A lipase mutant ΔLip4 and its encoding gene, preparation method and application

Abstract

The present invention discloses a lipase mutant ΔLip4, its encoding gene, preparation method, and application. The lipase mutant ΔLip4 is formed by mutating the amino acid at position 90 of the flexible region of the wild-type lipase Lip4 from a low-molecular-weight alanine to a high-molecular-weight, branched valine, and mutating the amino acid at position 140 of the rigid region from a high-molecular-weight, branched valine to a low-molecular-weight alanine. The amino acid sequence and nucleotide sequence are SEQ ID NO. 1 and SEQ ID NO. 2, respectively. The mutant enzyme provided by the present invention has significantly improved enzyme activity and heat resistance under alkaline conditions, solving the problems of low catalytic activity and insufficient thermal stability of the wild-type lipase under alkaline conditions, creating favorable conditions for the application of the enzyme in industries such as cotton and linen processing, leather degreasing, and industrial wastewater treatment.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology, and more specifically, relates to a lipase mutant ΔLip4 and its encoding gene, preparation method and application. Background Art

[0002] As a biocatalyst, lipase not only has the common characteristics of enzymes such as mild action conditions, high selectivity and specificity, environmental friendliness, safe use and recyclability, but also has the following unique properties: First, it is a special ester bond hydrolase that can catalyze the hydrolysis of natural substrate oils and fats (triglycerides) to produce fatty acids and glycerol; second, it can only act in heterogeneous systems (i.e., water-oil interface or organic phase) and has no catalytic effect on water-soluble substrates.

[0003] Currently, lipases are widely used in oil hydrolysis, food flavor and fragrance improvement, medical and pharmaceutical applications, waste paper deinking, low-grade oil modification, detergents, and cosmetics. A Chinese invention patent application with application number CN202011244303.X discloses a strain of Bacillus licheniformis expressing lipase and a fermentation method for producing the enzyme. The application specifically discloses that the fermentation broth produced by Bacillus licheniformis has an enzyme activity of up to 32,000 U / mL, and has a wide optimal range of action and significant low-temperature and alkali resistance, making it applicable to industrial production with different requirements. A Chinese invention patent application with application number CN202010594015.0 discloses a strain of Staphylococcus aureus (NCUS6) expressing lipase. The application specifically discloses that the fermentation broth has a lipase activity of up to 4,927.3 U / mL, an optimal pH of 8.0, and an optimal temperature of 37°C. The lipase expressed by the strain NCUS6 can significantly increase the yield of medium-chain oils prepared by enzymatic conversion of camphor seed kernel oil.

[0004] However, research on the application of lipases in the textile industry is relatively limited and currently remains at the experimental stage. Its application scope and targets are also relatively narrow, mainly used in wool shrinkage prevention, silk oil and wax removal, and improving the hydrophilicity of polyester. Therefore, it is necessary to screen for high-quality lipases suitable for industries such as cotton and linen processing and leather degreasing.

[0005] With the development of molecular biology techniques, the exploration of novel lipases derived from microorganisms and their simplified production processes is imperative. Cloning lipase genes from naturally high-yielding lipase-producing microorganisms and constructing superior mutant genetically engineered strains through molecular engineering techniques are of great practical significance for the fermentation production of high-quality lipases suitable for specific applications such as cotton and linen processing and leather degreasing.

[0006] So far, there has been no report on the mutation modification of lipase. Summary of the Invention

[0007] In view of this, the present invention provides a lipase mutant ΔLip4 and its encoding gene, preparation method and application.

[0008] To achieve the above object, the technical solution of the present invention is as follows:

[0009] The present invention provides a lipase mutant ΔLip4, in which the amino acid at position 90 of the flexible region of the wild-type lipase Lip4 is mutated from a small molecular weight alanine to a large molecular weight branched valine, and the amino acid at position 140 of the rigid region is mutated from a large molecular weight branched valine to a small molecular weight alanine.

[0010] Specifically, in the above technical scheme, the amino acid at position 90 in the flexible region (random coil) and the amino acid at position 140 in the rigid region (α-helix) are subjected to point mutations, specifically based on the lip4 gene sequence published in the NCBI database (GenBank accession number: OP222029.1), the amino acids encoded therein are replaced, and the amino acid substitution points are alanine at position 90 and valine at position 140.

[0011] Furthermore, in the above technical solution, the amino acid sequence of the lipase mutant ΔLip4 is shown in SEQ ID NO.1.

[0012] The present invention also provides a gene Δlip4 encoding the lipase mutant.

[0013] Furthermore, in the above technical solution, the nucleotide sequence of the gene Δlip4 is shown as SEQ ID NO.2.

[0014] The present invention also provides a vector containing the gene Δlip4.

[0015] The present invention also provides a host cell containing the gene Δlip4 or the vector.

[0016] The present invention also provides an engineered bacterium containing the gene Δlip4 or the vector.

[0017] In another aspect, the present invention also provides the use of the gene Δlip4 and the enzyme encoded by it in cotton and linen processing, leather degreasing and industrial wastewater treatment.

[0018] In another aspect, the present invention provides a method for producing the lipase mutant ΔLip4, comprising:

[0019] The nucleotide sequence shown in SEQ ID NO.2 is used as an expression vector using a plasmid capable of expressing the enzyme and a strain capable of expressing the enzyme as an expression host to achieve efficient expression of the mutant shown in SEQ ID NO.1.

[0020] In detail, in the above technical solution, the nucleotide sequence shown in SEQ ID NO.2 is used as an expression vector using pPICZαA or a plasmid capable of expressing the enzyme, and Pichia pastoris X33 or a strain capable of expressing the enzyme is used as an expression host to achieve efficient expression of the mutant gene Δlip4.

[0021] Specifically, in the above technical solution, the lipase gene lip4 comes from a lanuginous thermophilic hyphomycete Thermomyces lanuginosus; the pPICZαA expression unit used is inserted with a signal peptide, which can induce the expression of exogenous enzymes by methanol and has controllable expression; the encoding gene Δlip4 of the lipase mutant can be integrated into the genome of Pichia pastoris P. pastoris, making the engineered bacteria more stable.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] The mutant enzyme provided by the present invention has significantly improved enzyme activity and heat resistance under alkaline conditions, solving the problems of low catalytic activity and insufficient thermal stability of wild-type lipase under alkaline conditions, and creating good conditions for the application of the enzyme in industries such as cotton and linen processing, leather degreasing, and industrial wastewater treatment.

[0024] The present invention compares the degradation abilities of wild enzyme Lip4 and mutant enzyme ΔLip4 on p-nitrophenolate substrates under alkaline conditions. The results show that at a pH of 8.5, the lipase activity in the crude enzyme solution induced by the genetically engineered mutant strain pPICZαA-Δlip4 / X33 is a maximum of 8900 U / mL, which is 10 times the highest enzyme production of the original genetically engineered strain pPICZαA-lip4 / X33. After being incubated at 65°C for 2 hours, the residual enzyme activity of the mutant lipase is 95%, which is much higher than the residual enzyme activity (70%) of the original lipase under the same treatment conditions. That is, the mutant enzyme has the characteristics of high temperature resistance and high enzyme activity under alkaline conditions, indicating that it has important application prospects in industries such as cotton and linen processing, leather degreasing, and sewage treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Flowchart for the construction of lipase mutant engineered strains according to the embodiment of the present invention;

[0026] Figure 2 This is a construction map of the recombinant plasmid pPICZαA-Δlip4 in the embodiment of the present invention;

[0027] Figure 3 Schematic diagram of the principle of site-directed mutagenesis in an embodiment of the present invention;

[0028] Figure 4 The SDS-PAGE patterns of the wild-type and mutant enzymes induced in the examples of the present invention are shown;

[0029] Figure 5 Graph comparing the stable temperatures of the wild-type and mutant enzymes in the examples of the present invention. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to the embodiments.

[0031] It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] In the examples, unless otherwise specified, all means used are conventional means in the art.

[0033] As used herein, the terms "comprise," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a listed element is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0034] In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0035] Materials and reagents:

[0036] The expression vector pPICZαA and eukaryotic competent cells P. pastoris X33 were purchased from Novagen;

[0037] Speedy Cut SacI enzyme, Ni-NTA, and goat anti-mouse secondary antibody were purchased from Sangon Biological Company;

[0038] The UltraHiFidelity PCR Kit, prokaryotic cloning competent E. coli TOP10, RNA simple total RNA extraction kit, rapid site-directed mutagenesis kit, TIANScript II RT Kit, agarose gel DNA recovery kit, and plasmid extraction kit were all purchased from Tiangen Biotechnology Co., Ltd.

[0039] p-Nitrophenol phosphate, tryptone, yeast extract, and agar powder were purchased from Sigma;

[0040] The remaining chemical reagents were commercial products of analytical grade purchased from Sinopharm Group;

[0041] Primer synthesis and nucleic acid sequencing were performed by Qingke Biotechnology Co., Ltd.

[0042] like Figure 1 Shown is a flow chart for constructing a lipase mutant engineered strain in an embodiment of the present invention.

[0043] Example 1: Construction of recombinant plasmid of original lip4 gene

[0044] The mycelia of the wild-type strain T. lanuginosus were inoculated into 50 mL of LB liquid medium, cultured overnight at 40°C and 150 rpm, and the cells were collected by centrifugation. Total RNA was extracted according to the instructions of the RNA simple total RNA extraction kit, and the total RNA was reverse transcribed into cDNA according to the instructions of the TIANScript II RT Kit and stored at -20°C until use.

[0045] Based on the lipase gene lip4 sequence (Genbank accession number: OP222029.1), the following primers were designed using the bioinformatics software SnapGene:

[0046] F: 5'-C GAATTC GAGAAAAGAAGTCCTATTCGTCGAGAGGT-3' (containing EcoRI restriction enzyme cleavage site) (SEQ ID NO.3)

[0047] R: 5'-C GCGGCCGC GAGCTCGTCGACCCCTGTACAGAAATC-3' (containing NotI restriction enzyme cleavage site) (SEQ ID NO. 4).

[0048] PCR amplification was performed using T. lanuginosus cDNA as a template.

[0049] The PCR reaction system is as follows: cDNA template, 1 μL (about 50 ng); upstream and downstream primers, 2 μL each; 2×Pfu MasterMix, 50 μL; add ddH2O to a total volume of 100 μL; mix well and then perform PCR reaction.

[0050] The PCR reaction conditions were as follows: (1) pre-denaturation at 94°C for 5 min; (2) denaturation at 94°C for 50 s; (3) annealing at 58°C for 50 s; (4) extension at 72°C for 1 min; repeat steps (2)-(4) for 32 cycles; (5) extension at 72°C for 10 min.

[0051] The PCR products were confirmed by 1% agarose gel electrophoresis, and then recovered by gel excision and stored at -20°C for future use.

[0052] The pPICZαA plasmid and the target gene PCR product were double-digested with EcoRI and Not I. After gel purification, the digested products were ligated with T4 DNA ligase at 16°C overnight to obtain a recombinant plasmid. The recombinant plasmid was then transformed into E. coli Top10 using the heat shock method and plated on LB medium plates containing 25 μg / mL Zeocin and cultured overnight. Positive transformants were selected, cultured overnight, and plasmids were extracted and identified by PCR. Positive plasmids were submitted to Qingke Biotechnology for sequence determination. The recombinant plasmid with the correct target gene was pPICZαA-lip4( Figure 2 ).

[0053] Example 2: Site-directed mutagenesis

[0054] Principle of site-directed mutagenesis: The construction of point mutation plasmids uses the DpnI method ( Figure 3 ).

[0055] The PCR point mutation primers are designed according to the amino acid site to be mutated as follows:

[0056] F A90V :5′CTTCCTT GTT CTCGACAACA 3′ (SEQ ID NO. 5)

[0057] R A90V :5′AGTTGTCGAG CAA AAGGAAG 3′ (SEQ ID NO. 6)

[0058] FV 140V :5′GGCAGAAG GCG GAGGATGCT 3′ (SEQ ID NO. 7)

[0059] RV 140A :5′CCGTCTTC CGC CTCCTACDA 3′ (SEQ ID NO. 8)

[0060] The underlined parts represent the codons corresponding to the 90th valine and the 140th alanine encoded by the mutant gene.

[0061] The mutation site was introduced by whole-plasmid PCR using a rapid site-directed mutagenesis kit and the pPICZαA-lip4 recombinant plasmid as a template.

[0062] The PCR reaction system was as follows: 1 μL each of forward and reverse primers, 5 μL of 5×FastAlteration Buffer, 1 μL of plasmid DNA, 0.5 μL of FastAlteration DNA Polymerase, and the volume was made up to 25 μL with ddH2O.

[0063] The parameter settings are:

[0064] (1) Pre-denaturation at 95°C for 2 min; (2) Denaturation at 94°C for 20 s; (3) Annealing at 60°C for 10 s; (4) Extension at 68°C for 2.5 min; Repeat steps (2)-(4) for 18 cycles; (5) Incubate at 68°C for 5 min. The product was stored at 4°C.

[0065] Add 0.5 μL of restriction endonuclease Dpn I to 25 μL of the mutated PCR product, mix thoroughly, and digest at 37°C for 1 hour. Take 5 μL of the Dpn I digestion product and transform it into DH5α. Spread the transformed bacterial liquid evenly on an LB screening plate (containing 25 μg / mL Zeocin) and culture at 37°C overnight to obtain transformants of the relevant mutant strain. Extract the plasmid and sequence it to obtain the correct mutant plasmid.

[0066] Example 3: Construction, induced expression and electrophoresis analysis of recombinant Pichia pastoris

[0067] The correct mutant plasmid was linearized with Sac I, mixed with P. pastoris X33 competent cells at a volume ratio of 1:8, transferred to a pre-cooled electroporation cup, ice-bathed for 5 minutes, and then electroporated for 5ms; pre-cooled sorbitol was immediately added. In addition, the empty vector was electroporated into P. pastoris X33 as a negative control. The electroporated solution was placed in a 30°C incubator for 2 hours and then centrifuged. The bacteria were spread on YPDS (containing 100μg / mL Zeocin) plates and inverted at 30°C for incubation; after the formation of monoclonal colonies, the monoclonal clones were detected by PCR technology, and the PCR primers were yeast universal primers AOX-1. The positive clone colonies were inoculated into BMGY medium and cultured at 28.5°C until the OD 600 Change to BMMY medium for induction (1% methanol), incubate at 28.5°C for 70 hours, and then test.

[0068] Centrifuge 100 μL of fermentation broth at 12,000 rpm for 5 minutes. Transfer 80 μL of supernatant to a 1.5 mL centrifuge tube, add 20 μL of 5× Loading Buffer, and incubate in a boiling water bath for 10 minutes. Perform the same treatment on pPICZαA without the target gene as a negative control.

[0069] The secretion and expression of proteins were detected by SDS-PAGE and Western blot. The results showed that both the original genetically engineered strain pPICZαA-lip4 / X33 and the genetically engineered mutant strain pPICZαA-Δlip4 / X33 had specific target bands (such as Figure 4 shown).

[0070] Example 4: Enzyme activity comparison

[0071] In order to compare the biocatalytic ability of lipase contained in the crude enzyme solution of the original genetically engineered strain pPICZαA-lip4 / X33 and the genetically engineered mutant strain pPICZαA-Δlip4 / X33 under alkaline conditions, enzyme activity was determined under the same conditions.

[0072] Lipase activity was quantitatively determined using the p-nitrophenol (p-NP) method: p-nitrophenol phosphate was dissolved in isopropanol to prepare a 1 mM substrate solution A. The enzyme solution was diluted to an appropriate concentration of solution B using 100 mM Tris-C1 (pH 8.5). Solutions A and B were preheated at 45°C, mixed, and then reacted at 45°C for 5 minutes. After the reaction, 100 μL of 10% SDS was immediately added to the sample to terminate the enzymatic reaction. The absorbance OD was measured. 405 , converted into enzyme activity units based on the p-nitrophenol (p-NP) standard curve.

[0073] Unit enzyme activity is defined as the amount of enzyme required to release 1 μmol of p-nitrophenol (p-NP) per minute, expressed in U.

[0074] The results showed that under pH 8.5 conditions, when the induction culture time of the genetically engineered mutant strain pPICZαA-Δlip4 / X33 reached 192h, the lipase activity in the crude enzyme solution reached its peak, with a maximum of 8900U / mL, which was 10 times the highest enzyme production of the original genetically engineered strain pPICZαA-lip4 / X33, and its ability to catalyze the degradation of p-nitrophenol esters was greatly improved.

[0075] Example 5: Comparison of heat resistance

[0076] The fermentation broth of the original lipase Lip4 and the mutant lipase ΔLip4 induced by the engineered bacteria was kept at 30-80°C for 2h, and the residual enzyme activity of the crude enzyme solution was measured to characterize the thermal stability of the enzyme (such as Figure 5 shown).

[0077] The results showed that after being kept at 65°C for 2 hours, the residual enzyme activity of the mutant lipase was 95%, which was much higher than the residual enzyme activity of the original lipase (70%) under the same treatment conditions.

[0078] The above-mentioned embodiments only express several implementation methods of the present invention. The description thereof is relatively specific and detailed, but it should not be understood as limiting the scope of the invention patent.

[0079] It should be noted that those skilled in the art may make various modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims.

Claims

1. A lipase mutant ΔLip4, characterized in that: The amino acid at position 90 of the flexible region of wild-type lipase Lip4 was mutated from a small molecular weight alanine to a large molecular weight branched valine, and the amino acid at position 140 of the rigid region was mutated from a large molecular weight branched valine to a small molecular weight alanine; Its amino acid sequence is shown in SEQ ID NO.

1.

2. The gene encoding the lipase mutant according to claim 1 lip 4.

3. The gene according to claim 2 lip 4, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

2.

4. Containing the gene according to claim 2 or 3 lip 4 carriers.

5. Containing the gene according to claim 2 or 3 lip 4 or a host cell containing the vector according to claim 4.

6. Containing the gene according to claim 2 or 3 lip 4 or the engineered bacteria of the vector according to claim 4.

7. The gene according to claim 2 or 3 lip 4 and the enzymes it encodes in cotton and linen processing, leather degreasing and industrial wastewater treatment.

8. A method for producing the lipase mutant ΔLip4 according to claim 1, characterized in that: The nucleotide sequence shown in SEQ ID NO.2 is used as an expression vector using a plasmid capable of expressing the enzyme and a strain capable of expressing the enzyme as an expression host to achieve efficient expression of the mutant shown in SEQ ID NO.1.

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