Lipase mutant delta Lip4 as well as coding gene, preparation method and application thereof

Abstract

The invention discloses a lipase mutant delta Lip4 as well as a coding gene, a preparation method and application thereof. The lipase mutant delta Lip4 is obtained by mutating amino acid at the 90th position of a flexible region of wild lipase Lip4 from alanine with small molecular weight into valine with large molecular weight and branched chain, amino acid at the 140th site of the rigid region is mutated into alanine with small molecular weight from valine with large molecular weight and branched chain; the amino acid sequence and the nucleotide sequence of the gene are SEQ ID NO.1 and SEQ ID NO.2 respectively. The enzyme activity and the heat resistance of the mutant enzyme provided by the invention are obviously improved under the alkaline condition, the problems of low catalytic activity and insufficient heat stability of wild lipase under the alkaline condition are solved, and good conditions are created for application of the enzyme in the industries of cotton and linen processing, leather degreasing, industrial wastewater treatment and the like.

Description

Technical Field

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

[0002] As a biocatalyst, lipase has the following unique properties in addition to the common characteristics of enzymes, such as mild action conditions, high selectivity and specificity, environmental friendliness, safe use and recyclability: 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] At present, lipase is widely used in oil hydrolysis, improvement of food flavor and fragrance, medical medicine, waste paper deinking, modification of low-grade oil, detergent and cosmetics. The Chinese invention patent application with application number CN202011244303.X discloses a strain of Bacillus licheniformis expressing lipase and its fermentation enzyme production method, which specifically discloses that the fermentation broth enzyme activity of lipase produced by fermentation of Bacillus licheniformis reaches 32000U / mL, and has a wide range of optimal action range and significant low temperature resistance and alkali resistance, which can be applied to industrial production with different needs; the Chinese invention patent application with application number CN202010594015.0 discloses a strain of goat Staphylococcus aureus NCU S6 expressing lipase, and specifically discloses that the lipase activity of its fermentation broth is as high as 4927.3U / mL, the optimal action pH is 8.0, and the optimal action temperature is 37°C. The lipase expressed by the goat Staphylococcus aureus strain NCUS6 can significantly improve the yield of carbon chain structure oil in the preparation of camphor seed kernel oil by bioenzymatic conversion.

[0004] However, the application of lipase in the textile industry is relatively rare and is still in the experimental stage. Its application scope and objects are also relatively narrow, mainly used in wool shrinkage prevention, silk degreasing and waxing, and improving the hydrophilicity of polyester. Therefore, it is necessary to screen excellent lipase suitable for cotton and linen processing, leather degreasing and other industries.

[0005] With the development of molecular biology technology, it is imperative to explore new lipases from microorganisms and their simple production processes. Cloning lipase genes from natural high-yield lipase microorganisms and constructing excellent mutant genetic engineering strains through molecular modification methods for fermentation production of excellent lipases suitable for specific fields such as cotton and linen processing and leather degreasing has important practical production significance.

[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: The present invention provides a lipase mutant ΔLip4, wherein 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.

[0009] 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.

[0010] Furthermore, in the above technical solution, the amino acid sequence of the lipase mutant ΔLip4 is shown in SEQ ID NO.1.

[0011] The present invention also provides a gene Δlip4 encoding the lipase mutant.

[0012] Furthermore, in the above technical solution, the nucleotide sequence of the gene Δlip4 is shown in SEQ ID NO.2.

[0013] The present invention also provides a vector containing the gene Δlip4.

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

[0015] The present invention also provides an engineered bacterium containing the gene Δlip4 or the vector.

[0016] In another aspect, the present invention also provides the application of the gene Δlip4 and the enzyme encoded by it in cotton and linen processing, leather degreasing and industrial wastewater treatment.

[0017] In another aspect, the present invention also provides a method for producing the lipase mutant ΔLip4, comprising: The nucleotide sequence shown in SEQ ID NO.2 is used as an expression vector with 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.

[0018] In detail, in the above technical scheme, the nucleotide sequence shown in SEQ ID NO.2 is used as an expression vector with 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.

[0019] Specifically, in the above technical scheme, the lipase gene lip4 comes from a sparse cotton-like thermophilic hyphomycete Thermomyces lanuginosus; the pPICZαA expression unit used is inserted with a signal peptide, which can induce exogenous enzyme expression through methanol and has controllable expression; the encoding gene Δlip4 of the lipase mutant can be integrated into the genome of Pichia pastoris P. pastoris, and the engineered bacteria are more stable.

[0020] Compared with the prior art, the present invention has the following advantages: The mutant enzyme provided by the present invention has significantly improved enzyme activity and heat resistance under alkaline conditions, which solves the problems of low catalytic activity and insufficient thermal stability of wild-type lipase under alkaline conditions, and creates good conditions for the application of the enzyme in industries such as cotton and linen processing, leather degreasing, and industrial wastewater treatment.

[0021] The present invention compares the degradation ability of wild enzyme Lip4 and mutant enzyme ΔLip4 on p-nitrophenol ester substrate under alkaline conditions, and the results show that: under the condition of pH 8.5, the maximum lipase activity in the crude enzyme solution induced by the genetic engineering mutant strain pPICZαA-Δlip4 / X33 is 8900U / mL, which is 10 times the highest enzyme production of the original genetic engineering strain pPICZαA-lip4 / X33; when kept warm at 65°C for 2h, 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

[0022] Figure 1 The flowchart of the construction of the lipase mutant engineering strain in the embodiment of the present invention; Figure 2 This is a construction map of the recombinant plasmid pPICZαA-Δlip4 in the embodiment of the present invention; Figure 3 Schematic diagram of the principle of site-directed mutagenesis in an embodiment of the present invention; Figure 4 The SDS-PAGE patterns of the wild-type and mutant enzymes induced in the embodiments of the present invention are shown in FIG. Figure 5Graph comparing the stable temperatures of the wild-type and mutant enzymes in the examples of the present invention. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0024] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0025] In the examples, unless otherwise specified, all the means used are conventional means in the art.

[0026] As used herein, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

[0027] 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.

[0028] Materials and Reagents: The expression vector pPICZαA and eukaryotic competent cells P. pastoris X33 were purchased from Novagen; Speedy Cut SacI enzyme, Ni-NTA, and goat anti-mouse secondary antibody were purchased from Sangon Biological Company; 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. p-Nitrophenol phosphate, tryptone, yeast extract, and agar powder were purchased from Sigma; The remaining chemical reagents were all commercial products of analytical grade purchased from Sinopharm Group; Primer synthesis and nucleic acid sequencing were performed by Qingke Biotechnology Co., Ltd.

[0029] like Figure 1 Shown is a flow chart for constructing a lipase mutant engineering strain in an embodiment of the present invention.

[0030] Example 1: Construction of recombinant plasmid of original lip4 gene The mycelium of wild-type strain T. lanuginosus was inoculated into 50 mL LB liquid culture medium, cultured overnight at 40°C and 150 r / min, and the bacteria were collected by centrifugation; total RNA was extracted according to the instructions of RNA simple total RNA extraction kit, and the total RNA was reverse transcribed into cDNA according to the instructions of TIANScript II RT Kit kit, and stored at -20°C for future use.

[0031] According to the lipase gene lip4 sequence (Genbank accession number: OP222029.1), the following primers were designed using the bioinformatics software SnapGene: F: 5'-C GAATTC GAGAAAAGAAGTCCTATTCGTCGAGAGGT-3' (containing EcoRI restriction site) (SEQ ID NO.3) R: 5'-C GCGGCCGC GAGCTCGTCGACCCCTGTACAGAAATC-3' (containing NotI restriction site) (SEQ ID NO. 4).

[0032] PCR amplification was performed using T. lanuginosus cDNA as template.

[0033] The PCR reaction system is: 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.

[0034] 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.

[0035] The PCR products were identified by 1% agarose gel electrophoresis, recovered by gel excision, and stored at -20°C for future use.

[0036] 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 the recombinant plasmid, which was then transferred into E. coli Top10 using the heat shock method and plated on LB medium plates containing 25 μg / mL Zeocin for overnight culture. The positive transformants were selected for overnight culture and the plasmids were extracted for PCR identification. The positive plasmids were submitted to Qingke Biotechnology Company for sequence determination. The recombinant plasmid with the correct target gene was pPICZαA-lip4( Figure 2 ).

[0037] Example 2: Site-directed mutagenesis Principle of site-directed mutagenesis: The construction of point mutation plasmids uses the DpnI method ( Figure 3 ).

[0038] The PCR point mutation primers are designed according to the amino acid site to be mutated as follows: F A90V :5′CTTCCTT GTT CTCGACAACA 3′ (SEQ ID NO. 5) R A90V :5′AGTTGTCGAG CAA AAGGAAG 3′ (SEQ ID NO. 6) FV 140V :5′GGCAGAAG GCG GAGGATGCT 3′ (SEQ ID NO. 7) RV 140A :5′CCGTCTTC CGC CTCCTACDA 3′ (SEQ ID NO. 8) The underlined parts represent the codons corresponding to the 90th valine and the 140th alanine encoded by the mutant gene.

[0039] The mutation site was introduced by whole plasmid PCR using a rapid site-directed mutagenesis kit and pPICZαA-lip4 recombinant plasmid as a template.

[0040] The PCR reaction system was: 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 made up to 25 μL with ddH2O.

[0041] The parameter settings are: (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 and store the product at 4°C.

[0042] Add 0.5 μL of restriction endonuclease Dpn I to the mutated 25 μL PCR product, mix thoroughly, and digest at 37°C for 1 h; take 5 μL of Dpn I digestion product and transfer it into DH5α, spread the transformation liquid evenly on the LB screening plate (containing 25 μg / mL Zeocin), and culture at 37°C overnight to obtain transformants of the relevant mutant strains, extract the plasmid and sequence it to obtain the correct mutant plasmid.

[0043] Example 3: Construction, induced expression and electrophoresis analysis of recombinant Pichia pastoris 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 added immediately. In addition, the empty vector was electroporated to 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 a YPDS (containing 100μg / mL Zeocin) plate and inverted at 30°C for culture; after monoclonal colonies were formed, the monoclonal clones were detected using 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 OD 600 Change to BMMY medium for induction (1% methanol), incubate at 28.5°C for 70h and then test.

[0044] Take 100 μL of fermentation liquid and centrifuge at 12000 r / min for 5 minutes, take 80 μL of supernatant in a 1.5 mL centrifuge tube, add 20 μL of 5× Loading Buffer, and boil in water for 10 minutes. pPICZαA without the target gene was treated in the same way as a negative control.

[0045] 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).

[0046] Example 4: Enzyme activity comparison 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.

[0047] The quantitative determination of lipase activity was carried out using the p-nitrophenol (p-NP) method: p-nitrophenol phosphate was dissolved in isopropanol to prepare 1 mM substrate solution A. The enzyme solution was diluted to a solution B of appropriate concentration using 100 mM Tris-C1 (pH 8.5). Solutions A and B were preheated at 45°C and mixed, and then placed at 45°C for 5 min. After the reaction was completed, 100 μL of 10% SDS was immediately added to the sample to terminate the enzymatic reaction. The absorbance value OD was measured. 405 , converted into enzyme activity units based on the p-nitrophenol (p-NP) standard curve.

[0048] Unit enzyme activity definition: the amount of enzyme required to release 1 μmol of p-nitrophenol (p-NP) per minute, expressed as U.

[0049] 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.

[0050] Example 5: Comparison of heat resistance 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 2 h, 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).

[0051] The results showed that after being incubated 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 under the same treatment conditions (70%).

[0052] The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it should not be understood as limiting the scope of the invention patent.

[0053] It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these modifications and improvements all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A lipase mutant ΔLip4, characterized in that: The amino acid at position 90 of the flexible region of the 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.

2. The lipase mutant ΔLip4 according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO.

1.

3. The gene Δlip4 encoding the lipase mutant according to claim 2.

4. The gene Δlip4 according to claim 3, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

2.

5. A vector containing the gene Δlip4 according to claim 3 or 4.

6. A host cell containing the gene Δlip4 according to claim 3 or 4 or the vector according to claim 5.

7. An engineered bacterium containing the gene Δlip4 according to claim 3 or 4 or the vector according to claim 5.

8. Use of the gene Δlip4 and the enzyme encoded by it according to claim 3 or 4 in cotton and linen processing, leather degreasing and industrial wastewater treatment.

9. A method for producing the lipase mutant ΔLip4 according to claim 2, characterized in that: The nucleotide sequence shown in SEQ ID NO.2 is used as an expression vector with 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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