Lipase mutant and application thereof

Abstract

The invention discloses a lipase mutant and application thereof. The amino acid sequence of the lipase mutant is as shown in SEQ ID NO. 02. The catalyst has excellent chiral selectivity, catalytic activity, solvent tolerance and thermal stability, and has a wide application prospect; the method is used for chiral resolution of menthol, is simple in operation, short in time consumption and easy to amplify, solves the bottleneck of a traditional recrystallization method, promotes localization of menthol, has a wide application prospect, and can be expanded to the fields of resolution of other chiral compounds and enzyme engineering.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a lipase mutant and application thereof. BACKGROUND

[0002] Menthol is extracted from the leaves and stems of mint, a white crystal, and is the main component in mint and peppermint essential oil. There are generally two isomers of menthol (D and L types), and natural menthol is mainly the left-handed isomer (L-menthol). In the synthesis of L-menthol, the main method is to obtain a menthol mixture containing four pairs of stereoisomers by catalytic hydrogenation of thymol, and then to obtain racemic D, L-menthol by rectification, and then to separate the high-value L-menthol by chiral resolution technology. At present, the demand for menthol in China is highly dependent on imports, with an annual import of about 150,000 tons and an amount of 1.3 billion yuan. The chiral resolution step is the main technical bottleneck. The current chiral resolution process of L-menthol relies on the addition of seed crystals for recrystallization, which is time-consuming and difficult to scale up.

[0003] In the prior art, the limitations of chiral resolution technology have become a key factor restricting the production of menthol. Although the traditional method of adding seed crystals for recrystallization can achieve the separation of L-menthol, the operation process is complex, requiring multiple crystallization cycles, consuming a large amount of time and energy, and when the production scale is enlarged, problems such as uneven crystal purity and reduced yield are likely to occur. This leads to low overall production efficiency, which cannot meet the growing market demand. In particular, in China, the dependence on imports of menthol further highlights the urgency of local production, with an annual import of 150,000 tons and an economic cost of 1.3 billion yuan, exposing the constraints of chiral resolution technology on industrial development.

[0004] In modern industrial applications, the application range of lipase is continuously expanding, and it shows its unique value in the fields of oil processing, food industry, bioenergy, biomaterials, and environmental remediation. For example, lipase can catalyze the transesterification of oil and methanol to generate biodiesel and glycerol. This process is not only efficient and environmentally friendly, but also can achieve sustainable utilization of oil resources. Chiral compounds have important application value in the field of medicine, and lipase can be applied to the synthesis of chiral drugs and the resolution of optical isomers. These applications highlight the multifunctionality of lipase as a biological catalyst, which catalyzes esterification, hydrolysis, and transesterification reactions in non-aqueous environments, with the advantages of mild conditions and high selectivity. However, the performance of wild-type lipase in chiral resolution is often limited, and its stability is insufficient in high-concentration substrates or organic solvents, which cannot achieve efficient conversion.

[0005] Therefore, it is of great significance to improve lipase by genetic engineering, protein engineering and other means, develop lipase with excellent performance, and reduce its production cost, which promotes the application of the enzyme. The existing lipase modification technology includes site-directed mutagenesis, directed evolution and other methods, which can optimize the substrate binding site or catalytic center to improve stereoselectivity and catalytic activity. However, in the field of menthol chiral resolution, the existing enzyme modification still faces challenges, such as the need to further improve the thermal stability and solvent tolerance of the mutant enzyme to adapt to industrial conditions.

[0006] In addition, the application of lipase in the field of biological energy, such as catalyzing oil transesterification to produce biodiesel, reflects its environmental value, but the application of chiral resolution needs to pay more attention to the demand of pharmaceutical and fine chemical industry. The purity of chiral compounds directly affects the drug efficacy, so the role of lipase in the resolution of optical isomers is indispensable. Although there are reports of lipase used in the resolution of chiral alcohol compounds, there are few specific modifications for menthol, and there is a lack of efficient and scalable methods. This further emphasizes the necessity of improving lipase through engineering means to solve the time-consuming and scaling problems of traditional processes and promote the domestic production of chiral compounds such as menthol.

[0007] In summary, the existing technology has significant defects in the resolution of menthol chiral, including low selectivity, insufficient conversion rate and complex process. Through the combination of protein engineering and genetic engineering, it is expected to develop better lipase variants to achieve a simple and time-saving chiral resolution process. The progress in this field not only reduces the dependence on imports, but also promotes the wide application of lipase in many fields. SUMMARY

[0008] The present application aims to overcome the defects of the prior art and provide a lipase mutant.

[0009] Another object of the present application is to provide the application of the above-mentioned lipase mutant.

[0010] The technical solution of the present application is as follows:

[0011] A lipase mutant, whose amino acid sequence is shown in SEQ ID NO. 02.

[0012] An expression vector, which is a vector pPICZαA loaded with an amino acid sequence shown in SEQ ID NO. 02.

[0013] In a preferred embodiment of the present application, the α-MF signal peptide coding gene sequence carried by the vector pPICZαA is replaced by a gene sequence encoding a signal peptide with an amino acid sequence shown in SEQ ID NO. 04.

[0014] A lipase mutant expression bacterium, which is Pichia pastoris X33, is loaded with the above-mentioned expression vector after linearization.

[0015] In a preferred embodiment of the present application, the vector pGAPZ alpha A (the Zeocin resistance gene is replaced by the G418 resistance gene) is also loaded with the gene sequence of the chaperone encoding the amino acid sequence shown in SEQ ID NO. 06 after linearization.

[0016] A fermentation preparation method of the above-mentioned lipase mutant, which is carried out by using the above-mentioned lipase mutant expression bacterium.

[0017] The above-mentioned lipase mutant is used in the chiral resolution of menthol.

[0018] In a preferred embodiment of the present application, the lipase mutant is immobilized by a macroporous adsorption resin HPD450.

[0019] A menthol chiral resolution method, which is carried out by using the above-mentioned lipase mutant.

[0020] In a preferred embodiment of the present application, the lipase mutant is immobilized by a macroporous adsorption resin HPD450.

[0021] The present application has the following beneficial effects:

[0022] 1. The present application improves the selectivity of L enantiomer of DL-menthol from 52.5% to 99%. Under the conditions of 0.4M racemization substrate DL-menthol in n-hexane solvent at 50℃, L-menthol can be completely converted in 4h.

[0023] 2. The present application has excellent chiral selectivity, catalytic activity, solvent tolerance and thermal stability, and has a wide application prospect. The selectivity of L enantiomer and the catalytic activity are significantly improved. The selectivity ee is greater than 99%, and L-menthol is almost completely converted.

[0024] 3. The present application increases the expression amount of the lipase mutant by overexpression of the chaperone and fermentation optimization, so that the secretion concentration of the lipase mutant reaches 10g / L, which is convenient for large-scale production.

[0025] 4. The menthol chiral resolution method of the present application is simple in operation, short in time consumption and easy to scale up, solves the bottleneck of the traditional recrystallization method, promotes the domestication of menthol, has a wide application prospect, and can be extended to other chiral compound resolution and enzyme engineering fields. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The retention time diagram in Example 5 of the present application.

[0027] Figure 2The GC profile of the reaction product of Example 5 of the present application. In which L-menthol is almost completely reacted, and the by-product D-menthyl acetate is not detected. DETAILED DESCRIPTION

[0028] The technical solutions of the present application are further described and explained below through specific embodiments in conjunction with the accompanying drawings.

[0029] The experimental materials used in the following examples are shown below:

[0030] A. Strains and vectors:

[0031] Escherichia coli DH5α, Pichia pastoris X33, vector pPICZαA (Beijing Qikexing Biotechnology Co., Ltd., DOI: 10.1080 / 13102818.2016.1193442), vector pGAPZαA (Beijing Qikexing Biotechnology Co., Ltd., DOI: 10.5713 / ajas.16.0038).

[0032] B. Medium formula:

[0033] Escherichia coli culture medium (LB medium): 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0;

[0034] Yeast culture medium (YPD medium): 1% yeast extract, 2% peptone, 2% glucose;

[0035] Yeast screening medium (MD medium): 2% peptone, 2% agarose;

[0036] BMGY medium: 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 0.4 mg / L biotin, 1% glycerol;

[0037] BMMY medium: 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 0.4 mg / L biotin, 0.5% methanol;

[0038] Example 1 Construction of lipase mutant expression vector

[0039] (1) After codon optimization of the lipase mutant as shown in SEQ ID NO. 02, the whole gene synthesis was carried out, and the gene sequence encoding the lipase mutant as shown in SEQ ID NO. 03 was obtained;

[0040] SEQ ID NO. 02: APAAETLDRRAALPNPYDDPFYTTPSNIGTFAKGQVIQSRKVPTDIGNANNAASFQLQYRTTNTQNEAVADVATVWIPAKPASPPKIFSYQVYEDATALDCAPSYSYLTGLDQPNKVTAVLDTPIIIGWALQQGYYVVSSDHEGFKAAAIAGYEEGMAILDGIRALKNYQNLPSDSKVALEGFSGGAHATVWATSLAESYAPELNIVGASHGGTPVSAKDTFTFFNGGPFAGFALAGVSGLSLAHPDMESFIEARLNAKGQRTLKQIRGRGFCLPQVVLTYPFLNVFSLVNDTNLLNEAPIASILKQETVVQAEASYTVSVPKFPRFIWHAIPDEIVPYQPAATYVKEQCAKGANINFSPYPIAEHLTAEIFGLVPSLWFIKQAFDGTTPKVICGTPIPAIAGITTPSADQVLGSDLANQLRSLDGKQSAFGKPFGPITPP;

[0041] SEQ ID NO. 03:

[0042]

[0043] (2) The gene sequence obtained in step (1) is subjected to enzyme digestion, and then connected to the multiple cloning site of the vector pPICZαA, and the specific enzyme digestion sites are EcoR I and Not I;

[0044] (3) The gene sequence as shown in SEQ ID NO. 05 encoding the signal peptide of the amino acid sequence as shown in SEQ ID NO. 04 is substituted for the α-MF signal peptide encoding gene sequence originally in the vector pPICZαA in step (2) to obtain a recombinant vector;

[0045] SEQ ID NO. 04:

[0046] MKLLNFLLSFVTLFGLLSGSVFA;

[0047] SEQ ID NO. 05:

[0048] atgaagctgctaaatttcctgctgtccttcgttactttgtttggtctgctttctggttctgtgtttgca

[0049] (4) The recombinant vector constructed in step (3) is transformed into E. coli DH5α, and a single colony with correct sequencing is selected, cultured and plasmid is extracted, which is the lipase mutant expression vector.

[0050] Example 2 Construction of a lipase mutant expression strain

[0051] (1) The lipase mutant expression vector obtained in Example 1 is linearized using Sac I endonuclease, and then electroporated into Pichia pastoris X33 competent cells, and then selected using YPD plate medium containing 100 mg / L of bleomycin to obtain a single colony with correct sequencing;

[0052] (2) The single colony obtained in step (1) is cultured and verified, and the cells that can successfully express the lipase mutant are prepared into competent cells again;

[0053] (3) The vector pGAPZαA (Zeocin resistance gene is replaced by G418 resistance gene) with the gene sequence as shown in SEQ ID NO. 07 encoding the chaperone of the amino acid sequence as shown in SEQ ID NO. 06 is linearized, and then electroporated into the competent cells obtained in step (2), and then selected using YPD plate medium containing 200 mg / L of G418 to obtain a single colony with correct verification, which is the lipase mutant expression strain.

[0054] SEQ ID NO. 06: MPVDSSHKTASPLPPRKRAKTEEEKEQRRVERILRNRRAAHASREKKRRHVEFLENHVVDLESALQESAKATNKLKEIQDIIVSRLEALGGTVSDLDLTVPEVDFPKSSDLEPMSDLSTSSKSEKASTSTRRSLTEDLDEDDVAEYDDEEEDEELPRKMKVLNDKNKSTSIKQEKLNELPSPLSSDFSDVDEEKSTLTHLKLQQQQQQPVDNYVSTPLSLPEDSVDFINPGNLKIESDENFLLSSNTLQIKHENDTDYITTAPSGSINDFFNSYDISESNRLHHPAVMTDSSLHITAGSIGFFSLIGGGESSVAGRRSSVGTYQLTCIAIR.

[0055] SEQ ID NO. 07:

[0056] atgccagtcgactcatctcacaaaaccgcatctcccttaccccccagaaagagagcaaaaaccgaagaagagaaagagcaacgtagagttgaacgtattttgcgtaatagacgtgctgctcacgcctctagagagaagaaaagacgtcatgttgagttcttggaaaaccatgtagtcgacttggagtctgccctacaagaatctgctaaagccaccaataaattaaaagagatccaagacattattgtctcacgtttggaagcccttggtggaaccgttagtgatctggatctgactgtccctgaagtggactttccaaaatcctctgatttagagccaatgtctgacttgtccacatcctctaaaagtgaaaaggcttctactagtacaaggagatctttgaccgaggatcttgacgaagatgacgtggctgagtacgacgatgaggaagaagatgaggagttgcctagaaagatgaaggttctaaacgacaagaataaatccacctcaatcaaacaggagaagttaaacgaacttccatcccccttgtcctctgatttcagtgatgttgatgaggagaagtctactctgactcatttaaagttgcaacagcaacaacagcagccagtagacaattacgtcagtactccactttcactgccagaagactctgttgattttattaatccaggtaaccttaagatcgagtccgacgaaaacttcttgttgtcctctaataccttacagattaagcacgaaaacgataccgattacatcacaactgctccatctggttcaattaacgactttttcaacagttacgatatttccgaatcaaataggctgcatcatcccgctgtaatgactgattcctccttgcatataactgctggttccatcggatttttttctctgatcggcggaggtgaatcttctgtcgctggaagaagatcctcagttggcacctatcagttgacctgtattgccattagg

[0057] Example 3 Preparation of free lipase mutant

[0058] (1) Shake flask culture: the lipase mutant expression strain obtained in Example 2 was inoculated into BMGY medium, and cultured at 30°C with 200 rpm shaking for 1 day; then transferred into BMMY medium, and cultured at 30°C with 250 rpm shaking, and 1% methanol was added every day for 5 days of induction; then centrifuged at 9000 rpm for 10 min to remove the bacterial bodies, and the fermentation supernatant containing the lipase mutant was obtained;

[0059] (2) Fermentor culture: the lipase mutant expression strain obtained in Example 2 was inoculated into BMGY medium and cultured for 2 days, and 200 mL was inoculated into a 5 L fermentor with a liquid volume of 2 L; the culture temperature was 30°C, the rotation speed was 600 rpm, the aeration volume was 2 vvm, and the pH was controlled at pH 6.0 by ammonia; after the glycerol was consumed, methanol was added for induction, and the highest enzyme activity appeared at 144 h after fermentation, the wet weight of the bacterial bodies reached 480 g / L at 144 h of induction, and the extracellular protein concentration reached 10 g / L.

[0060] Example 4 Immobilization of the lipase mutant

[0061] Pre-treated macroporous adsorption resin HPD450 was added to the fermentation supernatant obtained in Example 3, and stirred and adsorbed at 20°C for 20 h; after adsorption equilibrium, the deionized water was rinsed twice, and then dried using a vacuum drying box to obtain the immobilized lipase mutant.

[0062] Example 5 Chiral resolution of DL-menthol by the lipase mutant

[0063] (1) 0.4M DL-menthol and 0.6M vinyl acetate were added to 100 mL of n-hexane, and the immobilized lipase mutant obtained in Example 4 was added to a final concentration of 5 g / L, and reacted at 50°C for 5 h (at the same time, a lipase wild type with an amino acid sequence as shown in SEQ ID NO. 01 was used as a control, and the preparation of the immobilized lipase wild type was the same as in Examples 1 to 4);

[0064] SEQ ID NO. 01: APAAETLDRRAALPNPYDDPFYTTPSNIGTFAKGQVIQSRKVPTDIGNANNAASFQLQYRTTNTQNEAVADVATVWIPAKPASPPKIFSYQVYEDATALDCAPSYSYLTGLDQPNKVTAVLDTPIIIGWALQQGYYVVSSDHEGFKAAFIAGYEEGMAILDGIRALKNYQNLPSDSKVALEGYSGGAHATVWATSLAESYAPELNIVGASHGGTPVSAKDTFTFLNGGPFAGFALAGVSGLSLAHPDMESFIEARLNAKGQRTLKQIRGRGFCLPQVVLTYPFLNVFSLVNDTNLLNEAPIASILKQETVVQAEASYTVSVPKFPRFIWHAIPDEIVPYQPAATYVKEQCAKGANINFSPYPIAEHLTAEIFGLVPSLWFIKQAFDGTTPKVICGTPIPAIAGITTPSADQVLGSDLANQLRSLDGKQSAFGKPFGPITPP;

[0065] It should be noted that the lipase mutant shown in SEQ ID NO. 02 of the present application is obtained by mutating the 149th phenylalanine in the amino acid sequence shown in SEQ ID NO. 01 to alanine, the 183rd tyrosine to phenylalanine, and the 225th leucine to phenylalanine;

[0066] (2) After the reaction of step (1) is completed, the conversion effect is detected using GC, and the specific detection method is as follows:

[0067] After the reaction is completed, 100 µL of the appropriately diluted reaction product is taken and analyzed by GC analysis. The analysis is performed on an Agilent 7890A gas chromatograph using Agilent J&W CP Cyclodextrin-β-2,3,6-M-19 (25 M x 0.25 mm x 0.25 µM) and a flame ionization detector (FID), and the retention time of each component is shown as Figure 1 .

[0068] The specific detection results of the present example are shown in Table 1 and Figure 2 : The L-enantiomer selectivity ee is > 99%, the conversion rate is 49.5%, and the L-menthol is almost completely converted.

[0069] Table 1

[0070] Lipase ee (%) Conversion (%) Wild type 52.5 30 Mutant 99.9 49.5

[0071] The above description is only the preferred embodiment of the present application, and therefore cannot limit the scope of the present application. Any equivalent changes and modifications made according to the patent scope and content of the present application should still be within the scope of the present application.

Claims

1. A lipase mutant, characterized in that: The amino acid sequence thereof is shown as SEQ ID NO.

02.

2. An expression vector, characterized by: The vector pPICZαA is loaded with the amino acid sequence shown as SEQ ID NO.

02.

3. The expression vector of claim 2, wherein: The α-MF signal peptide coding gene sequence originally provided in the vector pPICZαA is replaced by a gene sequence coding a signal peptide with the amino acid sequence shown as SEQ ID NO.

04.

4. A lipase mutant expression bacteria, characterized in that: The vector pPICZαA is loaded with the amino acid sequence shown as SEQ ID NO.

02.

5. The lipase mutant-expressing bacteria of claim 4, wherein the bacteria are selected from the group consisting of Escherichia coli, Pseudomonas sp., Bacillus sp., and Lactobacillus sp. The vector pGAPZαA is also loaded with a gene sequence coding a molecular chaperone with the amino acid sequence shown as SEQ ID NO.

06.

6. A process for the fermentative production of a lipase mutant according to claim 1, characterized in that: The lipase mutant expression strain of claim 4 or 5 is used.

7. Use of the lipase mutant of claim 1 in chiral resolution of menthol.

8. Use according to claim 7, characterized in that: The lipase mutant is immobilized by macroporous adsorption resin HPD450.

9. A method for chiral resolution of menthol, characterized by: The lipase mutant of claim 1 is used.

10. The method for chiral resolution of menthol according to claim 9, wherein: The lipase mutant is immobilized by macroporous adsorption resin HPD450.

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