Lipase mutants based on hinge region and tunnel engineering and their applications

Abstract

The present invention relates to a class of lipase mutations based on hinge region and tunnel engineering and their applications. Based on computer-aided design, the invention analyzes the amino acids in the hinge region of the lipase lid through molecular dynamics simulation and uses CAVER 3.0 software to analyze the amino acids in the amine channel. Using single-point mutagenesis, iterative, and combinatorial mutagenesis techniques, the obtained mutants contain one or more mutations at amino acids 37, 206, 207, 211, 212, and 213 of the amino acid sequence. The amino acid sequence of the lipase is shown in SEQ ID NO: 1. The mutants of the present invention can be used to catalyze the synthesis of corresponding aromatic amides from ortho-substituted anilines and have broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and in particular relates to a lipase mutant based on hinge region and tunnel engineering and applications thereof. Background Art

[0002] Amide compounds are among the most common small molecule pharmaceuticals. In fact, it's believed that up to 16% of reactions in pharmaceutical synthesis chemistry laboratories involve amide bond formation. Aromatic amides represent a significant portion of the numerous amide-based drug molecules. The core structure of amide drugs consists of an amide bond formed by an aromatic ring, such as a benzene ring or a pyridine ring, and an ester. Notably, most aromatic amides contain ortho-substituents on the benzene ring. These substituents contribute to the stability and solubility of aromatic amides, while also imparting unique chemical properties. Examples include the anti-cancer drugs dacomitinib and afatinib, as well as a series of caine-based local anesthetics, such as lidocaine and prilocaine.

[0003] With the development of biotechnology, enzyme catalysis has demonstrated unique advantages in amide synthesis. Enzymes exhibit high chemo-, regio-, and enantioselectivity, can function under mild conditions, and exhibit substrate promiscuity, thus holding enormous potential for application in amide synthesis. Currently, a range of enzymes for amide synthesis has been developed, including carboxylic acid reductases, acyltransferases, and lipases. Among these, lipases offer unique advantages in amide synthesis because they do not require expensive cofactors or activated esters. However, most lipases are unable to catalyze the amidation of aromatic amines, and lipases that can catalyze highly sterically hindered ortho-substituted aromatic amines are particularly rare. The commonly used Candida antarctica lipase (CALB) has very low catalytic activity towards bulky anilines, and is even less effective at catalyzing the amidation of ortho-substituted anilines. Furthermore, the aromatic amine-preferring lipase Ndbn also exhibits low activity towards ortho-substituted anilines, achieving a conversion rate of only 0.38% in the amide synthesis from o-toluidine and methyl 3-phenylpropionate. Therefore, utilizing protein engineering approaches to enhance the activity of lipases in the amidation reaction of o-toluidine with esters is of great significance. Summary of the Invention

[0004] The present invention takes the lipase Ndbn from Rhizorhabdus dicambivorans as the research object, uses AlphaFold2 for modeling, molecular docking and molecular dynamics simulation techniques to analyze the amino acids at the hinge of the lipase lid, and uses CAVER3.0 software to analyze the amino acids in the amine channel. After performing site-directed mutagenesis, a series of lipase mutants are obtained, which can efficiently catalyze the amidation reaction of o-toluidine with esters.

[0005] The specific technical solutions of the present invention are as follows:

[0006] A lipase mutant comprising one or more mutations in amino acids 37, 206, 207, 211, 212 and / or 213 of the lipase amino acid sequence, wherein the amino acid sequence of the lipase is shown in SEQ ID NO: 1.

[0007] The mutation at position 37 is: serine is mutated to alanine, arginine, valine, proline, asparagine, glutamine, leucine, lysine, isoleucine, histidine, glycine, phenylalanine, glutamic acid, threonine, tryptophan, methionine, tyrosine, aspartic acid or cysteine, preferably isoleucine and methionine.

[0008] The mutation at position 206 is: leucine is mutated to alanine, arginine, valine, proline, asparagine, glutamine, serine, lysine, isoleucine, histidine, glycine, phenylalanine, glutamic acid, threonine, tryptophan, methionine, tyrosine, aspartic acid or cysteine, preferably phenylalanine.

[0009] The mutation at position 207 is: glycine is mutated to alanine, arginine, valine, proline, asparagine, glutamine, leucine, lysine, isoleucine, histidine, serine, phenylalanine, glutamic acid, threonine, tryptophan, methionine, tyrosine, aspartic acid or cysteine, preferably serine.

[0010] The mutation at position 211 is: isoleucine is mutated to alanine, arginine, valine, proline, asparagine, glutamine, leucine, lysine, serine, histidine, glycine, phenylalanine, glutamic acid, threonine, tryptophan, methionine, tyrosine, aspartic acid or cysteine, preferably glycine.

[0011] The mutation at position 212 is: leucine is mutated to alanine, arginine, valine, proline, asparagine, glutamine, serine, lysine, isoleucine, histidine, glycine, phenylalanine, glutamic acid, threonine, tryptophan, methionine, tyrosine, aspartic acid or cysteine, preferably phenylalanine and methionine.

[0012] The mutation at position 213 is: serine is mutated to alanine, arginine, valine, proline, asparagine, glutamine, leucine, lysine, isoleucine, histidine, glycine, phenylalanine, glutamic acid, threonine, tryptophan, methionine, tyrosine, aspartic acid or cysteine, preferably aspartic acid.

[0013] Preferably, the lipase mutant described in the present invention is a single-site mutation, wherein the serine at position 37 is mutated to leucine or methionine (S37L, S37M), the leucine at position 206 is mutated to phenylalanine (L206F), the glycine at position 207 is mutated to serine (G207S), the isoleucine at position 211 is mutated to glycine (I211G), the leucine at position 212 is mutated to phenylalanine or methionine (L212F, L212M), and the serine at position 213 is mutated to aspartic acid (S213D).

[0014] Preferably, the lipase mutant of the present invention is a double-site mutation, in which the isoleucine at position 211 is mutated to glycine and the leucine at position 212 is mutated to phenylalanine or methionine.

[0015] Preferably, the lipase mutant of the present invention is a three-point mutation, wherein the leucine at position 206 is mutated to phenylalanine, and the isoleucine at position 211 is mutated to glycine and the leucine at position 212 is mutated to methionine; or, the glycine at position 207 is mutated to serine, and the isoleucine at position 211 is mutated to glycine and the leucine at position 212 is mutated to methionine; or, the isoleucine at position 211 is mutated to glycine, and the leucine at position 212 is mutated to methionine, and the serine at position 213 is mutated to aspartic acid.

[0016] Preferably, the lipase mutant of the invention is a four-site mutation, wherein the serine at position 37 is mutated to leucine or methionine, the leucine at position 206 is mutated to phenylalanine, the isoleucine at position 211 is mutated to glycine, and the leucine at position 212 is mutated to methionine.

[0017] Another object of the present invention is to provide a DNA molecule encoding the lipase mutant of the present invention.

[0018] Another object of the present invention is to provide an expression vector for a lipase mutant, which expresses the lipase mutant of the present invention. The expression vector contains a DNA molecule encoding the lipase mutant of the present invention.

[0019] The expression vector is a plasmid, a phage, a virus or a host cell.

[0020] The host cell is a prokaryotic cell or a eukaryotic cell, and can be Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus or Trichoderma, preferably Escherichia coli.

[0021] Another object of the present invention is to provide the use of the lipase mutant, DNA molecule or expression vector of the lipase mutant in amide synthesis.

[0022] Specifically, the lipase mutant, DNA molecule or expression vector of the lipase mutant of the present invention can catalyze the reaction of aromatic amine with aromatic ester to form aromatic amide. The aromatic amine is preferably an ortho-substituted aniline.

[0023] In a preferred embodiment of the present invention, o-toluidine and methyl 3-phenylpropionate are catalyzed to synthesize 3-phenyl-N-(o-tolyl)propionamide.

[0024]

[0025] Advantages of the present invention:

[0026] The present invention uses lipase Ndbn from Rhizorhabdus dicambivorans obtained by previous screening as a research object. By using techniques such as molecular docking and molecular dynamics, the amino acids at the hinge of the lipase lid are analyzed. Saturation mutagenesis is performed on eight sites L206-S213. It is found that single-point mutations of L206, G207, I211, L212 and S213 all result in mutants with significantly improved catalytic activity for the amidation of o-toluidine with methyl 3-phenylpropionate. Among them, I211G and L212F catalyze the synthesis of 3-phenyl-N-(o-tolyl)propionamide at yields that are 19.8 and 17.01 times that of the original enzyme, respectively. Iterative saturation mutagenesis of I211 and L212 reveals that double mutants I211G / L212M and I211G / L212F catalyze the synthesis of the target product at yields that are 51.01 and 32.01 times that of the original enzyme, respectively. Using this double mutant as a template, combined mutagenesis with other single-point optimal mutants, L206F, G207S, and S213D, revealed that the triple mutant, L206F / I211G / L212M, catalyzed the synthesis of the target product at a yield 93.56 times that of the original enzyme. CAVER3.0 software was then used to analyze the amino acids in the amine channel and subjected to saturation mutagenesis. S37L and S37M, respectively, catalyzed the synthesis of the target product at yields 13.42 and 10.38 times that of the original enzyme. Combined mutagenesis with the triple mutant yielded the quadruple mutant, S37M / L206F / I211G / L212M, which catalyzed the synthesis of the target product at a yield 132.32 times that of the original enzyme. This result demonstrates that the Ndbn lipase mutant of the present invention has higher catalytic activity than the original enzyme in the amidation reaction of o-toluidine with methyl 3-phenylpropionate, which is of great significance for the synthesis of ortho-substituted aromatic amides. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings constituting a part of this application are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0028] Figure 1It is the lid and hinge region of lipase Ndbn.

[0029] Figure 2 It is the Ndbn o-toluidine channel of lipase and its surrounding amino acids.

[0030] Figure 3 The SDS-PAGE electrophoresis diagram of Ndbn and its dominant mutant.

[0031] Figure 4 This is a graph showing the relative yield of 3-phenyl-N-(o-tolyl)propionamide catalyzed by saturated mutations of L206-L213, S37, L40, I99, L190 and L256 and the original enzyme Ndbn.

[0032] Figure 5 The bar graph shows the relative yield of the synthesis of 3-phenyl-N-(o-tolyl)propionamide catalyzed by the dominant mutant.

[0033] Figure 6 Figure 2 shows the enzymatic properties of Ndbn and its optimal mutant. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the present invention, the present invention will be further described below with reference to the accompanying drawings and implementation examples. It should be noted that this embodiment is only used to explain the present invention and is not intended to limit the scope of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0035] Example 1 Establishment of Lipase Hinge Region Amino Acids and Amine Channel Amino Acids

[0036] The lipase Ndbn selected by the present invention has a unique preference for aromatic amines in the synthesis of amide compounds, but its activity in catalyzing the synthesis of amides from ortho-substituted aniline and 3-phenylpropionic acid methyl ester is very low, with a conversion rate of only 0.38%. Therefore, the present invention improves the catalytic activity of lipase Ndbn in this reaction by modifying the hinge region amino acids and amine channel amino acids. Lipase has a typical lid structure that covers the active site of the enzyme. When the lipase is at the water-oil interface, the conformation of the protein molecule changes, the lid structure opens, the active center of the enzyme is exposed, and the enzyme catalyzes the reaction with the substrate. The hinge region can regulate the conformation of the lipase lid switch, thereby affecting the activity and stability of the lipase. The hinge region amino acids are determined to be L206-S213 ( Figure 1 ).

[0037] In the catalytic cycle of most lipases, the tunnel connecting the active site and the surrounding solvent plays a crucial role in ligand transport. For esterification reactions, the rapid elimination of water molecules and the transport rate of substrates are significantly affected by the tunnel. Similarly, for ester aminolysis reactions, the alcohol leaving rate directly affects the reaction rate. Therefore, the rational design of the corresponding lipase tunnel will accelerate the enzymatic amidation reaction. In view of the low activity of Ndbn in the amidation reaction of o-toluidine, the amine channel of Ndbn was analyzed using CAVER3.0 software ( Figure 2 ), DLKcat software was used to perform virtual saturation mutagenesis on amine channel amino acids, the Kcat values ​​of mutants for o-toluidine were predicted, and sites S37, L40, I99, L190, and L256, where the Kcat of the mutants was ≥ 1.5 times that of the wild-type, were selected for saturation mutagenesis.

[0038] Example 2 Construction of Lipase Ndbn Mutant Library

[0039] As described in Example 1, saturation mutagenesis was performed on the lipase Ndbn at positions L206-S213, S37, L40, I99, L190, and L256. The high-quality mutants I211G and L212F obtained from the single-point saturation mutagenesis were subjected to iterative saturation mutagenesis. The resulting advantageous double mutants were then combined with other advantageous single mutants at the hinge, and the resulting triple mutants were combined with advantageous mutants in the amine channel to obtain mutant strains with higher catalytic efficiency. Using L206 as an example, its construction method is as follows, and the primers used are shown in the table below:

[0040]

[0041] Using the pET-28a(+) plasmid with the lipase Ndbn gene sequence (SEQ ID NO: 1) as a template, referring to the Vazyme biological product and operation manual, a mutation primer pair was used to amplify the site-directed mutation sequence from the entire plasmid. The PCR product was treated with Dpn I. After the template digestion was completed, it was transformed into Escherichia coli competent cells E.coliBL21 (DE3) using the heat shock method and spread on LB agar plates containing 100μg / ml kanamycin sulfate. It was incubated inverted at 37°C overnight. The mutation results were verified by sequence determination by Anhui General Biological Company. Taking the L206 site as an example, the construction of the mutant was carried out using conventional PCR technology and the lipase Ndbn expression vector as a template for full plasmid amplification to introduce mutations. The resulting L206 mutant library was successfully constructed by sequencing verification.

[0042] Example 3 Fermentation of recombinant lipase Ndbn mutant in Escherichia coli

[0043] The mutant recombinant strains constructed in Example 1 were inoculated into 50 mL of LB liquid medium containing 100 μg / mL kanamycin sulfate and cultured overnight at 37°C and 180 rpm. The seed liquid was inoculated into 50 mL of fresh TB liquid medium at a 2% inoculum size and cultured at 37°C and 180 rpm until OD 600 When the pH value was between 0.6 and 1.0, the cells were taken out and cooled in an ice-water bath for 5 min, and the inducer IPTG (isopropyl-β-D-thiogalactopyranoside) (final concentration 0.3 mmol / L) was added and the expression was induced at 22°C and 180 rpm for 20 h.

[0044] Take the fermentation liquid of induced expression, centrifuge at 12000rpm for 20min, discard the supernatant, then resuspend and wash the bacteria with 50mM Tris-HCl (pH8.0) buffer, centrifuge at 12000rpm for 20min, discard the supernatant, resuspend again with buffer, and then ultrasonically disrupt. Centrifuge the disrupted liquid at 12000rpm for 20min, take the supernatant for SDS-PAGE electrophoresis detection, the concentration of the concentrated gel is 4%, the concentration of the separation gel is 12.5%, the sample and the loading buffer are mixed in a ratio of 3:1, and the reaction is carried out in a boiling water bath for 5min for loading electrophoresis. Set the initial voltage of the electrophoresis instrument to 120V. When the sample moves to the separation gel, increase the voltage to 230V until the sample moves to the bottom of the electrophoresis tank and end the electrophoresis.

[0045] The results of SDS-PAGE electrophoresis of the mutant crude enzyme solution are as follows Figure 3 As shown, the molecular weight of the target protein is 37 kDa, and each lane in the electrophoresis diagram has a clear band at 37 kDa, indicating that the target protein is successfully expressed in each mutant.

[0046] Example 4 Determination of the Activity of Lipase Ndbn Mutant in Catalyzing the Amidation Reaction of o-Toluidine and Methyl 3-Phenylpropionate

[0047] Referring to the methods of Examples 2 and 3, L206-S213, S37, L40, I99, L190, L256 and L256 mutants were constructed respectively, and the amidation reaction activity was determined using o-toluidine and methyl 3-phenylpropionate as substrates. The reaction conditions are as follows: 10 mM methyl 3-phenylpropionate and 20 mM o-toluidine were added to 3 mL of n-hexane, 30 mg of lipase Ndbn freeze-dried enzyme powder was added to activate the reaction, and the reaction was carried out at 30°C and 250 rpm for 24 hours. The inactivated enzyme was used as a blank control. The conversion rate was determined by HPLC. HPLC detection method: The chromatographic column was Diacel Chiralpak IA-3column (250×4.6 mm, 3 μm), the mobile phase was n-hexane:isopropanol = 90:10, the flow rate was 1.0 ml·min-1; UV detector (254 nm); column temperature: 25°C. By Figure 4Among the hinge region amino acid mutants, mutations of I211 to glycine and leucine significantly improved catalytic activity for the reaction, with the optimal mutation being I211G, which yielded 19.8 times the amide yield of the original enzyme. Mutations of L212 to phenylalanine and cysteine ​​also enhanced catalytic activity, with the optimal mutation being L212F, which yielded 17.01 times the amide yield of the original enzyme. Mutations of L206, G207, and S213 also exhibited enhanced catalytic activity, with the optimal mutations being L206F, G207S, and S213D, respectively. In the amine channel, mutations of S37 to leucine and methionine significantly enhanced catalytic activity, yielding amide yields of 13.42 and 10.38 times that of the original enzyme, respectively.

[0048] To obtain mutants with further enhanced catalytic activity, single-point mutations were iteratively and combined. Among the hinge region amino acids, mutations to I211 and L212 showed the most significant improvement in catalytic activity, and the proximity of the two sites suggests a synergistic effect. Therefore, using I211G as a template, iterative saturation mutagenesis was performed on the L212 site, and using L212F as a template, iterative saturation mutagenesis was performed on I211. The double mutant I211G / L212M was obtained, catalyzing amide synthesis with a yield 51.01-fold that of the original enzyme. Next, using the double mutant as a template, combined mutations were performed with other hinge region mutants with enhanced catalytic activity, L206F, G207S, and S213D, resulting in the triple mutant L206F / I211G / L212M, which catalyzed the target amide with a yield 93.56-fold that of the original enzyme. The triple mutant was used as a template and combined with the mutants S37M and S37L, which showed significantly improved catalytic activity in the amine channel, to obtain the quadruple mutant S37M / L206F / I211G / L212M as the optimal mutant. The amide yield of the enzyme catalyzing the synthesis of o-toluidine and methyl 3-phenylpropionate was 132.32 times that of the original enzyme ( Figure 5 ).

[0049] Example 5 Method for Determining the Enzyme Activity of Lipase Ndbn Original Enzyme and Mutants

[0050] The recombinant strain constructed in Example 2 was fermented and cultured according to the method of Example 3, and the obtained crude protein enzyme solution was used to measure the enzyme activity change using pNPG as a substrate. The measurement method is as follows:

[0051] Definition of enzyme activity unit: One unit of enzyme activity is the amount of enzyme required to catalyze the hydrolysis of pNPG to produce 1 μmol of pNP per minute at 45°C and pH 8.0. The specific steps are as follows:

[0052] (1) Solution A: 50 mM Tris-HCl buffer, pH 8.0, with 0.6% Triton X-100 and 0.1% gum arabic added;

[0053] (2) Preparation of solution B: Accurately weigh 3 mg of pNPP and dissolve it in 1 mL of isopropanol;

[0054] (3) Solution A and solution B were mixed at a volume ratio of 9:1 to prepare a pNP substrate solution with a concentration of 16.5 mM;

[0055] (4) Lipase activity assay: 10 μL of enzyme solution diluted appropriately was added to the reaction system, with inactivated enzyme solution as a blank control. 240 μL of substrate solution was then added and the reaction was carried out in a microplate reader. The reaction temperature was set at 30°C and the reaction time was 10 min. The amount of p-nitrophenol (pNP) generated at the end of the reaction was detected at 410 nm.

[0056] Example 6 Enzymatic Properties Analysis of Lipase Ndbn Original Enzyme and Mutants

[0057] In order to determine the optimal temperature of the mutant and the original enzyme, the enzyme solutions of the optimal mutant S37M / L206F / I211G / L212M and the original enzyme Ndbn obtained by screening in Example 4 were diluted according to a certain concentration and added to the substrate solution prepared in Example 5. The reactions were carried out at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C for 10 min, respectively. The absorption at a wavelength of 410 nm was measured, and the highest enzyme activity was taken as 100%. The relative enzyme activity was calculated in sequence, and a curve of enzyme activity changing with temperature was plotted.

[0058] In order to determine the temperature stability of the mutant and original enzymes, the enzyme solutions of the original enzyme Ndbn and the mutant S37M / L206F / I211G / L212M were incubated at the synthesis reaction temperature, i.e., 30°C, for 25 h, and samples were taken every 5 h to determine the residual activity.

[0059] The results are as follows Figure 6 As shown, the optimal temperature for both the original enzyme Ndbn and the mutant S37M / L206F / I211G / L212M is 45°C. After incubation at 30°C for 10-25 hours, the original enzyme Ndbn experienced a higher loss of enzyme activity than the mutant S37M / L206F / I211G / L212M, indicating that the mutant has better thermal stability than the original enzyme.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A lipase mutant, characterized in that The mutant is a lipase in which the isoleucine at position 211 in the amino acid sequence is mutated to glycine. The amino acid sequence of the lipase is shown in SEQ ID NO:

1.

2. A lipase mutant, characterized in that The mutant is a lipase in which the isoleucine at position 211 is mutated to glycine, and the leucine at position 212 is mutated to methionine. The amino acid sequence of the lipase is shown in SEQ ID NO:

1.

3. A lipase mutant, characterized in that The mutant is a lipase in which the isoleucine at position 211 is mutated to glycine, the leucine at position 212 is mutated to methionine, and the leucine at position 206 is mutated to phenylalanine. The amino acid sequence of the lipase is shown in SEQ ID NO:

1.

4. A lipase mutant, characterized in that The mutant is a lipase in which the isoleucine at position 211 is mutated to glycine, the leucine at position 212 is mutated to methionine, the leucine at position 206 is mutated to phenylalanine, and the serine at position 37 is mutated to methionine. The amino acid sequence of the lipase is shown in SEQ ID NO:

1.

5. A DNA molecule, characterized in that The DNA molecule encodes the lipase mutant according to any one of claims 1 to 4.

6. An expression vector for a lipase mutant, characterized in that Expressing the lipase mutant according to any one of claims 1 to 4.

7. The expression vector according to claim 6, characterized in that The expression vector is a plasmid, a phage, or a virus.

8. A host cell expressing the lipase mutant according to any one of claims 1 to 4, characterized in that The host cell is a prokaryotic cell or a eukaryotic cell.

9. The host cell according to claim 8, characterized in that The host cell is selected from Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus or Trichoderma.

10. Use of the lipase mutant according to any one of claims 1 to 4 in catalyzing the reaction of aromatic amines and aromatic esters to form aromatic amides.

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