A lipase mutant and its encoding gene and application

Abstract

The present invention discloses a lipase mutant and its application, which belongs to the field of enzyme engineering, and the amino acid sequence of the lipase mutant is shown in SEQ ID NO.2. The lipase mutant F206C-T226C has a tolerance of 70 min at 60 ° C and a tolerance of 30 min residual activity at 70 ° C of more than 65%, while the wild-type lipase has a tolerance of 70 min at 60 ° C and a tolerance of 30 min residual activity at 70 ° C of less than 50%. It can be seen that the thermal stability of the enzyme mutant obtained by the present invention is significantly improved, and the enzyme activity is also increased by more than 2 times relative to the starting lipase, which is of great value for industrial applications such as synthesis, hydrolysis, alcoholysis or transesterification of esters.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme engineering, and particularly relates to a polypeptide with lipase activity and a coding gene thereof, a vector and a host cell comprising the coding gene, and a preparation method and application of the polypeptide. Background Art

[0002] Lipase (EC3.1.1.3), also known as triacylglycerol hydrolase, catalyzes the hydrolysis of ester bonds in triglycerides, diglycerides, monoglycerides, other small molecule esters, polyol esters, and polyacid esters. It also catalyzes reactions such as acidolysis, alcoholysis, aminolysis, transesterification, and ester synthesis of esters. Fats are the natural substrates of lipase, and during the hydrolysis process, diglycerides and monoglycerides are produced, with glycerol and fatty acids as the final products. Fats are mostly hydrophobic substances, so the hydrolysis reaction occurs at the oil-water interface or in the organic phase. Lipase catalysis has the advantages of mild hydrolysis conditions, no need for coenzymes, low energy consumption, few by-products, and environmental friendliness. Therefore, it has shown great market prospects in the fields of oil processing, medicine, feed additives, and the chemical industry.

[0003] During industrial production, enzyme preparations often experience high temperatures. Natural lipases are susceptible to inactivation at high temperatures, a shortcoming that significantly increases the cost of industrial production and limits their industrial applications. When searching for lipases with improved thermal stability, due to the numerous sites available for mutation and the complexity of the protein structure, it is necessary to balance thermal stability with improved enzymatic activity. Obtaining lipase mutants with both improved thermal stability and higher enzymatic activity is of great value for industrial applications. Therefore, the present invention transforms existing lipases to produce lipases with both improved thermal stability and higher enzymatic activity. Summary of the Invention

[0004] One of the objectives of the present invention is to provide a lipase mutant with improved thermal stability and good enzyme activity.

[0005] The present invention is specifically implemented through the following technical solution: a lipase mutant, which is obtained by replacing the amino acid sequence of the lipase shown in SEQ ID NO.1.

[0006] Specifically, the lipase mutant is obtained by changing the phenylalanine (F) at position 206 in the lipase amino acid sequence SEQ ID NO.1 to cysteine ​​(C) and the threonine (T) at position 226 to cysteine ​​(C).

[0007] The amino acid sequence of the lipase mutant of the present invention is shown in SEQ ID NO.2.

[0008] Another object of the present invention is to provide a gene encoding the lipase mutant with improved thermal stability.

[0009] Specifically, the nucleotide sequence encoding the above lipase mutant is shown in SEQ ID NO. 3, or the reverse complementary sequence of the sequence.

[0010] The optimum temperature for the enzymatic reaction of the lipase mutant is 50° C.; and the lipase mutant can tolerate a relative enzyme activity of more than 65% at 60° C. for 70 min and a relative enzyme activity of about 45% at 70° C. for 45 min.

[0011] In another aspect of the present invention, a polypeptide obtained by modifying, deleting or adding one or more amino acids to the amino acid sequence SEQ ID NO.2 of the lipase mutant, and a sequence having 90% homology to the sequence shown in SEQ ID NO.2 is also within the protection scope of the present invention.

[0012] On the other hand, the nucleotide sequence encoding the same protein as the coding gene SEQ ID NO. 3 or its complementary sequence is also within the scope of protection of the present invention.

[0013] Another object of the present invention is to provide an expression cassette containing a gene encoding a lipase mutant and a recombinant expression vector containing the gene encoding the lipase mutant. Specifically, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector. More specifically, the expression vector comprises pGAPZα, pPIC9K, or pPICZα. Preferably, the recombinant expression vector is pPIC9K.

[0014] Another object of the present invention is to provide a recombinant engineered strain containing a gene encoding a lipase mutant. Specifically, the host cell is Escherichia coli, Pichia pastoris, or Saccharomyces cerevisiae.

[0015] Another object of the present invention is to provide applications of the lipase mutant with improved thermal stability, including synthesis, hydrolysis, alcoholysis or transesterification of esters.

[0016] Another object of the present invention is to provide a method for preparing a lipase mutant, which comprises expressing and purifying the above-mentioned recombinant engineered bacteria.

[0017] Another object of the present invention is to provide a method for preparing esters, which comprises using the above-mentioned lipase mutant or an immobilized enzyme thereof to catalyze the esterification reaction between fatty acids and glycerol.

[0018] The present invention is based on a lipase with high thermostability and enzyme activity, the amino acid sequence of which is shown in SEQ ID NO. 1. Structural analysis was first performed to determine the 3D molecular structure of the lipase and the potential amino acid residues that influence its thermostability and activity. Mutations were then used to obtain lipase mutants, which were then screened using thermostability and enzyme activity experiments to ultimately obtain the desired lipase mutants.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] Compared to the starting lipase (wild-type), this mutant exhibits improved thermostability and enzyme activity. Under the same conditions, the resulting lipase mutant F206C-T226C maintained a residual activity of over 65% at 60°C for 70 minutes and at 70°C for 30 minutes, while the wild-type lipase maintained a residual activity of under 50% at 60°C for 70 minutes and at 70°C for 30 minutes. This demonstrates that the enzyme mutant obtained by the present invention exhibits significantly improved thermostability and over 2-fold increased enzyme activity compared to the starting lipase. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The optimum temperature determination results of the lipase mutant (after enzyme mutation) and the initial lipase (before enzyme mutation) in the present invention;

[0022] Figure 2 The results of the thermal stability test of the lipase of the present invention (after enzyme mutation) and the initial lipase (before enzyme mutation) at 60°C are shown;

[0023] Figure 3 These are the results of thermal stability tests at 70° C. of the lipase mutant (after enzyme mutation) and the initial lipase (before enzyme mutation) in the present invention. DETAILED DESCRIPTION

[0024] The following will be combined with specific embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] The experimental techniques and methods used in this example are all conventional methods unless otherwise specified. The materials, reagents, and biological materials used in this example can all be obtained through regular commercial channels unless otherwise specified.

[0026] The experimental methods in the present invention are all conventional methods unless otherwise specified. For molecular biology experimental methods not specifically described, please refer to "Molecular Cloning Experiment Guide" edited by J. Sambrook et al., or follow the methods in the instructions of commercial kits.

[0027] Through modeling and analysis of the spatial structure of a known lipase protein, the mutation sites were ultimately identified as phenylalanine at position 206 and threonine at position 226 in SEQ ID NO. 1. A specific approach involves using the starting lipase gene as a template and using site-directed mutagenesis to mutate potential amino acid residues in the lipase gene. This generated a novel lipase gene. This mutant gene was then linked to a vector to construct a recombinant plasmid, which was then transformed into a suitable host bacterium for heterologous expression to produce a lipase mutant. This mutant has been shown to exhibit ideal heat resistance and enzyme activity, making it suitable for industrial production and application.

[0028] The amino acid sequence of the mutant obtained by mutation of the lipase in the embodiment of the present invention is shown in SEQ ID NO.2.

[0029] The nucleotide sequence of the lipase mutant of the present invention is shown in SEQ ID NO.3.

[0030] Experimental materials and reagents

[0031] Expression host bacteria and vectors: GS115 and pPIC9K were purchased from Novagen.

[0032] Main reagents: DNA Marker, Protein Marker (TaKaRa); Plasmid Kit (QIAGEN).

[0033] Experimental instruments: centrifuge (Eppendorf); PCR amplifier (Bio-Rad); nucleic acid electrophoresis instrument (Bio-Rad); protein electrophoresis instrument (Amersham Bioscience); gel imager (Bio-Rad).

[0034] Mainly used culture media: YPD and LB culture media are commonly used culture media in this field.

[0035] Determination of lipase activity: Under certain conditions, the amount of enzyme required to hydrolyze the substrate p-nitrophenyl palmitate (p-NPP) to produce 1 μmol p-nitrophenol (p-NP) per minute is one enzyme activity unit, expressed in U.

[0036] Experimental instruments: constant temperature water bath; pH instrument; microplate reader (Bio-Rad), etc.

[0037] Solution preparation:

[0038] pH buffer: 0.1 mol / L monohydrate citric acid buffer and 0.1 mol / L phosphate buffer; 0.1 mol / L Tris-HCl buffer; 0.1 mol / L glycine-NaOH buffer.

[0039] Substrate solution: 10 mmol / L p-nitrophenol palmitate (Sigma).

[0040] The p-nitrophenol method uses a 500μL volume containing 420μL of 50mmol / L buffer, 30μL of 10mmol / L p-NPP substrate, and 50μL of diluted enzyme solution. The substrate and buffer are mixed and preheated at the reaction temperature for 2 minutes. The diluted enzyme solution is then added and mixed thoroughly. The reaction is allowed to react for 5 minutes. 50μL of 1.0mol / L SDS is added to terminate the reaction, and 500μL of 1.0mol / L Na2CO3 is added for color development. The OD value is measured at 405nm.

[0041] Lipase thermal stability assay

[0042] The purified mutant enzyme and the starting lipase solution were diluted to the same protein concentration and then subjected to different temperature tolerance tests: 60°C for 20 minutes, 30 minutes, 50 minutes, 70 minutes, and 90 minutes; and 70°C for 10 minutes, 20 minutes, 30 minutes, 45 minutes, and 60 minutes. The residual enzyme activity was then measured and the percentage of residual activity was calculated. The control was an enzyme solution that had not been subjected to the temperature tolerance test.

[0043] Example 1 Preparation of lipase mutants

[0044] The method for obtaining the lipase mutant of the present invention comprises the following steps:

[0045] (1) Site-directed mutagenesis: Using the recombinant plasmid containing the starting lipase-encoding gene linked to the vector as a template, PCR amplification was performed using a primer pair containing the specific mutation. PCR amplification conditions were: 95°C for 5 min; 30 cycles of 98°C for 10 s, 55°C for 25 s, and 72°C for 3 min; and extension at 72°C for 5 min. Reaction system: 1 μL of each primer, 12.5 μL of Primer Star (2X), 1 μL of plasmid template, and 9.5 μL of ddH2O.

[0046] (2) Mutation PCR Verification: Take 10 μL of the amplified product and perform electrophoresis on 1% agarose gel. If the band is correct, recover and purify it.

[0047] (3) Transformation: Introduce the purified product into competent cells, flick gently to mix, and place on ice for 30 minutes; heat shock at 42°C for 40 seconds and immediately cool on ice for 10 minutes; add 500 μL LB medium and culture on a shaker at 37°C for 45 minutes; centrifuge at 7000 rpm for 3 minutes, then add 100 μL LB medium to resuspend the cells, spread on a plate, and culture at 37°C overnight.

[0048] (4) Verification of positive clones: After overnight culture on the plate, single colonies were picked and placed in 500 μL of LB medium containing resistance. After culture at 200 rpm for 3 h, PCR identification was performed. The selected positive clones were sequenced and the sequencing results were compared with the starting sequence to obtain the target sequence.

[0049] (5) Find the recombinant plasmid with the correct mutation; extract the recombinant plasmid with the correct mutation and transfer it into Pichia pastoris for expression, and then measure the enzyme activity and thermal stability after fermentation.

[0050] Among them, some site-directed mutagenesis primers are as follows: F: tcacgctggttgcctttcctcatacaacca (SEQ ID NO: 4); R: tatagtcagggtaagcgcacaattggtcttgaa (SEQ ID NO: 5).

[0051] After mutation according to the above method, sequencing was performed, and screening was performed in combination with thermal stability and enzyme activity experiments to finally obtain the target lipase mutant. The amino acid sequence of the lipase mutant is shown in SEQ ID NO.2, and the encoding gene sequence is shown in SEQ ID NO.3. The enzymatic properties of the lipase mutant were determined.

[0052] Example 2 Enzyme activity determination

[0053] One unit of lipase activity is defined as the amount of enzyme that releases 1 μmol of p-nitrophenol in 1 minute under standard assay conditions. Enzyme activity units, U / mL.

[0054] The total assay volume is 500 μL, containing 420 μL of 50 mmol / L buffer, 30 μL of 10 mmol / L p-NPP substrate, and 50 μL of diluted enzyme solution. The substrate and buffer are mixed and preheated at the reaction temperature for 2 minutes. The diluted enzyme solution is then added and mixed thoroughly. The reaction is allowed to react for 5 minutes, and then 50 μL of 1.0 mol / L SDS is added to terminate the reaction. The OD value is measured at a wavelength of 405 nm.

[0055] The enzyme activity of lipase and its mutants was determined according to the above method, and the enzyme activity of the initial lipase was used as a control to calculate the relative enzyme activity. The results are shown in Table 1. The enzyme activity of the mutant was increased by 2.64 times compared with the original strain without modification.

[0056] Table 1

[0057] Relative enzyme activity mutation site Initial lipase (wild type) 1 / Enzyme mutants 2.64 F206C, T226C

[0058] Example 3 Determination of the optimal action temperature of lipase

[0059] 1 mg of the lipase mutant was added to 10 mL of 50 mM Tris-HCl buffer (pH 8.0) and pre-incubated at gradient temperatures of 20, 30, 40, 50, 60, and 70°C for 60 min. The relative lipase activity of the sample was then tested according to the method disclosed in the present invention to obtain the optimal temperature of the lipase mutant. The results are shown in FIG. Figure 1 The optimal temperature of the lipase mutant is 50°C, while the optimal temperature of the original lipase (wild type) is 40°C.

[0060] Example 4: Thermal stability test of lipase mutants

[0061] The purified lipase and its mutant enzyme solutions were placed in different temperatures: 60°C for 20 min, 30 min, 50 min, 70 min, and 90 min; and 70°C for 10 min, 20 min, 30 min, 45 min, and 60 min. The p-NPP hydrolysis ability was immediately measured after the insulation period, and the residual enzyme activity was calculated using the enzyme solution that had not been heat treated as a control.

[0062] The tolerance of lipase at different temperatures is as follows Figure 2 、 Figure 3 As shown in the figure, the relative enzyme activity shows a gradual decrease with increasing temperature and time, but the relative enzyme activity of the lipase after mutation is always higher than that before mutation. The enzyme mutant has a residual activity of over 65% at 60°C for 70 minutes and 70°C for 30 minutes, while the wild-type lipase has a residual activity of under 50% at 60°C for 70 minutes and 70°C for 30 minutes. This shows that the enzyme mutant obtained by the present invention has significantly improved thermal stability.

[0063] Sequence Listing

[0064] SEQ ID NO.1:

[0065] Three-letter form:

[0066] Val Pro Val Ala Gly His Lys Gly Ser Val Lys Ala Thr Asn Gly Thr AspPhe Gln Leu Pro Pro Leu Ile Ser Ser Arg Cys Thr Pro Pro Ser His Pro Glu ThrThr Gly Asp Pro Asp Ala Glu Ala Tyr Tyr Ile Asn Lys Ser Val Gln Trp Tyr GlnAla His Gly Gly Asn Tyr Thr Ala Leu Ile Lys Arg Asp Thr Glu Thr Val Gly GlyMet Thr Leu Asp Leu Pro Glu Asn Pro Pro Pro Ile Pro Ala Thr Ser Thr Ala ProSer Ser Asp Ser Gly Glu Val Val Thr Ala Thr Ala Ala Gln Ile Lys Glu Leu ThrAsn Tyr Ala Gly Val Ala Ala Thr Ala Tyr Cys Arg Ser Val Val Pro Gly Thr AsnTrp Asp Cys Lys Gln Cys Leu Lys Tyr Val Pro Asp Gly Lys Leu Ile Lys Thr PheThr Ser Leu Leu Thr Asp Thr Asn Gly Phe Ile Leu Arg Ser Asp Ala Gln Lys ThrIle Tyr Val Thr Phe Arg Gly Thr Asn Ser Phe Arg Ser Ala Ile Thr Asp Met ValPhe Thr Phe Thr Asp Tyr Ser Pro Val Lys Gly Ala Lys Val His Ala Gly Phe LeuSer Ser Tyr Asn Gln Val Val Lys Asp Tyr Phe Pro Val Val Gln Asp Gln Leu ThrAla Tyr Pro Asp Tyr Lys Val Ile Val Thr Gly His Ser Leu Gly Gly Ala Gln AlaLeu Leu Ala Gly Met Asp Leu TyrGln Arg Glu Lys Arg Leu Ser Pro Lys Asn LeuSer Ile Tyr Cys Val Gly Cys Pro Arg Val Gly Asn Asn Ala Phe Ala Tyr Tyr ValAsp Ser Thr Gly Ile Pro Phe His Arg Thr Val His Lys Arg Asp Ile Val Pro HisVal Pro Pro Gln Ala Phe Gly Tyr Leu His Pro Gly Val Glu Ser Trp Ile Lys GluAsp Pro Ala Asp Val Gln Ile Cys Thr Ser Asn Ile Glu Thr Lys Gln Cys Ser AsnSer Ile Val Pro Phe Thr Ser Ile Ala Asp His Leu Thr Tyr Phe Gly Ile Asn GluGly Ser Cys Leu

[0067] One-letter form:

[0068] VPVAGHKGSVKATNGTDFQLPPLISSRCTPPSHPETTGDPDAEAYYINKSVQWYQAHGGNYTALIKRDTETVGGMTLDLPENPPPIPATSTAPSSDSGEVVTATAAQIKELTNYAGVAATAYCRSVVPGTNWDCKQCLKYVPDGKLIKTFTSLLTDTNGFILRSDAQKTIYVTFRGTNSFRSAITDMVFTFTDYSPVKGAKVHAGFLSSYNQVVKDYFPVVQDQLTAYPDYKVIVTGHSLGGAQALLAGMDLYQREKRLSPKNLSIYCVGCPRVGNNAFAYYVDSTGIPFHRTVHKRDIVPHVPPQAFGYLHPGVESWIKEDPADVQICTSNIETKQCSNSIVPFTSIADHLTYFGINEGSCL

[0069] SEQ ID NO.2:

[0070] Three-letter form:

[0071] Val Pro Val Ala Gly His Lys Gly Ser Val Lys Ala Thr Asn Gly Thr AspPhe Gln Leu Pro Pro Leu Ile Ser Ser Arg Cys Thr Pro Pro Ser His Pro Glu ThrThr Gly Asp Pro Asp Ala Glu Ala Tyr Tyr Ile Asn Lys Ser Val Gln Trp Tyr GlnAla His Gly Gly Asn Tyr Thr Ala Leu Ile Lys Arg Asp Thr Glu Thr Val Gly GlyMet Thr Leu Asp Leu Pro Glu Asn Pro Pro Pro Ile Pro Ala Thr Ser Thr Ala ProSer Ser Asp Ser Gly Glu Val Val Thr Ala Thr Ala Ala Gln Ile Lys Glu Leu ThrAsn Tyr Ala Gly Val Ala Ala Thr Ala Tyr Cys Arg Ser Val Val Pro Gly Thr AsnTrp Asp Cys Lys Gln Cys Leu Lys Tyr Val Pro Asp Gly Lys Leu Ile Lys Thr PheThr Ser Leu Leu Thr Asp Thr Asn Gly Phe Ile Leu Arg Ser Asp Ala Gln Lys ThrIle Tyr Val Thr Phe Arg Gly Thr Asn Ser Phe Arg Ser Ala Ile Thr Asp Met ValPhe Thr Phe Thr Asp Tyr Ser Pro Val Lys Gly Ala Lys Val His Ala Gly Cys LeuSer Ser Tyr Asn Gln Val Val Lys Asp Tyr Phe Pro Val Val Gln Asp Gln Leu CysAla Tyr Pro Asp Tyr Lys Val Ile Val Thr Gly His Ser Leu Gly Gly Ala Gln AlaLeu Leu Ala Gly Met Asp Leu Tyr<h2 style=";text-align:left;direction:ltr">Gln Arg Glu Lys Arg Leu Ser Pro Lys Asn LeuSer Ile Tyr Cys Val Gly Cys Pro Arg Val Gly Asn Asn Ala Phe Ala Tyr Tyr ValAsp Ser Thr Gly Ile Pro Phe His Arg Thr Val His Lys Arg Asp Ile Val Pro HisVal Pro Pro Gln Ala Phe Gly Tyr Leu His Pro Gly Val Glu Ser Trp Ile Lys GluAsp Pro Ala Asp Val Gln Ile Cys Thr Ser Asn Ile Glu Thr Lys Gln Cys Ser AsnSer Ile Val Pro Phe Thr Ser Ser Ile Ala Asp His Leu Thr Tyr Phe Gly Ile Asn GluGly Ser Cys Leu<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0072] <h2 style=";text-align:left;direction:ltr"> More information:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0073] <h2 style=";text-align:left;direction:ltr"> VPVAGHKGSVKATNGTDFQLPPLISSRCTPPSHPETTGDPDAEAYYINKSVQWYQAHGGNYTALIKRDTETVGGMTLDLPENPPPIPATSTAPSSDSGEVVTATAAQIKELTNYAGVAATAYCRSVVPGTNWDCKQCLKYVPDGKLIKTFTSLLTDTNGFILRSDAQKTIYVTFRGTNSFR SAITDMVFTFTDYSPVKGAKVHAGCLSSYNQVVKDYFPVVQDQLCAYPDYKVIVTGHSLGGAQALLAGMDLYQREKRLSPKNLSIYCVGCPRVGNNAFAYYVDSTGIPFHRTVHKRDIVPHVPPQAFGYLHPGVESWIKEDPADVQICTSNIETKQCSNSIVPFTSIADHLTYFGINEGSCL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0074] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.3:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0075]

[0076] SEQ ID NO:4:

[0077] Tcacgctggttgccttctctcatacaacca

[0078] SEQ ID NO:5:

[0079] Tatagtcagggtaagcgcacaattggtcttgaa

Claims

1. A lipase mutant, characterized in that: The amino acid sequence of the lipase mutant is shown in SEQ ID NO.

2.

2. A polynucleotide molecule, characterized in that It is a nucleotide sequence encoding the lipase mutant according to claim 1 or its complementary sequence.

3. The polynucleotide molecule according to claim 2, wherein Its nucleotide sequence is shown in SEQ ID NO.

3.

4. An expression cassette or recombinant expression vector containing the polynucleotide molecule according to claim 2 or 3.

5. The recombinant expression vector according to claim 4, wherein The vector is pGAPZα, pPIC9K or pPICZα.

6. A host cell containing the lipase mutant according to claim 1, the polynucleotide molecule according to claim 2 or 3, or the expression cassette or recombinant expression vector according to claim 4.

7. The host cell according to claim 6, wherein The host cell is Escherichia coli or yeast.

8. A method for preparing a lipase mutant, characterized in that: The method comprises expressing and purifying the host cell according to claim 6 or 7.

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