Lipase mutant and application thereof in improving ester flavoring substances in baijiu

Abstract

The application relates to the technical field of genetic engineering and protein engineering, and particularly provides a lipase mutant and application thereof. The mutant has significantly improved enzyme activity level and tolerance under acidic conditions, can be widely applied to the field of liquor brewing, and can greatly increase the content of ester flavoring substances in liquor, wherein the content of ethyl acetate, ethyl butyrate and ethyl hexanoate is respectively increased by 26.1%, 125.9% and 108.1% compared to the wild-type lipase treatment group, and an unexpected technical effect is achieved.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and protein modification technology, specifically to a lipase mutant and its application in enhancing the flavor of esters in baijiu (Chinese liquor). Background Technology

[0002] Lipases, also known as triglyceride hydrolases, are a class of enzymes that specifically act on ester bonds, catalyzing ester hydrolysis, esterification, and transesterification (also known as transesterification). In early research, researchers generally believed that lipases could only hydrolyze glycerides. However, subsequent studies have shown that lipases can effectively act on polyhydroxy esters, polycarboxylic esters, and thioesters, exhibiting broad substrate specificity. Lipases typically exhibit reversibility and the reaction direction is determined by the system medium during catalysis. Furthermore, lipases possess positional and isomerist characteristics, showing good application potential in the resolution and synthesis of chiral compounds. Currently, lipases are widely used in energy, food processing, agriculture, biopharmaceuticals, and chemical industries, and have also been researched and applied in the field of liquor brewing in recent years.

[0003] Baijiu (Chinese white liquor) is made primarily from grains, using daqu (large koji), xiaoqu (small koji), bran koji, enzymes, and yeast as saccharification and fermentation agents. The process involves cooking, saccharification, fermentation, distillation, aging, and blending. Due to differences in soil, climate, microbial flora, raw materials, and brewing techniques, baijiu from different regions exhibits various aroma types. Sauce aroma, strong aroma, and light aroma are considered the three main aroma types of traditional daqu baijiu. Different aroma types of baijiu have different main aroma components. The characteristic ester flavor compounds of light aroma baijiu are ethyl acetate and ethyl lactate, while the characteristic ester flavor compound of strong aroma baijiu is ethyl hexanoate. These ester compounds impart aroma, freshness, and mellowness to baijiu, and are extremely important flavor substances in the baijiu brewing process, forming the basis for the grading and classification of raw baijiu.

[0004] Esters in baijiu brewing are mainly produced by esterification reactions catalyzed by lipases. However, the low content of natural lipases in the brewing environment affects the content and abundance of esters in baijiu. Therefore, screening for specific lipases and applying them to the baijiu brewing system can not only increase the content of esters in baijiu and enrich its aroma components, giving it a better flavor, but also significantly improve the yield of high-quality baijiu. Summary of the Invention

[0005] To address the problems of existing technologies, this invention provides a lipase mutant and its applications. The lipase mutant exhibits high enzyme activity, strong tolerance to acidic environments, and can significantly increase the content of ester flavor compounds in baijiu (Chinese liquor), making it widely applicable in the baijiu brewing industry.

[0006] The present invention provides a lipase mutant with enhanced lipase activity, comprising an amino acid sequence having at least 90% identity with SEQ ID NO:2, and comprising, compared with SEQ ID NO:2, at least one substitution containing an amino acid at a position selected from the group consisting of: 93, 128, 168.

[0007] In some embodiments of the invention, the amino acid sequence of the mutant has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity with SEQ ID NO:2.

[0008] In some more specific embodiments, the amino acid sequence of the mutant has at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% identity with SEQ ID NO:2.

[0009] In some embodiments of the present invention, the mutant comprises a substitution of at least one amino acid from the group consisting of: A93K, Q128D, and R168Y.

[0010] In some embodiments of the present invention, the mutant contains a substitution or combination of substitutions selected from the following substitutions and combinations of substitutions: A93K, Q128D, R168Y, A93K / Q128D, Q128D / R168Y, A93K / R168Y, A93K / Q128D / R168Y.

[0011] The present invention also relates to DNA molecules encoding the above-mentioned lipase mutants.

[0012] The present invention also relates to recombinant expression plasmids comprising the above-described DNA molecules.

[0013] The present invention also relates to a host cell comprising the above-described recombinant expression plasmid.

[0014] This invention also provides the application of the above-mentioned lipase mutant in the brewing of baijiu (Chinese liquor).

[0015] This invention provides mutants CLT3, CLT9, and CLT16, respectively, containing single-point mutations of A93K, Q128D, and R168Y, based on the wild-type lipase CL. The enzyme activity and acid tolerance of these mutants under acidic conditions are significantly higher than those of the wild-type.

[0016] The optimal pH for the wild-type lipase CL is 6.0, while the optimal pH for the mutants CLT3 and CLT9 is 5.5, and the optimal pH for the mutant CLT16 is 5.0. Furthermore, the relative enzyme activity of all mutants is increased under pH conditions ranging from 2.0 to 5.0. Notably, the relative enzyme activity of mutant CLT16 at pH 2.0 is increased to 60%, significantly higher than that of the wild-type.

[0017] Wild-type lipase CL exhibits good stability within the pH range of 4.0-7.0, but when the pH drops below 4.0, the residual enzyme activity gradually decreases with decreasing pH. Lipase mutants CLT3, CLT9, and CLT16 all show varying degrees of improved tolerance to acidic conditions. Among them, lipase mutant CLT16 maintains a residual enzyme activity exceeding 90% within the pH range of 3.0-7.0, and nearly 70% at pH 2.0, significantly higher than the wild-type.

[0018] The lipase mutant described in this invention can be widely used in the field of baijiu brewing. It can significantly increase the content of ester flavor substances in baijiu. The contents of ethyl acetate, ethyl butyrate and ethyl hexanoate are increased by 26.1%, 125.9% and 108.1% respectively compared with the wild-type lipase CL treatment group, which is more in line with the flavor of strong-aroma baijiu and has achieved unexpected technical effects. Attached Figure Description

[0019] Figure 1 This is a temperature-relative enzyme activity change curve for the lipase mutant;

[0020] Figure 2 This is a graph showing the pH-relative enzyme activity change of the lipase mutant.

[0021] Figure 3 This is a graph showing the stability changes of the lipase mutant under different pH conditions. Detailed Implementation

[0022] The method of the present invention will be further illustrated below with examples. Experimental methods in the following examples that do not specify specific conditions can generally be operated under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual* by J. Sambrook et al., or according to the manufacturer's recommendations. Those skilled in the art can better understand and master the present invention through these examples. However, the protection and scope of the claims of the present invention are not limited to the specific examples provided, but should include the scope of protection that can be extended by those skilled in the art based on this specification without inventive effort.

[0023] Experimental materials and reagents:

[0024] Strains and vectors: Escherichia coli DH5α, Pichia pastoris GS115, and vector pPIC9k were purchased from Invitrogen.

[0025] Enzymes and kits: PCR enzymes and ligases were purchased from Takara, restriction endonucleases from Fermentas, plasmid extraction kits and gel purification and recovery kits from Omega, and GeneMorph II random mutation kits from Beijing Bomais Biotechnology Co., Ltd.

[0026] Culture medium formulation:

[0027] Enrichment medium for lipase-producing strains: 3g yeast extract, 10g beef extract, 10g peptone, 5g glucose, 0.5g cysteine ​​hydrochloride, 3g NaCl, 3g NaAc, 12mL olive oil emulsion, 1000mL water, pH 7.0;

[0028] Lipase-producing strain screening plates:

[0029] Escherichia coli culture medium (LB medium): 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0;

[0030] LB+Amp medium: LB medium with 100 μg / mL ampicillin;

[0031] LB+Kanamycin medium: LB medium supplemented with 50 μg / mL kanamycin;

[0032] The present invention will now be described in detail with reference to the embodiments.

[0033] Example 1: Cloning of the lipase gene

[0034] A lipase-producing strain was isolated and screened from the cellar mud of strong-aroma baijiu (Chinese liquor), and identified as *Clostridium kluyveri*. Using the genomic DNA of this strain as a template, PCR amplification was performed using the following primers to obtain the lipase gene of *Clostridium kluyveri*, named CL, with its nucleotide sequence SEQ ID NO: 1 and its encoding amino acid sequence SEQ ID NO: 2.

[0035] The primer sequences used are as follows:

[0036] F: ATGGCAAATCATTTAGAA;

[0037] R:CTAAATTCTTTTTTCAAT.

[0038] The PCR amplification system consisted of: 1 μL template, 1 μL upstream primer F, 1 μL downstream primer R, 10 μL 5×PS Buffer, 4 μL dNTPs (2.5 mM), 1 μL Primer-Star DNA polymerase, and 32 μL ddH2O, with a total reaction volume of 50 μL.

[0039] The PCR cycling program was as follows: 95℃ pre-denaturation for 5 min, 30 cycles: 94℃ for 30 sec, 56℃ for 30 sec, 72℃ for 1 min, and 72℃ for 10 min.

[0040] Example 2: Recombinant expression of lipase gene and determination of enzyme activity

[0041] The lipase CL gene was expressed in Escherichia coli BL21(DE3).

[0042] First, the lipase CL gene was cloned into the expression vector pET-28a. The primer sequences are as follows:

[0043] 28a-F: ATA CCATGG CAAATCATTTAGAA (underlined is the NcoI restriction site);

[0044] 28a-R: GCA CTCGAG CTAAATTCTTTTTTCAAT (The underlined part is the XhoI restriction site).

[0045] Using the lipase CL gene as a template, PCR amplification was performed using the primers described above.

[0046] The PCR amplification system consisted of: 1 μL template, 1 μL upstream primer 28a-F, 1 μL downstream primer 28a-R, 10 μL 5×PS Buffer, 4 μL dNTPs (2.5 mM), 1 μL Primer-Star DNA polymerase, and 32 μL ddH2O, for a total reaction volume of 50 μL.

[0047] The PCR cycling program was as follows: 95℃ pre-denaturation for 5 min, 30 cycles: 94℃ for 30 sec, 56℃ for 30 sec, 72℃ for 1 min, and 72℃ for 10 min.

[0048] The PCR product was recovered from the gel, digested with NcoI and XhoI, and then ligated with the pET-28a vector that had been digested with the same enzymes. The ligation was performed overnight at 16°C and transformed into E. coli DH5α. The transformed product was plated on LB+Kana plates and incubated upside down at 37°C. After the transformants appeared, the positive clones were verified by colony PCR. After sequencing verification, the correct recombinant plasmid pET-CL was finally obtained.

[0049] The recombinant plasmid pET-CL was transformed into BL21(DE3), plated on LB+Kana plates, and incubated upside down at 37°C. Once transformants appeared, they were considered positive transformants.

[0050] Pick one positive transformant into a 5 mL LB+Kana liquid test tube and incubate overnight at 37°C. Transfer 500 μL of fermentation broth to a 50 mL LB+Kana liquid shake flask and incubate at 37°C for about 4 h until the OD value reaches approximately 0.6–1.0. Add 250 μL of 100 mM IPTG and incubate overnight at 16°C.

[0051] Collect bacterial cells by centrifugation, add 10 mL of 50 mM phosphate buffer, sonicate to disrupt the cell wall, centrifuge again, and the supernatant is the crude enzyme solution. The lipase activity was measured to be 499 U / mL.

[0052] Lipase activity assay:

[0053] 1) Definition of enzyme activity: The amount of 1 μmol of titratable fatty acid produced by the hydrolysis of substrate by 1 mL of enzyme solution at 40℃ and pH 7.5 for 1 min is defined as one unit of enzyme activity, expressed as U / mL.

[0054] 2) Substrate preparation: Weigh 40g of polyvinyl alcohol (PVA), add 800mL of water, heat in a boiling water bath, stir until completely dissolved, cool, and bring the volume to 1000mL. Filter through clean double-layered gauze and collect the filtrate for later use. Measure 150mL of the above filtrate, add 50mL of olive oil, and process using a high-speed homogenizer for 6 minutes (divided into two processes, 5 minutes apart, each process lasting 3 minutes). This yields a milky white PVA emulsion substrate. The substrate solution should be prepared fresh before use.

[0055] 3) Determination Procedure: Take two 100mL Erlenmeyer flasks. Add 4mL of substrate solution and 5mL of pH 7.5 phosphate buffer to each flask (A) and (B), respectively. Add 15mL of 95% ethanol to flask A and preheat in a water bath at 40℃±0.2℃ for 5min. Then add 1.00mL of the enzyme solution to each flask (A and B), mix immediately, and start timing. After reacting for 15min, immediately add 15mL of 95% ethanol to flask B to terminate the reaction. Transfer the reaction solution to a 50mL beaker. Add 5mL of deionized water to the Erlenmeyer flask and rinse before transferring to the 50mL beaker. Under stirring, add 0.05mol / L sodium hydroxide solution to the beaker and titrate until the pH reaches 9.92. The titration endpoint is reached when the pH does not change for 20s. Record the volume of sodium hydroxide standard solution consumed.

[0056] 4) Enzyme activity calculation:

[0057] Calculate lipase activity using the formula below, and round the result to the nearest integer.

[0058]

[0059] X — Enzyme activity of the sample, U / mL;

[0060] V1—Volume of sodium hydroxide standard solution consumed during sample titration, in mL;

[0061] V2—Volume of sodium hydroxide standard solution consumed during blank titration, in mL;

[0062] c — Concentration of the sodium hydroxide standard solution, mol / L;

[0063] 1.00 mL of 50-0.05 mol / L sodium hydroxide solution is equivalent to 50 μmol of fatty acid;

[0064] n—Enzyme solution dilution factor;

[0065] 0.05 — Conversion factor for sodium hydroxide standard solution concentration;

[0066] 15 — Reaction time: 15 min.

[0067] Example 4: Screening of lipase mutants

[0068] To further enhance the enzyme activity of wild-type lipase CL, a large number of mutation sites were randomly screened for in this gene without damaging the protein's secondary structure and active site.

[0069] 1) Construction of mutant library

[0070] Using the lipase CL gene SEQ ID NO: 1 obtained by PCR amplification as a template, the above primers were used to perform error-prone PCR amplification with 28a-F and 28a-R, respectively.

[0071] 100 μL error-prone PCR reaction system: Mg-free 2+ The buffer, 0.2 μM primer, 7 mM MgCl2, 0.2 mM MnCl2, 0.2 mM dGTP, 0.2 mM dATP, 1.0 mM dCTP, 1.0 mM dTTP, 50 ng template, and 5 U Taq enzyme.

[0072] The PCR cycling program was as follows: 95℃ pre-denaturation for 5 min, 30 cycles: 94℃ for 30 sec, 56℃ for 30 sec, 72℃ for 1 min, and 72℃ for 10 min.

[0073] The PCR products were recovered from the gel, digested with NcoI and XhoI, and then ligated with the pET-28a vector that had been digested with the same enzymes. The ligation was carried out overnight at 16°C and transformed into E. coli DH5α. The transformed products were plated on LB+Kana plates and incubated upside down at 37°C. After the transformants appeared, all colonies were washed with water and the plasmids were extracted to obtain the lipase mutant library.

[0074] 2) Mutant expression

[0075] The plasmid of the above lipase mutant library was transformed into BL21(DE3), plated on LB+Kana plates, and incubated upside down at 37°C. Once the transformants appeared, they were considered positive transformants.

[0076] Pick positive transformants into 5 mL LB+Kana liquid test tubes and incubate overnight at 37°C.

[0077] Transfer 500 μL of the culture to a 50 mL LB+Kana liquid shake flask and incubate at 37°C for approximately 4 hours until the OD value reaches approximately 0.6–1.0. Add 250 μL of 100 mM IPTG and incubate overnight at 16°C. Centrifuge the fermentation broth, collect the cells, add 10 mL of 50 mM phosphate buffer, sonicate to disrupt the cell walls, centrifuge again, and the supernatant is the crude enzyme solution.

[0078] 3) Mutant screening

[0079] Add 180 μL of substrate (10 mM potassium phosphate buffer, pH 7.2, 0.01% bromophenol blue, 100 mM CNDE) to the microplate; take 20 μL of crude lipase solution and add it to 180 μL of pH 8.0 buffer to dilute it 10 times. Then, take 20 μL of the diluted enzyme solution and add it to the microplate containing 180 μL of substrate and mix well. Use wild-type lipase CL as a control. The rate at which the reaction solution changes from blue to yellow is used as a screening indicator. A faster change from blue to yellow indicates higher lipase activity.

[0080] After multiple rounds of error-prone PCR screening, three mutants with significantly increased enzyme activity compared to the wild-type lipase CL were finally selected and named CLT3, CLT9, and CLT16, respectively. Enzyme activity assays showed that the enzyme activities of mutants CLT3, CLT9, and CLT16 were 698 U / mL, 823 U / mL, and 897 U / mL, respectively, representing increases of 39.9%, 64.9%, and 79.8% compared to the wild type.

[0081] Lipase sequences were cloned and sequenced from mutants CLT3, CLT9, and CLT16, respectively. Sequencing results showed that mutants CLT3, CLT9, and CLT16 contained the following single-point mutations: A93K, Q128D, and R168Y, respectively.

[0082] The amino acid sequences of the lipase single-point mutants CLT3, CLT9, and CLT16 are SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively.

[0083] Example 5: Determination of the enzymatic properties of lipase

[0084] The enzymatic properties of crude enzyme solutions of wild-type lipase CL and its mutants CLT3, CLT9, and CLT16 were determined.

[0085] (1) Optimal operating temperature

[0086] The enzyme activities of lipases CL, CLT3, CLT9, and CLT16 in crude enzyme solutions were measured at 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃, and pH 7.5, respectively. The relative enzyme activities of lipases under different temperature conditions were calculated with the initial enzyme activity as 100%, and temperature-relative enzyme activity curves were plotted.

[0087] The results are as follows Figure 1 As shown, the optimal operating temperature of wild-type lipase CL and mutants CLT3, CLT9, and CLT16 is 40℃, indicating that the mutation has little effect on the optimal operating temperature of wild-type lipase CL.

[0088] (2) Optimal pH

[0089] Crude lipase solutions of CLT16 (CLT3, CLT9, and CLT16) were diluted to appropriate concentrations with phosphate-citrate buffer solutions at pH values ​​of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. The enzyme activity of lipase in the crude enzyme solutions was then measured at 40°C. The relative enzyme activity of lipase under different pH conditions was calculated as 100% of the initial enzyme activity, and pH-relative enzyme activity curves were plotted.

[0090] The results are as follows Figure 2 As shown, the optimal pH for the wild-type lipase CL is 6.0, while the optimal pH for the mutants CLT3 and CLT9 is 5.5, and the optimal pH for the mutant CLT16 is 5.0. The relative enzyme activity of the mutants is increased under pH conditions of 2.0-5.0. The relative enzyme activity of the mutant CLT16 at pH 2.0 is increased to 60%, which is significantly higher than that of the wild type.

[0091] (3) pH tolerance

[0092] Wild-type lipase CL and mutant lipases CLT3, CLT9, and CLT16 crude enzyme solutions were diluted to appropriate concentrations using phosphate-citrate buffer solutions at pH 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0, respectively. The solutions were then treated at 40°C under different pH conditions for 2 days. The enzyme activity of the lipases before and after treatment was measured. The enzyme activity before treatment was taken as 100%. The residual enzyme activity rate of the lipases after treatment under different pH conditions was calculated, and a pH-residual enzyme activity bar chart was plotted.

[0093] The results are as follows Figure 3 As shown, the wild-type lipase CL exhibits good stability within the pH range of 4.0-7.0. However, when the pH drops below 4.0, the residual enzyme activity gradually decreases with decreasing pH. In contrast, the lipase mutants CLT3, CLT9, and CLT16 all demonstrate varying degrees of improved tolerance to acidic conditions. Specifically, the lipase mutant CLT16 maintains a residual enzyme activity exceeding 90% within the pH range of 3.0-7.0, and approaches 70% at pH 2.0.

[0094] The above results show that, compared with the wild type, the lipase mutant provided by the present invention has significantly enhanced tolerance to acidic environments, achieving unexpected technical effects.

[0095] Example 6: Application of the lipase mutant CLT16 in enhancing the ester flavor of Baijiu (Chinese liquor)

[0096] Yellow water is a high-concentration brewing wastewater generated during the solid-state fermentation of strong-aroma baijiu, containing large amounts of organic acids such as lactic acid and acetic acid. Firstly, a compound hexanoic acid bacteria were used to pre-treat the yellow water through fermentation, reducing the lactic acid content and increasing the butyric and hexanoic acid content. After fermentation pre-treatment, the yellow water had a pH of 3.5 and an alcohol concentration of 60 g / L.

[0097] In this embodiment, two treatment groups were set up. Wild-type lipase CL and mutant CLT16 crude enzyme solution were added to the pretreated yellow water, respectively, so that the enzyme activity of lipase in the reaction system was 2000 U / L. The reaction was carried out at 40℃ with stirring for 24 h to obtain esterified liquid. The content of major ester flavor substances in the esterified liquid was determined according to the method described in GB / T10345-2007 Baijiu Analysis Method. Yellow water without any added enzyme was used as a blank control group. Each group was performed in triplicate. The specific results are shown in Table 1.

[0098] As shown in Table 1, compared with the blank control group, the addition of lipase to catalyze the esterification reaction significantly increased the content of the main ester flavor compounds, ethyl acetate, ethyl butyrate, and ethyl hexanoate, in the esterification solution of the treated group, indicating that lipase can effectively catalyze the conversion of organic acids into esters. Compared with the wild-type lipase CL treatment group, the content of ethyl acetate, ethyl butyrate, and ethyl hexanoate in the esterification solution of the lipase mutant CLT16 treatment group increased by 26.1%, 125.9%, and 108.1%, respectively, which is more consistent with the flavor of strong-aroma baijiu. This demonstrates that the lipase mutant CLT16 provided by this invention has stronger tolerance to the acidic environment of yellow water and higher catalytic efficiency, achieving unexpected technical results.

[0099] Table 1 Comparison of the content of major ester flavor compounds.

[0100]

[0101] In summary, the lipase mutant provided by this invention has significantly enhanced tolerance to acidic environments, high efficiency in catalyzing esterification reactions, and can significantly increase the content of ester flavor substances in baijiu. It can be widely used in the baijiu brewing field and has broad prospects.

Claims

1. A lipase mutant, characterized in that, The amino acid sequence of the lipase mutant is SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO:

5.

2. A DNA molecule encoding the lipase mutant of claim 1.

3. A recombinant expression plasmid comprising the DNA molecule of claim 2.

4. A host cell, characterized in that, The host cell comprises the recombinant expression plasmid of claim 3; the host cell is a non-plant cell or a non-animal cell.

5. The host cell of claim 4, wherein The host cell is E. coli (Escherichia coli) Escherichia coli ).

6. The lipase mutant of claim 1 in the application of Baijiu brewing.

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