Pectinase as well as preparation method and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to pectinase as well as a preparation method and application thereof. The amino acid sequence of the pectinase is shown as SEQ ID NO: 1. The nucleotide sequence of the pectinase coding gene is shown as SEQ ID NO: 2. The invention further provides application of the pectinase or the recombinant expression vector or the recombinant expression transformant thereof to degradation of tobacco pectin, release of aroma components and improvement of tobacco aroma quality. The pectinase disclosed by the invention shows efficient tobacco pectin degradation capacity, the content of 20 flavor substances in tobacco treated by the pectinase is remarkably increased, especially compounds such as solanone, 4, 7, 9-megastigmatriene-3-ketone, dihydroactinidiolide, roriolate and the like, and the flavor enhancement effect on smoke is remarkable.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a pectinase, its preparation method, and its application. Background Technology

[0002] Pectin is a key binding component of the intercellular matrix in tobacco, playing a crucial role in maintaining the integrity and mechanical toughness of tobacco leaf tissue. An appropriate amount of pectin ensures the structural stability of tobacco leaves and enhances their hygroscopic properties through osmotic regulation. However, excessive pectin content in tobacco not only leads to incomplete combustion but also significantly reduces the smoking quality of tobacco due to the small molecules such as methanol produced during pyrolysis. Furthermore, during the natural aging process of tobacco leaves, pectin can gradually convert into acetic acid, causing discomfort such as coughing and sore throat in smokers, and producing an irritating odor, severely damaging the purity and sensory comfort of the smoke. During combustion, pectin also decomposes to generate various volatile harmful components, posing a potential threat to human health. In addition, although some pectin degrades after initial and re-drying processes, a large amount of pectin remains difficult to decompose due to structural changes such as molecular chain cross-linking polymerization and the removal of esterification groups. This phenomenon results in stiff, less elastic, and less brittle tobacco leaves after curing, along with a significant reduction in filling value, severely affecting the processing suitability of the tobacco leaves.

[0003] Pectinase is a general term for a class of enzymes that break down pectin, mainly including protopectinase, polygalacturonase, pectin lyase, and pectin esterase. Among them, oligogalacturonate lyase (OGL), also known as pentinlyases (PL), belongs to both the pectin lyase family and the polysaccharide lyase (PL) family. OGL mainly acts in the later stages of pectin degradation, catalyzing the degradation of deesterified pectin or oligogalacturonic acid via exocleation. It specifically cleaves the α-1,4-glycosidic bond of oligogalacturonic acid through β-trans elimination, generating unsaturated oligogalacturonic acid, thereby participating in the degradation metabolism of pectin polysaccharides. It is worth noting that processed tobacco pectin typically forms a unique and stable conformation. Conventional microbial pectinases and commercially available pectinase preparations often fail to achieve efficient degradation of tobacco pectin due to substrate specificity mismatch, insufficient catalytic efficiency, and inability to effectively penetrate the substrate's binding site. Therefore, developing specialized pectinase formulations with both high specificity and strong adaptability, targeting the complex structural characteristics and transformation patterns of tobacco pectin, has become a key technological breakthrough for precisely regulating the physical properties of tobacco leaves and improving their sensory quality.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a pectinase, its preparation method, and its application, to solve the problem of low degradation efficiency of tobacco pectin. This pectinase can efficiently degrade tobacco pectin, release aroma components, and improve the aroma quality of tobacco.

[0006] In a first aspect, the present invention provides a pectinase having the amino acid sequence shown in SEQ ID NO:1.

[0007] In a second aspect, the present invention provides a pectinase encoding gene that encodes the pectinase described above.

[0008] Preferably, the nucleotide sequence of the pectinase encoding gene is shown in SEQ ID NO:2.

[0009] A third aspect of the present invention provides a recombinant expression vector comprising the vector and the pectinase encoding gene.

[0010] Specifically, the pectinase-encoding gene of the present invention is constructed by ligating it into various suitable vectors using conventional methods in the art. The vector can be any conventional vector in the art, and the expression vector is a plasmid, preferably a pET-28a(+) series expression plasmid. The pectinase-encoding gene can be operatively ligated downstream of a suitable regulatory sequence in the selected vector to achieve constitutive or inducible expression of pectinase.

[0011] Preferably, the recombinant expression vector has resistance to kanamycin or tetracycline.

[0012] In a fourth aspect, the present invention provides a recombinant expression transformant comprising the aforementioned recombinant expression vector.

[0013] Specifically, the recombinant expression transformant is obtained by transforming the recombinant expression vector of the present invention into a host cell. The host cell can be any conventional host cell in the art, provided that it can stably replicate on its own using the recombinant expression vector and that the pectinase encoding gene it carries can be effectively expressed.

[0014] Preferably, the host cell is Escherichia coli; more preferably, the host cell is Escherichia coli BL21(DE3) competent cells.

[0015] In a fifth aspect, the present invention provides the application of the pectinase, the pectinase encoding gene, the recombinant expression vector, and the recombinant expression transformant, wherein the pectinase, the pectinase encoding gene, the recombinant expression vector, or the recombinant expression transformant is used to degrade tobacco pectin, release aroma components, and improve the aroma quality of tobacco.

[0016] A sixth aspect of the present invention provides a method for preparing pectinase, comprising the following steps: (1) The pectinase encoding gene was introduced into the vector plasmid pET-28a(+) with a histidine tag to obtain a recombinant expression vector; the recombinant expression vector was transformed into Escherichia coli BL21(DE3) competent cells to obtain recombinant expression transformants; (2) The recombinant expression transformant was inoculated into LB liquid medium containing kanamycin and cultured overnight with shaking; then transferred to fresh LB medium and cultured until OD600 reached 0.8, then IPTG was added to induce expression. (3) After induction, the bacterial cells were collected by centrifugation. The bacterial cells were washed with sterile water and centrifuged to collect the precipitate. The bacterial cells were resuspended in lysis buffer and broken up in an ice bath. The crude enzyme solution after breaking up was purified to obtain pectinase.

[0017] A seventh aspect of the present invention provides a method for enhancing the flavor of tobacco, comprising the following steps: (1) Mix pectinase with tobacco at a mass ratio of 1:2000-1:50; (2) Static treatment at a temperature of 30-55℃ for 1-10 hours; (3) Inactivate the tobacco at a temperature of 50-200℃ for 1-60 minutes to obtain the flavored tobacco.

[0018] Preferably, in step (1), pectinase is mixed with tobacco at a mass ratio of 1:1000; In step (2), the static treatment is carried out at a temperature of 40°C for 6 hours; in step (3), the inactivation is carried out at a temperature of 160°C for 1-2 minutes.

[0019] The beneficial effects of this invention are: The pectinase provided by this invention can effectively degrade pectin in tobacco, release key aroma components, and significantly improve the aroma and sweetness of tobacco. Moreover, the pectinase provided by this invention has high enzymatic activity and stability. In addition, the preparation method of the pectinase of this invention is green, efficient, and sustainable, which is in line with the development trend of modern tobacco industry. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 The diagram shows the construction of the recombinant plasmid provided by the present invention; wherein, a) is a schematic diagram of the recombinant plasmid construction method; and b) is a diagram of the colony PCR verification results.

[0022] Figure 2 The SDS-PAGE analysis results provided by this invention are shown in the figure; wherein, M-standard protein; 1-pre-induction bacterial lysate; 2-post-induction bacterial lysate; 3-crude enzyme solution; 4-0% B solution elution flow-through solution; 5-20% B solution eluted protein solution.

[0023] Figure 3 The nickel column chromatography chromatogram provided by this invention.

[0024] Figure 4 The image shows the enzyme activity results provided by this invention. Detailed Implementation

[0025] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0026] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form includes the plural form unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0027] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: Preparation of pectinase (oligogalacturonate lyase) (1) According to Klebsiella variegata ( Klebsiella variicola GB3 Dedicated primers were designed for the oligogalacturonic acid lyase gene (OGL) of [a specific gene]. The upstream primer F: CATCGCAACTACTTCTTAT (SEQ ID NO: 3), and the downstream primer R: CGATATCAGCAGATAGTA (SEQ ID NO: 4), containing restriction enzyme sites NcoI and XhoI for subsequent cloning. OGL was amplified by PCR using the pET-28a(+) vector with kanamycin resistance. The purified PCR product and pET-28a(+) vector were digested with restriction endonucleases NcoI and XhoI, respectively. The gene fragment was ligated to the vector fragment using T4 DNA ligase to construct a recombinant plasmid, as shown below. Figure 1 As shown in a) of the diagram. The recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells, plated on LB agar plates containing 50 μg / mL kanamycin, and incubated overnight at 37°C. Single colonies were picked for colony PCR verification, as shown in the diagram. Figure 1 As shown in b), a bright band after colony PCR amplification indicates successful construction of the recombinant strain. Positive clones are sent for sequencing confirmation. After correct sequencing, the recombinant strain is stored at -80°C for later use.

[0029] (2) The preserved recombinant strain was inoculated at a rate of 1% into 15 ml of LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking at 180 rpm. The inoculum was then transferred to 1 L of fresh LB medium at a rate of 2% and cultured at 37°C until the OD600 reached about 0.8. IPTG was then added to a final concentration of 1 mM and expression was induced at 16°C for 16 h.

[0030] (3) After induction, the bacterial cells were collected by centrifugation at 4°C and 4000 rpm for 30 min. The bacterial cells were washed twice with sterile water and then centrifuged to collect the precipitate. The bacterial cells were resuspended in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.5) at a ratio of 1 g: 15 mL. The cells were then disrupted in an ice bath using an ultrasonic disruptor (60% operating power, 5 s working and 5 s rest, total disruption time 20 min). The supernatant was the crude enzyme solution.

[0031] (4) The crude enzyme solution was separated and purified using a Ni-NTA affinity chromatography column and the AKTA purifier rapid protein purification system. The specific steps are as follows: Solution preparation: 20% ethanol, 20 mmol / L pH 8.0 Tris-HCl (solution A), and 20 mmol / L pH 7.5 Tris-HCl containing 1 mol / L imidazole (solution B) were filtered through a 0.22 μm microporous membrane and pre-cooled to 4℃. Purification: The system was washed at 0.3 MPa and 3.0 mL / min until the baseline was stable, equilibrated with solution A, and the target component was collected by gradient elution with solution B. The salt ion concentration of the crude purified protein solution was too high, so it was desalted using a HiTrap™ Desating column and stored at 4℃.

[0032] SDS-PAGE gel electrophoresis was performed on the bacterial lysates before and after induction, such as... Figure 2 As shown, the sample had no obvious bands before induction, while the bands were clearly visible after induction. The apparent molecular weight of the protein was approximately between 45 and 66.2 kDa, consistent with the theoretical molecular weight of 44.2 kDa.

[0033] like Figure 3As shown, after gradient elution with a nickel column, two absorption peaks were observed at 280 nm: the flow-through peak of the 0% B solution and the target protein peak of the 20% B solution. The absorption peak at 280 nm using the 20% B solution reached 2813 mAu. SDS-PAGE analysis of each component showed bands in both the crude enzyme solution and the 20% elution buffer. Figure 2 The target protein band was clearly visible with few impurities in the 20% elution buffer, consistent with the theoretical molecular weight, indicating good nickel affinity chromatography and successful heterologous expression of oligogalacturonate lyase. After purification by Ni-NTA affinity chromatography, an enzyme with a purity greater than 92% was obtained. The enzyme concentration was measured to be 18.63 mg / mL, yielding approximately 246 mg of enzyme per liter of recombinant bacterial culture, indicating a high compatibility between the *E. coli* expression system and oligogalacturonate lyase.

[0034] Example 2: Characteristics of pectinase (oligogalacturonate lyase) The amino acid sequence of oligogalacturonic acid lyase is as follows: MAKGMRVKLNYQVSHDPDTGAEVTRLTPPEVTCHRNYFYQKCFFNDGSHLLFAGEFDGHWNYYLLNIASAEAIQLTEGAGDNTFGGFLSPDDKSLYYVKNDRTLLEVNLTTLVEREVYRVSDDWVGYGTWVANSDCSKLVGIEIAKSDWTPLNDWQIFHDFFHKGPHCRLLRVDLHSGESQVIHEEKIWLGH PIYRPFDDHTVAFCHEGPHDLVDARMWLVNEDGSHVRKVKTHAPGESCTHEFWVPDGSALIYVSYLKGQQGRTIYRFDPESGVNEALMTMPACSHLMSNFDGTLL VGDGGSGTPVDVKDTGGYSIDNDPYLYVFNVAQKRYFRVARHDTSWATVANSRQVTHPHPSFTPDDSAILFSSDKDGKPAIYIAKLPEHPPMLSA (SEQ ID NO: 1).

[0035]

[0036] The specific method for enzyme activity assay is as follows: Take 200 μL of oligogalacturonic acid lyase diluted to 5 mg / mL and 2 mL of 10 mM Tris-HCl buffer (initial pH 7.5), mix with 1.8 mL of 1 mg / mL oligogalacturonic acid substrate (with 1 mM CaCl2), and react at an initial temperature of 40℃ for 30 min. Terminate the reaction at 100℃, cool to room temperature with running water, and monitor the absorbance at 235 nm. Use the reaction system with the enzyme solution inactivated by boiling as the control group. Enzyme activity is defined as: under the assay conditions, the amount of enzyme required to catalyze the cleavage of oligogalacturonic acid substrate to generate 1 μmol of double bonds per minute is defined as 1 U. The specific methods for determining enzyme substrate specificity are as follows: Using 1 mg / mL pectin, GalA3, and GalA4 as substrates, the enzymes were reacted at an initial pH of 7.5 and an initial temperature of 40℃ for 30 min. The highest activity was taken as 100%, and the relative enzyme activity was calculated. For the optimum temperature determination, using the optimal substrate, the enzyme activity was measured at different temperatures (30℃-70℃), and the highest activity was taken as 100%, and the relative activity was calculated. For the optimum temperature determination, the enzyme activity was measured at the optimum temperature using different pH buffers (e.g., pH 3-11), using the same method as above. For the enzyme stability determination, the enzyme was placed in buffers at different temperatures (20℃-80℃) and incubated for 0.5, 1, 1.5, 2, 2.5, and 3 h, and the relative enzyme activity was measured to test its thermal stability. Similarly, the enzyme was incubated in buffer solutions with different pH values ​​(5.0-9.0) for 0.5, 1, 1.5, and 2 h, and the remaining enzyme activity was measured to test pH stability. To determine the effect of metal ions on enzyme activity, the enzyme solution was placed in 10 mmol / L buffer solutions containing different metal ions (optimal pH) and incubated at 45 ℃ for 30 min. Enzyme activity was measured at the optimal temperature, with the reaction system without enzyme as the control group. Enzyme kinetic parameters were determined as follows: under the optimal pH and temperature conditions, the relative enzyme activity was measured using 0-1 mg / mL GalA3 as substrate. The maximum reaction rate (Vmax) and Michaelis constant (Km) of OGL were calculated by double reciprocal plotting.

[0037] Enzyme activity increases continuously when the temperature is between 30 and 45℃; it gradually decreases when the temperature is between 45 and 70℃, with the optimal temperature for enzyme activity at 45℃. At 40-50℃, enzyme activity remains at approximately 80% or higher of its maximum, exhibiting good stability. Enzyme activity is low at pH 3.0-4.0; it increases sharply at pH above 4.0; and reaches its maximum at pH 6.0. However, as the pH continues to increase, enzyme activity begins to decrease, with the optimal pH being 6.0. The enzyme exhibits good stability between pH 6 and 8.

[0038] The enzyme activity was detected using tobacco pectin, pectin, galacturonic acid trisaccharide (GalA3), and galacturonic acid tetrasaccharide (GalA4) as substrates, respectively. Figure 4 As shown, the enzyme exhibits the highest activity when using GalA3 as a substrate, followed by GalA4. Pectin also produces a certain amount of galacturonic acid and oligogalacturonic acid when used as a substrate, while tobacco pectin shows the lowest activity. The relatively low enzyme activity when using pectin and tobacco pectin as substrates is due to the high degree of methylation of pectin (over 80%), making it a weak substrate for oligogalacturonic acid lyases. Tobacco pectin, due to its complex structure resulting from purification methods, contains fewer low-esterification regions. The relatively high enzyme activity when using GalA3 and GalA4 as substrates indicates that the enzyme has the ability to degrade oligogalacturonic acid, and that oligogalacturonic acid lyases show stronger specificity for GalA3.

[0039] Based on the double reciprocal curve method, the kinetic equation for the accelerated reaction was obtained as: y = 3.232x + 1.031, R² = 0.9912. The measured Km and Vm of OGL for GalA3 were 3.135 mg / mL and 0.970 U / mL, respectively. This indicates that oligogalacturonate lyase has a high affinity for the substrate and strong substrate specificity, providing a new approach for the decomposition of tobacco pectin.

[0040] Example 3: Application of pectinase (oligogalacturonate lyase) in tobacco leaf quality improvement The tobacco leaves (China Tobacco 100 initial-cured tobacco leaves) after initial curing and stem removal were rehydrated using a steamer and then shredded using a shredder. The shredded leaves were stored in resealable plastic bags for later use. The oligogalacturonic acid lyase prepared in Example 1 was evenly sprayed onto the tobacco shreds at a ratio of 1:1000 by weight, mixed thoroughly, and then statically treated in a constant temperature and humidity incubator at 40°C and 60% humidity for 6 hours. After treatment, the shredded leaves were sterilized at 160°C and dried.

[0041] The effects of enzymatic fermentation on the aroma components of tobacco are shown in Table 1. After OGL treatment, the content of 20 aroma substances increased significantly, with 9 substances increasing by 15%–30% and the other 9 increasing by more than 30%. In particular, four compounds, namely solanone, 4,7,9-macrostigmatrien-3-one, dihydroactinolone, and lolioester, showed significant effects on enhancing the aroma of tobacco, increasing by 43.95%, 62.86%, 47.37%, and 37.44%, respectively. Furan derivatives such as furfural and 5-hydroxymethylfurfural, as products of sugar thermal degradation and Maillard reaction, impart caramelization characteristics to the smoke; the natural lactone lolio ester provides fruity aromas and a subtle alcoholic note, adding sweetness; solanone, a characteristic tobacco component, enhances the fermented feel and body, while 4,7,9-mega-stigmatrien-3-one contributes woody notes, improving smoothness and synergistically enhancing sweetness; phytone enhances cigarette aroma and reduces off-flavors; in addition, phenylacetaldehyde and 4-hydroxy-β-dihydrodamascone together provide violet and rose notes. These compounds work synergistically to shape the complex and unique aroma of tobacco.

[0042] Table 1 Comparison of chemical components in tobacco leaves before and after OGL treatment

[0043] The tobacco leaves were processed into cigarettes, and a panel of professional judges conducted a sensory evaluation, with scores averaged. The sensory evaluation results before and after treatment are shown in Table 2. The results indicate that OGL treatment significantly improved the aroma quality and quantity of the tobacco leaves, increased their concentration, and slightly enhanced their smoothness. This demonstrates that the added aroma components from OGL-treated tobacco leaves can significantly improve cigarette quality.

[0044] Table 2 Sensory evaluation of tobacco leaves before and after OGL treatment

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pectinase, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. A pectinase-encoding gene, characterized in that, The pectinase described in claim 1 is encoded.

3. The pectinase encoding gene according to claim 2, characterized in that, The nucleotide sequence of the pectinase encoding gene is shown in SEQ ID NO:

2.

4. A recombinant expression vector, characterized in that, Includes the vector and the pectinase encoding gene as described in any one of claims 2-3.

5. The recombinant expression vector according to claim 4, characterized in that, The carrier is pET-28a(+).

6. A recombinant expression transformant, characterized in that, It includes the recombinant expression vector according to any one of claims 4-5.

7. The application of the pectinase according to claim 1, the pectinase encoding gene according to any one of claims 2-3, the recombinant expression vector according to any one of claims 4-5, and the recombinant expression transformant according to claim 6, characterized in that, The pectinase of claim 1, the pectinase encoding gene of any one of claims 2-3, the recombinant expression vector of any one of claims 4-5, or the recombinant expression transformant of claim 6 are used to degrade tobacco pectin, release aroma components, and improve the aroma quality of tobacco.

8. A method for preparing pectinase, characterized in that, Includes the following steps: (1) The pectinase encoding gene was introduced into the vector plasmid pET-28a(+) with a histidine tag to obtain a recombinant expression vector; the recombinant expression vector was transformed into Escherichia coli BL21(DE3) competent cells to obtain recombinant expression transformants; (2) The recombinant expression transformant was inoculated into LB liquid medium containing kanamycin and cultured overnight with shaking; then transferred to fresh LB medium and cultured until OD600 reached 0.8, then IPTG was added to induce expression. (3) After induction, the bacterial cells were collected by centrifugation. The bacterial cells were washed with sterile water and centrifuged to collect the precipitate. The bacterial cells were resuspended in lysis buffer and broken up in an ice bath. The crude enzyme solution after breaking up was purified to obtain pectinase.

9. A method for enhancing the flavor of tobacco, characterized in that, Includes the following steps: (1) Mix pectinase with tobacco at a mass ratio of 1:2000-1:50; (2) Static treatment at a temperature of 30-55℃ for 1-10 hours; (3) Inactivate the tobacco at a temperature of 50-200℃ for 1-60 minutes to obtain the flavored tobacco.

10. The tobacco flavoring method according to claim 9, characterized in that, In step (1), pectinase and tobacco are mixed at a mass ratio of 1:1000; In step (2), the sample is statically treated at 40°C for 6 hours. In step (3), the virus is inactivated at a temperature of 160°C for 1-2 minutes.

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