Lipase mutant as well as coding gene, expression plasmid and application thereof

Abstract

The invention belongs to the field of biological enzymes, and particularly relates to a lipase mutant as well as a coding gene, an expression plasmid and application thereof, and the lipase mutant is an amino acid sequence with at least one mutation of M81C and F329R in a wild amino acid sequence as shown in SEQ ID NO.1. According to the invention, Met at the 81 site of a geotrichum candidum Y162 lipase GCL protein sequence (wild amino acid sequence) is innovatively mutated into Cys (M81C) and / or Phe at the 329 site of the geotrichum candidum Y162 lipase GCL protein sequence (wild amino acid sequence) is innovatively mutated into Arg (F329R); therefore, the enzyme activity, stability and other properties of the mutant can be effectively improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of enzymes, and particularly relates to the field of lipase mutations. BACKGROUND

[0002] Lipase (EC 3.1.1.3) is a highly efficient biological catalyst, and its core catalytic property is its high specificity in catalyzing ester hydrolysis. This process not only covers basic reaction types such as hydrolysis, alcoholysis and acidolysis, but also drives complex conversion processes such as ester synthesis and ester exchange. From the perspective of the mechanism of action, lipase activates the catalytic triad through a "substrate-induced fitting" mode, and exhibits a significant "interface activation" effect at the oil-water interface. This property makes it irreplaceable in industrial fields such as food processing, biodiesel production, and synthesis of chiral drugs.

[0003] With the rapid development of synthetic biology, scientists have begun to explore the use of microbial systems such as Escherichia coli and Pichia pastoris for the heterologous expression of lipases. These expression systems have gradually attracted attention due to their high efficiency and operability, providing a new way 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 capacity and ability to correctly fold complex proteins, the system also faces some challenges in practical application. For example, the expression level of some lipase genes in Pichia pastoris may be low, affecting the yield of target proteins; proteins may be misfolded during expression, leading to reduced activity or the formation of inclusion bodies, increasing the difficulty of subsequent purification; in addition, proteases in host cells may degrade foreign proteins, not only weakening the stability and activity of enzymes, but also negatively affecting production efficiency and increasing production costs.

[0004] To solve these problems, scientists have adopted a series of strategies. Optimizing expression conditions is one of the important means to improve the yield and activity of lipases. By adjusting parameters such as medium composition, temperature, pH value and inducer concentration, the expression efficiency of lipases can be significantly improved. Increasing the gene copy number is also an effective method. By constructing a multi-copy plasmid, the expression of the lipase gene can be increased, thereby increasing the yield of the enzyme. In addition, rational design and modification of the amino acid sequence is also a key strategy for improving the performance of lipases. Through techniques such as site-directed mutagenesis, gene editing and protein engineering, it is possible to optimize the amino acid sequence of lipases, improving their catalytic efficiency, stability and substrate specificity.

[0005] Several lipase mutations have been reported in the prior art. For example, U.S. Patent Publication No. US5726048A discloses a novel enzyme system produced by a Geotrichum candidum mutant that selectively hydrolyzes triglycerides. Furthermore, Patent Publication No. WO2023035357A1 discloses a lipase mutant and its application, specifically one or more mutations selected from the group consisting of A262H, A338V, V364I, A158P / V, and I159N, based on the amino acid sequence of SEQ ID NO: 1. For example, patent document No. US10167456B2 discloses a novel bifunctional enzyme mutant and its application in noodle processing, wherein 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, and the substitution is relative to the parent amino acid sequence such as SEQ ID NO: 1; the lipase mutant maintains triglyceride and phospholipid hydrolysis activity and good thermal stability.

[0006] In summary, although there are some methods for lipase mutation in the existing technology, there are many types of wild-type lipases, and there are many sites at which wild-type lipases can theoretically mutate. The functions and effects of mutations in different sequences are unpredictable. Therefore, the development of highly active and highly stable mutants remains an industry-wide challenge. Summary of the Invention

[0007] In response to the problems of numerous lipase mutation methods and unpredictable mutation effects, the present invention provides a lipase mutant, aiming to provide a new mutant that mutates the wild-type enzyme of Geotrichum candidum Y162 lipase to improve its enzymatic activity.

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

[0009] The third object of the present invention is to provide the use of the lipase mutant in the preparation of glycerides.

[0010] There are many different types of wild-type lipases, and the number of amino acid sequences in each wild-type lipase is enormous. Finding highly active mutants from this vast array of mutations is extremely difficult. In response to the current state of lipase mutations, the present invention, after in-depth research, provides the following solutions:

[0011] A lipase mutant has an amino acid sequence in which at least one of the mutations M81C and F329R exists in the wild amino acid sequence shown in SEQ ID NO.1.

[0012] The present application innovatively mutates Met at position 81 of the Geotrichum candidum Y162 lipase GCL protein sequence (wild-type amino acid sequence; also referred to as GCL-WT) to Cys (M81C) and / or mutates Phe at position 329 to Arg (F329R); thus, the enzyme activity, stability, and other properties of the mutant can be effectively improved.

[0013] Preferably, in the present application, the lipase mutant is a mutant having both M81C and F329R mutations. The present application shows that the simultaneous presence of the two mutants can optimize the activity domain of the obtained enzyme and further strengthen the enzyme activity and stability.

[0014] The M81C mutant of the present application is also referred to as GCL-M81C. The F329R mutant of the present application is also referred to as GCL-F329R. The M81C-F329R mutant of the present application is also referred to as GCL-M81C-F329R.

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

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

[0017] The present application also comprises a coding gene of the lipase mutant, which has the gene sequence of SEQ ID NO. 4.

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

[0019] The mutant amino acid sequence, coding gene, and expression plasmid of the present application can be prepared based on known processes.

[0020] The present application also provides a use of the lipase mutant as an enzyme catalyst for ester group hydrolysis of ester compounds.

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

[0022] In the present application, the ester compound can be oil and fat (e.g., glyceride), and the ester group hydrolysis can obtain fatty acid and glycerol.

[0023] In the present application, the pH of the ester group hydrolysis is 7.0-7.5.

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

[0025] Beneficial effects

[0026] The present invention provides a novel lipase mutant with excellent enzyme activity and stability. The present invention also shows that a mutant with both M81C and F329R mutations can achieve even better performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The enzyme activities of different mutants are shown in Figure 2 . DETAILED DESCRIPTION

[0028] In the present invention, the mutant can be prepared based on conventional amino acid mutation methods.

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

[0030] Based on the GCL protein sequence of Geotrichum candidum Y162 lipase in the UniProt database (accession number: P22394; SEQ ID NO. 1), Beijing Qingke Biotechnology was commissioned to use artificial synthesis methods and optimize the sequence according to the codon preference of Pichia pastoris to construct it on the plasmid pPIC9K, thus obtaining the wild-type pPIC9K-GCL expression plasmid.

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

[0032] Site-directed mutagenesis was used to construct mutants GCL-M81C, GCL-F329R, GCL-P132D, GCL-V409L, and GCL-M81C-F329R. Primers were designed to encompass the target mutation sites (see below). Full-length plasmid fragments containing the target amino acid mutation sites were amplified using pPIC9K-GCL as a template using PCR. PCR amplification conditions were: 95°C for 5 minutes, followed by 30 cycles of 95°C for 15 seconds, 50°C for 15 seconds, and 72°C for 3 minutes, and finally 72°C for 5 minutes. PCR products were purified using a DNA purification kit and transformed into competent E. coli DH5α cells. The products were then plated onto LB agar plates (50 μg / mL kan). The transformants were picked and sent to Beijing Qingke Biotechnology for sequencing. The results showed that expression plasmids for GCL-M81C, GCL-F329R, GCL-P132D, GCL-V409L, and GCL-M81C-F329R mutants were obtained.

[0033] The primer sequences for the above PCR 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] AAGTTTGTGGGTACAAAGACAAgagTCTATCGATGGATGCTTCGCTAGCA.

[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 endonuclease Sal I, a portion of the sample and the original circular plasmid were subjected to agarose gel electrophoresis. The electrophoresis band length that matched the linearized plasmid confirmed the successful linearization of the recombinant plasmid pPIC9K-GCL. The recombinant plasmids could be transformed into Pichia pastoris GS115 and screened on MD plates (Pichia pastoris MD plates). The resulting single colonies were further streaked on YPD plates (yeast extract peptone dextrose agar plates) containing different concentrations of the antibiotic G418 for high copy screening. The single colonies grown on the high-concentration plates could be used for shake flask expression verification.

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

[0049] Example 4: Determination of enzyme activity of lipase GCL and its mutants

[0050] The lipase activity was determined by acid-base titration. A 4% aqueous solution of PVA and tributyrin were mixed at a volume ratio of 3:1 and homogenized in a high-pressure homogenizer to form a tributyrin emulsion, which was used as a substrate.

[0051] Take 50mL stoppered Erlenmeyer flasks, label them blank (A) and sample (B), and add 5.00mL of pH 7.0 phosphate buffered saline (PBS) and 4.00mL of olive oil emulsion to each. Add 15mL of 95% ethanol to bottle A. Preheat the mixture in a 40°C water bath at 150 rpm / min for 5 minutes. Add 1mL of the enzyme solution to be tested (appropriately diluted supernatant from the centrifugation after induction in Example 3) to each of bottles A and B. Immediately mix and time the reaction. After an accurate reaction time of 15 minutes, immediately add 15mL of 95% ethanol to bottle B to terminate the reaction. Remove the bottle and cool to room temperature. Add 2 drops of phenolphthalein indicator to each of the blank and sample bottles. Titrate with 0.05M NaOH standard solution until the solution turns slightly red and does not fade for 30 seconds. Record the volume of NaOH standard solution consumed.

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

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

[0054] pass Figure 1 It can be seen that the M81C and / or F329R mutations described in the present invention can unexpectedly improve the enzymatic activity of phospholipase.

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

[0056] Using pPICZαA-GCL-M81C-F329R as a template, BamH I was used to digest the linearized fragment, which already contained a single copy of the GCL-M81C-F329R expression cassette. pPICZαA-GCL-M81C-F329R was then digested with BamH I and Bgl II to create a smaller fragment, also containing a single copy of the GCL-M81C-F329R expression cassette. These two fragments were ligated with T4 ligase and transformed into DH5a cells. Transformants were screened by plating on LB plates with 50 μg / mL zeocin. Transformants were selected and shaken to extract the plasmid, which was then digested with Bgl II and BamH I for verification. Repeat these steps to construct multiple copies of the 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, and plated on YPD + 100 μg / mL zeocin (bleomycin) for 3 to 4 days. The grown single clones were then shake-flask expression verified according to the method provided in Example 3, and then enzyme activity was detected according to the method provided in Example 4. It was found that the enzyme activity was increased by 1.75 times on the basis of the 5.96-fold increase.

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

[0058] 原始GCL核酸序列(SEQ ID NO.2)

[0059] 氨基酸序列(SEQ ID NO.3) MVSKSLFLAAAVNLAGVLAQAPRPSLNGNEVISGVLEGKVDTFKGIPFADPPLNDLRFKHPQPFTGSYQGLKANDFSPACCQLDPGNSLTLLDKALGLAKVIPEEFRGPLYDMAKGTVSMNEDCLYLNVFRPAGTKPDAKLPVMVWIYGGAFVYGSSAAYPGNSYVKESINMGQPVVFVSINYRTGPFGFLGGDAITAEGNTNAGLHDQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLIAYGGDNTYNGKKLFHSAILQSGGPLPYHDSSSVGPDISYNRFAQYAGCDTSASANDTLECLRSKSSSVLHDAQNSYDLKDLRGLLPQFLGFGPRPDGNIIPDAAYELFRSGRYAKVPYISGNQEDEGTAFAPVALNATTTPHVKKWLQYIFYDASEASIDRVLSLYPQTLSVGSPFRTGILNALTPQFKRVAAILSDMLFQSPRRVMLSATKDVNRWTYLSTHLHNLVPFLGTFHGNELIFQFNVNIGPANSYLRYFISFANHHDPNVGTNLLQWDQYTDEGKEMLEIHMTDNVMRTDDYRIEGISNFETDVNLYG。

[0060] GCL-M81C-F329R核酸序列:(SEQ ID NO.4)

[0061] GCL-M81C氨基酸序列(SEQ ID NO.5) MVSKSLFLAAAVNLAGVLAQAPRPSLNGNEVISGVLEGKVDTFKGIPFADPPLNDLRFKHPQPFTGSYQGLKANDFSPACCQLDPGNSLTLLDKALGLAKVIPEEFRGPLYDMAKGTVSMNEDCLYLNVFRPAGTKPDAKLPVMVWIYGGAFVYGSSAAYPGNSYVKESINMGQPVVFVSINYRTGPFGFLGGDAITAEGNTNAGLHDQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLIAYGGDNTYNGKKLFHSAILQSGGPLPYHDSSSVGPDISYNRFAQYAGCDTSASANDTLECLRSKSSSVLHDAQNSYDLKDLRGLLPQFLGFGPRPDGNIIPDAAYELFRSGRYAKVPYISGNQEDEGTAFAPVALNATTTPHVKKWLQYIFYDASEASIDRVLSLYPQTLSVGSPFRTGILNALTPQFKRVAAILSDMLFQSPRRVMLSATKDVNRWTYLSTHLHNLVPFLGTFHGNELIFQFNVNIGPANSYLRYFISFANHHDPNVGTNLLQWDQYTDEGKEMLEIHMTDNVMRTDDYRIEGISNFETDVNLYG。

[0062] GCL-F329R氨基酸序列(SEQ ID NO.6) MVSKSLFLAAAVNLAGVLAQAPRPSLNGNEVISGVLEGKVDTFKGIPFADPPLNDLRFKHPQPFTGSYQGLKANDFSPACCQLDPGNSLTLLDKALGLAKVIPEEFRGPLYDMAKGTVSMNEDCLYLNVFRPAGTKPDAKLPVMVWIYGGAFVYGSSAAYPGNSYVKESINMGQPVVFVSINYRTGPFGFLGGDAITAEGNTNAGLHDQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLIAYGGDNTYNGKKLFHSAILQSGGPLPYHDSSSVGPDISYNRFAQYAGCDTSASANDTLECLRSKSSSVLHDAQNSYDLKDLRGLLPQFLGFGPRPDGNIIPDAAYELFRSGRYAKVPYISGNQEDEGTAFAPVALNATTTPHVKKWLQYIFYDASEASIDRVLSLYPQTLSVGSPFRTGILNALTPQFKRVAAILSDMLFQSPRRVMLSATKDVNRWTYLSTHLHNLVPFLGTFHGNELIFQFNVNIGPANSYLRYFISFANHHDPNVGTNLLQWDQYTDEGKEMLEIHMTDNVMRTDDYRIEGISNFETDVNLYG。

[0063] GCL-P132D氨基酸序列(SEQ ID NO.7) MVSKSLFLAAAVNLAGVLAQAPRPSLNGNEVISGVLEGKVDTFKGIPFADPPLNDLRFKHPQPFTGSYQGLKANDFSPACCQLDPGNSLTLLDKALGLAKVIPEEFRGPLYDMAKGTVSMNEDCLYLNVFRDAGTKPDAKLPVMVWIYGGAFVYGSSAAYPGNSYVKESINMGQPVVFVSINYRTGPFGFLGGDAITAEGNTNAGLHDQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLIAYGGDNTYNGKKLFHSAILQSGGPLPYHDSSSVGPDISYNRFAQYAGCDTSASANDTLECLRSKSSSVLHDAQNSYDLKDLRGLLPQFLGFGPRPDGNIIPDAAYELFRSGRYAKVPYISGNQEDEGTAFAPVALNATTTPHVKKWLQYIFYDASEASIDRVLSLYPQTLSVGSPFRTGILNALTPQFKRVAAILSDMLFQSPRRVMLSATKDVNRWTYLSTHLHNLVPFLGTFHGNELIFQFNVNIGPANSYLRYFISFANHHDPNVGTNLLQWDQYTDEGKEMLEIHMTDNVMRTDDYRIEGISNFETDVNLYG。

[0064] GCL-V409L氨基酸序列(SEQ ID NO.8) 。

Claims

1. A lipase mutant, characterized in that: The amino acid sequence has at least one mutation of M81C and F329R in the wild amino acid sequence shown in SEQ ID NO.

1.

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

3.

3. The lipase mutant according to claim 1 or 2, characterized in that: It is a multi-copy amino acid sequence having M81C and F329R mutations in the wild amino acid sequence shown in SEQ ID NO.

1.

4. A gene encoding the lipase mutant according to any one of claims 1 to 3, characterized in that: It is a gene sequence with SEQID NO.

4.

5. An expression plasmid for a lipase mutant, characterized in that: It is a plasmid capable of expressing the coding gene according to claim 4.

6. Use of the lipase mutant according to any one of claims 1 to 3, characterized in that: It is used as an enzyme catalyst for the hydrolysis of ester groups in ester compounds.

7. The use of the lipase mutant according to claim 6, characterized in that: The ester compound is oil, and fatty acid and glycerol are obtained through the ester hydrolysis reaction.

8. The use of the lipase mutant according to claim 7, characterized in that: The pH of the ester hydrolysis reaction is 7.0~7.

5.

9. The use of the lipase mutant according to claim 7, characterized in that: 25~40℃ for ester hydrolysis reaction.

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