Surfactant-tolerant lipase mutant and application thereof

Abstract

The invention discloses a lipase mutant tolerant to a surfactant and application of the lipase mutant. According to the invention, yarrowia lipolytica lipase 2 (YLL2) with an amino acid sequence as shown in SEQ ID NO.1 is used as a parent, and aspartic acid (D) is mutated into isoleucine (I) through site-specific mutagenesis, so that the lipase mutant D97I capable of tolerating various surfactants is obtained. Compared with a lipase YLL2 parent, the lipase mutant D97I has the advantages that the tolerance of the lipase mutant D97I to AEO-9, SDS, Triton X-80, Tween-100, AOS and AES is improved by 32.3%, 25.2%, 12.7%, 14.11%, 10.5% and 14.2% respectively, and the lipase mutant D97I can be better applied to the detergent industry and the food processing industry.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme engineering technology and more specifically relates to a surfactant-tolerant lipase mutant and its application. Background Art

[0002] Lipase (EC 3.1.1.3), as an important industrial enzyme, is widely present in animals, plants and microorganisms. It can be used to catalyze reactions such as hydrolysis, esterification and transesterification of oils and fats. It is a key biocatalyst in biosynthesis, biotransformation and biodegradation processes.

[0003] Surfactants are widely used in detergents, biodiesel production, food processing, and environmental management. Their core function is to improve the contact efficiency between oils and enzymes through emulsification, dispersion, or solubilization. However, their physical and chemical properties (such as amphiphilic structure and charge characteristics) may disrupt the active center or overall conformation of lipases, leading to enzyme inactivation. The importance of lipase tolerance to surfactants lies in its direct influence on the efficiency and stability of enzymes in practical industrial applications. For example, in the detergent industry, lipases can efficiently break down grease stains. When they have the ability to tolerate surfactants, they can maintain long-term activity in complex washing environments containing surfactants, significantly enhancing their oil removal ability while reducing detergent dosage and washing temperature. In the food processing field, lipases are used to improve taste and enhance nutritional value. Surfactant-tolerant lipases can operate stably in the presence of surfactants commonly used in food processing, ensuring the stability of product quality. Therefore, it is necessary to develop lipases that can tolerate surfactants so that they can maintain catalytic activity in complex environments with high surfactant concentrations, thereby reducing production costs and broadening their application range.

[0004] Yarrowia lipolytica lipase 2 (YLL2) has attracted much attention in the industrial field due to its efficient hydrolysis ability of long-chain fatty acids and alkali resistance, but the enzyme has poor tolerance to surfactants. Summary of the Invention

[0005] Aiming at the shortcoming that Yarrowia lipolytica lipase 2 has poor tolerance to surfactants, the present invention uses Yarrowia lipolytica lipase 2 as a parent to provide a surfactant-tolerant lipase mutant.

[0006] The first object of the present invention is to provide a surfactant-tolerant lipase mutant.

[0007] The second object of the present invention is to provide a gene.

[0008] The third object of the present invention is to provide a recombinant vector.

[0009] The fourth object of the present invention is to provide a recombinant bacterium.

[0010] The fifth object of the present invention is to provide a biocatalyst.

[0011] A sixth object of the present invention is to provide a detergent.

[0012] The seventh object of the present invention is to provide use of the lipase mutant, the recombinant bacterium or the biocatalyst in catalyzing a reaction containing a surfactant.

[0013] The eighth object of the present invention is to provide use of the lipase mutant, the gene, the recombinant vector, the recombinant bacteria or the biocatalyst in preparing a preparation for catalyzing a reaction containing a surfactant.

[0014] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0015] The present invention obtains a surfactant-tolerant lipase mutant, D97I, by mutating the amino acid 97 of Yarrowia lipolytica lipase 2 (YLL2) from aspartic acid (D) to isoleucine (I). Compared to the parent lipase YLL2, the enzymatic activity of the lipase mutant D97I remains unchanged, while its tolerance to AEO-9, SDS, Triton X-80, Tween-100, AOS, and AES is significantly improved. Therefore, the present invention seeks protection for the lipase mutant D97I and its related applications.

[0016] The present invention provides a surfactant-tolerant lipase mutant, which is obtained by taking Yarrowia lipolytica lipase 2 with an amino acid sequence as shown in SEQ ID NO.1 as a parent and mutating the 97th amino acid from aspartic acid to isoleucine.

[0017] Specifically, the amino acid sequence of the lipase mutant is shown in SEQ ID NO.3.

[0018] The present invention also provides a gene encoding the lipase mutant.

[0019] As an optional embodiment, the nucleotide sequence of the gene is shown as SEQ ID NO.4.

[0020] The present invention also provides a recombinant vector containing the gene of the present invention.

[0021] The present invention also provides a recombinant bacterium containing the gene or the recombinant vector of the present invention.

[0022] The present invention also provides a biocatalyst containing the lipase mutant and / or the recombinant bacteria of the present invention.

[0023] The present invention also provides a detergent comprising the lipase mutant of the present invention and a surfactant.

[0024] Specifically, the surfactant is an anionic surfactant and / or a nonionic surfactant.

[0025] Specifically, the anionic surfactant includes sodium dodecylbenzenesulfonate (SDS), sodium α-olefinsulfonate (AOS) and sodium fatty alcohol polyoxyethylene ether sulfate (AES); the nonionic surfactant includes Triton X, Tween and fatty alcohol polyoxyethylene ether (AEO).

[0026] More specifically, the Triton X is Triton X-80; the Tween is Tween-100; and the AEO is AEO-9.

[0027] The present invention claims protection for the use of the lipase mutant, the recombinant bacteria or the biocatalyst in catalyzing a reaction containing a surfactant.

[0028] The present invention also claims protection for the use of the lipase mutant, the gene, the recombinant vector, the recombinant bacteria or the biocatalyst in preparing a preparation for catalyzing a reaction containing a surfactant.

[0029] Specifically, the surfactant is an anionic surfactant and / or a nonionic surfactant.

[0030] Specifically, the anionic surfactant includes SDS, AOS and AES; the nonionic surfactant includes Triton X, Tween and AEO.

[0031] More specifically, the Triton X is Triton X-80; the Tween is Tween-100; and the AEO is AEO-9.

[0032] The present invention has the following beneficial effects:

[0033] The present invention uses Yarrowia lipolytica lipase 2 (YLL2) as a parent, and mutates its amino acid position 97 from aspartic acid (D) to isoleucine (I), resulting in a lipase mutant D97I that is tolerant to multiple surfactants. Compared to the parent lipase YLL2, the lipase mutant D97I exhibits increased tolerance to AEO-9, SDS, Triton X-80, Tween-100, AOS, and AES by 32.3%, 25.2%, 12.7%, 14.11%, 10.5%, and 14.2%, respectively, making it suitable for applications in the detergent and food processing industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The figure is an agarose gel electrophoresis diagram of the expression vectors of various lipase mutants constructed by PCR amplification; M in the figure is a molecular weight marker; lanes 1 to 6 are lipase mutants H89N, L91I, E92V, T96V, D97I and R99L, respectively.

[0035] Figure 2 The SDS-PAGE detection results of recombinantly expressed lipase YLL2 and its mutants; M in the figure is the molecular weight marker; lanes 1 to 7 are lipase YLL2 and lipase mutants H89N, L91I, E92V, T96V, D97I and R99L, respectively.

[0036] Figure 3 The results of the tolerance test of lipase YLL2 and its mutants to AEO-9 and SDS; ** indicates P < 0.01, indicating extremely significant differences.

[0037] Figure 4 The results of the tolerance test of lipase YLL2 and its mutant D97I to CTAB, Triton X-80, Tween-100, AOS and AES; ** indicates P < 0.01, indicating extremely significant differences. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0039] Unless otherwise specified, all reagents and materials used in the following examples were commercially available.

[0040] The amino acid sequence of the Yarrowia lipolytica lipase 2 (YLL2) described in the Examples of the present invention is shown in SEQ ID NO. 1, and the nucleotide sequence of the gene encoding the lipase is shown in SEQ ID NO. 2. The amino acid sequence of the lipase mutant D97I is shown in SEQ ID NO. 3, and the nucleotide sequence of the gene encoding the lipase is shown in SEQ ID NO. 4.

[0041] Example 1 Preparation of lipase YLL2 and its mutants

[0042] In order to improve the tolerance of lipase YLL2 to surfactants, the present invention selected 6 sites for site-directed mutagenesis, and obtained 6 different lipase mutants, namely lipase mutants H89N, L91I, E92V, T96V, D97I and R99L.

[0043] 1. Construction of expression vector for lipase YLL2

[0044] Sangon Biotech (Shanghai) Co., Ltd. was commissioned to perform complete gene synthesis of the gene encoding lipase YLL2 (shown in SEQ ID NO. 2), and the expression vector pPICZαA-Lip2 was constructed using EcoRI and NotⅠ restriction endonuclease sites as entry points.

[0045] 2. Construction of expression vector for lipase mutant

[0046] The present invention uses the constructed pPICZαA-Lip2 expression vector as a template and employs PCR site-directed mutagenesis technology to introduce mutation sites through PCR amplification to construct an expression vector for the aforementioned lipase mutant. The primers used for PCR amplification are shown in Table 1.

[0047] Table 1 Primers used to construct lipase mutant expression vectors

[0048]

[0049]

[0050] The PCR amplification reaction system was as follows: DNA template (pPICZαA-Lip2) 50 ng, 2× Primer start 10 μL, upstream and downstream primers 1 μL each, dimethyl sulfoxide 1 μL, and ddH2O supplemented to 20 μL.

[0051] The PCR amplification reaction program was as follows: 98°C for 3 min; 30 cycles of 98°C for 30 s, 55°C for 30 s, and 72°C for 4 min; and extension at 72°C for 7 min.

[0052] After PCR amplification, the amplified products were detected by 1% agarose electrophoresis. Figure 1 As shown. Figure 1 It can be seen that the present invention successfully constructed the expression vector of the lipase mutant.

[0053] The PCR amplification product was purified and recovered. 1 μL of Dpn I, 1 μL of buffer, and 8 μL of the PCR amplification product were mixed and digested with Dpn I at 37°C for 1 h to remove the unmutated template DNA. The Dpn I digestion product was evenly mixed with 50 μL of Escherichia coli Top10 competent cells. The heat-shock transformed bacterial solution was spread on an LLB resistance plate containing 25 μg / mL zeocin and cultured at 37°C overnight. Positive clones were selected, and the plasmids were extracted and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to confirm the accuracy of the mutation site.

[0054] After sequencing, the mutation sites of each lipase mutant were accurate and consistent with expectations. The sequenced expression vectors were used to recombinantly express lipase YLL2 and its mutants.

[0055] 3. Recombinant expression of lipase YLL2 and its mutants

[0056] The expression vector verified to be correct by sequencing was linearized using Pme I restriction endonuclease, and a high-purity linear DNA product was obtained by purification and recovery; 5 μg of the purified product was mixed with Pichia pastoris X-33 competent cells and transformed by electroporation; the transformed bacterial liquid was evenly spread on a YPD screening plate containing 100 μg / mL Zeocin and placed in a 30°C constant temperature incubator for 72 hours; a single colony was picked and inoculated into 2 mL of YPD screening medium containing 200 μg / mL Zeocin, and the culture was shaken at a constant temperature of 37°C for 12 to 16 hours to achieve strain amplification; the bacteria were collected by centrifugation, resuspended in 100 mL of BMGY basal medium, and cultured at 30°C and 200 rpm; when the bacteria grew to the logarithmic phase, they were transferred to 400 mL BMMY induction medium was added with methanol at a final concentration of 1%, and the expression was induced and cultured at 25°C and 200 rpm for 96 h. During the induced expression culture, anhydrous methanol was added every 24 h to continuously activate the AOX1 promoter, and the final concentration of the added methanol was 1%.

[0057] After the induction expression culture is completed, the fermentation enzyme solution is centrifuged at 4°C and 7000g / min for 10 minutes, the supernatant crude enzyme solution is collected, and purified using a nickel column. The purification process is as follows: the nickel column is equilibrated with buffer A (20nm Tris-HCl, pH 7.0, containing 60mM imidazole, 500mM sodium chloride); the crude enzyme solution is mixed with the nickel column, and the nickel column is rinsed with buffer A 15 times the volume of the nickel filler to fully remove impurities; the target protein is eluted with 15mL of buffer B (20nm Tris-HCl, pH 7.0, containing 500mM imidazole); 20nm Tris-HCl (pH 7.0) is used as the salt exchange buffer, and the eluted target protein is salt-exchanged using a 10kDa ultrafiltration tube, and the salt exchange buffer is 20nm Tris-HCl (pH 7.0), thereby obtaining the recombinant expressed lipase YLL2 and its mutants, i.e., the recombinant enzyme.

[0058] The purity of the recombinant enzyme was verified by 12% SDS-PAGE vertical electrophoresis. Figure 2 As shown. Figure 2 It can be seen that all recombinant enzyme samples are single bands and can be used for subsequent determination.

[0059] Example 2 Surfactant tolerance determination of lipase mutants

[0060] The recombinant enzyme sample was diluted with 20mM Tris-HCl (pH 7.0), and the nonionic surfactant fatty alcohol polyoxyethylene ether (AEO-9) and the anionic surfactant sodium dodecyl sulfate (SDS) were added to the sample to a final concentration of 0.1mg / mL. The final concentrations of both AEO-9 and SDS were 20mM. A control group was maintained without surfactant. After incubation at 4°C for 4 hours, the residual enzyme activity of the surfactant-treated recombinant enzyme was determined using the pNP colorimetric assay, with the activity of the enzyme treated without surfactant as 100%. The residual enzyme activity of the lipase mutants with increased residual enzyme activity was further analyzed using ANOVA, with LSD used for multiple comparisons. P values less than 0.01 were considered significant.

[0061] The pNP colorimetric method is as follows: the standard reaction system consists of 915 μL Tris-HC buffer (50 mM, pH 7.0), 80 μL 4-nitrophenyl palmitate (final concentration 5 mM) dissolved in a mixed solvent of acetonitrile / ethanol (1:1), and 5 μL purified lipase (i.e., recombinant enzyme); after reacting at 30°C for 10 minutes, 500 μL 20% (w / v) trichloroacetic acid is added to terminate the reaction; 500 μL 20% (w / v) sodium carbonate solution is added to the reaction mixture for color development, and its absorbance at 405 nm is measured.

[0062] The specific enzyme activity is defined as the amount of enzyme required to produce 1 micromole of fatty acid per minute under the above reaction conditions.

[0063] The results of the tolerance test of lipase YLL2 and its mutants to surfactant AEO-9 and SDS are shown in Figure 2. Figure 3 As shown. Figure 3 The results show that under AEO-9 and SDS treatment, the residual enzyme activity of lipase YLL2 was 34.8% and 20.4%, respectively, while that of mutant D97I was 67.1% and 45.6%, respectively. Compared with lipase YLL2, the residual enzyme activity of mutant D97I increased by 32.3% and 25.2%, respectively, which is a highly significant difference.

[0064] Example 3: Test of the specific activity of lipase mutants and their tolerance to other surfactants

[0065] 1. Enzyme activity determination

[0066] The specific enzyme activity of lipase YLL2 and its mutant D97I was determined using the pNP colorimetric method. For specific methods, see Example 2. Statistical analysis was performed using ANOVA, and multiple comparisons were performed using LSD. P values less than 0.05 were considered significant. The results of the specific enzyme activity determination of lipase YLL2 and its mutant D97I are shown in Table 2. As shown in Table 2, the specific activities of lipase YLL2 and mutant D97I were 382.3 and 364.8 U / mg, respectively. Compared with lipase YLL2, the specific enzyme activity of lipase mutant D97I was not significantly reduced.

[0067] Table 2 Specific enzyme activities of lipase YLL2 and its mutant D97I

[0068]

[0069] Note: The same shoulder letters indicate P>0.05, indicating no significant difference; different shoulder letters indicate P<0.05, indicating significant difference.

[0070] 2. Tolerance test to other surfactants

[0071] The tolerance of lipase YLL2 and its mutant D97I to other surfactants (cationic surfactant (CTAB), nonionic surfactant (Triton X-80 and Tween-100) and anionic surfactant (AOS and AES)) was determined by referring to the method described in Example 2 and the results were analyzed for significance. Figure 4 As shown in the figure, under CTAB treatment, the residual enzyme activities of lipase YLL2 and mutant D97I were similar, with no significant difference. However, under treatment with Triton X-80, Tween-100, AOS, and AES, the residual enzyme activity of mutant D97I increased by 12.7%, 14.11%, 10.5%, and 14.2%, respectively, compared to lipase YLL2. Mutant D97I showed significantly improved tolerance to these surfactants.

[0072] 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A surfactant-tolerant lipase mutant, characterized in that: The mutant is obtained by using the Yarrowia lipolytica lipase 2 with an amino acid sequence as shown in SEQ ID NO. 1 as a parent and mutating the 97th amino acid from aspartic acid to isoleucine.

2. A gene, characterized in that The gene encodes the lipase mutant according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

4. A recombinant vector, characterized in that Containing the gene according to claim 2 or 3.

5. A recombinant bacterium, characterized in that Containing the gene according to claim 2 or 3 or the recombinant vector according to claim 4.

6. A biocatalyst, characterized in that Containing the lipase mutant according to claim 1 and / or the recombinant bacteria according to claim 5.

7. A detergent, characterized in that Contains the lipase mutant according to claim 1 and a surfactant; the surfactant is an anionic surfactant and / or a nonionic surfactant.

8. Use of the lipase mutant according to claim 1, the recombinant bacterium according to claim 5, or the biocatalyst according to claim 6 in catalyzing a reaction containing a surfactant, characterized in that: The surfactant is an anionic surfactant and / or a nonionic surfactant.

9. Use of the lipase mutant according to claim 1, the gene according to claim 2 or 3, the recombinant vector according to claim 4, the recombinant bacterium according to claim 5, or the biocatalyst according to claim 6 in preparing a preparation for catalyzing a reaction containing a surfactant, characterized in that: The surfactant is an anionic surfactant and / or a nonionic surfactant.

10. The use according to claim 8 or 9, characterized in that: The anionic surfactants include sodium dodecylbenzene sulfonate, sodium α-olefin sulfonate and sodium fatty alcohol polyoxyethylene ether sulfate; the nonionic surfactants include Triton X, Tween and fatty alcohol polyoxyethylene ether.

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