Nicotine-tolerant pectate lyase mutant and application thereof in tobacco leaf treatment

Abstract

The invention discloses a nicotine-tolerant pectate lyase mutant and an application thereof in tobacco leaf treatment. According to the invention, pectin lyase PL6 is taken as a starting enzyme, mutation sites are designed and optimized, and single-site saturated mutation is designed according to two sites Lys-107 and Asp-84 of hydrogen bonds formed by enzyme and nicotine, so that a mutant is constructed. The enzyme activity of the pectate lyase mutants D84A and K107R still remains 83.63% and 81.30% under the condition that the nicotine concentration is 1%; d84A and K107R enzyme solutions are sprayed on the tobacco leaves, and the pectin is degraded, so that the quality of the tobacco leaves is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the field of biological enzymes, and particularly relates to a nicotine-tolerant pectin lyase mutant and application thereof in tobacco leaf processing. Background Art

[0002] Biological enzymes play a vital role in tobacco leaf processing. They can promote the rapid decomposition of high-molecular substances such as starch, protein, cellulose, pectin, etc. in tobacco leaves, and accelerate the conversion of pigments, polyphenols, and terpenes in tobacco leaves into small-molecule aroma substances, thereby increasing the content of ketones, aldehydes, acids, esters, terpenes, and low-level fatty acids in tobacco products. However, due to the low endogenous enzyme content in tobacco leaves, these chemical transformations cannot be achieved in a short period of time, so the decomposition of macromolecular substances and the generation of small-molecule aroma substances are at a relatively low level. In order to overcome this challenge, external enzymes such as exogenous proteases, amylases, cellulases, pectinases, and glucanases are introduced into tobacco leaf processing. They can quickly catalyze chemical reactions, promote changes in the chemical composition of tobacco leaves, and help accumulate aroma substances. The action of these key biological enzymes can significantly improve the internal chemical composition of tobacco leaves in a short period of time, reduce impurities, increase the attractiveness of tobacco, and highlight the unique flavor style of cigarettes.

[0003] Pectin is a polysaccharide whose main component is a plant cell wall polysaccharide rich in galacturonic acid. Its basic structure consists of homopolymers connected by α-1,4 glycosidic bonds. Pectin content has an important influence on the quality of tobacco leaves because it can have multiple effects on the properties of tobacco leaves. High levels of pectin may affect the production and properties of tobacco smoke, because higher pectin content may lead to more smoke production, thereby affecting the combustion performance and smoke composition of tobacco. In addition, the degradation of pectin may release flavor compounds that can affect the flavor and smell of tobacco products. The content and characteristics of pectin may also affect the flavor of tobacco leaves, so controlling the pectin content in tobacco leaves has become an important research direction in the field of tobacco leaf processing.

[0004] Pectinase is a comprehensive enzyme that specifically degrades pectin-like substances. Specifically, pectinase lyase can specifically cut the α-1,4 glycosidic bonds in pectin to form oligosaccharides with unsaturated galacturonic acid residues at the non-reducing end. Most of these pectin lyases are derived from microorganisms, mainly including fungi such as Penicillium, Aspergillus, and Fusarium. The application of pectin lyase to treat tobacco leaves has a series of advantages, such as high specificity, high efficiency, mild operating conditions, and no introduction of pollutants. This makes pectin lyase have a wide range of application value in tobacco leaf processing. However, the pectin lyase preparations currently available on the market have certain challenges in dealing with tobacco leaf environments with high nicotine content, because they may cause conformational changes in the enzyme or reduce enzyme activity, thereby weakening the pectin lyase's ability to degrade pectin and having a negative impact on the treatment effect of tobacco leaves.

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

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a nicotine-tolerant pectin lyase mutant and its application in tobacco leaf processing. The present invention uses pectin lyase PL6 as the starting enzyme, designs and optimizes the mutation site, designs single-site saturation mutations based on the two sites Lys-107 and Asp-84 where the enzyme forms hydrogen bonds with nicotine, and constructs a mutant library.

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

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

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

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

[0011] The present invention also provides an engineering strain expressing the pectin lyase mutant D84A, wherein the engineering strain is obtained by transforming the recombinant expression vector into a host bacterium.

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

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

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

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

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

[0017] The present invention also provides an engineering strain expressing the pectin lyase mutant K107R, wherein the engineering strain is obtained by transforming the recombinant expression vector into a host bacterium.

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

[0019] Application of the pectin lyase mutant D84A or K107R of the invention in tobacco leaf processing.

[0020] Specifically, 100 g of tobacco leaves were weighed, and 20 mL of enzyme solution containing pectin lyase mutants (100 units / mL) was sprayed on the surface of the tobacco leaves. During the spraying process, the tobacco leaves were often turned over to mix the enzyme solution evenly. The leaves were sealed in a sealed bag and fermented at 45° C. and 65% humidity for 60-72 hours.

[0021] The pectin lyase mutants D84A and K107R of the present invention still have 83.63% and 81.30% of enzyme activities remaining at 1% nicotine concentration; when tobacco leaves are sprayed with D84A and K107R enzyme solutions, pectin is degraded so that the quality of tobacco leaves is significantly improved.

[0022] After testing, it was found that the pectin degradation rate of tobacco leaves reached 42.6%, and the reducing sugar content increased by 21.1%, indicating that the quality of tobacco leaves has been significantly improved. The degradation of pectin not only relaxes the cell wall structure of tobacco leaves, but also promotes the release of soluble substances, significantly improving the aroma and taste of tobacco leaves. The increased reducing sugar improves the sweetness and burning performance of tobacco leaves, making the tobacco leaves softer, smoother, and burning more smoothly. Overall, enzymatic fermentation technology effectively improves the quality of tobacco leaves, can increase the complexity of flavor and the comfort of smoking, and provides an innovative optimization solution for tobacco processing with good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Figure A is a schematic diagram of the protein as a whole, Figure B is a schematic diagram of the whole with docking results, and Figure C is a detailed display of docking.

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

[0025] Figure 3 Effect of nicotine on the activity of pectin lyase. The black columns are commercial pectin lyase, and the gray columns are pectin lyase PL6.

[0026] Figure 4 Nicotine tolerance of mutants.

[0027] Figure 5 Changes of pectin degradation rate with fermentation time. DETAILED DESCRIPTION

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

[0029] (I) Mutation site design

[0030] According to the protein structure, double docking was performed with pectin molecules and nicotine molecules. The conformation of the docking result was selected as the initial conformation of the dynamics. Gromacs2019.6 was selected as the dynamics simulation software, and amber14sb was selected as the protein force field. The TIP3P water model was added to the complex system using the TIP3P water model, a water box was established, and a sodium ion equilibrium system was added. Under the elastic simulation, the Verlet and cg algorithms and PME were used to deal with electrostatic interactions, and the steepest descent method was used to minimize the energy with the maximum number of steps (50,000 steps). The Coulomb force cutoff distance and the van der Waals radius cutoff distance were both 1.4nm. The canonical system (NVT) and the isothermal and isobaric system (NPT) equilibrium systems were used for 30ps equilibrium simulations, respectively, and then 100ns of MD simulations were performed at room temperature and pressure. During the MD simulation process, the hydrogen bonds involved were constrained by the LINCS algorithm, and the integration step was 2fs. The PL6rticle-mesh Ewald (PME) method was used for calculation, and the cutoff value was set to 1.2nm. The non-bonded interaction cutoff value was set to The V-rescale temperature coupling method was used to control the simulation temperature to 300K, and the Berendsen method was used to control the pressure to 1 bar. HBNUM was used to describe the hydrogen bond contacts between two proteins, proteins and small molecules, and proteins and water molecules. Gmx_MMPBSA calculated the binding free energy.

[0031] like Figure 1As shown in the figure, the active cavity of pectin lyase PL6 for binding small molecules is located outside the cavity formed by multiple groups of β-sheets and loops in the middle of the protein. This study calculated the hydrogen bonds between the protein and the two small molecules respectively. From a quantitative point of view, the binding between protein and pectin is tighter. The free energy of protein binding to nicotine reaches -12.41kcal / mol, indicating that it has the potential for binding. Based on the three-dimensional structure, molecular dynamics analysis of protein and nicotine was performed. 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 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, and the others around are hydrophobic amino acids, such as Ser-35. The results show that the polar effect corresponding to the hydrogen bond is the best, and the hydrophobic effect contributes less. According to the two sites Lys-107 and Asp-84 where the enzyme forms hydrogen bonds with nicotine, single-site saturation mutations are 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, and the mutant sequences containing D84A and K107R were sent to Qingke Biotechnology Co., Ltd. (Qingdao) for gene synthesis, and the two mutant target genes were connected to the pET-22b (+) vector to prepare a plasmid powder containing the recombinant vector and transform it into Escherichia coli Top10. The plasmid powder was transformed into Escherichia coli BL21 (DE3): the synthesized plasmid powder was dissolved in 80 μL of Eliution buffer (pH 7.5 10 mM Tris-HCl), 10 μL of the dry powder solution was mixed with 20 μL of BL21 (DE3) competent cells, ice-bathed for 10 minutes, and then the mixture was heat-stimulated for 45 seconds in a 42°C water bath, and then ice-bathed for 2 minutes, 1 mL of LB medium was added and incubated at 37°C for 1 hour. The cells were plated on an LB solid plate containing ampicillin and cultured for 16 h, and 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 a 250 mL conical flask containing 50 mL of culture medium at 37 ° C and 160 rpm until the OD600 of the culture medium was 0.6. A 0.2 M IPTG solution was prepared in advance, and 50 uL was added to the culture medium to a final concentration of 0.2 mM. The culture was continued at 20 ° C and 160 rpm for 24 hours, and the cells were collected by centrifuge at 10000 rpm for 10 min. The collected cells were resuspended in 10 mL of PBS buffer solution, and ultrasonicated at 120 w power with an interval of 3 s and 4 s. The supernatant was centrifuged to obtain two crude enzyme solutions. Ammonium sulfate was used for gradient salting out, and the 30-50% ammonium sulfate precipitation part was collected, dissolved and dialyzed into glycine-sodium hydroxide buffer. A CM-Sepharose Fast Flow cation exchange chromatography column was used for further separation and purification. After loading, the unadsorbed protein was first eluted with an equilibrium buffer, and then eluted with a gradient of 20mmol / LTris-HCl, pH 7.6 buffer with different NaCl concentrations at a flow rate of 1.5ml / min. The active part was collected, concentrated by ultrafiltration, and finally passed through a HiPrep 26 / 10Desalting column at a flow rate of 3mL / min. The first elution peak was collected to obtain a pure enzyme solution. The molecular weight of the recombinant enzyme PL6 is about 41.0kDa.

[0035] (III) Determination of nicotine tolerance of 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 water bath at 40°C for 10 minutes. After taking it out, add 3 mL of DNS reagent and mix well, inactivate it in a boiling water bath for 10 minutes, and finally make up to 25 mL. Use a microplate reader to measure the OD value at a wavelength of 540 nm. According to the definition, pectinase activity is the ability to hydrolyze pectin substrate to produce 1 μg of galacturonic acid per minute at 40°C and pH 7.5, and the unit is U. The pectinase activity of the 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, and use the experimental group without nicotine as a control. Figure 3 As shown in the figure, in the presence of nicotine concentrations above 0.6%, the activity of the commercial enzyme and the original enzyme PL6 decreased significantly. At a concentration of 1%, the activity of the two was 65% and 72% of that in the absence of nicotine, respectively. Figure 4As shown, multiple single-site mutants constructed based on two potential nicotine-binding sites have good tolerance to 1% nicotine concentration. Among them, D84A and K107R have the largest residual activity at a nicotine concentration of 1%, and the enzyme activity still remains at 83.63% and 81.30%. Further combined mutations, the double mutation D84A / K107R has 78.3% residual activity at a nicotine concentration of 1%, and its tolerance to nicotine is lower than that of a single mutation, indicating that the effects of the two mutations cannot be superimposed. Therefore, K107R and D84A are the best mutants for nicotine tolerance.

[0037] (IV) Application of mutants in tobacco leaf processing

[0038] 100g tobacco leaves were placed in a constant temperature and humidity chamber, 20ml of D84A, K107R enzyme solution (100 units / ml) was sprayed evenly on the surface, and fermented for 72 hours at 45℃ and 65% humidity. After measurement, it was found that the pectin degradation rate reached 42.6%, and the reducing sugar content increased by 21.1%. The relationship between fermentation time and degradation rate is shown in the figure. Figure 5 As shown. In addition, when the two enzyme solutions D84A and K107R were sprayed in the same volume, the pectin degradation rates were 39.4% and 37.8%, respectively; the reducing sugar content increased by 19.7% and 21.2%, respectively. This shows that the quality of tobacco leaves has been significantly improved. The degradation of pectin not only relaxes the cell wall structure of tobacco leaves, but also promotes the release of soluble substances, significantly improving the aroma and taste of tobacco leaves. The increased reducing sugar improves the sweetness and burning performance of tobacco leaves, making them softer, smoother, and burning more smoothly. Overall, enzymatic fermentation technology effectively improves the quality of tobacco leaves, increases the complexity of flavor and the comfort of smoking, and provides an innovative optimization solution for tobacco processing with good application prospects.

[0039] SEQ ID NO: 1 (original sequence, amino acid sequence of PL6 after removing the signal peptide)

[0040] APTVVHETIRVPAGQTFDGKGQTYVANPNTLGDGSQAENQKPIFRLEAGASLKNVVIGAPA

[0041] ADGVHCYGDCTITNVIWEDVGEDALTLKSSGTVNISGGAAYKAYDKVFQINAAGTINIRNF

[0042] RADDIGKLVRQNGGTTYKVVMNVENCNISRVKDAILRTDSSTSTGRIVNTRYSNVPTLFKG

[0043] FKSGNTTASGNTQY

[0044] YHSLDVDKDHYDGLFDAKKDSQYITFSWNYVHDAWKSMLMGNSDSDNNNRTITFHHNW

[0045] FENLNSRVPAFRFGEGHIYNNYYSNIIESGINSRMGARIKIENNLFENAKDPIVSWYSDKPGY

[0046] WDVSNNKFVNSTGSMPTSSTTTYQPPYKYSLDSVDNVKAVVTQNAGVGKINPSEQ ID NO:2(D84A)

[0047] APTVVHETIRVPAGQTFDGKGQTYVANPNTLGDGSQAENQKPIFRLEAGASLKNVVIGAPA

[0048] ADGVHCYGDCTITNVIWEDVGEAALTLKSSGTVNISGGAAYKAYDKVFQINAAGTINIRNF

[0049] RADDIGKLVRQNGGTTYKVVMNVENCNISRVKDAILRTDSSTSTGRIVNTRYSNVPTLFKG

[0050] FKSGNTTASGNTQY

[0051] YHSLDVDKDHYDGLFDAKKDSQYITFSWNYVHDAWKSMLMGNSDSDNNNRTITFHHNW

[0052] FENLNSRVPAFRFGEGHIYNNYYSNIIESGINSRMGARIKIENNLFENAKDPIVSWYSDKPGY

[0053] WDVSNNKFVNSTGSMPTSSTTTYQPPYKYSLDSVDNVKAVVTQNAGVGKINPSEQ ID NO:3(K107R)

[0054] APTVVHETIRVPAGQTFDGKGQTYVANPNTLGDGSQAENQKPIFRLEAGASLKNVVIGAPA

[0055] ADGVHCYGDCTITNVIWEDVGEDALTLKSSGTVNISGGAAYKAYDRVFQINAAGTINIRNF

[0056] RADDIGKLVRQNGGTTYKVVMNVENCNISRVKDAILRTDSSTSTGRIVNTRYSNVPTLFKG

[0057] FKSGNTTASGNTQY

[0058] YHSLDVDKDHYDGLFDAKKDSQYITFSWNYVHDAWKSMLMGNSDSDNNNRTITFHHNW

[0059] FENLNSRVPAFRFGEGHIYNNYYSNIIESGINSRMGARIKIENNLFENAKDPIVSWYSDKPGY

[0060] WDVSNNKFVNSTGSMPTSSTTTYQPPYKYSLDSVDNVKAVVTQNAGVGKINPSEQ ID NO:4

[0061] tatcatagcctggatgtggataaagatcattatgatggcctgtttgatgcgaaaaaagatagccagtatattacctttagctggaactatgtgcatgat

[0062] gcgtggaaaagcatgctgatgggcaacagcgatagcgataacaacaaccgcaccattacctttcatcataactggtttgaaaacctgaacagcc

[0063] gcgtgccggcgtttcgctttggcgaaggccatatttataacaactattatagcaacattattgaaagcggcattaacagccgcatgggcgcgcgc

[0064] attaaaattgaaaacaacctgtttgaaaacgcgaaagatccgattgtgagctggtatagcgataaaccgggctattgggatgtgagcaacaacaa

[0065] atttgtgaacagcaccggcagcatgccgaccagcagcaccaccacctatcagccgccgtataaatatagcctggatagcgtggataacgtgaa

[0066] agcggtggtgacccagaacgcgggcgtgggcaaaattaacccg

[0067] SEQ ID NO:5

[0068] tatcatagcctggatgtggataaagatcattatgatggcctgtttgatgcgaaaaaagatagccagtatattacctttagctggaactatgtgcatgat

[0069] gcgtggaaaagcatgctgatgggcaacagcgatagcgataacaacaaccgcaccattacctttcatcataactggtttgaaaacctgaacagcc

[0070] gcgtgccggcgtttcgctttggcgaaggccatatttataacaactattatagcaacattattgaaagcggcattaacagccgcatgggcgcgcgc

[0071] attaaaattgaaaacaacctgtttgaaaacgcgaaagatccgattgtgagctggtatagcgataaaccgggctattgggatgtgagcaacaacaa

[0072] atttgtgaacagcaccggcagcatgccgaccagcagcaccaccacctatcagccgccgtataaatatagcctggatagcgtggataacgtgaa

[0073] agcggtggtgacccagaacgcgggcgtgggcaaaattaacccg

Claims

1. A nicotine-tolerant pectate 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 is abbreviated as K107R, and its amino acid sequence is shown in SEQ ID NO:

3.

2. The gene encoding the pectin lyase mutant according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the pectin lyase mutant D84A is shown in SEQ ID NO: 4; The nucleotide sequence of the gene encoding the pectin lyase mutant K107R is shown in SEQ ID NO:

5.

3. A recombinant expression vector containing the gene encoding the pectin lyase mutant according to claim 2.

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

5. An engineered strain expressing the pectin lyase mutant according to claim 1, characterized in that: The engineered strain is obtained by transforming the recombinant expression vector according to claim 3 or 4 into a host bacterium.

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

7. Use of the pectin lyase mutant according to claim 1 in tobacco leaf processing.

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

9. The use 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 15-25 mL / 100 g tobacco leaves.

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