencing was obtained. The correct expression strain was induced to express according to Example 3 and the quantitative mutant protein was purified for enzyme activity determination. A total of 14 double-site superposition mutant strains were constructed, and the enzyme activity results are shown in Table 4 below.

[0046] Table 4: Enzyme activity assay of double-site superposition mutants

[0047]

[0048]

[0049] Results: The data showed that the thermal stability of the double-site superimposed mutants was generally improved compared with the wild-type LST, among which K45Y / Y85F, K45Y / N120G, K45Y / S190A, N120G / S190A, Y85F / S190A, Y85F / N120G, Y85F / M131L and Y85F / S190W had more obvious improvement in thermal stability, and the enzyme activity loss was reduced by 38.0%, 26.2%, 32.9%, 31.1%, 28.4%, 27.1%, 36.2% and 28.4% respectively compared with the wild-type LST, but their enzyme activities were basically equivalent to that of the wild type. Moreover, compared with the corresponding single-site mutants in Table 3 , the thermal stability of the double-site superposition mutants was further improved, for example, the enzyme activity loss of K45Y / Y85F was reduced by 38.0%, while the enzyme activity loss of K45Y or Y85F was reduced by 2.8% and 15.5%, respectively, indicating that the effective site superposition played a synergistic role.

[0050] Example 5: Construction of three-site superposition mutant strain and enzyme activity determination

[0051] Since the results of Example 4 show that the superposition of effective sites can further enhance the thermal stability of LST, a triple-site superposition mutant was constructed based on the double-site mutant in order to obtain a mutant with better thermal stability. K45Y,Y85F,M131L Taking the construction of expression vector as an example, other mutants were constructed in sequence. The specific operation is as follows: K45Y,Y85F -pET30a + As a template, the primer pair M131L-f / M131L-r was used to amplify the fragment, and the recombination, transformation and screening were carried out in the same manner as in Example 4, and finally the LST with correct sequencing was obtained. K45Y,Y85F,M131L The other three-site superposition mutant expression strains were constructed in the same way, and a total of 16 mutants were obtained, among which the mutant LST K45Y,N120G,S190W and LST K45Y,Y85F,M131L Due to clarification after induction, no enzyme activity data was obtained. The reason may be that the expression of the mutant caused toxicity to E. coli cells or had the activity of cleaving its cell wall, resulting in failure to grow or clarification. The enzyme activity and thermal stability test data of the other 14 mutants are shown in Table 5 below.

[0052] Table 5: Enzyme activity and thermal stability determination of three-site superposition mutants

[0053]

[0054]

[0055] Results: The data showed that the thermal stability of most three-site superposition mutants was improved to varying degrees compared with the wild-type LST, among which K45Y / Y85F / N120G, Y85F / M131L / S190A, Y85F / N120G / M131L and Y85F / N120G / S190A had a more obvious improvement in thermal stability, with enzyme activity loss reduced by 28.1%, 31.9%, 32.9% and 25.9% respectively compared with the wild-type. However, compared with the corresponding double-site mutants in Table 4, there was no further improvement, for example: the enzyme activity loss of Y85F / M131L / S190A was reduced by 31.9%, and the enzyme activity loss of Y85F / M131L was reduced by 36.2%, indicating that superposition of mutations on the basis of double-site mutants did not produce a superposition effect. Therefore, the site superposition was no longer continued.

[0056] Example 6: Tm value determination

[0057] Tm values ​​were measured for the mutants containing effective sites K45Y, Y85F, N120G, M131L, S190W and S190A in Example 3, the mutants containing effective mutation sites K45Y / Y85F, K45Y / N120G, K45Y / S190A, N120G / S190A, Y85F / S190A, Y85F / N120G, Y85F / M131L and Y85F / S190W in Example 4, and the mutants containing mutation sites K45Y / Y85F / N120G, Y85F / M131L / S190A, Y85F / N120G / M131L and Y85F / N120G / S190A in Example 5. The instrument used was UNcle Client (V4.01). The Tm values ​​of the mutants are shown in Table 6 below.

[0058] Table 6 Tm values ​​of mutants

[0059] Mutants T-value △Tm value WT 62.3 K45Y 62.6 0.3 Y85F 63.3 1.0 N120G 62.6 0.3 M131L 62.8 0.5 S190W 63.7 1.4 S190A 63.4 1.1 K45Y / Y85F 68.9 6.6 K45Y / N120G 62.6 0.3 K45Y / S190A 63.7 1.4 N120G / S190A 59.9 -2.4 Y85F / S190A 63.3 1.0 Y85F / N120G 63.1 0.8 Y85F / M131L 69.3 7.0 Y85F / S190W 63.5 1.2 K45Y / Y85F / N120G 63.4 1.1 Y85F / M131L / S190A 63.5 1.2 Y85F / N120G / M131L 63.7 1.4 Y85F / N120G / S190A 63.1 0.8

[0060] Results: Through the determination of the Tm values ​​of 18 mutants, the Tm values ​​of most mutants increased by 0.3-7.0°C, among which the mutants containing the mutation sites K45Y / Y85F and Y85F / M131L showed the most obvious improvement in thermal stability, increasing by 6.6°C and 7.0°C, respectively. The results of Tm value determination were basically consistent with the results of enzyme activity determination, and the two confirmed each other.

[0061] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A highly thermostable lysostaphin mutant, characterized in that: The amino acid sequence is shown in SEQ ID NO.1, and one or more of the following mutation sites exist: K45Y, Y85F, N120G, M131L, S190A and S190W.

2. The lysostaphin mutant according to claim 1, characterized in that The mutation sites are selected from the following sites, including but not limited to the following mutants: K45Y / Y85F; K45Y / N120G; K45Y / S190A; Y85F / S190A; Y85F / S190W; Y85F / M131L; Y85F / N120G; N120G / S190A; K45Y / Y85F / N120G; Y85F / N120G / S190A; Y85F / M131L / S190A; Y85F / N120G / M131L.

3. A nucleic acid encoding the lysostaphin mutant according to claim 1 or 2.

4. An expression vector containing the encoding nucleic acid as claimed in claim 3, preferably the expression vector is a prokaryotic expression vector, including but not limited to pET30a + .

5. A recombinant strain containing the encoding nucleic acid according to claim 3 or the expression vector according to claim 4, specifically, it is a fungus or a bacterium.

6. The recombinant strain according to claim 5, characterized in that The fungus is Pichia pastoris, and the bacteria are Lactococcus lactis, Bacillus subtilis, Staphylococcus and Streptococcus or Escherichia coli.

7. The recombinant strain according to claim 6, characterized in that The Escherichia coli was BL21 (DE3).

8. Use of the lysostaphin mutant according to claim 1 or 2, comprising the encoding nucleic acid according to claim 3, the expression vector according to claim 4, and the recombinant strain according to claim 5 or 6 in the preparation of an anti-staphylococcal product.

9. The use according to claim 8, characterized in that The staphylococci include, but are not limited to, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugtownensis, Staphylococcus saprophyticus or Staphylococcus aureus.

10. The use according to claim 8 or 9, characterized in that: The products are food, feed, detergents, medicines, cosmetics and medical devices.

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