Lipase mutant as well as coding gene and application thereof

Abstract

The invention discloses a lipase mutant and application thereof, and belongs to the field of enzyme engineering, and the amino acid sequence of the lipase mutant is shown as SEQ ID NO. 2. The lipase mutant F206C-T226C can tolerate 70 minutes at the temperature of 60 DEG C and can tolerate 65% or more of residual activity at the temperature of 70 DEG C after 30 minutes, while wild lipase can tolerate 70 minutes at the temperature of 60 DEG C and can tolerate 50% or less of residual activity at the temperature of 70 DEG C after 30 minutes. Therefore, the thermal stability of the obtained enzyme mutant is remarkably improved, the enzyme activity is also improved by more than two times compared with that of initial lipase, and the enzyme mutant has important value in industrial application 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 (triacylglycerol lipase, EC3.1.1.3), also known as triacylglycerol hydrolase, can catalyze the hydrolysis of ester bonds of triglycerides, diglycerides, monoglycerides, other small molecule esters, polyol esters and polyacid esters. It can also catalyze the acidolysis, alcoholysis, aminolysis, transesterification and ester synthesis of esters. Fat is the natural substrate of lipase. During the hydrolysis process, diglycerides and monoglycerides are produced, and the final products of hydrolysis are glycerol and fatty acids. Fats are mostly hydrophobic substances, so the hydrolysis reaction occurs at the oil-water interface or in the organic phase. Lipase catalysis has the characteristics 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 chemical industry.

[0003] In the process of industrial production, enzyme preparations often experience high temperature environments, and the disadvantage that natural lipases are easily inactivated at high temperatures causes a substantial increase in the cost of industrial production, limiting the application of lipases in industry. When looking for lipases with good thermal stability, due to the large number of sites that can be mutated and the complexity of the protein structure, it is necessary to take into account both thermal stability and good enzyme activity. Obtaining a lipase mutant with thermal stability and higher enzyme activity is of great value for industrial application. Therefore, the present invention transforms the existing lipase to make it a lipase with higher thermal stability and enzyme activity. Summary of the invention

[0004] One of the purposes 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 scheme: 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 to cysteine ​​(C) and the threonine (T) at position 226 to cysteine ​​(C) in the lipase amino acid sequence SEQ ID NO.1.

[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 is 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 minutes and a relative enzyme activity of about 45% at 70°C for 45 minutes.

[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 with 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 protection scope of the present invention.

[0013] Another object of the present invention is to provide an expression cassette containing a lipase mutant encoding gene and a recombinant expression vector containing the lipase mutant encoding gene. Specifically, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector. More specifically, the expression vector includes 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 engineering 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 thermal stability and enzyme activity obtained in the prior art, and the amino acid sequence of the lipase is shown in SEQ ID NO. 1. The lipase is first structurally analyzed to determine the 3D molecular structure model of the lipase and the potential amino acid residues that affect the thermal stability and enzyme activity of the lipase, and then a lipase mutant is obtained by mutation, and screening is performed in combination with thermal stability and enzyme activity experiments to finally obtain the target lipase mutant.

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

[0020] Compared with the starting lipase (wild type), the mutant has better thermal stability and enzyme activity. Under the same conditions, the obtained lipase mutant F206C-T226C has a residual activity of more than 65% at 60°C for 70 minutes and 70°C for 30 minutes, while the wild-type lipase has a residual activity of less than 50% at 60°C for 70 minutes and 70°C for 30 minutes. 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 more than 2 times higher than that of the starting lipase. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the optimum temperature determination result 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;

[0023] Figure 3 These are the results of the thermal stability test of the lipase mutant (after enzyme mutation) and the initial lipase (before enzyme mutation) at 70°C 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 solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The experimental techniques and methods used in this example are all conventional technical methods unless otherwise specified. The materials, reagents and biological materials used in this example can 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" compiled by J. Sambrook et al., or perform according to the methods in the instructions of commercial kits.

[0027] By simulating and analyzing the spatial structure of the protein of the known lipase, it was finally determined that the mutation sites were phenylalanine at position 206 and threonine at position 226 in SEQID NO.1. The specific implementation scheme is to use the starting lipase gene as a template, mutate the gene of the potential amino acid residue of the lipase by the method of gene site-directed mutagenesis, obtain a new lipase gene, connect the mutant gene with a vector to construct a recombinant plasmid, and transfer it into the corresponding host bacteria for heterologous expression to obtain a lipase mutant. The mutant has been verified to have ideal heat resistance and enzyme activity, and is suitable for industrial production and application.

[0028] The amino acid sequence of the mutant obtained after 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 amplification instrument (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; ELISA 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] Specific scheme of p-nitrophenol method: The total system is 500μL, which contains 420μL of 50mmol / L buffer, 30μL of 10mmol / L substrate p-NPP and 50μL of diluted enzyme solution. After mixing the substrate and buffer, preheat them at the reaction temperature for 2min, add the diluted enzyme solution and mix well, react for 5min, add 50μL of 1.0mol / L SDS to terminate the reaction, and add 500μL of 1.0mol / L Na2 CO3 for color development; measure its OD value at a wavelength of 405nm.

[0041] Lipase thermal stability assay

[0042] The purified enzyme mutant and the starting lipase enzyme solution were diluted to the same protein concentration, and then placed in different temperatures: 60°C for 20min, 30min, 50min, 70min, 90min; and 70°C for 10min, 20min, 30min, 45min, 60min, and then the residual enzyme activity was measured, and the residual activity percentage was further calculated. The control was the enzyme solution that was not subjected to the temperature tolerance experiment.

[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: The recombinant plasmid containing the starting lipase encoding gene connected to the vector was used as a template, and a primer pair containing a specific mutation was used for PCR amplification. The PCR amplification conditions were: 95°C for 5 min; 98°C for 10 s, 55°C for 25 s, 72°C for 3 min, 30 cycles; 72°C for 5 min extension. 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 1% agarose gel electrophoresis. After the correct band is detected, recover and purify it.

[0047] (3) Transformation: Introduce the purified product into competent cells, flick to mix, and place on ice for 30 min; heat shock at 42°C for 40 s and immediately cool on ice for 10 min; add 500 μL LB medium and culture on a shaker at 37°C for 45 min; centrifuge at 7000 rpm for 3 min, 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, a single colony was picked and placed in 500 μL of LB medium containing resistance, cultured at 200 rpm for 3 h, and then PCR identification was performed; the screened 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 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 unit, U / mL.

[0054] The total system for determination is 500μL, which contains 420μL of 50mmol / L buffer, 30μL of 10mmol / L substrate p-NPP and 50μL of diluted enzyme solution. After mixing the substrate and buffer, preheat at the reaction temperature for 2min, add the diluted enzyme solution and mix well, react for 5min, add 50μL of 1.0mol / L SDS to terminate the reaction, and measure its OD value at a wavelength of 405nm.

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

[0057] Example 3 Determination of the optimal action temperature of lipase

[0058] 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. Then, the relative lipase activity of the sample was tested according to the method disclosed in the present invention to obtain the optimal action 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.

[0059] Example 4: Thermal stability test of lipase mutants

[0060] The purified lipase and its mutant enzyme solutions were placed in different temperatures: 60°C for 20 min, 30 min, 50 min, 70 min, 90 min; and 70°C for 10 min, 20 min, 30 min, 45 min, 60 min. The p-NPP hydrolysis ability was immediately measured after the insulation, and the enzyme solution that had not been heat treated was used as a control to calculate the residual enzyme activity rate.

[0061] The tolerance of lipase at different temperatures is shown in Figure 2. Figure 2 , Figure 3 As shown. As can be seen from the figure, with the increase of temperature and time, the relative enzyme activity shows a trend of gradual decrease, but the relative enzyme activity of the lipase after mutation is always higher than that before mutation. The enzyme mutant tolerates 70 minutes at 60°C and 30 minutes at 70°C with a residual activity of more than 65%, while the wild-type lipase tolerates 70 minutes at 60°C and 30 minutes at 70°C with a residual activity of less than 50%. It can be seen that the thermal stability of the enzyme mutant obtained by the present invention is significantly improved. Sequence Listing SEQ ID NO.1: Three-letter form: 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 Single-letter form: VPVAGHKGSVKATNGTDFQLPPLISSRCTPPSHPETTGDPDAEAYYINKSVQWYQAHGGNYTALIKRDTETVGGMTLDLPENPPPIPATSTAPSSDSGEVVTATAAQIKELTNYAGVAATAYCRSVVPGTNWDCKQCLKYVPDGKLIKTFTSLLTDTNGFILRSDAQKTIYVTFRGTNSFRSAITDMVFTFTDYSPVKGAKVHAGFLSSYNQVVKDYFPVVQDQLTAYPDYKVIVTGHSLGGAQALLAGMDLYQREKRLSPKNLSIYCVGCPRVGNNAFAYYVDSTGIPFHRTVHKRDIVPHVPPQAFGYLHPGVESWIKEDPADVQICTSNIETKQCSNSIVPFTSIADHLTYFGINEGSCL SEQ ID NO.2: Three-letter form: 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"> More information:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VPVAGHKGSVKATNGTDFQLPPLISSRCTPPSHPETTGDPDAEAYYINKSVQWYQAHGGNYTALIKRDTETVGGMTLDLPENPPPIPATSTAPSSDSGEVVTATAAQIKELTNYAGVAATAYCRSVVPGTNWDCKQCLKYVPDGKLIKTFTSLLTDTNGFILRSDAQKTIYVTFRGTNSFR SAITDMVFTFTDYSPVKGAKVHAGCLSSYNQVVKDYFPVVQDQLCAYPDYKVIVTGHSLGGAQALLAGMDLYQREKRLSPKNLSIYCVGCPRVGNNAFAYYVDSTGIPFHRTVHKRDIVPHVPPQAFGYLHPGVESWIKEDPADVQICTSNIETKQCSNSIVPFTSIADHLTYFGINEGSCL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.3:<h2 style=";text-align:left;direction:ltr"> SEQ ID NO:4: Tcacgctggttgccttctctcatacaacca SEQ ID NO:5: 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, characterized in that 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, characterized in that: 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, characterized in that 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.

9. A method for preparing oily esters, characterized in that: The lipase mutant as claimed in claim 1 is used to catalyze the esterification reaction between fatty acids and glycerol.

10. Use of the lipase mutant according to claim 1 in the synthesis, hydrolysis, alcoholysis or transesterification of esters.

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