Lipase mutant, its encoding gene, expression plasmid and application

Abstract

This invention belongs to the field of bioenzymes, specifically relating to lipase mutants, their encoding genes, expression plasmids, and applications. The lipase mutant is an amino acid sequence containing at least one mutation, either M81C or F329R, in the wild-type amino acid sequence shown in SEQ ID NO.1. This invention innovatively mutates Met at position 81 (M81C) and / or Phe at position 329 (F329R) of the Geotrichum candida Y162 lipase GCL protein sequence (wild-type amino acid sequence); this effectively improves the enzyme activity, stability, and other properties of the mutant.

Description

Technical Field

[0001] This invention belongs to the field of enzymes, specifically to the field of lipase mutation. Background Technology

[0002] Lipase (EC3.1.1.3), as a highly efficient biocatalyst, possesses the core catalytic characteristic of highly specific catalytic esterification reactions. This process encompasses not only basic reaction types such as hydrolysis, alcoholysis, and acidolysis, but also drives complex transformation processes such as ester synthesis and transesterification. From a mechanistic perspective, lipase activates the catalytic tertiary pathway through a substrate-induced fit, exhibiting a significant interfacial activation effect at the oil-water interface. This characteristic makes it irreplaceable in industrial fields such as food processing, biodiesel production, and chiral drug synthesis.

[0003] With the rapid development of synthetic biology, scientists have begun to explore the heterologous expression of lipases using microbial systems such as *E. coli* and *Pichia pastoris*. These expression systems have gradually gained attention due to their high efficiency and operability, providing new avenues for the large-scale production and application of lipases. However, although the *Pichia pastoris* expression system is widely used in the production of recombinant proteins due to its high expression efficiency and ability to correctly fold complex proteins, it also faces some challenges in practical applications. For example, the expression level of certain lipase genes in *Pichia pastoris* may be low, affecting the yield of the target protein; proteins may misfold during expression, leading to reduced activity or the formation of inclusion bodies, increasing the difficulty of subsequent purification; furthermore, proteases within the host cell may degrade exogenous proteins, which not only weakens enzyme stability and activity but also negatively impacts production efficiency and increases production costs.

[0004] To address these challenges, scientists have employed a range of strategies. Optimizing expression conditions is a crucial method for enhancing lipase yield and activity. By adjusting parameters such as culture medium composition, temperature, pH, and inducer concentration, lipase expression efficiency can be significantly improved. Increasing gene copy number is also an effective approach; constructing multi-copy plasmids can increase lipase gene expression, thereby boosting enzyme yield. Furthermore, rational design and modification of the amino acid sequence are key strategies for improving lipase performance. Techniques such as site-directed mutagenesis, gene editing, and protein engineering can potentially optimize the amino acid sequence of lipases, enhancing their catalytic efficiency, stability, and substrate specificity.

[0005] Existing technologies also report some lipase mutations. For example, US Patent Publication No. US5726048A discloses a novel enzyme system that selectively hydrolyzes triglycerides using a *Geotrichum candida* mutant. Furthermore, Patent Publication No. WO2023035357A1 also discloses a lipase mutant and its applications, specifically involving one or more mutations selected from A262H, A338V, V364I, A158P / V, and I159N based on the amino acid sequence shown in SEQ ID NO: 1. For example, patent document US10167456B2 discloses a novel bifunctional enzyme mutant and its application in the processing of flour products. The lipase amino acid sequence has one of the following amino acid substitutions: p298t, p298t / h317p, p298t / h317p / v326s, p298t / t218s / s234f, p298t / h317p / p168l / a129s, p298t / s234f / k161r / v326s, where the substitution is relative to the parent amino acid sequence as shown in SEQ ID NO: 1. The lipase mutant maintains triglyceride and phospholipid hydrolysis activity and good thermal stability.

[0006] In summary, although there are some existing techniques for mutating lipases, there are many types of wild-type lipases, and theoretically, there are many sites that can be mutated in wild-type lipases. The functions and effects of different sequence mutations are unpredictable. Therefore, developing highly active and highly stable mutants remains an industry-wide challenge. Summary of the Invention

[0007] To address the issues of numerous lipase mutation methods and unpredictable mutation effects, this invention provides a lipase mutant, aiming to provide a novel mutant that improves the enzyme activity of the wild-type lipase of Geotrichum candida Y162 by mutating it.

[0008] The present invention also provides a coding gene and expression plasmid capable of encoding the lipase mutant.

[0009] A third objective of this invention is to provide the application of the lipase mutant in the preparation of glycerides.

[0010] Wild-type lipases are diverse, and each wild-type lipase contains a large number of amino acid sequences, making it extremely difficult to find highly active mutants from this vast mutation landscape. In response to the current state of lipase mutations, this invention, after in-depth research, provides the following solution:

[0011] A lipase mutant having an amino acid sequence having at least one mutation in M81C or F329R in the wild-type amino acid sequence shown in SEQ ID NO.1.

[0012] This invention innovatively mutates the Met at position 81 of the GCL protein sequence (wild-type amino acid sequence; also known as GCL-WT) of Geotrichum candida Y162 lipase to Cys (M81C) and / or mutates the Phe at position 329 to Arg (F329R); thus effectively improving the enzyme activity, stability and other properties of the mutant.

[0013] Preferably, in this invention, the lipase mutant is a mutant possessing both M81C and F329R mutations. Research in this invention shows that the simultaneous presence of these two mutants can optimize the active domain of the enzyme, further enhancing enzyme activity and stability.

[0014] The mutant of M81C in this invention is also labeled as GCL-M81C. The mutant of F329R in this invention is also labeled as GCL-F329R. The mutant of M81C-F329R in this invention is also labeled as GCL-M81C-F329R.

[0015] In this invention, the lipase mutant has the amino acid sequence shown in SEQ ID NO.3.

[0016] The lipase mutant of the present invention is a multicopy amino acid sequence having the M81C and F329R mutations in the wild-type amino acid sequence shown in SEQ ID NO.1.

[0017] The present invention also includes a gene encoding the lipase mutant described above, which is a gene sequence having SEQ ID NO.4.

[0018] The present invention also provides an expression plasmid for the lipase mutant described above, which is a plasmid capable of expressing the coding gene.

[0019] The mutant amino acid sequence, encoding gene, and expression plasmid described in this invention can all be prepared using known processes.

[0020] The present invention also provides an application of the lipase mutant described above, using it as an enzyme catalyst for the ester hydrolysis reaction of ester compounds.

[0021] In this invention, thanks to the innovative use of the lipase mutant, the enzyme activity, catalytic activity, and stability of the ester hydrolysis reaction can be effectively improved.

[0022] In this invention, the ester compound can be an oil (e.g., a glycerol ester), which undergoes the ester group hydrolysis reaction to yield fatty acids and glycerol.

[0023] In this invention, the pH of the ester hydrolysis reaction is 7.0~7.5.

[0024] In this invention, the ester hydrolysis reaction is carried out at 25~40 °C.

[0025] Beneficial effects

[0026] This invention provides a novel lipase mutant with excellent enzyme activity and stability. The research also shows that the M81C / F329R double mutant achieves even better performance. Attached Figure Description

[0027] Figure 1 Enzyme activity diagrams for different mutants. Detailed Implementation

[0028] In this invention, the mutant can be prepared using conventional amino acid mutation methods.

[0029] Example 1: Construction of a wild-type lipase GCL expression vector from Pichia pastoris

[0030] Based on the GCL protein sequence of Geotrichum y162 lipase from the UniProt database (accession number: P22394; SEQ ID NO.1), Beijing Qingke Biotechnology Co., Ltd. was commissioned to use artificial synthesis methods and optimize the sequence according to the codon preference of Pichia pastoris to construct the wild-type pPIC9K-GCL expression plasmid.

[0031] Example 2: Construction of GCL amino acid site mutants for lipase

[0032] The mutants GCL-M81C, GCL-F329R, GCL-P132D, GCL-V409L, and GCL-M81C-F329R were constructed using site-directed mutagenesis. Primers were designed to embed the target mutation sites, as shown below. PCR was used with pPIC9K-GCL as a template to amplify the full-length plasmid fragment containing the target amino acid mutation sites. PCR amplification conditions were: 95℃ for 5 min; 95℃ for 15 s, 50℃ for 15 s, 72℃ for 3 min, 30 cycles; 72℃ for 5 min. The PCR products were purified using a DNA purification kit and transformed into *E. coli* DH5α competent cells. The cells were plated on LB agar plates (50 μg / mL kan). Transformants were selected and sent to Beijing Qingke Biotechnology for sequencing. The results showed that expression plasmids of GCL-M81C, GCL-F329R, GCL-P132D, GCL-V409L, and GCL-M81C-F329R mutants were obtained.

[0033] The primer sequences for the PCR described above are as follows:

[0034] MC-F: tgcCAATTGGACCCAGGTAACTCCTTGA;

[0035] MC-R:

[0036] AGGAGTTACCTGGGTCCAATTGgcaACAAGCTGGACTAAAATCATTAGC.

[0037] PD-F: gacGCTGGAACGAAGCCAGATGCTAA.

[0038] PD-R:

[0039] TAGCATCTGGCTTCGTTCCAGCgtcTCTGAAAACGTTCAAATAAAGACAGT.

[0040] FR-F: cgaGGTTTGTTGCCACAATTCCTGGGA.

[0041] FR-R:

[0042] CCAGGAATTGTGGCAACAAACCtcgCAAATCCTTCAAATCGTAAGAGTTTTGAG.

[0043] VL-F: ctcTTGTCTTTGTACCCACAAACTTTGTCAG.

[0044] VL-R:

[0045] AAGTTTGTGGGTACAAAGACAAgTCTATCGATGGATGCTTCGCTAGCA.

[0046] Example 3: Recombinant expression of lipase GCL and its mutants

[0047] Plasmids pPIC9K-GCL, pPIC9K-GCL-M81C, and pPIC9K-GCL-M81C-F329R were obtained. After digestion with the restriction enzyme Sal I, a portion of the sample and the original circular plasmid were subjected to agarose gel electrophoresis. The corresponding length of the electrophoretic bands of the linearized plasmids proved the successful linearization of the recombinant plasmid pPIC9K-GCL. It can be transformed into Pichia pastoris GS115 and screened on MD plates (Pichia pastoris MD plates). The resulting single clones were further streaked on YPD plates (yeast extract peptone dextrose agar plates) containing different concentrations of antibiotic G418 for high copy selection. The single clones that grew on the high concentration plates can be verified by shake-flask expression.

[0048] The strain was inoculated into BMGY medium and pre-cultured at 30℃ and 220 rpm / min for 24 hours. After centrifugation at 5000 rpm / min, the cells were collected, washed twice with sterile water, and resuspended in BMGY medium until the OD600 was 1. The cells were then induced at 25℃ and 220 rpm, with methanol added every 24 hours to a final concentration of 1%. Induction was completed after 120 hours, and the supernatant was collected by centrifugation to determine the lipase activity.

[0049] Example 4: Enzyme activity assay of lipase GCL and its mutants

[0050] The method for determining lipase activity is as follows: the hydrolytic activity of lipase is determined by acid-base titration. A 4% PVA aqueous solution and glyceryl tributyrate are mixed at a volume ratio of 3:1 and homogenized under high pressure to form a glyceryl tributyrate emulsion, which is then used as the substrate.

[0051] Take 50 mL Erlenmeyer flasks with stoppers, label them blank flask (A) and sample flask (B). Add 5.00 mL of phosphate-buffered saline (PBS) at pH 7.0 and 4.00 mL of olive oil emulsion to each flask. Add 15 mL of 95% ethanol to flask A and preheat in a 40°C water bath at 150 rpm for 5 min. Add 1 mL of the enzyme solution to be tested (appropriately diluted supernatant from the centrifugation at the end of induction in Example 3) to each flask A and B, mix immediately, and start timing. After reacting for 15 min, immediately add 15 mL of 95% ethanol to flask B to terminate the reaction. Remove and cool to room temperature. Add 2 drops of phenolphthalein indicator to each flask, and titrate with 0.05 M NaOH standard solution until the solution turns slightly pink and remains so for 30 s. Record the volume of NaOH standard solution consumed.

[0052] Enzyme activity is defined as the amount of enzyme required to hydrolyze tricresyl ester to release 1 μmol of fatty acid per minute (1.00 mL of 0.05 mol / L NaOH standard solution is equivalent to 50 μmol of fatty acid). The enzyme activity of GCL is calculated using the following formula: Hydrolytic activity (U / mL) = (V - V0) × c × 50 × n / (0.05 × t). Where V is the volume (mL) of NaOH standard solution consumed when titrating the sample; V0 is the volume (mL) of NaOH standard solution consumed when titrating the control; c is the concentration of NaOH standard solution (mol / L); 50 is the equivalent of 50 μmol of fatty acid in 1.00 mL of 0.05 mol / L NaOH standard solution; n is the dilution factor of the sample; 0.05 is the conversion factor for NaOH standard solution concentration; and t is the reaction time (15 min).

[0053] Enzyme activity see Figure 1The wild-type lipase (GCL-WT) activity was 10.35 U / mL, while the mutants GCL-M81C and GCL-M81C-F329R were 2.48 times and 5.96 times that of the wild-type, respectively. GCL-F329R was 2.14 times that of the wild-type. Similarly, GCL-P132D (containing the P132D mutant amino acid sequence in the wild-type amino acid sequence shown in SEQ ID NO.1) and GCL-V409L (containing the V409L mutant amino acid sequence in the wild-type amino acid sequence shown in SEQ ID NO.1) were 1.21 times and 0.82 times that of the wild-type, respectively.

[0054] pass Figure 1 It is known that the mutations of M81C and / or F329R described in this invention can unexpectedly improve the enzyme activity of phospholipase.

[0055] Example 5: Construction of a multi-copy pPICZαA-GCL-M81C-F329R mutant plasmid

[0056] A linear fragment digested with BamHI was obtained using pPICZαA-GCL-M81C-F329R as a template. This linearized fragment already contained one copy of the GCL-M81C-F329R expression cassette. A smaller fragment digested with BamHI and BglII also contained one copy of the GCL-M81C-F329R expression cassette. These two fragments were ligated using T4 ligase and transformed into DH5α. The transformed individuals were plated on LB agar plates with 50 μg / mL zeocin for culture and screening. The resulting transformants were picked, cultured, and the plasmid extracted was verified by double digestion with BglII and BamHI. Repeating these steps allowed for the construction of a multi-copy pPICZαA-GCL-M81C-F329R mutant plasmid. The obtained multi-copy pPICZαA-GCL-M81C-F329R mutant plasmid was transformed into the strain with a 5.96-fold increase in lipase activity in Example 4. The plating was spread on YPD + 100 μg / mL zeocin and cultured for 3-4 days. The resulting single clones were then expressed and verified by shaking flask according to the method provided in Example 3. The enzyme activity was then detected according to the method provided in Example 4, and it was found that the enzyme activity increased by 1.75 times on top of the 5.96-fold increase.

[0057] Original GCL (SEQ ID NO.1)

[0058] MVSKSLFLAAAVNLAGVLAQAPRPSLNGNEVISGVLEGKVDTFKGIPFADPPLNDLRFKHPQPFTGSYQGLKANDFSPACMQLDPGNSLTLLDKALGLAKVIPEEFRGPLYDMAKGTVSMNEDCLYLNVFRPAGTKPDAKLPVMVWIYGGAFVYGSSAAYPGNSYVKESINMGQPVVFVSINYRTGPFGFLGGDAITAEGNTNAGLHDQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLIAYGGDNTYNGKKLFHSAILQSGGPLPYHDSSSVGPDISYNRFAQYAGCDTSASANDTLECLRSKSSSVLHDAQNSYDLKDLFGLLPQFLGFGPRPDGNIIPDAAYELFRSGRYAKVPYISGNQEDEGTAFAPVALNATTTPHVKKWLQYIFYDASEASIDRVLSLYPQTLSVGSPFRTGILNALTPQFKRVAAILSDMLFQSPRRVMLSATKDVNRWTYLSTHLHNLVPFLGTFHGNELIFQFNVNIGPANSYLRYFISFANHHDPNVGTNLLQWDQYTDEGKEMLEIHMTDNVMRTDDYRIEGISNFETDVNLYG。

[0059] Original GCL nucleic acid sequence (SEQ ID NO.2)

[0060]

[0061] Amino acid sequence (SEQ ID NO.3)

[0062] MVSKSLFLAAAVNLAGVLAQAPRPSLNGNEVISGVLEGKVDTFKGIPFADPPLNDLRFKHPQPFTGSYQGLKANDFSPACCQLDPGNSLTLLDKALGLAKVIPEEFRGPLYDMAKGTVSMNEDCLYLNVFRPAGTKPDAKLPVMVWIYGGAFVYGSSAAYPGNSYVKESINMGQPVVFVSINYRTGPFGFLGGDAITAEGNTNAGLHDQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLIAYGGDNTYNGKKLFHSAILQSGGPLPYHDSSSVGPDISYNRFAQYAGCDTSASANDTLECLRSKSSSVLHDAQNSYDLKDLRGLLPQFLGFGPRPDGNIIPDAAYELFRSGRYAKVPYISGNQEDEGTAFAPVALNATTTPHVKKWLQYIFYDASEASIDRVLSLYPQTLSVGSPFRTGILNALTPQFKRVAAILSDMLFQSPRRVMLSATKDVNRWTYLSTHLHNLVPFLGTFHGNELIFQFNVNIGPANSYLRYFISFANHHDPNVGTNLLQWDQYTDEGKEMLEIHMTDNVMRTDDYRIEGISNFETDVNLYG。

[0063] GCL-M81C-F329R nucleic acid sequence: (SEQ ID NO.4)

[0064]

[0065] Amino acid sequence of GCL-M81C (SEQ ID NO.5)

[0066] MVSKSLFLAAAVNLAGVLAQAPRPSLNGNEVISGVLEGKVDTFKGIPFADPPLNDLRFKHPQPFTGSYQGLKANDFSPACCQLDPGNSLTLLDKALGLAKVIPEEFRGPLYDMAKGTVSMNEDCLYLNVFRPAGTKPDAKLPVMVWIYGGAFVYGSSAAYPGNSYVKESINMGQPVVFVSINYRTGPFGFLGGDAITAEGNTNAGLHDQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLIAYGGDNTYNGKKLFHSAILQSGGPLPYHDSSSVGPDISYNRFAQYAGCDTSASANDTLECLRSKSSSVLHDAQNSYDLKDLRGLLPQFLGFGPRPDGNIIPDAAYELFRSGRYAKVPYISGNQEDEGTAFAPVALNATTTPHVKKWLQYIFYDASEASIDRVLSLYPQTLSVGSPFRTGILNALTPQFKRVAAILSDMLFQSPRRVMLSATKDVNRWTYLSTHLHNLVPFLGTFHGNELIFQFNVNIGPANSYLRYFISFANHHDPNVGTNLLQWDQYTDEGKEMLEIHMTDNVMRTDDYRIEGISNFETDVNLYG.

[0067] Amino acid sequence of GCL-F329R (SEQ ID NO.6)

[0068] MVSKSLFLAAAVNLAGVLAQAPRPSLNGNEVISGVLEGKVDTFKGIPFADPPLNDLRFKHPQPFTGSYQGLKANDFSPACCQLDPGNSLTLLDKALGLAKVIPEEFRGPLYDMAKGTVSMNEDCLYLNVFRPAGTKPDAKLPVMVWIYGGAFVYGSSAAYPGNSYVKESINMGQPVVFVSINYRTGPFGFLGGDAITAEGNTNAGLHDQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLIAYGGDNTYNGKKLFHSAILQSGGPLPYHDSSSVGPDISYNRFAQYAGCDTSASANDTLECLRSKSSSVLHDAQNSYDLKDLRGLLPQFLGFGPRPDGNIIPDAAYELFRSGRYAKVPYISGNQEDEGTAFAPVALNATTTPHVKKWLQYIFYDASEASIDRVLSLYPQTLSVGSPFRTGILNALTPQFKRVAAILSDMLFQSPRRVMLSATKDVNRWTYLSTHLHNLVPFLGTFHGNELIFQFNVNIGPANSYLRYFISFANHHDPNVGTNLLQWDQYTDEGKEMLEIHMTDNVMRTDDYRIEGISNFETDVNLYG。

[0069] Amino acid sequence of GCL-P132D (SEQ ID NO.7)

[0070] MVSKSLFLAAAVNLAGVLAQAPRPSLNGNEVISGVLEGKVDTFKGIPFADPPLNDLRFKHPQPFTGSYQGLKANDFSPACCQLDPGNSLTLLDKALGLAKVIPEEFRGPLYDMAKGTVSMNEDCLYLNVFRDAGTKPDAKLPVMVWIYGGAFVYGSSAAYPGNSYVKESINMGQPVVFVSINYRTGPFGFLGGDAITAEGNTNAGLHDQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLIAYGGDNTYNGKKLFHSAILQSGGPLPYHDSSSVGPDISYNRFAQYAGCDTSASANDTLECLRSKSSSVLHDAQNSYDLKDLRGLLPQFLGFGPRPDGNIIPDAAYELFRSGRYAKVPYISGNQEDEGTAFAPVALNATTTPHVKKWLQYIFYDASEASIDRVLSLYPQTLSVGSPFRTGILNALTPQFKRVAAILSDMLFQSPRRVMLSATKDVNRWTYLSTHLHNLVPFLGTFHGNELIFQFNVNIGPANSYLRYFISFANHHDPNVGTNLLQWDQYTDEGKEMLEIHMTDNVMRTDDYRIEGISNFETDVNLYG。

[0071] Amino acid sequence of GCL-V409L (SEQ ID NO.8)

[0072] 。

Claims

1. A lipase mutant, characterized in that: The wild-type amino acid sequence shown in SEQ ID NO.1 contains at least one mutated amino acid sequence of M81C or F329R.

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

3.

3. The encoding gene of a lipase mutant according to any one of claims 1 to 2, characterized in that: The gene sequence is SEQ ID NO.

4.

4. An expression plasmid for a lipase mutant, characterized in that: It is a plasmid expressing the encoding gene of claim 3.

5. The application of the lipase mutant according to any one of claims 1 to 2, characterized in that: It can be used as an enzyme catalyst for the hydrolysis of ester groups in ester compounds; The ester compounds are fats and oils, which, through the ester hydrolysis reaction, yield fatty acids and glycerol.

6. The application of the lipase mutant as described in claim 5, characterized in that: The pH for ester hydrolysis is 7.0~7.

5.

7. The application of the lipase mutant as described in claim 5, characterized in that: The ester hydrolysis reaction is carried out at 25-40 °C.

Images