A Nicotine-Tolerant Pectin Lysase Mutant and Its Application in Tobacco Leaf Processing

Abstract

This invention discloses a nicotine-resistant pectin lyase mutant and its application in tobacco leaf treatment. Using pectin lyase PL6 as the starting enzyme, this invention designs and optimizes mutation sites, designing single-site saturation mutations based on the two hydrogen bond formation sites between the enzyme and nicotine, Lys-107 and Asp-84, to construct mutants. The pectin lyase mutants D84A and K107R of this invention retain 83.63% and 81.30% of their enzyme activities at a 1% nicotine concentration, respectively. Spraying tobacco leaves with D84A and K107R enzyme solutions significantly improves tobacco leaf quality due to pectin degradation.

Description

Technical Field

[0001] This invention belongs to the field of biological enzymes, specifically relating to a nicotine-resistant pectin lyase mutant and its application in tobacco processing. Background Technology

[0002] Bioenzymes play a crucial role in tobacco processing. They rapidly decompose macromolecules such as starch, protein, cellulose, and pectin in tobacco leaves, while simultaneously accelerating the conversion of pigments, polyphenols, and terpenoids into smaller aroma compounds, thereby increasing the content of ketones, aldehydes, acids, esters, terpenes, and lower fatty acids in tobacco products. However, due to the low content of endogenous enzymes in tobacco leaves, these chemical transformations cannot be achieved in a short time, resulting in relatively low levels of both macromolecular decomposition and small-molecule aroma compound formation. To overcome this challenge, exogenous enzymes such as proteases, amylases, cellulases, pectins, and glucans are introduced into tobacco processing. These enzymes can rapidly catalyze chemical reactions, altering the chemical composition of tobacco leaves and contributing to the accumulation of aroma compounds. The action of these key bioenzymes can significantly improve the internal chemical composition of tobacco leaves in a short time, reduce impurities, enhance the attractiveness of tobacco, and highlight the unique aroma and style of cigarettes.

[0003] Pectin is a polysaccharide, primarily composed of galacturonic acid-rich plant cell wall polysaccharides, its basic structure consisting of homopolymers linked by α-1,4 glycosidic bonds. Pectin content significantly impacts tobacco quality due to its multiple effects on tobacco properties. High pectin content may affect tobacco smoke production and properties, potentially leading to increased smoke generation and thus influencing combustion performance and smoke composition. Furthermore, pectin degradation can release flavor compounds that can affect the flavor and aroma of tobacco products. The content and characteristics of pectin also influence tobacco flavor; therefore, controlling pectin content in tobacco leaves has become an important research direction in tobacco processing.

[0004] Pectinases are a class of complex enzymes that specifically degrade pectin-like substances. Specifically, pectinase lyases can specifically cleave the α-1,4 glycosidic bonds in pectin, forming oligosaccharides with non-reducing-terminated unsaturated galacturonic acid residues. Most of these pectinase lyases are derived from microorganisms, primarily including Penicillium, Aspergillus, and Fusarium fungi. Using pectinases to treat tobacco leaves offers several advantages, such as high specificity, high efficiency, mild operating conditions, and no introduction of contaminants. This makes pectinases widely applicable in tobacco leaf treatment. However, currently available commercially available pectinase preparations face certain challenges when dealing with tobacco environments with high nicotine content, as they may cause conformational changes or reduced enzyme activity, thereby weakening the pectin-degrading ability of the pectin lyase and negatively impacting the treatment effect on tobacco leaves.

[0005] Therefore, the development of nicotine-tolerant pectin lyases has the potential to become a new biological enzyme resource in the tobacco processing field, which will effectively help control the level of pectin degradation in tobacco leaves. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention provides a nicotine-tolerant pectin lyase mutant and its application in tobacco processing. This invention uses pectin lyase PL6 as the starting enzyme, and through the design and optimization of mutation sites, single-site saturation mutations are designed based on the two sites Lys-107 and Asp-84 where the enzyme forms hydrogen bonds with nicotine, to construct a mutant library.

[0007] One of the technical solutions of the present invention provides a nicotine-resistant pectin lyase mutant D84A, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0008] The present invention also provides the encoding gene of the pectin lyase mutant D84A, the nucleotide sequence of which is shown in SEQ ID NO: 4.

[0009] The present invention also provides a recombinant expression vector containing the gene encoding the pectin lyase mutant D84A.

[0010] The recombinant expression vector is further preferably the pET-22b(+) vector.

[0011] The present invention also provides an engineered strain expressing the pectin lyase mutant D84A, which is obtained by transforming the engineered strain into host bacteria through the recombinant expression vector.

[0012] Furthermore, the host bacteria is preferably Escherichia coli BL21(DE3).

[0013] The second technical solution of the present invention provides a nicotine-resistant pectin lyase mutant K107R, the amino acid sequence of which is shown in SEQ ID NO: 3.

[0014] The present invention also provides the encoding gene of the pectin lyase mutant K107R, the nucleotide sequence of which is shown in SEQ ID NO: 5.

[0015] The present invention also provides a recombinant expression vector containing the gene encoding the pectin lyase mutant K107R.

[0016] The recombinant expression vector is further preferably the pET-22b(+) vector.

[0017] The present invention also provides an engineered strain expressing the pectin lyase mutant K107R, which is obtained by transforming the engineered strain into host bacteria through the recombinant expression vector.

[0018] Furthermore, the host bacteria is preferably Escherichia coli BL21(DE3).

[0019] The application of pectin lyase mutants D84A or K107R in tobacco leaf treatment according to the present invention.

[0020] Specifically, weigh 100g of tobacco leaves, spray 20mL of enzyme solution (100 units / mL) containing a pectin lyase mutant onto the surface of the tobacco leaves, and frequently stir the tobacco leaves during the spraying process to ensure that the enzyme solution is mixed evenly. Seal the tobacco leaves in a sealed bag and ferment them for 60-72 hours at 45℃ and 65% humidity.

[0021] The pectin lyase mutants D84A and K107R of this invention still retain 83.63% and 81.30% of their enzyme activity at a 1% nicotine concentration; spraying tobacco leaves with D84A and K107R enzyme solutions significantly improves the quality of tobacco leaves by degrading pectin.

[0022] Measurements revealed that the pectin degradation rate of tobacco leaves reached 42.6%, while the reducing sugar content increased by 21.1%, indicating a significant improvement in tobacco leaf quality. Pectin degradation not only loosened the cell wall structure of the tobacco leaves but also promoted the release of soluble substances, significantly enhancing the aroma and taste. The increased reducing sugar content improved the sweetness and combustion performance of the tobacco leaves, making them softer, smoother, and burning more steadily. Overall, enzymatic fermentation technology effectively improves the quality of tobacco leaves, increasing flavor complexity and smoking comfort, providing an innovative optimization solution for tobacco processing, and showing promising application prospects. Attached Figure Description

[0023] Figure 1 Figure A is a schematic diagram of the protein as a whole, Figure B is a schematic diagram of the overall docking results, and Figure C shows the docking details.

[0024] Figure 2 Electrophoresis image of recombinant PL6. M: protein marker; 1: unpurified protein solution; 2: purified pectinase PL6.

[0025] Figure 3 The effect of nicotine on the activity of pectin lyase. The black bars represent commercial pectin lyase, and the gray bars represent pectin lyase PL6.

[0026] Figure 4 Nicotine tolerance in mutants.

[0027] Figure 5 The change in pectin degradation rate with fermentation time. Detailed Implementation

[0028] The technical solution of the present invention will be further analyzed and explained through specific embodiments below.

[0029] (I) Mutation site design

[0030] Double docking was performed between the protein structure and pectin and nicotine molecules. The resulting docking conformation was selected as the initial conformation for dynamics. Gromacs 2019.6 was chosen as the kinetic simulation software, and amber14sb was used for the protein force field. A TIP3P water model was added to the complex system to establish a water box and a sodium ion equilibrium system was added. Electrostatic interactions were handled using Verlet and CG algorithms, respectively, and the steepest descent method was used for energy minimization at the maximum number of steps (50,000 steps). The Coulomb force cutoff distance and van der Waals radius cutoff distance were both 1.4 nm. Equilibrium simulations were performed for 30 ps using canonical system (NVT) and isothermal-isobaric system (NPT) equilibrium systems, followed by 100 ns of molecular dynamics (MD) simulations at room temperature and pressure. During the MD simulations, hydrogen bonds were constrained using the LINCS algorithm with an integration step size of 2 fs. The PL6rticle-mesh Ewald (PME) method was used for calculations, with a cutoff value set to 1.2 nm. The cutoff value for non-bonded interactions was set to... The simulation temperature was controlled at 300 K using the V-rescale temperature coupling method, and the pressure was controlled at 1 bar using the Berendsen method. HBNUM was used to describe the hydrogen bonding contacts between two proteins, between a protein and a small molecule, and between a protein and a water molecule. Gmx_MMPBSA was used to calculate the binding free energy.

[0031] like Figure 1As shown, the active cavity for the pectin lyase PL6 binding to small molecules is located outside a cavity formed by multiple β-sheets and loops in the middle of the protein. This study calculated the hydrogen bonds between the protein and the two small molecules; quantitatively, the binding between the protein and pectin is stronger. The free energy for nicotine binding to the protein reaches -12.41 kcal / mol, indicating its binding potential. Based on the three-dimensional structure, molecular dynamics analysis was performed on the protein and nicotine binding. Lys-107, Asp-84, Gln-110, Glu-104, Asn-134, Asn-112, Asn-39, Glu-83, and Asp-63 form hydrogen bonds with small molecules at wavelengths of 0.322, 0.328 / 0.296 / 0.281, 0.324 / 0.312 / 0.316, 0.295, 0.302, 0.297 / 0.290, 0.292, 0.311 / 0.295, and 0.290 nm, respectively. The surrounding molecules are all hydrophobic amino acids, such as Ser-35. The results indicate that the polar interactions corresponding to the hydrogen bonds are optimal, while the hydrophobic interactions contribute less. Based on the two sites where the enzyme forms hydrogen bonds with nicotine, Lys-107 and Asp-84, single-site saturation mutations were designed to construct a mutant library.

[0032] (II) Gene Optimization and Recombinant Expression

[0033] The amino acid sequence of PL6 after removing the signal peptide is shown in SEQ ID NO: 1. The gene sequence of alkaline pectinase PL6 was optimized. The D84A and K107R mutant sequences were sent to Qingke Biotechnology Co., Ltd. (Qingdao) for gene synthesis. Qingke was entrusted to ligate the two mutated target genes into the pET-22b(+) vector to prepare a plasmid powder containing the recombinant vector, which was then transformed into *E. coli* Top10. The plasmid powder was transformed into *E. coli* BL21(DE3): The synthesized plasmid powder was dissolved in 80 μL of solution buffer (pH 7.5, 10 mM Tris-HCl). 10 μL of the powder solution was mixed with 20 μL of BL21(DE3) competent cells, incubated on ice for 10 min, then heat-stimulated in a 42℃ water bath for 45 s, incubated on ice again for 2 min, and then 1 mL of LB medium was added and incubated at 37℃ for 1 h. The samples were spread on LB solid plates containing ampicillin and cultured for 16 h. Transformants were picked to obtain two recombinant strains, BL21(DE3) / pET22b-PL6-D84A and BL21(DE3) / pET22b-PL6-K107R.

[0034] The recombinant strain was cultured in LB medium containing 50 μg / mL ampicillin, and cultured in 250 mL Erlenmeyer flasks containing 50 mL of medium at 37 °C and 160 rpm with shaking until the OD600 of the medium reached 0.6. 0.2 M IPTG solution was prepared in advance, and 50 μL was added to the medium to a final concentration of 0.2 mM. The cells were cultured at 20 °C and 160 rpm for 24 hours, and then collected by centrifugation at 10,000 rpm for 10 min. The collected cells were resuspended in 10 mL of PBS buffer and then sonicated at 120 W with 3-second sonication followed by 4-second pauses. The supernatant was collected by centrifugation to obtain two crude enzyme solutions. Gradient salting out was performed with ammonium sulfate, and the 30-50% ammonium sulfate precipitate was collected, dissolved, and dialyzed into glycine-sodium hydroxide buffer. For further separation and purification, a CM-Sepharose Fast Flow cation exchange chromatography column was used. After loading the sample, unadsorbed proteins were eluted with equilibration buffer until baseline was reached. Then, a gradient elution was performed with different NaCl concentrations of 20 mmol / L Tris-HCl at pH 7.6 at a flow rate of 1.5 mL / min. The active fraction was collected, concentrated by ultrafiltration, and finally desalted using a HiPrep 26 / 10 Desalting column at a flow rate of 3 mL / min. The first elution peak was collected to obtain the pure enzyme solution. The molecular weight of the recombinant enzyme PL6 was approximately 41.0 kDa.

[0035] (III) Determination of nicotine tolerance in pectin lyase mutants

[0036] Take 1.8 mL of 1% pectin substrate solution (citric acid-phosphate buffer, pH 5.0), add 0.2 mL of diluted crude enzyme solution, and react in a 40℃ water bath for 10 minutes. After removing from the water bath, add 3 mL of DNS reagent, mix well, and inactivate by boiling in a water bath for 10 minutes. Finally, adjust the volume to 25 mL. Measure the OD value at 540 nm using a microplate reader. By definition, pectinase activity is the ability to hydrolyze pectin substrate to produce 1 μg of galacturonic acid per minute at 40℃ and pH 7.5, expressed in U. The pectinase activity of fermentation broth BL21(DE3) / pET22b-PL6 is 263.5 U / mL. Add different concentrations of nicotine (0.2%, 0.4%, 0.6%, 0.8%, 1.0%) to the mixed crude enzyme solution and substrate reaction system. Measure the enzyme activity after the nicotine is completely dissolved, using the experimental group without nicotine as a control. Figure 3 As shown, in the presence of nicotine at concentrations above 0.6%, the activities of both the commercial enzyme and the original enzyme PL6 decreased significantly. At a concentration of 1%, their activities were 65% and 72% of those without nicotine, respectively. Figure 4As shown, several single-point mutants constructed based on two potential nicotine-binding sites exhibited good tolerance to 1% nicotine concentration. Among them, D84A and K107R showed the highest residual activity at 1% nicotine concentration, with enzyme activities remaining at 83.63% and 81.30%, respectively. Further combined mutations revealed that the double-mutant D84A / K107R retained 78.3% activity at 1% nicotine concentration, but its nicotine tolerance was actually lower than that of a single mutation, indicating that the effects of the two mutations are not additive. Therefore, K107R and D84A are the optimal mutants for nicotine tolerance.

[0037] (iv) Application of mutants in tobacco leaf treatment

[0038] 100 grams of tobacco leaves were placed in a constant temperature and humidity chamber, and 20 ml of D84A and K107R enzyme solution (100 units / ml) was evenly sprayed onto the surface. Fermentation was carried out at 45℃ and 65% humidity for 72 hours. After testing, it was found that the pectin degradation rate reached 42.6%, while the reducing sugar content increased by 21.1%. The relationship between fermentation time and degradation rate is as follows: Figure 5 As shown in the figure. Furthermore, when the same volume of the two enzyme solutions, D84A and K107R, was sprayed, the pectin degradation rates were 39.4% and 37.8%, respectively; the reducing sugar content increased by 19.7% and 21.2%, respectively. This indicates a significant improvement in tobacco quality. Pectin degradation not only loosens the cell wall structure of tobacco leaves but also promotes the release of soluble substances, significantly improving the aroma and taste of the tobacco. The increased reducing sugar improves the sweetness and combustion performance of the tobacco, making it softer, smoother, and burning more steadily. Overall, enzymatic fermentation technology effectively improves the quality of tobacco leaves, increases flavor complexity and smoking comfort, and provides an innovative optimization solution for tobacco processing, showing promising application prospects.

Claims

1. A nicotine-resistant pectin lyase mutant, characterized in that: The pectin lyase mutant is abbreviated as D84A, and its amino acid sequence is shown in SEQ ID NO: 2; Alternatively, the pectin lyase mutant may be abbreviated as K107R, and its amino acid sequence is shown in SEQ ID NO:

3.

2. The encoding gene of the pectin lyase mutant according to claim 1.

3. A recombinant expression vector containing the gene encoding the pectin lyase mutant as described in claim 2.

4. The recombinant expression vector according to claim 3, characterized in that: The recombinant expression vector is the pET-22b(+) vector.

5. An engineered strain expressing the pectin lyase mutant of claim 1, characterized in that: The engineered strain was obtained by transforming it into host bacteria using the recombinant expression vector described in claim 3 or 4.

6. The engineered strain according to claim 5, characterized in that: The host bacteria is Escherichia coli BL21(DE3).

7. The application of the pectin lyase mutant of claim 1 in the degradation of tobacco pectin.

8. The application according to claim 7, characterized in that: Spray the surface of tobacco leaves with an enzyme solution containing a pectin lyase mutant. During the spraying process, frequently turn the tobacco leaves to ensure that the enzyme solution is mixed evenly. Seal the leaves in a sealed bag and ferment for 60-72 hours at 45°C and 65% humidity.

9. The application according to claim 8, characterized in that: The concentration of the pectin lyase mutant in the enzyme solution is 100 units / mL, and the spraying amount is 15-25mL / 100g tobacco leaves.

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