A Candida antarctica lipase b mutant and a method for preparing the same

Abstract

The application discloses a Candida antarctica lipase B mutant and a preparation method thereof, and belongs to the technical field of biology. Eleven mutants with improved enzyme activity are obtained, including six unit point mutants and five combined mutants; compared with a wild type, the enzyme activity of the mutants Q11L, F71N, F118Y, V149T, L219N, T244D, S31T / F71N, F71N / F118Y, F71N / L219N, S31T / F71N / F118Y and F71N / F118Y / L219N is improved, and is respectively increased by 18.45% to 240.88%; the CALB mutant with high enzyme activity is obtained, and has great industrial application potential and economic value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an enzyme-active Candida antarcticis lipase B mutant and its preparation method. Background Technology

[0002] Lipases (EC 3.1.1.3) catalyze the hydrolysis of lipids to produce fatty acids, glycerol, and mono- or diglycerides. Lipase B (CALB), derived from Candida antarctica, possesses excellent catalytic performance and has been commercially available via eukaryotic expression (Novozym 435), finding wide application in the food, flavor, and biofuel industries.

[0003] However, CALB still faces some challenges in actual industrial production. For example, its poor tolerance to harsh industrial environments such as high temperatures and extreme pH levels, low expression levels, and limited catalytic activity on non-natural substrates restrict its further industrial application. Therefore, improving the catalytic activity and stability of CALB to make it more suitable for industrial applications is urgent. Modifying the enzyme through protein engineering is the main strategy to solve this problem. Summary of the Invention

[0004] To overcome the shortcomings and deficiencies of existing technologies, the present invention aims to provide a CALB mutant with enhanced enzyme activity and its preparation method. 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 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. This invention yields a CALB mutant with high enzyme activity, possessing significant industrial application potential and economic value.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A mutant of Candida antarctica lipase B, whose amino acid sequence is SEQ ID NO.1, is obtained by any 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; except for S31T / F71N / L219N.

[0009] In Q11L, the 11th amino acid is mutated from Q to L, and the same applies to the others.

[0010] Furthermore, the Candida antarcticis lipase B mutant, whose amino acid sequence is SEQ ID NO.1, is 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] The encoding gene of a mutant of Candida antarcticis lipase B.

[0014] Preferably, an enhanced enzyme activity Candida antarcticus lipase B mutant F71N / L219N, the amino acid sequence of which is shown in SEQ ID NO.3, is described above. The mutant has amino acids 71 ​​and 219 mutated from phenylalanine (F) and leucine (L) to asparagine (N) and asparagine (N), respectively.

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

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

[0017] (a) An expression cassette containing the above-mentioned encoded genes;

[0018] (b) A recombinant expression vector containing the above-mentioned coding genes;

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

[0020] (d) Recombinant microorganisms containing the above-mentioned encoding genes;

[0021] (e) Recombinant microorganisms containing the expression cassette described in (a);

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

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

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

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

[0026] Furthermore, the host microorganisms corresponding to the recombinant microorganisms mentioned in (d), (e), and (f) are selected from prokaryotes or yeasts, etc.; 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 yeasts are Pichia pastoris GS115.

[0027] The application of the above-mentioned mutants, coding genes, or mutant-related biological materials in the preparation of highly active Candida antarcticis lipase B mutants.

[0028] Furthermore, the aforementioned mutants, coding genes, or mutant-related biomaterials are applied in fields such as food, medicine, daily chemicals, and chemicals.

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

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

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

[0032] 1. Construction of CALB mutant recombinant expression vector: 1) The wild-type gene of Candida antarctica lipase B synthesized after codon optimization and the pelB signal peptide from Bacteroides lysodeoxycholica were ligated into the pET-28a(+) vector containing the Pt7 promoter to construct the expression plasmid pET-28a(+)-Pt7-pelB-CALBWT; 2) The site-directed mutant of wild-type CALB was amplified by overlap PCR to obtain the corresponding CALB mutant expression plasmid.

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

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

[0035] The present invention has the following advantages and effects compared with the prior art:

[0036] This invention yielded 11 mutants with enhanced enzyme activity, including 6 single-point mutants and 5 combined mutants. Compared to 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 were all 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. This invention provides CALB mutants with high enzyme activity, which have significant industrial application potential and economic value. Attached Figure Description

[0037] Figure 1 The images show colony PCR electrophoresis images of wild-type CALB and mutant expression vectors; where M is the DL5000 DNA Marker, and lanes 1-24 are, in order: 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 Bar chart showing the relative enzyme activity of wild-type CALB and mutants.

[0039] Figure 3 Bar chart showing the residual enzyme activity of wild-type CALB and mutants. Detailed Implementation

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

[0041] In this embodiment, the CALB used was derived from Candida antarctica, whose amino acid sequence is shown in SEQ ID NO.1. The nucleotide sequence of the coding gene obtained after optimization of the Aspergillus niger codon is shown in SEQ ID NO.2. At the same time, a 6*His tag sequence (CATCATCATCATCATCAC) was introduced at the 3' end, which was synthesized by a commercial company.

[0042] Example 1

[0043] Construction of Antarctic Candida lipase B and its mutant expression vector

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

[0045] Using pET-28a(+) plasmid as a template, primers ZF / R and KOD One were used. TM Linearized vector fragments were amplified using PCR Master Mix. The CALB encoding gene nucleotide sequence (6*His+SEQ ID NO.2), optimized with a 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 the high-fidelity enzyme Primer Start Mix. The pelB signal peptide sequence was introduced into the CALBWT fragment using primers. The amplified fragments were homologously fused using In-Fusion homologous recombination. The ligation product was transformed into E. coli Match1 T1 competent cells using a chemical method. Positive transformants were selected and sequenced for identification. Finally, the wild-type CALB expression vector pET-28a(+)-Pt7-pelB-CALBWT was obtained.

[0046] The amino acid sequence of the pelB signal peptide is shown as 1–22aa in GenBank No. QGS70241.2; the gene sequence encoding the pelB signal peptide is shown as 1–66bp 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-type CALB expression vector pET-28a(+)-Pt7-pelB-CALBWT as a template, linear vector fragments containing corresponding mutation sites were amplified using amplification primers containing the corresponding mutation sites.

[0053] The specific amplification primers are shown in the table below (lowercase letters indicate the replaced codons):

[0054]

[0055]

[0056] The construction of plasmids with multiple mutations requires multiple primer pairs for amplification. Taking the F71N / L219N mutant as an example, fragment 1 needs to be amplified using primer pair F71N-F / L219N-R, and fragment 2 needs to be amplified using L219N-F / F71N-R. The amplified fragments are then homologously fused using in-fusion. The ligation product is then chemically transformed into E. coli Match1 T1 competent cells. Positive transformants are selected and sequenced for identification, finally yielding the CALB mutant expression vector.

[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 *E. coli* Rosetta (DE3) competent cells via chemical transformation. LB plates containing kanamycin and chloramphenicol were used as selection plates. Transformants were picked from the selection plates and identified by colony PCR using primers YZ-F / R. Positive samples were identified as the correct recombinant expression strains. The identification results are as follows: Figure 1 As shown, the amplified band size was 992 bp, and the corresponding recombinant expression strain was correctly identified.

[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) Inoculate the CALB and mutant recombinant expression strains that were correctly identified in step (1) into 10 mL of liquid LB medium (containing 50 μg / mL kanamycin and 50 μg / mL chloramphenicol), and culture at 37℃ and 220 rpm for 8–14 h as seed culture;

[0065] b) Inoculate the seed culture from step a) into TB medium (containing 50 μg / mL kanamycin and 50 μg / mL chloramphenicol) at an inoculation volume of 3%, with a liquid volume of 30 mL / 100 mL. Incubate at 37°C and 220 rpm until the bacterial concentration reaches OD500. 600 =0.8~1, add IPTG to the fermentation broth to a final concentration of 0.1M, and ferment at 20℃ for 24h;

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

[0067] d) Filter the CALB crude enzyme solution obtained in step c) using a 0.22 μm filter. Purify the filtrate by nickel column affinity chromatography. Wash the nickel column with 1.5 column volumes of Buffer B (500 mM NaCl, 500 mM imidazole, 20 mM Tris-HCl, pH 8.0), and rinse with Buffer A (500 mM NaCl, Tris-HCl, pH 8.0) to the UV baseline. Load the supernatant into a HisTRAP™ HP Ni-NTA pre-packaged column and perform gradient elution using a gradient mixing of phases B and A. The target protein elutes in 30% Buffer B. Remove the collected eluent by ultrafiltration (20 mM Tris-HCl, pH 8.0) to remove imidazole and other contaminating proteins, obtaining the target protein.

[0068] Example 3

[0069] CALB and mutant enzyme activity analysis

[0070] This invention employs a microporous colorimetric method to determine the hydrolytic activity of CALB lipase. The principle is that lipase hydrolyzes the substrate 4-nitrophenylbutyrate to generate p-nitrophenol (pNP) under alkaline conditions. p-Nitrophenol exhibits maximum absorption near a wavelength of 405 nm. By measuring the absorbance and calculating the amount of p-nitrophenol, the enzyme activity can be determined. Enzyme activity is defined as the amount of enzyme required to release 1 μmol of p-nitrophenol per minute at 45°C and pH = 8. The enzyme protein concentration is determined using the BCA method, with bovine serum albumin as the standard.

[0071] The reaction system is as follows:

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

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

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

[0075]

[0076] Incubate at 45℃ for 5 min, add 1000 μL of pre-cooled stop solution, and measure A. 405 Zero it according to the comparison.

[0077] Enzyme activity calculation:

[0078]

[0079] The measurement results 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 all 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. This invention yields CALB mutants with high enzyme activity, which have significant industrial application potential and economic value.

[0080] Example 4

[0081] Thermal stability analysis of CALB and mutants

[0082] This invention characterizes the thermostability of CALB and its mutants using residual enzyme activity after heat treatment. Experimental procedure: 2 mL of CALB and mutant fermentation broth was centrifuged at 10,000 rpm for 20 min at 4°C. 200 μL of the supernatant was transferred to a 1.5 mL Eppendorf tube, incubated in a 50°C water bath for 10 min, and then placed on ice for 5 min. The residual enzyme activity of CALB and its mutants was then determined using the method described in Example 3. Definition of residual enzyme activity: The hydrolytic activity of CALB and its mutants was determined using a microwell colorimetric method. The hydrolytic activity of CALB and its mutants before heat treatment was defined as the initial enzyme activity. The ratio of the hydrolytic activity of CALB and its mutants after heat treatment to the initial enzyme activity was defined as the residual enzyme activity.

[0083] Calculation of residual enzyme activity:

[0084]

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

[0086] In summary, compared with wild-type CALB, the mutant obtained by this invention significantly improves its enzyme activity and has great application value in the pharmaceutical, chemical and other industrial fields.

[0087] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A Candida antarcticis lipase B mutant, characterized in that: The amino acid sequence of the Candida antarcticis lipase B mutant is obtained by any one of the following mutations of SEQ ID NO.1: F71N; or S31T / F71N; or F71N / F118Y; or F71N / L219N; or S31T / F71N / F118Y; or F71N / F118Y / L219N; The amino acid sequence of the mutant F71N / L219N is shown in SEQ ID NO.

3.

2. The gene encoding the Candida antarcticis lipase B mutant as described in claim 1.

3. The gene according to claim 2, characterized in that: The nucleotide sequence of the gene encoding the mutant F71N / L219N is shown in SEQ ID NO.

4.

4. The biomaterial related to the Antarctic Candida lipase B mutant as described in claim 1, characterized in that: It can be any one or more combinations of the following biological materials: (a) An expression cassette containing the gene of claim 2 or 3; (b) A recombinant expression vector containing the gene of claim 2 or 3; (c) A recombinant expression vector containing the expression cassette described in (a); (d) A recombinant microorganism containing the gene described in claim 2 or 3; (e) Recombinant microorganisms containing the expression cassette described in (a); (f) Recombinant microorganisms 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 described in (a) are: the T7 promoter, the pelB signal peptide, and the T7 terminator; The starting vector for the recombinant expression vectors described in (b) and (c) is a pET series vector; The host microorganisms corresponding to the recombinant microorganisms described in (d), (e), and (f) are selected from prokaryotes or yeast.

6. The use of the gene according to any one of claims 2 to 3 or the biological material according to any one of claims 4 to 5 in the preparation of the Candida antarcticis lipase B mutant according to claim 1.

7. The application of the Antarctic Candida lipase B mutant according to claim 1, the gene according to any one of claims 2 to 3, or the biological material according to any one of claims 4 to 5 in the food, daily chemical, or chemical industries.

8. A method for obtaining the Candida antarcticis lipase B mutant of claim 1, characterized in that: The method includes the following steps: by designing primers containing mutation sites to perform site-directed mutagenesis on the gene encoding the amino acid sequence shown in SEQ ID NO.1 of Candida antarcticis lipase B, and then expressing the mutant to obtain the Candida antarcticis lipase B mutant as described in claim 1.

9. The method according to claim 8, characterized in that: Primers containing the mutation site were designed to introduce a mutation into the gene encoding the amino acid sequence shown in SEQ ID NO.1 of Candida antarcticis lipase B. After the sequence was confirmed to be correct, the gene was transformed into Escherichia coli Rosetta (DE3) for expression, thus obtaining the Candida antarcticis lipase B mutant as described in claim 1.

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