Lipase mutant lipm1718 and use thereof

Abstract

The application discloses a lipase mutant LipM1718, the amino acid sequence of the lipase mutant LipM1718 is shown as SEQ ID NO. 2, and the lipase mutant LipM1718 comprises eleven mutation sites of E61D, Q72N, N73A, P86A, N116A, A117P, I126V, Y134F, Q194R, A260I and S277T; the nucleotide sequence of a gene coding the lipase mutant LipM1718 is shown as SEQ ID NO. 1. The application constructs a recombinant carrier pET22b- LipM1718 containing the lipase mutant LipM1718, which is expressed in a prokaryotic host cell Escherichia coli BL21 (DE3). Experiment proves that the obtained lipase has high efficient catalytic activity of hydrolysis of ester substrates after the gene is expressed in the prokaryotic host cell, and has great application potential in the harmless treatment and resource utilization field of kitchen waste oil.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering. More specifically, this invention relates to a lipase mutant LipM1718 and its applications. Background Technology

[0002] Lipases have wide applications in food, detergent, oil and fat industries, bioremediation, and biodiesel production. Microbial lipases, due to their wide pH and substrate adaptability, hold and maintain a dominant position in the lipase market. According to research by Beizhes Consulting, the global microbial lipase market size was approximately RMB 6.544 billion in 2025, and is projected to maintain steady growth, reaching nearly RMB 13.35 billion by 2032, with a compound annual growth rate (CAGR) of 10.72% over the next six years. The expanding application scenarios and market growth of lipases have also spurred research in areas such as lipase-producing strain isolation, novel lipase heterologous high-efficiency expression, and targeted lipase modification.

[0003] Acinetobacter spp. are an important source of lipases, but research reports on highly active and specific lipases are relatively few. Acinetobacter lipases, however, possess advantages such as high pH stability and low substrate selectivity, making them an important source of industrial microbial lipases. This invention is based on previously discovered strains... Acinetobacter junii lipase gene of WCO-9 (GDMCC No: 61851) AjLip1718 A novel lipase mutant, LipM1718, with higher activity and better stability was obtained through random mutation. This lipase mutant shows broad potential application prospects. Summary of the Invention

[0004] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0005] To achieve these objectives and other advantages of the present invention, a lipase Ajlip1718 is provided, the amino acid sequence of which is shown in SEQ ID NO.4.

[0006] A gene encoding the lipase Ajlip1718 as described above, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0007] The present invention also provides a lipase mutant LipM1718, which is the lipase Ajlip1718 with the amino acid sequence SEQ ID NO.4, wherein the 61st amino acid is changed from Glu to Asp, the 72nd amino acid is changed from Gln to Asn, the 73rd amino acid is changed from Asn to Ala, the 86th amino acid is changed from Pro to Ala, the 116th amino acid is changed from Asn to Ala, the 117th amino acid is changed from Ala to Pro, the 126th amino acid is changed from Ile to Val, the 134th amino acid is changed from Tyr to Phe, the 194th amino acid is changed from Gln to Arg, the 260th amino acid is changed from Ala to Ile, and the 277th amino acid is changed from Ser to Thr.

[0008] The amino acid sequence of a lipase mutant LipM1718 as described above is shown in SEQ ID NO.2.

[0009] A gene encoding the lipase mutant LipM1718 as described above, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0010] The present invention also provides a recombinant vector containing the genes described above.

[0011] The present invention also provides the application of the recombinant vector described above in the cultivation of lipase-producing microorganisms.

[0012] The present invention also provides a recombinant engineered strain obtained by transformation of the recombinant vector as described above.

[0013] Preferably, the recombinant vector is transferred into the prokaryotic host cell *Escherichia coli* to obtain a recombinant engineered strain.

[0014] The present invention also provides the application of the recombinant engineered strain described above in the cultivation of lipase-producing microorganisms.

[0015] Preferably, the expression of lipase produced after fermentation is induced for use in catalyzing p-nitrobenzene esters of different chain lengths.

[0016] Preferably, the recombinant engineered strain is induced to produce a lipase mutant gene. LipM1718 It can be expressed in large quantities in recombinant engineered strains; the supernatant was collected by cell disruption and centrifugation as crude enzyme solution for lipase activity detection.

[0017] Preferably, the recombinant engineered strain is induced to produce a lipase mutant gene. LipM1718 It can be expressed in large quantities in recombinant engineered strains; the supernatant collected by cell disruption and centrifugation is used as crude enzyme solution for catalyzing p-nitrobenzene esters of different chain lengths.

[0018] The present invention also provides the application of the lipase mutant LipM1718 as described above in the catalytic degradation of lipids.

[0019] The present invention also provides an application of the lipase mutant LipM1718 as described above in the catalytic degradation of oils in a high-temperature, alkaline environment.

[0020] This invention provides at least the following beneficial effects: It offers a lipase mutant, LipM1718, containing eleven mutation sites: E61D, Q72N, N73A, P86A, N116A, A117P, I126V, Y134F, Q194R, A260I, and S277T, which significantly improves its enzyme activity, thermostability, and alkali resistance. This invention also constructs a lipase containing… LipM1718 The gene recombination vector pET22b- LipM1718 The gene was expressed in the prokaryotic host cell Escherichia coli BL21(DE3). Experiments showed that the lipase obtained after expression in the prokaryotic host cell had highly efficient catalytic activity for the hydrolysis of ester substrates, and had great application potential in the field of harmless treatment and resource utilization of kitchen waste oil.

[0021] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0022] Figure 1 Prediction of transmembrane structure for the lipase mutant LipM1718;

[0023] Figure 2 Prediction of the signal peptide for the lipase mutant LipM1718;

[0024] Figure 3 For PCR amplification and purification LipM1718 Gene fragment; where M is the Trans 2K Plus II DNA Marker, and lane 1 is the amplified and purified DNA fragment. LipM1718 Gene fragments; lane 2 is a blank control.

[0025] Figure 4 For recombinant plasmid pET22b- LipM1718 Construction and validation; where M is the Trans 2K PlusII DNA marker, lane 1 is the positive control for WCO-9 genomic DNA template PCR, lane 2 is the blank control, lane 3 is the plasmid PCR result, and lane 4 is the recombinant plasmid pET22b- LipM1718 ;

[0026] Figure 5This is an SDS-PAGE electrophoresis analysis of the lipase mutant LipM1718 after massive induction; where M represents the pre-stained protein marker, and lanes 1-2 are for BL21-22b and pET22b- LipM1718 / BL21 strain fermentation supernatant, lanes 3-4 contain induced expression of BL21-22b and pET22b- LipM1718 Unpurified lysed supernatant of strain BL21;

[0027] Figure 6 This is a protein standard curve plotted according to the standard curve plotting method in the instructions for the protein quantification kit.

[0028] Figure 7 Standard curve plotted for lipase substrate specificity analysis;

[0029] Figure 8 To analyze the differences in hydrolytic activity of lipase mutant LipM1718 and lipase AjLip1718 on substrates of different chain lengths. Detailed Implementation

[0030] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0031] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0032] The plasmids, strains, and reagents involved in the examples are as follows:

[0033] Strains and plasmids: Acinetobacter junii WCO-9 was isolated and deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) by the laboratory, accession number GDMCC No: 61851, disclosed in patent CN202111193972.3; the top 10 Escherichia coli clones and the Escherichia coli expression strain BL21(DE3) were preserved in the laboratory; plasmid pET22b was preserved in the laboratory, and the recombinant expression plasmid pET22b- LipM1718 and recombinant expression strain pET22b- LipM1718 / BL21 is constructed by the present invention.

[0034] Biochemical reagents: Restriction endonucleases were purchased from NEB; dNTPs, high-fidelity Primestar HS DNA polymerase, T4 DNA ligase, etc., were purchased from TaKaRa (Dalian Treasure Biotech); agarose gel DNA recovery kit, general DNA product purification kit, general plasmid mini-preparation kit, and bacterial genome extraction kit were all purchased from TIANGEN (Tiangen Biotech); Ni-NTA resin chromatography column and Bradford method protein quantification kit were purchased from TransGen Biotech; IPTG, antibiotics, etc., were purchased from Aladdin; p-nitrophenyl ester substrate (ρ-NPC) 8-18 The reagents were purchased from SIGMA, USA; all other biochemical reagents were of analytical grade. Primer synthesis and sequencing used in the experiment were performed by BGI Genomics and Beijing Biomarker Biotechnology Co., Ltd.

[0035] LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, and agar powder 15 g / L added to the solid medium. High-pressure steam (121℃, 1.034×10⁻⁶) 5 Sterilize for 30 minutes (Pa).

[0036] Ampicillin (Amp) (50 mg / mL): Weigh 0.5 g of ampicillin powder, dissolve it in 10 mL of double-distilled water, filter to remove impurities, dispense into individual containers, and store at -20 ℃.

[0037] IPTG (0.1 mol / L): Weigh 0.24 g IPTG, dissolve it in 10 mL of double-distilled water, filter it through a membrane to sterilize it, dispense it into individual containers, and store it in a -20 ℃ refrigerator.

[0038] This invention was discovered through the following research:

[0039] (1) Analysis Acinetobacter junii Lipase gene from WCO-9 strain AjLip1718 mutant homologous gene sequence LipM1718 The nucleotide sequence of the gene, as shown in SEQ ID NO.1, was found to be high catalytic activity. LipM1718 is a novel lipase mutant, and its amino acid sequence is shown in SEQ ID NO.2. This lipase mutant gene is of great significance in the discovery, research, and application of novel lipase mutants, and also provides a gene source for cultivating lipase-producing engineered microorganisms.

[0040] (2) The amino acid sequence of LipM1718 was predicted online using http: / / web.expasy.org / protparam / . The protein consists of 410 amino acids, of which the total number of negatively charged amino acid residues (Asp+Glu) is 31 and the total number of positively charged amino acid residues (Arg+Lys) is 33. The protein molecular formula is C 1960 H 3084 N 520 O 598 S 15 Its molecular weight is 44.1 kDa, its theoretical isoelectric point is 8.23, and its instability index is 35.38, classifying it as a stable protein. Signal peptide and transmembrane structure analysis of LipM1718 revealed that this lipase lacks a transmembrane structure and is classified as an intracellular protein. Figure 1 The first 19 amino acids form the signal peptide region. Figure 2 Secondary structure prediction revealed that the protein has 8 α-helices and 6 β-sheets in its secondary structure.

[0041] (3) Sequence alignment of lipase mutant LipM1718

[0042] Sequencing results showed a specific mutant lipase gene LipM1718 The protein LipM1718 encoded by this gene has an amino acid sequence identity of 96.59%, and is a novel lipase mutant.

[0043] (4) Obtain the gene containing the lipase mutant. LipM1718 recombinant strains

[0044] 1) Amplification using lipase mutant genes obtained from previous random mutations as templates LipM1718 Gene fragments ( Figure 3 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, with a gene sequence length of 1233 bp. After enzyme digestion and ligation, it was recombined into the vector pET22b to construct a gene containing the complete lipase mutant. LipM1718 Recombinant plasmid pET22b- LipM1718 ( Figure 4 );

[0045] 2) Containing LipM1718 The recombinant plasmid pET22b- of the gene LipM1718 The recombinant engineered strain pET22b- was obtained by transferring the strain into the prokaryotic host cell *Escherichia coli* BL21(DE3). LipM1718 / BL21.

[0046] (5) Recombinant engineered strain pET22b- LipM1718 / Inducible expression of BL21 lipase mutant LipM1718 ( Figure 5 )

[0047] 1) Recombinant engineered strain pET22b- LipM1718 / BL21 was induced with 0.5 mM IPTG, and pET22b empty vector strain was used as control. SDS-PAGE electrophoresis and enzyme activity assay showed that the lipase mutant LipM1718 could be expressed in large quantities in the recombinant strain.

[0048] 2) After the recombinant strain was induced to express large amounts of the protein by IPTG, the supernatant was collected by cell disruption and centrifugation.

[0049] (6) Activity assay and substrate specificity analysis of lipase mutant LipM1718

[0050] The supernatant crude enzyme solution was used for lipase activity determination. The lipase mutant LipM1718 was determined according to the national standard method (GB / T 23535—2009). The results showed that the activity of LipM1718 was 2810.85 U / L. Substrate specificity analysis showed that the lipase mutant LipM1718 had extremely high hydrolytic activity and good broad-spectrum substrate activity for fatty acid substrates of different chain lengths. However, its catalytic hydrolytic activity decreased with the extension of the C10~C18 fatty acid substrate chain length. Figure 8 Overall, the enzyme activity of the lipase mutant LipM1718 is significantly better than that of the unmodified AjLip1718, showing broad prospects in industrial applications.

[0051] (7) Analysis of the high temperature and alkali resistance characteristics of lipase mutant LipM1718

[0052] The lipase mutant LipM1718 exhibited a residual enzyme activity of 78.25% after treatment at 90°C for 3 min, with residual activity ranging from 60.41% to 69.62% within a pH range of 9–10, and from 47.38% to 55.22% within a pH range of 10.5–11. In contrast, the lipase AjLip1718 showed a residual enzyme activity of 34.67% after treatment at 90°C for 3 min, with residual activity ranging from only 40.38% to 52.79% within a pH range of 9–10, and below 40% within a pH range of 10.5–11. Compared to the original lipase AjLip1718, the lipase mutant LipM1718 of this invention possesses superior high-temperature and alkali-resistant properties, making it suitable for use in high-temperature and alkaline environments.

[0053] Example 1

[0054] Recombinant plasmid pET22b- LipM1718 The process of constructing and transforming the strain into an expression strain:

[0055] One originating from Acinetobacter junii The amino acid sequence of WCO-9 lipase AjLip1718 is shown in SEQ ID NO.4, and the nucleotide sequence of the gene encoding lipase Ajlip1718 is shown in SEQ ID NO.3. To further improve the performance of lipase AjLip1718, a large number of mutations were screened for, resulting in a lipase mutant. The amino acid sequence of this mutant is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding this mutant is shown in SEQ ID NO.1. This lipase mutant was named LipM1718.

[0056] according to Acinetobacter junii WCO-9 genome AjLip1718 Gene sequence design for amplifying the target gene LipM1718 The universal primers are shown in Table 1:

[0057] Table 1

[0058]

[0059] Using the mutant LipM1718 gene fragment as a template, the full-length target gene was amplified using primers 1718-F / R listed in Table 1. The amplification system (50 μL) consisted of: 25 μL 2×phanta Max Master Mix; 0.5 μL template; 1 μL each of primers 1718-F / R; and 22.5 μL sterile ddH2O. After addition, the mixture was thoroughly mixed and centrifuged. PCR conditions were: 95 ℃ for 10 min; 94 ℃ for 30 sec; 57 ℃ for 30 sec; 72 ℃ for 1.5 min; 34 cycles; 72 ℃ for 10 min. The lipase mutant gene was purified and recovered. LipM1718 Excerpt ( Figure 3 ).

[0060] Use restriction endonucleases ( Bam H Ⅰ and Hin d III) Digest the target gene fragment and pET22b plasmid. The enzyme digestion system is 20 μL: 10×Cutsmart Buffer 2 μL; target fragment 10 μL. Bam H Ⅰ and Hin 1 μL each of dIII; 6 μL of sterile ddH2O; mix well and digest at 37°C for 12 h. The digested fragments were purified using a standard DNA product purification kit.

[0061] Establishment of the ligation system (20 μL): 10×T4 DNA Ligase Buffer 2 μL; T4 DNA Ligase 1 μL; pET22b digestion fragment 10 μL; target gene digestion fragment 7 μL; mix well and ligate at 16℃ for 4 h. Take 10 μL of the ligation product and mix it with 50 μL of *E. coli* Top10 competent cells in a sterile centrifuge tube. Incubate on ice for 30 min, then incubate at 42℃ for 1-2 min, followed immediately on ice for 2-3 min. Add 800 μL of antibiotic-free LB liquid medium to the centrifuge tube. Incubate at 37℃ and 200 rpm for 1 h with shaking. Centrifuge at 4000 rpm for 5 min, remove excess supernatant, resuspend the cells in the remaining 150 μL of supernatant, and spread on LB solid medium containing the corresponding antibiotic (Amp). Incubate at 37℃ for 12-16 h. Pick single colonies for verification and use as negative and positive controls. The results show that the transformed strain amplified to... LipM1718 The fragment indicates that the target gene was successfully ligated into the pET22b vector.

[0062] Select successfully validated colonies for amplification culture, and extract recombinant plasmid pET22b- using a standard plasmid miniprep kit. LipM1718 After successful verification by PCR and enzyme digestion, the recombinant plasmid was sent to BGI for sequencing. Figure 4 Sequencing results confirmed successful construction of the recombinant plasmid. The recombinant plasmid pET22b- LipM1718 The recombinant expression strain pET22b- was obtained by transforming the expression strain BL21(DE3) into competent E. coli cells using the method described above. LipM1718 / BL21.

[0063] Example 2

[0064] Inducible expression of the lipase mutant LipM1718:

[0065] (1) IPTG-induced lipase mutant LipM1718 at different concentrations

[0066] Select recombinant expression strain pET22b- LipM1718 A single colony of / BL21 was inoculated into 20 mL of LB liquid medium (containing Amp) and cultured at 37°C with shaking at 200 rpm for 12–16 h to prepare a seed culture. The OD of the bacterial culture was then measured. 600 and according to the initial OD 600 =0.1 was inoculated into fresh 50 mL LB liquid medium (containing Amp) and incubated at 37°C with shaking at 200 rpm until OD. 600Different amounts of IPTG were added to each culture flask to achieve final IPTG concentrations of 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, and 1.0 mM, respectively. Induction was performed at 16℃ and 180 rpm for 48 h, with a 0.1 mM-induced pET22b / BL21 empty vector expression strain serving as a control. Bacterial cells were collected by centrifugation at 8000×g for 10 min at 4℃. The cells were resuspended in 50 mM Tris-HCl (pH=8.0) and washed 2-3 times. After washing, the cells were resuspended in 2 mL of 50 mM Tris-HCl (pH=8.0). The expression differences of the lipase LipM1718 mutant were detected by SDS-PAGE electrophoresis. Finally, a 0.5 mM IPTG concentration was selected for inducing the expression of the lipase mutant LipM1718.

[0067] (2) Massive induction of lipase mutant LipM1718

[0068] Select recombinant expression strain pET22b- LipM1718 / BL21 single colonies were inoculated into 20 mL LB liquid medium (containing Amp) and cultured at 37℃ with shaking at 200 rpm for 12–16 h to prepare seed culture. The culture was then analyzed according to the initial OD... 600 =0.1 Inoculate the seed culture into a fresh 500 mL LB liquid medium (containing Amp) and incubate at 37°C with shaking at 200 rpm until OD. 600 =0.5, add IPTG to a final concentration of 0.5 mM, and induce at 16℃ and 180 rpm for 20 h. Collect bacterial cells by centrifugation at 8000×g for 10 min at 4℃. Wash the cells 2-3 times with 50 mM Tris-HCl (pH=8.0), and then resuspend the cells in 20 mL of 50 mM Tris-HCl (pH=8.0). Use an ultrasonic disruptor to disrupt the cells with 25% power, sonication on for 2 s, sonication off for 3 s, followed by ice bath disruption for 10 min. Centrifuge at 12000×g for 20 min at 4℃, and collect the supernatant, which is the crude LipM1718 enzyme solution.

[0069] (3) Protein concentration determination

[0070] Plot a protein standard curve according to the standard curve plotting method in the protein quantification kit instructions. Calculate the protein concentration in the sample. The protein standard curve plotting is as follows: Figure 6 The above;

[0071] Add an appropriate volume of sample (100 μL) to a 1.5 mL centrifuge tube. For samples with excessively high concentrations, dilute with H2O. Add 1.0 mL of Coomassie Brilliant Blue staining solution to the tube, mix well, and incubate at room temperature for 5-10 minutes. Measure the absorbance at 595 nm. Use imidazole without LipM1718 protein as a blank control. Calculate the protein concentration in the sample based on the standard curve.

[0072] Example 3

[0073] The application of a lipase mutant, LipM1718, in the catalytic degradation of lipids includes:

[0074] Activity assay and substrate specificity analysis of lipase mutant LipM1718:

[0075] (1) Assay of lipase mutant LipM1718

[0076] Prepare all reagents required for lipase activity determination according to the indicator titration method shown in GB / T 23535—2009. Specific procedures for the indicator titration method for lipase activity determination are as follows: a) Take two 100 mL Erlenmeyer flasks. Add 4.0 mL of substrate solution (an emulsion obtained by homogenizing 4% polyvinyl alcohol and olive oil in a 3:1 ratio using a high-speed homogenizer, freshly prepared) and phosphate buffer (pH=7.5) to each of the blank flask (A) and sample flask (B), respectively. Add 15.0 mL of 95% ethanol to flask A and preheat in a water bath at 40℃±0.2℃ for 5 min. Then add 1.0 mL of the enzyme solution to each of flasks A and B, mix immediately, and start timing. After reacting accurately for 15 min, immediately add 15.0 mL of 95% ethanol to flask B to terminate the reaction and remove the flask. b) Add two drops of phenolphthalein indicator solution to each of the blank and sample solutions, and titrate with sodium hydroxide standard solution until a faint red color is maintained for 30 minutes. The titration endpoint is determined by the absence of color fading. The volume of sodium hydroxide standard solution consumed is recorded. The activity of the lipase mutant LipM1718 is calculated based on the given lipase activity calculation formula and the sample protein concentration.

[0077] (2) Substrate specificity analysis of lipase mutant LipM1718

[0078] Construction of the standard curve: Weigh 0.08346 g of p-nitrophenol, dissolve it in a small amount of 95% ethanol, and then dilute to 100 mL with water to a concentration of 6 mmol / L. Add different amounts of p-nitrophenol solution and 50 mmol / L Tris-HCl (pH 8.0) buffer according to Table 2. Then add 0.25 mL of 10% trichloroacetic acid to each tube, followed by 0.25 mL of 10% Na2CO3 solution, for a total volume of 1.5 mL. Measure the absorbance at 410 nm and construct the standard curve. Figure 7 ), with 3 repetitions per group.

[0079] Using isopropanol as the solvent, prepare the reaction substrate solutions (substrate should be prepared fresh for use). Weigh 45 mg of each of the following substrates (p-nitrophenyloctanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, and stearate) into 15 mL of isopropanol. The reaction system is as follows: First, add 900 μL of 50 mmol / L Tris-HCl (pH 8.0) buffer, then 90 μL of the reaction substrate, mix well, then add 10 μL of enzyme solution. Incubate at 37°C for 10 min, then quickly add 250 μL of 10% trichloroacetic acid to terminate the reaction. Finally, add 250 μL of 10% Na₂CO₃ solution for color development, and measure the absorbance at 410 nm. The blank control is a 200 mM imidazole solution without the lipase mutant LipM1718. One enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol / min of p-nitrophenol under the experimental conditions. The enzyme activity calculation formula is:

[0080] A= ([A1-A0] × K+C0) × V1× n / (V2× t)

[0081] In the formula, A -- sample enzyme activity (U / L); A1 -- absorbance OD value of sample enzyme solution; A0 -- absorbance OD value of corresponding blank enzyme solution; K -- slope of p-nitrophenol standard curve; C0 -- intercept of p-nitrophenol standard curve; n -- dilution factor; V1 -- volume of reaction solution / mL; V2 -- volume of enzyme solution / mL; t -- reaction time / min.

[0082] Lipase AjLip1718 and lipase mutant LipM1718 exhibit hydrolytic activities against substrates of different chain lengths, such as... Figure 8 As shown, the results indicate that the lipase mutant LipM1718 possesses extremely high enzyme activity and good substrate broad-spectrum activity. Compared with the original lipase AjLip1718, the mutant LipM1718 exhibits improved catalytic activity for long-chain p-nitrophenyl ester substrates, with C10-C16 enzyme activities ranging from 1.25 to 3.5 times that of the original enzyme. It can be applied to catalyze the degradation of oils and has great application potential in the harmless treatment and resource utilization of kitchen waste oil.

[0083] Table 2

[0084]

[0085] Example 4

[0086] Application of a lipase mutant, LipM1718, in the catalytic degradation of lipids under high temperature and alkaline conditions.

[0087] Analysis of the high temperature and alkali resistance characteristics of lipase mutant LipM1718:

[0088] The crude enzyme solutions of AjLip1718 and LipM1718 were diluted with 50 mmol / L Tris-HCl (pH 8.0) buffer and treated at 90°C for 3 min. Residual enzyme activity (substrate: p-nitrobenzodecanoic acid) was then measured. The enzyme activity residual rate was calculated with the enzyme activity of the untreated sample as 100%. The residual enzyme activity of the lipase mutant LipM1718 of this invention was 78.25%, and the residual enzyme activity of the lipase AjLip1718 was 34.67%.

[0089] The crude enzyme solutions of AjLip1718 and LipM1718 were diluted with 50 mmol / L Tris-HCl buffer (pH = 9, 9.5, 10, 10.5, 11), respectively, and incubated at 37°C for 30 min. The residual enzyme activity (substrate: p-nitrobenzoic acid) was then measured. The enzyme activity residual rate was calculated with the enzyme activity of the untreated sample as 100%. The residual enzyme activity of the lipase mutant LipM1718 of this invention was 60.41%–69.62% in the pH range of 9–10 and 47.38%–55.22% in the pH range of 10.5–11. The residual enzyme activity of lipase AjLip1718 was only 40.38%–52.79% in the pH range of 9–10 and below 40% in the pH range of 10.5–11.

[0090] The results show that, compared with the original lipase AjLip1718, the lipase mutant LipM1718 of this invention has superior high temperature and alkali resistance properties and can be applied in high temperature and alkaline environments.

[0091] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A lipase mutant, LipM1718, characterized in that, The lipase mutant LipM1718 is the lipase Ajlip1718 with the amino acid sequence SEQ ID NO.4, where amino acid position 61 is changed from Glu to Asp, amino acid position 72 is changed from Gln to Asn, amino acid position 73 is changed from Asn to Ala, amino acid position 86 is changed from Pro to Ala, amino acid position 116 is changed from Asn to Ala, amino acid position 117 is changed from Ala to Pro, amino acid position 126 is changed from Ile to Val, amino acid position 134 is changed from Tyr to Phe, amino acid position 194 is changed from Gln to Arg, amino acid position 260 is changed from Ala to Ile, and amino acid position 277 is changed from Ser to Thr.

2. The lipase mutant LipM1718 as described in claim 1, characterized in that, The amino acid sequence of the lipase mutant LipM1718 is shown in SEQ ID NO.

2.

3. A gene encoding the lipase mutant LipM1718 of claim 1.

4. The gene as described in claim 3, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. A recombinant vector containing the gene of claim 4.

6. A recombinant engineered strain obtained by transformation of the recombinant vector according to claim 5.

7. The recombinant engineered strain as described in claim 6, characterized in that, The recombinant vector was transferred into the prokaryotic host cell Escherichia coli to obtain the recombinant engineered strain.

8. The use of a recombinant engineered strain as described in claim 6 or 7 in the production of the lipase mutant LipM1718.

9. The use of a lipase mutant LipM1718 as described in claim 1 or 2 in the catalytic degradation of p-nitrophenyloctanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, or stearate.

Images