Candida antarctica lipase B mutant and preparation method thereof

Abstract

The invention discloses a Candida antarctica lipase B mutant and a preparation method thereof, and belongs to the technical field of biology. According to the invention, 11 mutants with improved enzyme activity are obtained, including 6 single site mutants and 5 combined mutants; compared with a wild type, the enzyme activities of the mutants Q11L, F71N, F118Y, V149T, L219N, T244D, S31T / F71N, F71N / F118Y, F71N / L219N, S31T / F71N / F118Y and F71N / F118Y / L219N are all improved, and are respectively improved by 18.45%-240.88%; according to the invention, the CALB mutant with high enzymatic activity is obtained, and the CALB mutant 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 Candida antarctica lipase B mutant with improved enzyme activity and a preparation method thereof. Background Art

[0002] Lipase (EC 3.1.1.3) can catalyze the hydrolysis of oils and fats to produce fatty acids, glycerol and mono- or di-glycerides. Lipase B (CALB) from Candida antarctica has good catalytic performance and is commercially available (Novozym 435) through eukaryotic expression hosts. It is widely used in the food industry, spice industry, biofuels and other fields.

[0003] However, at present, CALB still faces some difficulties in actual industrial production, such as poor tolerance to harsh industrial production environments such as high temperature and extreme pH, low expression level, and limited catalytic activity on non-natural substrates, which restricts the further industrial application of lipase. Therefore, it is urgent to improve the catalytic activity and stability of CALB to make it more suitable for industrial production applications. Modifying the enzyme through protein engineering is the main strategy to solve this problem. Summary of the invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a CALB mutant with improved enzyme activity and a preparation method thereof. Among them, the enzyme activities of mutants Q11L, F71N, F118Y, V149T, L219N, T244D, S31T / F71N, F71N / F118Y, F71N / L219, S31T / F71N / F118Y and F71N / F118Y / L219N are all improved, which are increased by 76.39%, 34.07%, 20.97%, 20.29%, 18.45%, 34.06%, 127.41%, 99.86%, 240.88%, 172.80% and 192.47% respectively; the present invention obtains a CALB mutant with high enzyme activity, which has great industrial application potential and economic value.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A Candida antarctica lipase B mutant, wherein the amino acid sequence is SEQ ID NO.1 obtained by any one of the following mutations:

[0007] 1) at least one of Q11L, F71N, F118Y, V149T, L219N and T244D;

[0008] 2) S31T and at least one of Q11L, F71N, F118Y, V149T, L219N and T244D; but excluding S31T / F71N / L219N.

[0009] Among them, Q11L means that the 11th amino acid mutates from Q to L, and the same applies to the others;

[0010] Furthermore, the Candida antarctica lipase B mutant has an amino acid sequence of SEQ ID NO.1 obtained by any of the following mutations:

[0011] Q11L; or F71N; or F118Y; or V149T; or L219N; or T244D; or S31T / F71N; or F71N / F118Y; or F71N / L219N; or S31T / F71N / F118Y; or F71N / F118Y / L219N.

[0012] The gene sequence encoding the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.2.

[0013] A gene encoding the Candida antarctica lipase B mutant.

[0014] Preferably, a Candida antarctica lipase B mutant F71N / L219N with improved enzyme activity has an amino acid sequence as shown in SEQ ID NO. 3. The amino acid at position 71 and the amino acid at position 219 of the mutant are mutated from phenylalanine F and leucine L to asparagine N and asparagine N, respectively.

[0015] A gene encoding an Antarctic Candida lipase B mutant F71N / L219N with improved enzyme activity, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0016] The mutant-related biological material is any one or more combinations of the following biological materials:

[0017] (a) an expression cassette containing the above encoding gene;

[0018] (b) a recombinant expression vector containing the above encoding gene;

[0019] (c) a recombinant expression vector containing the expression cassette described in (a);

[0020] (d) a recombinant microorganism containing the above encoding gene;

[0021] (e) a recombinant microorganism containing the expression cassette described in (a);

[0022] (f) A recombinant microorganism containing the recombinant expression vector described in (b) or (c).

[0023] Furthermore, the expression elements used in the expression cassette in (a) are: T7 promoter (Pt7), pelB signal peptide and T7 terminator (Tt7).

[0024] The pelB signal peptide is derived from Paenibacillus chitinolyticus, and its amino acid sequence is shown in 1-22aa in GenBank No. QGS70241.2; the gene sequence encoding the pelB signal peptide is shown in 1-66bp in GenBank No. MN121846.2.

[0025] Furthermore, the starting vector of the recombinant expression vector in (b) and (c) is a pET series vector, etc.; preferably, it is a pET-28a(+) vector.

[0026] Furthermore, the host microorganism corresponding to the recombinant microorganism in (d), (e), and (f) is selected from prokaryotes or yeasts; the prokaryotes include bacteria such as Escherichia; the yeasts include yeasts such as Pichia pastoris. More specifically, the prokaryotes are Escherichia coli, specifically Escherichia coli Rosette (DE3) and Match1T1; the yeast is Pichia pastoris GS115.

[0027] Application of the mutant, encoding gene or mutant-related biological materials in the preparation of an Antarctic Candida albicans lipase B mutant with high enzyme activity.

[0028] Furthermore, the mutants, encoding genes or mutant-related biomaterials are applied in the fields of food, medicine, daily chemicals, chemical industry, etc.

[0029] A method for obtaining the above mutant comprises the following steps: performing site-directed mutagenesis on the gene encoding the amino acid sequence of Candida antarctica lipase B as shown in SEQ ID NO.1 by designing primers containing mutation sites and then expressing the site-directed mutagenesis to obtain the Candida antarctica lipase B mutant.

[0030] Furthermore, primers containing mutation sites were designed to introduce mutations into the gene encoding the amino acid sequence of Candida antarctica lipase B as shown in SEQ ID NO.1. After correct sequencing, the gene was transformed into Escherichia coli Rosetta (DE3) for expression to obtain a Candida antarctica lipase B mutant.

[0031] The specific implementation steps of the present invention are as follows:

[0032] 1. Construction of CALB mutant recombinant expression vector: 1) The wild-type gene of Antarctic Candida lipase B synthesized after codon optimization and the pelB signal peptide from Bacteroides solani were connected to the pET-28a(+) vector containing the Pt7 promoter to construct the expression plasmid pET-28a(+)-Pt7-pelB-CALBWT; 2) The wild-type CALB was site-directedly mutated by overlapping PCR amplification to obtain the corresponding CALB mutant expression plasmid.

[0033] 2. Construction of CALB mutant recombinant bacteria and preparation of CALB with high enzyme activity: The CALB mutant expression plasmid was transformed into Escherichia coli Rosetta (DE3) to obtain recombinant bacteria; and then the CALB mutant was obtained by fermentation.

[0034] 3. Characterization of the enzymatic properties of the CALB mutant: The enzymatic properties of the mutant were determined by microwell colorimetry using 4-nitrophenylbutyrate as a substrate.

[0035] Compared with the prior art, the present invention has the following advantages and effects:

[0036] The present invention obtains 11 mutants with improved enzyme activities, including 6 single-site mutants and 5 combined mutants; compared with the wild type, the enzyme activities of mutants Q11L, F71N, F118Y, V149T, L219N, T244D, S31T / F71N, F71N / F118Y, F71N / L219N, S31T / F71N / F118Y and F71N / F118Y / L219N are all improved, which are increased by 76.39%, 34.07%, 20.97%, 20.29%, 18.45%, 34.06%, 127.41%, 99.86%, 240.88%, 172.80% and 192.47%, respectively; the present invention obtains a CALB mutant with high enzyme activity, which has great industrial application potential and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is the PCR electrophoresis of wild-type CALB and mutant expression vector colonies; M is DL5000 DNA Marker, and lanes 1-24 are: Q11L, S31T, S56L, S56A, F71N, F118Y, V149T, A151P, S201T, F205H, L219N, Q231M, T244D, L261K, M298K, S31T / F71N, F71N / F118Y, F71N / L219, S31T / F71N / F118Y, S31T / F71N / L219N and F71N / F118Y / L219N.

[0038] Figure 2 It is a bar graph of the relative enzyme activities of wild-type CALB and mutants.

[0039] Figure 3 It is a bar graph of the residual enzyme activity of wild-type CALB 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 examples, the CALB used is derived from Candida antarctica, and its amino acid sequence is shown in SEQ ID NO.1. The nucleotide sequence of the encoding gene obtained after Aspergillus niger codon optimization is shown in SEQ ID NO.2. At the same time, a 6*His tag sequence (CATCATCATCATCATCAC) is introduced at the 3' end, which is synthesized by a commercial company.

[0042] Example 1

[0043] Construction of expression vectors for Candida antarctica lipase B and its mutants

[0044] (1) Construction of wild-type CALB expression vector

[0045] Using pET-28a(+) plasmid as template and primers ZF / R, KOD One TM The linearized vector fragment was amplified by PCR Master Mix enzyme; the CALB encoding gene nucleotide sequence (6*His+SEQ ID NO.2) obtained after codon optimization of the 6*His tag sequence introduced at the 3' end was used as a template, the CALBWT gene sequence was amplified using primers WT-F / R and high-fidelity enzyme Primer Start Mix, the pelB signal peptide sequence was introduced into the CALBWT fragment through primers, the amplified fragments were homologously fused through In-Fusion homologous recombination, the ligation products were chemically transformed into Escherichia coli Match1 T1 competent cells, the positive transformants were picked for sequencing and identification, and finally the wild CALB expression vector pET-28a(+)-Pt7-pelB-CALBWT was obtained.

[0046] The amino acid sequence of the pelB signal peptide is shown in 1 to 22 aa in GenBank No. QGS70241.2; the gene sequence encoding the pelB signal peptide is shown in 1 to 66 bp in GenBank No. MN121846.2.

[0047] ZF: 5′-CTAGCATAACCCCTTGGGGCC-3′;

[0048] ZR: 5′-GTCGGCAGGTATTTCATGGTATATCTCCTTCTTAAAGTT-3′;

[0049] WT-F: 5′-ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCCTCCCTTCCGGTAGCGATCC-3′;

[0050] WT-R: 5′-GCCCCAAGGGGTTATGCTAGTCAGTGATGATGATGATGATGGG-3′.

[0051] (2) Construction of CALB mutant expression vector

[0052] Using the constructed wild CALB expression vector pET-28a(+)-Pt7-pelB-CALBWT as a template, a linear vector fragment containing the corresponding site mutation was amplified using amplification primers containing the corresponding mutation site.

[0053] The specific amplification primers are shown in the following table (lowercase indicates the replaced codons):

[0054]

[0055]

[0056] Plasmid construction for multi-site mutations requires multiple primer pairs for amplification. Taking the F71N / L219N mutant as an example, the primer pairs F71N-F / L219N-R are used to amplify fragment 1, and L219N-F / F71N-R is used to amplify fragment 2. The amplified fragments are homologously fused by In-Fusion, 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 CALB mutant expression vector is obtained.

[0057] Example 2

[0058] Construction, fermentation and protein purification of wild-type CALB and mutant recombinant expression strains

[0059] (1) Construction of wild-type CALB and mutant recombinant expression strains

[0060] The expression vectors of CALB and mutants constructed in Example 1 were transformed into competent E. coli Rosetta (DE3) cells by chemical transformation. LB plates containing kanamycin and chloramphenicol were used as screening plates. Transformants were selected from the screening plates and identified by colony PCR using primers YZ-F / R. The positive identified transformants were the correct recombinant expression strains. The identification results are shown in Figure 1 As shown, the size of the amplified band was 992 bp, and the corresponding recombinant expression strain was correctly identified and obtained.

[0061] YZ-F: 5′-CTCCCTTCCGGTAGCGATC-3′;

[0062] YZ-R: 5′-GCCCCAAGGGGTTATGCTAG-3′.

[0063] (2) Fermentation and protein purification of wild-type CALB and mutant recombinant expression strains

[0064] a) The CALB and mutant recombinant expression strains correctly identified in step (1) were inoculated into 10 mL of liquid LB medium (containing 50 μg / mL kanamycin and 50 μg / mL chloramphenicol), and cultured at 37° C. and 220 rpm for 8 to 14 h to serve as seed solution;

[0065] b) The seed solution in step a) was inoculated into TB medium (containing 50 μg / mL kanamycin and 50 μg / mL chloramphenicol) with an inoculum volume of 3% and a liquid volume of 30 mL / 100 mL. The culture was carried out at 37°C and 220 rpm until the bacterial concentration reached OD 600 =0.8~1, add IPTG with a final concentration of 0.1M to the fermentation broth, and ferment at 20℃ for 24h;

[0066] c) The fermentation liquid obtained in step b) is centrifuged (10000 g, 10 min) to remove the bacterial cells, and the obtained supernatant is the CALB crude enzyme liquid.

[0067] d) The crude CALB enzyme solution obtained in step c) was filtered with a 0.22 μm filter head. The filtrate was purified by nickel column affinity chromatography, and the nickel 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 ultraviolet baseline level, and the supernatant was loaded into HisTRAPTM HP Ni-NTA pre-packed column, and the B phase and the A phase were gradient mixed for gradient elution, and the target protein was eluted at 30% buffer B. The collected eluate was replaced by ultrafiltration (20 mM Tris-HCl, PH = 8.0) to remove imidazole and impurities, and obtain the target protein.

[0068] Example 3

[0069] Analysis of enzyme activity of CALB and mutants

[0070] The present invention adopts microporous colorimetry to determine the hydrolysis activity of CALB lipase. The principle is that lipase can hydrolyze the substrate 4-nitrophenylbutyrate to generate p-nitrophenol (pNP) under alkaline conditions. P-nitrophenol has a maximum absorption near a wavelength of 405nm. The absorption value is measured and the amount of p-nitrophenol is converted to obtain the enzyme activity. Enzyme activity definition: Under the conditions of 45°C and pH=8, the amount of enzyme required to release 1 μmol of p-nitrophenol per minute is 1 unit of enzyme activity. The enzyme protein concentration is determined by the BCA method, with bovine serum albumin as the standard.

[0071] The reaction system is as follows:

[0072] (1) Buffer: 50 mM Tris-HCl (pH 8.0).

[0073] (2) Substrate: 25 mM 4-nitrophenol butyrate emulsion.

[0074] (3) Stop solution: 90% ethanol.

[0075]

[0076] Incubate at 45°C for 5 min, add 1000 μL of pre-cooled stop solution, and measure A 405 , compare and adjust to zero.

[0077] Enzyme activity calculation:

[0078]

[0079] The results of the test are as follows Figure 2As shown, compared with the wild type, the enzyme activities of mutants Q11L, F71N, F118Y, V149T, L219N, T244D, S31T / F71N, F71N / F118Y, F71N / L219, S31T / F71N / F118Y and F71N / F118Y / L219N were improved by 76.39%, 34.07%, 20.97%, 20.29%, 18.45%, 34.06%, 127.41%, 99.86%, 240.88%, 172.80% and 192.47%, respectively; the present invention obtains a CALB mutant with high enzyme activity, which has great industrial application potential and economic value.

[0080] Example 4

[0081] Thermal stability analysis of CALB and its mutants

[0082] The present invention uses the residual enzyme activity after heat treatment to characterize the thermal stability of CALB and mutants. Experimental steps: Take 2 mL of CALB and mutant fermentation broth and centrifuge at 4°C and 10,000 rpm for 20 minutes, take 200 μL of the supernatant to a 1.5 mL Eppendorf tube, water bath at 50°C for 10 minutes, and place on ice for 5 minutes, then use the method in Example 3 to determine the residual enzyme activity of CALB and mutants. Definition of residual enzyme activity: The hydrolysis activity of CALB and mutants is determined by microporous colorimetry, and the hydrolysis activity of CALB and mutants without heat treatment is defined as the initial enzyme activity, and the ratio of the hydrolysis activity of CALB and mutants after heat treatment to the initial enzyme activity is defined as the residual enzyme activity.

[0083] Calculation of residual enzyme activity:

[0084]

[0085] The measurement results are as follows Figure 3 As shown, compared with the wild type, the thermal stability of mutants S31T, F118Y, V149T, L219N, T244D, and L261K were improved by 31.76%, 16.51%, 25.04%, 5.25%, 21.51%, and 3.81%, respectively.

[0086] In summary, compared with the wild-type CALB, the mutant obtained in the present invention significantly improves its own enzyme activity and has great application value in industrial fields such as medicine and chemical industry.

[0087] 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 Candida antarctica lipase B mutant, characterized in that: The amino acid sequence of the Candida antarctica lipase B mutant is obtained by subjecting SEQ ID NO.1 to any of the following mutations: 1) at least one of Q11L, F71N, F118Y, V149T, L219N and T244D; 2) S31T and at least one of Q11L, F71N, F118Y, V149T, L219N and T244D; but excluding S31T / F71N / L219N.

2. The Candida antarctica lipase B mutant according to claim 1, characterized in that: The amino acid sequence of the Candida antarctica lipase B mutant is obtained by subjecting SEQ ID NO.1 to any of the following mutations: Q11L; or F71N; or F118Y; or V149T; or L219N; or T244D; or S31T / F71N; or F71N / F118Y; or F71N / L219N; or S31T / F71N / F118Y; or F71N / F118Y / L219N; Among them, the amino acid sequence of the mutant F71N / L219N is shown in SEQ ID NO.

3.

3. The Candida antarctica lipase B mutant according to claim 1 or 2, characterized in that: The gene sequence encoding the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.

2.

4. A gene encoding the Candida antarctica lipase B mutant according to any one of claims 1 to 3.

5. The gene according to claim 4, characterized in that: The nucleotide sequence of the gene encoding the mutant F71N / L219N is shown in SEQ ID NO.

4.

6. The biological material related to the Candida antarctica lipase B mutant according to any one of claims 1 to 3, characterized in that: Any one or more combinations of the following biological materials: (a) an expression cassette containing the gene according to claim 4 or 5; (b) a recombinant expression vector containing the gene according to claim 4 or 5; (c) a recombinant expression vector containing the expression cassette described in (a); (d) a recombinant microorganism containing the gene according to claim 4 or 5; (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).

7. The biomaterial according to claim 6, characterized in that: The expression elements used in the expression cassette in (a) are: T7 promoter, pelB signal peptide and T7 terminator; The starting vector of the recombinant expression vector in (b) and (c) is a pET series vector; The host microorganism corresponding to the recombinant microorganisms in (d), (e) and (f) is selected from prokaryotes or yeast.

8. Use of the Candida antarctica lipase B mutant according to any one of claims 1 to 3, the gene according to any one of claims 4 to 5, or the biomaterial according to any one of claims 6 to 7, characterized in that: One of the following applications: 1) Application in the preparation of an Antarctic Candida lipase B mutant with high enzyme activity; 2) Application in food, medicine, daily chemicals or chemical industry.

9. A method for obtaining the Candida antarctica lipase B mutant according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: performing site-directed mutagenesis on a gene encoding an amino acid sequence of Candida antarctica lipase B as shown in SEQ ID NO. 1 by designing primers containing mutation sites, and then expressing the site-directed mutagenesis to obtain the Candida antarctica lipase B mutant according to any one of claims 1 to 3.

10. The method according to claim 9, characterized in that: Primers containing mutation sites are designed to introduce mutations into the gene encoding the Antarctic Candida lipase B with an amino acid sequence as shown in SEQ ID NO.1, and after correct sequencing, the gene is transformed into Escherichia coli Rosetta (DE3) for expression to obtain the Antarctic Candida lipase B mutant according to any one of claims 1 to 3.

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