A mutant of Streptomyces rhodochrous phospholipase A2 and preparation method thereof

Abstract

The invention discloses a phospholipase A2 mutant of Streptomyces purpurogenus and a preparation method thereof, and belongs to the field of biotechnology. The invention obtains 6 mutants with single-site mutations and 5 combined mutants; compared with the wild type, the enzyme activities of the mutants N21E, Q47K, F53H, Y68H, I84L, Q116M, N21E / I84L, Q47K / I84L, I84L / Q116M, N21E / I84L / Q116M and Q47K / I84L / Q116M are all improved, which are increased by 32.3%, 20.2%, 27.9%, 20.5%, 69.7%, 60.5%, 106.6%, 92.9%, 213.2%, 406.5% and 304.3% respectively. The enzyme activity of the phospholipase A2 mutant obtained by the invention is significantly improved, and has great industrial application potential and economic value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a mutant of Streptomyces rhodochrous phospholipase A2 with improved enzyme activity and a preparation method thereof. Background Art

[0002] Phospholipids are the main components of all biological membranes. They are widely present in nature, play a variety of roles, and have important physiological significance. Due to the unique physical and chemical properties and nutritional value of phospholipids, phospholipids have been widely used in industries such as food, health products, medicine and feed. However, in most applications, naturally occurring phospholipids either lack structural diversity or are insufficient in quantity; therefore, new methods must be developed to synthesize new molecules or modify the natural structure of phospholipids.

[0003] Enzymatic modification is an important direction for the preparation of functional phospholipids. Phospholipase A2 (PLA2, EC 3.1.1.4) hydrolyzes the ester bond at the sn-2 position of phospholipids to generate free fatty acids and corresponding lysophospholipids. Compared with natural lecithin, lecithin prepared by PLA2 not only shows enhanced emulsification properties, but also can form stable emulsions under various process conditions. Therefore, lysophosphatidylcholine has a wide range of applications in industrial fields such as food, cosmetics and pharmaceuticals. In particular, PLA2 can be widely used in enzymatic degumming, which is a key process in the refining of vegetable oils and other edible oils.

[0004] At present, phospholipase A2 is sometimes still limited in industrial applications. For example, natural phospholipases do not meet the requirements of practical applications in terms of activity, specificity and stability, and the phospholipase yield of natural production bacteria is not high. Therefore, it is urgent to obtain high-yield phospholipases that meet food safety for functional oil processing applications. Transforming the enzyme through protein engineering is the main strategy to solve this problem. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a phospholipase A2 mutant of Streptomyces purpurogenus with improved enzyme activity and a preparation method thereof. Among them, the enzyme activities of the mutants N21E, Q47K, F53H, Y68H, I84L, Q116M, N21E / I84L, Q47K / I84L, I84L / Q116M, N21E / I84L / Q116M and Q47K / I84L / Q116M are all improved, which are increased by 32.3%, 20.2%, 27.9%, 20.5%, 69.7%, 60.5%, 106.6%, 92.9%, 213.2%, 406.5% and 304.3% respectively; the present invention obtains a phospholipase A2 mutant with high enzyme activity, which has great industrial application potential and economic value.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A mutant of phospholipase A2 from Streptomyces purpurogenus, wherein the amino acid sequence of SEQ ID NO.1 is obtained by any one of the following mutations:

[0008] At least one of N21E, Q47K, F53H, Y68H, I84L and Q116M; wherein N21E, i.e. the 21st amino acid is mutated from N to E, and the same applies to the others;

[0009] Furthermore, the mutant of phospholipase A2 of Streptomyces rhodochrous is obtained by subjecting the amino acid sequence of SEQ ID NO.1 to any of the following mutations:

[0010] or N21E; or Q47K; or F53H; or Y68H; or I84L; or Q116M; or N21E / I84L; or Q47K / I84L; or I84L / Q116M; or N21E / I84L / Q116M; or Q47K / I84L / Q116M.

[0011] The gene sequence encoding the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.2.

[0012] A gene encoding the phospholipase A2 mutant of Streptomyces rhodochrous.

[0013] Preferably, a mutant N21E / I84L / Q116M of phospholipase A2 from Streptomyces rhodochrous with improved enzyme activity has an amino acid sequence as shown in SEQ ID NO. 3. The 21st, 84th and 116th amino acids of the mutant are mutated from asparagine N, isoleucine I and glutamine Q to glutamic acid E, leucine L and methionine M, respectively.

[0014] A gene encoding a phospholipase A2 mutant N21E / I84L / Q116M of Streptomyces rhodochrous with improved enzyme activity, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0015] The mutant-related biological material is any one or more combinations of the following biological materials:

[0016] (a) an expression cassette containing the above encoding gene;

[0017] (b) a recombinant expression vector containing the above encoding gene;

[0018] (c) a recombinant expression vector containing the expression cassette described in (a);

[0019] (d) a recombinant microorganism containing the above encoding gene;

[0020] (e) a recombinant microorganism containing the expression cassette described in (a);

[0021] (f) A recombinant microorganism containing the recombinant expression vector described in (b) or (c).

[0022] Furthermore, the expression elements used in the expression cassette in (a) are: P43 and PgntK dual promoters, AprE signal peptide and Ter terminator.

[0023] The sequence of the PgntK promoter is shown in bp 1-182 in SEQ ID NO.5.

[0024] The AprE signal peptide is derived from Bacillus subtilis, and its amino acid sequence is shown in 1-29aa in GenBank No. WP_015383267.1; the gene sequence encoding the AprE signal peptide is shown in 183-269bp in SEQ ID NO.5.

[0025] Furthermore, the starting vector of the recombinant expression vector in (b) and (c) is a pBE series vector, etc.; preferably a pBE2-R vector, a pBE vector containing a dual promoter of P43 and PgntK, etc.

[0026] Further, the host microorganism corresponding to the recombinant microorganism in (d), (e), and (f) is selected from prokaryotes or yeasts, etc.; the prokaryotes include bacteria such as Bacillus; the yeasts include yeasts such as Pichia pastoris. More specifically, the prokaryotes are Bacillus subtilis and Bacillus amyloliquefaciens, specifically Bacillus subtilis ATCC6051 and Bacillus amyloliquefaciens LB1ba02; the yeast is Pichia pastoris GS115.

[0027] The application of the mutant, coding gene or mutant-related biological materials in the preparation of phospholipase A2 mutants with high enzyme activity.

[0028] Application of the above mutants, encoding genes or mutant-related biological materials in the production of functional phospholipids.

[0029] Furthermore, the mutants, encoding genes or mutant-related biomaterials are used in the fields of health care products, food, medicine, daily chemicals and oil refining.

[0030] A method for obtaining the mutant comprises the following steps: performing site-directed mutagenesis on the gene encoding the amino acid sequence of Streptomyces purpurogenous phospholipase A2 as shown in SEQ ID NO.1 by designing primers containing mutation sites, and then expressing the site-directed mutagenesis to obtain the mutant of Streptomyces purpurogenous phospholipase A2.

[0031] Furthermore, primers containing mutation sites were designed to introduce mutations into the gene encoding the phospholipase A2 of Streptomyces purpurogenum whose amino acid sequence is shown in SEQ ID NO.1. After correct sequencing, the gene was transformed into Bacillus subtilis ATCC6051 for expression to obtain a mutant of the phospholipase A2 of Streptomyces purpurogenum.

[0032] The specific implementation steps of the present invention are as follows:

[0033] 1. Construction of a recombinant expression vector for a phospholipase A2 mutant: 1) The wild-type gene of phospholipase A2 synthesized from Streptomyces rhodopsus after codon optimization and the AprE signal peptide from Bacillus subtilis were connected to a pBE vector containing a dual promoter of P43 and PgntK to construct an expression plasmid pBE-P43-PgntK-AprE-SvPLA2WT; 2) The wild-type phospholipase A2 was site-directedly mutated by overlapping PCR amplification to obtain the corresponding phospholipase A2 mutant expression plasmid.

[0034] 2. Construction of phospholipase A2 mutant recombinant engineering bacteria and preparation of phospholipase A2 with high enzyme activity: The phospholipase A2 mutant expression plasmid is transformed into Bacillus subtilis ATCC6051 to obtain recombinant engineering bacteria; then the phospholipase A2 mutant is obtained by fermentation.

[0035] 3. Characterization of the enzymatic properties of the phospholipase A2 mutant: The enzymatic properties of the mutant were determined by microwell colorimetry using 2-thiohexadecanoylethylphosphocholine as a substrate.

[0036] Compared with the prior art, the present invention has the following advantages and effects:

[0037] The present invention obtains 11 mutants with improved enzyme activities, including 6 mutants with single-site mutations and 5 combined mutants; compared with the wild type, the enzyme activities of the mutants N21E, Q47K, F53H, Y68H, I84L, Q116M, N21E / I84L, Q47K / I84L, I84L / Q116M, N21E / I84L / Q116M and Q47K / I84L / Q116M are all improved, which are increased by 32.3%, 20.2%, 27.9%, 20.5%, 69.7%, 60.5%, 106.6%, 92.9%, 213.2%, 406.5% and 304.3% respectively. The enzyme activities of the mutants obtained by the present invention are significantly improved, and the improvement of the enzyme activity of phospholipase A2 further improves the industrial utilization value of the enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the PCR electrophoresis diagram of wild phospholipase A2 and mutant expression vector colonies; wherein, M is DL5000 DNA Marker, and lanes 1-12 are: WT, N21E, Q47K, F53H, Y68H, I84L, Q116M, N21E / I84L, Q47K / I84L, I84L / Q116M, N21E / I84L / Q116M and Q47K / I84L / Q116M.

[0039] Figure 2 It is a bar graph showing the relative enzyme activities of wild-type phospholipase A2 and mutants. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. The operating steps or conditions not particularly noted in the following examples can be performed with reference to conventional techniques.

[0041] In the embodiment, the phospholipase A2 used is derived from Streptomyces violaceoruber, and its amino acid sequence is shown in SEQ ID NO.1. The nucleotide sequence of the encoding gene obtained after codon optimization is shown in SEQ ID NO.2. At the same time, a 6*His tag sequence (CACCACCACCACCACCAC) is introduced at the 3' end, which is synthesized by a commercial company.

[0042] Example 1

[0043] Construction of expression vectors for wild-type phospholipase A2 and its mutants

[0044] (1) Construction of wild phospholipase A2 expression vector

[0045] Using pBEP43-Pcd-SamyQ-SplB-Fc plasmid as a template, the Pcd-SamyQ-SplB-Fc-Ter part in the template was replaced with PgntK-AprE-SvPLA2WT-6*His-Ter (sequence see SEQ ID NO.5, wherein 1-182bp: PgntK, 183-269bp: AprE signal peptide, 270-635bp: SvPLA2WT, 636-656bp: 6*His, 657-695bp: Ter terminator), and the plasmid was transformed into Escherichia coli Match1 T1 competent cells by chemical method, and the positive transformants were picked for sequencing and identification, and finally the wild phospholipase A2 expression vector pBE-P43-PgntK-AprE-SvPLA2WT was obtained.

[0046] Among them, the pBEP43-Pcd-SamyQ-SplB-Fc plasmid is disclosed in the document "CN113957071A, a combined DNA fragment with dual promoter and dual secretion signal functions and its application".

[0047] The amino acid sequence of the AprE signal peptide is shown in 1-29aa of GenBank No. WP_015383267.1.

[0048] (2) Construction of phospholipase A2 mutant expression vector

[0049] Using the constructed wild phospholipase A2 expression vector pBE-P43-PgntK-AprE-SvPLA2WT as a template, a linear vector fragment containing the corresponding site mutation was amplified using amplification primers containing the corresponding mutation site.

[0050] The specific amplification primers are shown in the following table (lowercase indicates the replaced codons):

[0051]

[0052] Plasmid construction for multi-site mutations requires multiple primer pairs for amplification. Taking the N21E / I84L mutant as an example, the primer pairs N21E-F / I84L-R are required to amplify fragment 1, and I84L-F / N21E-R is required to amplify fragment 2. The amplified fragments are homologously fused using the Gib-seamless cloning (multi-fragment) kit, and the ligation products are chemically transformed into Escherichia coli Match1 T1 competent cells. Positive transformants are selected for sequencing and identification, and finally the phospholipase A2 mutant expression vector is obtained.

[0053] Example 2

[0054] Construction, fermentation and protein purification of wild phospholipase A2 and its mutant recombinant expression strains

[0055] (1) Construction of wild-type phospholipase A2 and mutant recombinant expression strains

[0056] The expression vectors of phospholipase A2 and mutants constructed in Example 1 were transformed into competent cells of Bacillus subtilis ATCC6051 by electroporation (2500V, 4.3-5.5ms), and LB plates containing kanamycin were used as screening plates. Transformants were picked from the screening plates for colony PCR identification (complete expression cassette of phospholipase A2), and the positive identification products obtained were the correct recombinant expression strains. The identification results are as follows: Figure 1 As shown, the size of the amplified band was 847 bp, and the corresponding recombinant expression strain was correctly identified and obtained.

[0057] (2) Fermentation and protein purification of wild phospholipase A2 and mutant recombinant expression strains

[0058] a) The phospholipase A2 and mutant recombinant expression strains correctly identified in (1) were inoculated into 5 mL of liquid LB medium (containing 50 μg / mL kanamycin) and cultured at 37° C. and 220 rpm for 8-14 h as seed solution;

[0059] b) inoculating the seed solution in a) into LB medium (containing 50 μg / mL kanamycin), with an inoculum size of 1% and a liquid volume of 50 mL / 250 mL, and fermenting at 37° C. and 220 rpm for 24-48 h;

[0060] c) The fermentation liquid obtained in b) is centrifuged (8000 g, 5 min) to remove the bacteria, and the obtained supernatant is the crude phospholipase A2 enzyme liquid.

[0061] d) The crude phospholipase A2 enzyme solution obtained in c) was filtered with a 0.22 μm filter. The filtrate was purified by nickel affinity chromatography, and the column was washed with 1.5 column volumes of buffer B (500 mM NaCl, 500 mM imidazole, 20 mM Tris-HCl, PH 8.0), and rinsed with buffer A (500 mM NaCl, Tris-HCl, PH 8.0) to the UV baseline level. The supernatant was loaded into a HisTRAPTM HP Ni-NTA pre-packed column, and the B phase and the A phase were gradient mixed for gradient elution. The target protein was eluted at 30% buffer B. The collected eluate was replaced by ultrafiltration (500 mM NaCl, 20 mM Tris-HCl, 3 mM DTT (dithiothreitol), PH = 8.0) to remove imidazole and impurities, and obtain the target protein.

[0062] Example 3

[0063] Analysis of enzymatic properties of phospholipase A2 and its mutants

[0064] The present invention adopts microporous colorimetry to determine the enzyme activity of phospholipase A2. Its principle is based on the sulfhydryl colorimetric method established by Aarsman et al. in 1976, that is, a phospholipid analog 2-thiohexadecanoylthio-l-ethyl-phosphorylcholine (HEPC) is used as a substrate. The sn-2 position of the substrate is replaced by a thioester bond, which can still be hydrolyzed by phospholipase A2 and release free sulfhydryl groups. It has a maximum absorption near the wavelength of 414nm with 5,5'-dithionitrobenzoic acid (DTNB). The absorption value is measured and the amount of sulfhydryl groups is converted to obtain the enzyme activity. Enzyme activity definition: at 37°C, the amount of enzyme that releases 1 nmol of free sulfhydryl groups per minute is 1 unit of enzyme activity.

[0065] The reaction system is as follows:

[0066] (1) Basic solution: 200 mM Tris-HCl (pH 8.0), 200 mM CaCl2, 5 mM DTNB, and 5 mM sodium deoxycholate were prepared in a volume ratio of 4.5:1:1:1 to prepare the basic solution.

[0067] (2) Working solution: add 1 mL of 5 mM HEPC to 15 mL of base solution and mix well.

[0068] (3) Control solution: add 15 mL of base solution to 1 mL of distilled water and mix well.

[0069]

[0070] Incubate at 37°C for 30 min, measure A410, and adjust blank to zero.

[0071] Enzyme activity calculation:

[0072] a) Measure the change in absorbance per minute: ΔA410 / min = Atest - Ad / 30, and use the ΔA410 / min of the blank well to adjust to zero.

[0073] b) Calculate the enzyme activity using the following formula: The reaction rate at 410 nm can be determined using the extinction coefficient of DTNB. The actual extinction coefficient of DTNB at 414 nm is 13.6 mM -1 cm -1 , the extinction coefficient of DTNB at 410nm is about 13.5mM - 1 cm -1 The depth of the reaction solution with a volume of 200 μL in the 96-well ELISA plate is 0.606 cm.

[0074]

[0075] The results of the test are as follows Figure 2 As shown, compared with the wild type, the enzyme activities of mutants N21E, Q47K, F53H, Y68H, I84L, Q116M, N21E / I84L, Q47K / I84L, I84L / Q116M, N21E / I84L / Q116M and Q47K / I84L / Q116M were all improved by 32.3%, 20.2%, 27.9%, 20.5%, 69.7%, 60.5%, 106.6%, 92.9%, 213.2%, 406.5%, 304.3%, respectively. The mutant enzyme activity obtained by the present invention is significantly improved, further improving the industrial utilization value of the enzyme.

[0076] In summary, compared with the wild-type phospholipase A2, the mutant obtained by the present invention significantly improves its own enzyme activity and has great application value in industrial fields such as food and medicine.

[0077] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A phospholipase A2 mutant of Streptomyces rhodochrous, characterized in that: The amino acid sequence of the Streptomyces rhodochrous phospholipase A2 mutant is obtained by subjecting SEQ ID NO.1 to any of the following mutations: I84L; or N21E / I84L; or Q47K / I84L; or I84L / Q116M; or N21E / I84L / Q116M; or Q47K / I84L / Q116M; Among them, the amino acid sequence of the mutant N21E / I84L / Q116M is shown in SEQ ID NO.

3.

2. A gene encoding the mutant of phospholipase A2 of Streptomyces rhodochrous according to claim 1.

3. The gene according to claim 2, characterized in that: The nucleotide sequence of the gene encoding the mutant N21E / I84L / Q116M is shown in SEQ ID NO.

4.

4. The biological material related to the phospholipase A2 mutant of Streptomyces rhodochrous according to claim 1, characterized in that: Any one or more combinations of the following biological materials: (a) an expression cassette containing the gene according to claim 2 or 3; (b) a recombinant expression vector containing the gene according to claim 2 or 3; (c) a recombinant expression vector containing the expression cassette described in (a); (d) a recombinant microorganism containing the gene according to claim 2 or 3; (e) a recombinant microorganism containing the expression cassette described in (a); (f) A recombinant microorganism containing the recombinant expression vector described in (b) or (c).

5. The biomaterial according to claim 4, characterized in that: The expression elements used in the expression cassette in (a) are: P43 and PgntK dual promoters, AprE signal peptide and Ter terminator; The starting vector of the recombinant expression vector described in (b) and (c) is a pBE series vector; The host microorganism corresponding to the recombinant microorganisms in (d), (e) and (f) is selected from prokaryotes or yeast.

6. Use of the mutant of phospholipase A2 of Streptomyces rhodochrous according to claim 1, the gene according to any one of claims 2 to 3, or the biomaterial according to any one of claims 4 to 5, characterized in that: One of the following applications: 1) Application in the preparation of phospholipase A2 mutants with high enzyme activity; 2) Application in the production of functional phospholipids; 3) Application in food, medicine, daily chemical fields or oil refining.

7. The use according to claim 6, characterized in that: Application in the field of health care products.

8. A method for obtaining the phospholipase A2 mutant of Streptomyces rhodochrous according to claim 1, characterized in that: The method comprises the following steps: performing site-directed mutagenesis on a gene encoding a phospholipase A2 of Streptomyces purpurogenum having an amino acid sequence as shown in SEQ ID NO.1 by designing primers containing mutation sites, and then expressing the site-directed mutagenesis to obtain the phospholipase A2 mutant of Streptomyces purpurogenum according to claim 1.

9. The method according to claim 8, characterized in that: Primers containing mutation sites are designed to introduce mutations into the gene encoding the phospholipase A2 of Streptomyces purpurogenum whose amino acid sequence is shown in SEQ ID NO.

1. After correct sequencing, the gene is transformed into Bacillus subtilis ATCC6051 for expression to obtain the mutant of the phospholipase A2 of Streptomyces purpurogenum according to claim 1.

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