A lipase mutant with improved heat resistance and its preparation method and application

Abstract

The present invention belongs to the field of protein engineering technology, and specifically discloses a lipase mutant with improved heat resistance, and a preparation method and application thereof. The lipase mutant TLL-NDTQ provided by the present invention is obtained by mutating the E, S, and G amino acids at the 56th, 58th, and 59th positions of the wild-type lipase TLL to N, T, and Q, respectively, and its amino acid sequence is shown in SEQ ID NO.6. Compared with the wild-type lipase TLL, the lipase mutant provided by the present invention has an optimum temperature of 45 ° C, an optimum pH of 10.0, an initial activity of more than 80% in a pH range of 7.0-11.0, and a higher stability in a 10% methanol solution. The mutant provided by the present invention has potential application value in the production of feed granulation, leather wrinkle resistance, and food baking with high heat environment requirements.

Description

Technical Field

[0001] The invention relates to the technical field of protein engineering, and in particular to a lipase mutant with improved heat resistance, and a preparation method and application thereof. Background Art

[0002] As a widely used biocatalyst, lipase is widely used in various industrial fields. In order to cope with the harsh conditions such as high temperature, high pressure, extreme acid and alkali in the industrial production process, the activity, thermal stability and pH tolerance of lipase are optimized, which is conducive to reducing the loss of enzyme resources caused by industrial production. The lipase TLL produced by Thermomyces lanuginosus is known for its excellent thermal stability, substrate specificity and tolerance to organic solvents. These characteristics make TLL widely used in many fields such as feed processing, food processing, drug development and biofuel manufacturing, and environmental pollutant treatment. Although TLL comes from thermophilic fungi, the application conditions in most industrial fields (such as high-temperature pelleting process of feed, baking food processing, and strong acid or strong alkali conditions) will cause enzyme loss of TLL, resulting in increased production costs.

[0003] Protein engineering technology, as an emerging technology that has flourished in the past two decades, provides more possibilities for optimizing the properties of TLL. Based on this, the present invention intends to transform lipase TLL with the help of protein engineering to make it have better biological activity and heat resistance, so that it can play a greater role in fields with high tolerance requirements. Summary of the invention

[0004] The purpose of the present invention is to provide a lipase mutant with better heat resistance, so that it has higher application value in production with high temperature requirements.

[0005] In order to achieve the above object, the present invention provides a lipase mutant with improved heat resistance, the amino acid sequence of the mutant is shown in SEQ ID NO.6.

[0006] The present invention also provides a coding gene for the lipase mutant, and the nucleotide sequence thereof is shown as SEQ ID NO.5.

[0007] The present invention also provides a method for obtaining the above-mentioned mutant encoding gene, which is obtained by amplifying the encoding gene of the wild-type lipase TLL using mutant primers with nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4, wherein the nucleotide sequence of the wild-type lipase TLL encoding gene is as shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant plasmid containing the mutant encoding gene, and the recombinant plasmid is preferably selected from pPIC9K.

[0009] The present invention also provides a recombinant expression bacterium expressing the above lipase mutant, which is obtained by transferring the above recombinant plasmid into a host expression bacterium, wherein the recombinant expression bacterium is preferably selected from GS115.

[0010] The present invention also provides a method for preparing the lipase mutant, which is obtained by culturing and inducing recombinant expression bacteria and purifying the mutant.

[0011] The lipase mutant provided by the invention can be used in the industrial production of lipase, and can be particularly used in feed granulation, leather wrinkle resistance or food baking.

[0012] The lipase mutant of the present invention has the following advantages:

[0013] Compared with the wild enzyme TLL, the lipase mutant provided by the present invention has improved optimum temperature and optimum pH, and improved pH stability and high temperature tolerance, shows higher stability and activity retention rate under high temperature conditions and alkaline conditions, and has potential application value in the industrial production and application of lipase. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the protein crystal structure of the wild-type lipase TLL of the lipase family in the present invention.

[0015] Figure 2 It is the PCR electrophoresis diagram of the mutant TLL-NDTQ of the present invention.

[0016] Figure 3 It is the plasmid electrophoresis diagram of TLL and mutant TLL-NDTQ of the present invention.

[0017] Figure 4 The figure shows the SDS-PAGE analysis of the purified enzymes of TLL and mutant TLL-NDTQ of the present invention.

[0018] Figure 5 The optimum temperature and optimum pH of TLL and mutant TLL-NDTQ of the present invention are measured, wherein: Figure 5 A in the figure is the optimum pH measurement result of the wild enzyme and mutant. Figure 5 B in the figure is the optimum temperature determination result of the wild enzyme and the mutant.

[0019] Figure 6 The temperature tolerance and pH stability test results of TLL and mutant TLL-NDTQ of the present invention are shown in FIG. Figure 6 A in the figure is the enzyme activity test result of the wild type enzyme and the mutant after endurance for 1 hour at different pH. Figure 6 B in the figure is the enzyme activity test result of the wild enzyme and the mutant after endurance at 80℃ for different time periods.

[0020] Figure 7 The figure shows the stability of the TLL and mutant TLL-NDTQ of the present invention in methanol solutions of different concentrations. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0022] Note: All methods not specifically described in this application are conventional methods in the art, and all reagents and consumables not specifically given are conventional reagents and consumables in the art.

[0023] Example 1 Construction of lipase mutants and determination of enzymatic properties

[0024] It is known that TLL belongs to the lipase family, and its amino acid sequence is shown in SEQ ID NO.1. The nucleotide sequence encoding the wild enzyme TLL is shown in SEQ ID NO.2. The specific sequence is shown below. The protein crystal structure simulation diagram of the wild enzyme TLL is shown in Figure 1 As shown, it has the ability to catalyze the hydrolysis of triacylglycerol. Multiple mutants were obtained by selecting multiple sites on the TLL sequence for mutation modification, and the optimal temperature of the obtained mutants was determined. When the E, S, and G amino acids at the 56th, 58th, and 59th sites of the TLL wild enzyme were mutated to N, T, and Q, respectively, it was recorded as the mutant TLL-NDTQ, and its optimal temperature changed and increased to 45°C compared to 40°C of the TLL wild enzyme. The specific mutant construction and determination are as follows:

[0025] Among them, the amino acid sequence of the wild enzyme TLL is:

[0026] EVSQDLFNQFNLFAQYSAAAYCGKNNDAPAGTNITCTGNACPEVEKADATFLYSFEDSGVGDVTGFLALDNTNKLIVLSFRGSRSIENWIGNLNFDLKEINDICSGCRGHDGFTSSWRSVADTLRQKVEDAVREH PDYRVVFTGHSLGALATVAGADLRGNGYDIDVFSYGAPRVGNRAFAEFLTVQTGGTLYRITHTNDIVPRLPPREFGYSHSSPEYWIKSGTLVPVTRNDIVKIEGIDATGGNNQPNIPDIPAHLWYFGLIGTCL.

[0027] The nucleotide sequence encoding the wild enzyme TLL is as follows:

[0028] gaggtctcgcaggatctgtttaaccagttcaatctctttgcacagtattctgcagccgcatactgcggaaaaaacaatgatgccccagctggtacaaacattacgtgcacgggaaatgcctgcccagaggtagagaaggccgatgcaacgtttctctactcgtttgaagactctggagtgggagatgtcaccggattccttgctctcgacaacacgaacaaattgatcgtcctctctttcagaggatctagatccattgagaactggatcggaaatcttaacttcgacttgaaagagatcaatgacatttgctccggatgcagaggacatgacggtttcacttcgtcctggagatctgtagccgatacgttaagacagaaggtggaggatgctgtgagagagcatccagactatagagtggtgtttaccggacatagcttgggtggtgcattggcaactgttgccggagcagacctgagaggaaatggttatgatatcgacgtgttttcatatggagcccctagagtcggaaacagagcttttgcagagttcctgaccgtacagaccggaggaacactctacagaattacccacaccaatgatattgtccctagactccctccacgcgagttcggttacagacattctagcccagagtactggatcaaatctggaacccttgtcccagtcaccagaaacgatatcgtgaagattgaaggaatcgatgccaccggaggaaacaaccagcctaacattcctgatatccctgcccacctatggtacttcggtttaattggaacatgtctt。

[0029] 1. Primer design and construction of mutants

[0030] Using the sequence of the TLL wild enzyme encoding gene as a template, the mutation primers of the mutant TLL-NDTQ were designed using DNAMAN and BioXM 2.7.1 according to the instructions of the rapid site-directed mutagenesis kit, as shown in Table 1 below, and all primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0031] Table 1 Primers for mutant TLL-NDTQ

[0032]

[0033] The mutation primers in Table 1 were used to perform mutation amplification using the sequence of the TLL wild enzyme encoding gene as a template, wherein the PCR reaction system and amplification program are shown in Tables 2 and 3 below, respectively.

[0034] Table 2 PCR reaction system

[0035]

[0036] Table 3 PCR mutagenesis program

[0037]

[0038] After mutation amplification is completed, the PCR product is subjected to electrophoresis, and the electrophoresis result is as follows: Figure 2 As shown, lane M is the control marker of DL2000, and lane 1 is the PCR amplification product. The amplification product was connected to the vector pPIC9K to obtain TLL-pPIC9K, and was transferred into DMT competent cells by heat shock method, and then spread on LB culture dishes with resistance and cultured in a 37°C incubator overnight.

[0039] At the same time, the nucleotide sequence of wild-type TLL was constructed into the same vector using conventional construction methods, and transferred into competent cells for plasmid sequencing verification, thereby obtaining a recombinant plasmid and recombinant expression bacteria that can express wild-type TLL lipase.

[0040] The next day, single colonies were randomly picked from the plates, marked and sent for sequencing, and those with successful sequencing were selected as single colonies with successful mutations, thus obtaining recombinant expression bacteria expressing the lipase mutant TLL-NDTQ.

[0041] The wild-type lipase strain and the lipase mutant strain obtained above were routinely cultured, and their recombinant plasmids were extracted respectively. After the recombinant plasmids were linearized, they were electro-transformed into the host bacteria Pichia pastoris GS115 for heterologous expression, and the wild-type lipase TLL and the lipase mutant TLL-NDTQ were purified. The recombinant plasmid was sequenced and verified to obtain the nucleotide sequence of the lipase mutant TLL-NDTQ as shown in SEQ ID NO.5, and the amino acid sequence of the lipase mutant was obtained as shown in SEQ ID NO.6. The specific sequence is shown below. Among them, the electrophoresis diagram of the plasmid that can express TLL and the mutant TLL-NDTQ is shown in Figure 3 As shown, lane M is a control marker for DL15000, and lanes 1 and 2 are recombinant plasmids that can express wild-type TLL and mutant TLL-NDTQ, respectively. The purified wild-type enzyme and mutant were subjected to SDS gel electrophoresis, and the SDS-PAGE electrophoresis results of purified enzymes of TLL and mutant TLL-NDTQ were obtained as shown in FIG. Figure 4 As shown, lane M is a protein control marker, and lanes 1 and 2 are recombinant plasmids of wild-type TLL lipase and mutant lipase, respectively.

[0042] Among them, the nucleotide sequence encoding the mutant TLL-NDTQ is (SEQ ID NO.5): gaggtctcgcaggatctgtttaaccagttcaatctctttgcacagtattctgcagccgcatactgcggaaaaaacaatgatgccccagctggtacaaacattacgtgcacgggaaatgcctgcccagaggtagagaaggccgatgcaacgtttctctactcgtttaatgacactcaagtgggagatgtcaccggattccttgctctcgacaacacgaacaaattgatcgtcctctctttcagaggatctagatccattgagaactggatcggaaatcttaacttcgacttgaaagagatcaatgacatttgctccggatgcagaggacatgacggtttcacttcgtcctggagatctgtagccgatacgttaagacagaaggtggaggatgctgtgagagagcatccagactatagagtggtgtttaccggacatagcttgggtggtgcattggcaactgttgccggagcagacctgagaggaaatggttatgatatcgacgtgttttcatatggagcccctagagtcggaaacagagcttttgcagagttcctgaccgtacagaccggaggaacactctacagaattacccacaccaatgatattgtccctagactccctccacgcgagttcggttacagacattctagcccagagtactggatcaaatctggaacccttgtcccagtcaccagaaacgatatcgtgaagattgaaggaatcgatgccaccggaggaaacaaccagcctaacattcctgatatccctgcccacctatggtacttcggtttaattggaacatgtctt。

[0043] The amino acid sequence of the mutant TLL-NDTQ is (SEQ ID NO.6):

[0044] EVSQDLFNQFNLFAQYSAAAYCGKNNDAPAGTNITCTGNACPEVEKADATFLYSFNDTQVGDVTGFLALDNTNKLIVLSFRGSRSIENWIGNLNFDLKEINDICSGCRGHDGFTSSWRSVADTLRQKVEDAVREH PDYRVVFTGHSLGALATVAGADLRGNGYDIDVFSYGAPRVGNRAFAEFLTVQTGGTLYRITHTNDIVPRLPPREFGYSHSSPEYWIKSGTLVPVTRNDIVKIEGIDATGGNNQPNIPDIPAHLWYFGLIGTCL.

[0045] 2. Determination of the properties of lipase

[0046] (1) Determination of optimum pH and temperature

[0047] In order to explore the optimal pH of TLL and mutant TLL-NDTQ, enzyme activity tests were performed in buffers with different pH values ​​(4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0). Figure 5 As shown in A. It can be seen that the optimal pH value of the wild-type TLL is 9.0, and the optimal pH of the mutant TLL-NDTQ is 10, which is higher than the optimal pH of the wild-type enzyme.

[0048] The optimum temperature of the wild-type lipase (TLL) and the mutant TLL-NDTQ obtained above was determined according to the lipase activity determination method. Under the optimum pH condition, the reaction system was placed at different temperatures (30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C) for reaction. After determination, the optimum temperature of the enzymatic reaction of the lipase mutant was obtained as follows: Figure 5 As shown in B, it can be seen that the optimum temperature of the Thermomyces lanuginosus lipase mutant TLL-NDTQ is 45°C, which is 5°C higher than that of the wild-type lipase.

[0049] (2) pH tolerance and temperature tolerance determination

[0050] In order to explore the pH stability of TLL and its mutant TLL-NDTQ, the enzyme was tolerated for 1 h in buffers with different pH values ​​(5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0), and then the enzyme activity was tested at the optimal temperature and optimal pH. Figure 6As shown in A, different enzymes have significant differences in activity at different pH values. Under conditions where the relative residual enzyme activity is higher than 80%, the wild-type TLL maintains high activity in the pH range of 8.0 to 11.0, and the TLL-NDTQ mutant has high activity in the pH range of 7.0 to 10.0.

[0051] The obtained enzyme solution was diluted to the corresponding multiples and placed at 80°C for 10 min, 20 min, 30 min, 45 min, 60 min, 75 min, and 90 min, respectively. Then, the temperature tolerance of wild-type lipase (TLL) and lipase mutant TLL-NDTQ was determined according to the lipase activity determination method at the optimal pH and temperature. Figure 6 As shown in B, it can be seen that with the increase of time, the relative enzyme activity of lipase gradually decreases, and the relative enzyme activity of mutant TLL-NDTQ remains 77.59% after tolerating 90 minutes at 80°C. Compared with the wild-type lipase, the relative enzyme activity of mutant TLL-NDTQ increased by 36.88% after tolerating 90 minutes at 80°C. It shows that after the optimum temperature is increased, the thermal stability of the mutant TLL-NDTQ is also greatly improved compared with the wild type, and this lipase mutant with improved thermal stability can be used in industrial production, including feed granulation, leather wrinkle resistance and food baking production with high heat environment requirements, and has great application value.

[0052] Experimental Example 1 Determination of the stability of lipase in methanol

[0053] To evaluate the stability of TLL protein and its mutant TLL-NDTQ in 10% methanol solution, the enzyme solution was incubated in 10% methanol buffer system for 1 hour, and then the enzyme activity was quantitatively analyzed under their respective optimal temperature and optimal pH conditions. Figure 7 As shown in the figure, after treatment with methanol, the residual enzyme activity of wild-type TLL was 85.50%, while that of mutant TLL-NDTQ was 94.57%. Compared with wild-type TLL, the residual enzyme activity of TLL-NDTQ was significantly increased by 9.07%, indicating that the stability of TLL-NDTQ in 10% methanol solution was enhanced.

[0054] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

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

6.

2. The gene encoding the lipase mutant according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.

5.

3. A method for obtaining the coding gene as claimed in claim 2, characterized in that: The coding gene is obtained by amplifying the coding gene of the wild-type lipase TLL using mutant primers with nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4, wherein the nucleotide sequence of the coding gene of the wild-type lipase TLL is as shown in SEQ ID NO.

2.

4. A recombinant plasmid containing the coding gene as claimed in claim 2.

5. The recombinant plasmid according to claim 4, characterized in that The recombinant plasmid is selected from pPIC9K.

6. A recombinant expression bacterium expressing the lipase mutant according to claim 1, characterized in that: The recombinant expression bacteria are obtained by transferring the recombinant plasmid as described in any one of claims 4 or 5 into host expression bacteria.

7. The recombinant expression bacteria according to claim 6, characterized in that: The host expression bacteria is selected from yeast GS115.

8. A method for preparing the lipase mutant according to claim 1, characterized in that: The method is obtained by culturing the recombinant expression bacteria as described in any one of claims 6 or 7 and purifying the recombinant expression bacteria.

9. Use of the lipase mutant according to claim 1 in industrial production of lipase.

10. The use according to claim 9, characterized in that: The applications include feed pelleting, leather wrinkle reduction and / or food baking.

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