Novel thermophilic beta-1, 4-endomannase

Abstract

The invention belongs to the technical field of enzyme biochemical engineering, and particularly relates to novel thermophilic beta-1, 4-endo-mannase, the amino acid sequence of the enzyme is as shown in SEQ ID No.1, and the optimum reaction temperature is 90-100 DEG C. The thermophilic beta-mannase is applied to a tobacco material, and the thermophilic beta-1, 4-endo-mannase is obtained after the thermophilic beta-1, 4-endo-mannase is applied to the tobacco material. The integrated process of on-line addition, low-temperature pre-catalysis, high-temperature efficient catalysis and synchronous inactivation is realized, and the bottleneck that the existing enzyme preparation cannot be applied to the high-temperature link of the silk thread production line is broken through. By applying the enzyme, the filling value of tobacco shreds can be remarkably increased, the physical indexes and sensory quality of cigarettes are optimized, the process is green and efficient, and an additional inactivation step is not needed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of enzyme biochemical engineering, and particularly relates to a novel thermophilic beta-1, 4-endomannanase. BACKGROUND

[0002] In the tobacco industry, the further improvement of raw material quality is often limited by the structure of cell wall. Especially, the non-starch polysaccharides such as mannan contained therein can significantly enhance the rigidity of tobacco tissue, thereby leading to the decrease of filling value, the increase of resistance, and the adverse effects on the fullness of combustion and the efficiency of aroma release.

[0003] Beta-mannanase can specifically degrade mannan and is an effective biological treatment means for improving the above problems. However, the existing commercial enzyme preparations generally have the defect of insufficient heat resistance, and the optimum action temperature thereof is usually not more than 60℃, which is difficult to withstand the subsequent high-temperature processing process. In order to avoid the instant inactivation of the enzyme, the current application usually adopts the "off-line treatment" mode, that is, the enzyme preparation is added in the feeding or leaf storage link at room temperature. Although this mode can play a role to a certain extent, it also has the following significant drawbacks: first, the reaction efficiency is low, the enzyme activity is insufficient at low temperature, and it usually takes several hours to show effect, which seriously slows down the production rhythm;

[0004] second, there is a quality risk, and long-term mild environment is easy to breed microorganisms, causing mildewing of tobacco; and third, the process controllability is poor, and the treatment effect is easily affected by production scheduling fluctuations, leading to unstable product quality.

[0005] It is worth noting that the modern tobacco primary processing technology has generally adopted gradient baking technology, which includes a key 90℃-100℃ medium-high temperature pretreatment stage for promoting the conversion of aroma precursor substances, and then enters the high-temperature drying and setting stage above 120℃. This precise temperature control strategy not only can improve the sensory quality of cigarettes, but also provides a new breakthrough point for solving the bottleneck of enzyme application.

[0006] Based on this, an ideal process mode can be conceived: the enzyme preparation is added in the feeding stage, so that it can play a certain catalytic role at a lower temperature in the storage stage, thereby realizing the preliminary degradation of the substrate and the reaction preparation; then, when the material enters the 90℃-100℃ pretreatment stage, the enzyme activity rapidly increases to the peak value, and the efficient catalysis is completed in a short time; and then, in the subsequent drying stage above 120℃, the enzyme is naturally and completely inactivated. Through the integrated process of "on-line addition-low temperature pre-catalysis-high temperature efficient catalysis

[0007] —synchronous inactivation", the treatment efficiency can be significantly improved, the quality risk can be effectively avoided, and the process can be precisely controlled.

[0008] However, the core obstacle of the implementation of this path is that the existing beta-mannanase is rapidly inactivated above 60℃, and cannot work in the pretreatment window of 90-100℃. Therefore, developing a new type of heat-resistant beta-mannanase with an optimal action temperature highly matched with the pretreatment stage is the key to solving the above problems.

[0009] To solve the above problems, the present application is proposed. SUMMARY

[0010] The technical solutions adopted by the present application are as follows:

[0011] The present application provides a novel thermophilic beta-1, 4-endomannanase in the first aspect, and the amino acid sequence thereof is shown in SEQ ID No. 1.

[0012] Preferably, the optimal reaction temperature thereof is 90-100℃. The optimal reaction temperature of the thermophilic beta-1, 4-endomannanase C4111 is 95℃, and the optimal pH is 5.5.

[0013] The present application provides a DNA molecule encoding the thermophilic beta-1, 4-endomannanase in the second aspect.

[0014] Preferably, the nucleotide sequence thereof is optimized for expression in E. coli.

[0015] The present application provides a recombinant expression vector containing the DNA molecule in the third aspect.

[0016] Preferably, the recombinant expression vector is plasmid pET28a.

[0017] The present application provides a host cell containing the recombinant expression vector in the fourth aspect.

[0018] Preferably, the host cell is E. coli BL21 (DE3).

[0019] The present application provides a tobacco processing method in the fifth aspect, which comprises: applying the thermophilic beta-1, 4-endomannanase in claim 1 or 2 to tobacco material, and then subjecting the tobacco material to a treatment stage with a temperature of 90-100℃, and then to a baking stage with a temperature higher than 120℃. The beta-1, 4-endomannanase C4111 is inactivated at 120℃.

[0020] The present application has the following beneficial effects:

[0021] 1. The optimal temperature of the novel thermophilic beta-mannanase C4111 of the application is as high as 95℃, and it can still maintain more than 80% of the enzyme activity after 2 hours of treatment at 90℃. This thermal stability enables it to directly withstand and efficiently act on the key 90℃-100℃ pretreatment stage in the tobacco gradient curing process, breaking through the technical bottleneck of existing enzyme preparations (optimal temperature ≤60℃) inactivating instantly in this high-temperature window, and laying a material foundation for realizing online application.

[0022] 2. Based on the unique activity range of the enzyme, the application first proposes a new enzyme treatment method of "online addition-low temperature pre-catalysis-high temperature efficient catalysis-synchronous inactivation" seamlessly integrated into the current feeding, storage and gradient curing process. This method discards the traditional time-consuming and energy-consuming independent offline treatment step, which has quality risks, and directly embeds the enzyme treatment into the main process of the tobacco processing line, realizes the continuous production, and significantly reduces the comprehensive cost. The process utilizes the catalytic peak of the enzyme in the pretreatment stage (90-100℃), and naturally utilizes the subsequent curing stage above 120℃ to completely inactivate the enzyme, which is precise and green, and does not require additional intervention.

[0023] 3. The enzyme can efficiently act on the mannan in the cell wall under the high-temperature pretreatment environment of tobacco processing, realizing efficient initiation and completion of the reaction. This direct action on the key substrate at the optimal temperature point of the process ensures the high efficiency of mannan degradation, which can effectively solve the problems of low filling value, high resistance, poor combustion and other problems caused by mannan. Ultimately, this technology can significantly optimize the physical indicators and sensory quality of cigarettes, which is manifested as smoother smoke, more complete combustion, improved aroma release, and fundamental guarantee of product batch quality stability.

[0024] 4. The comprehensive characteristics of the enzyme of the application meet the harsh requirements of online tobacco processing, which other enzymes with only general heat resistance do not have. The novel thermophilic beta-mannanase C4111 of the application is screened from a specific high-temperature hot spring environment by metagenomic technology, and its comprehensive characteristics (such as 95℃ optimal activity, 90℃ long-term stability, highest activity near pH 5.5, etc.) constitute a unique and mutually supporting technical combination, enabling it to perform "high-temperature efficient catalysis and subsequent complete inactivation" in this specific process path. There may be other heat-resistant beta-mannanases in the field, but their comprehensive performance spectrum (such as optimal temperature, pH adaptability, thermal stability balance point) may not completely match the entire dynamic process of tobacco gradient curing, so the application provides a complete technical solution to solve a specific technical problem, rather than a simple replacement of a single attribute.

[0025] 5、After the high-temperature beta-mannanase C4111 is treated on line according to the application, the filling value of the cut tobacco can be increased by more than 15%, and the suction resistance is effectively reduced. This shows that it can still maintain good catalytic activity and substrate specificity in the tobacco system containing various complex chemical components. The professional evaluation results show that the sensory quality of the treated cigarette product is effectively improved, mainly in that the smoothness of smoke is improved, the irritation is reduced, and the aftertaste is pure. These results collectively show that the enzyme treatment can effectively target the degradation of mannan in a complex system, achieving the purpose of improving the processing performance and smoking experience while not introducing obvious negative effects. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0027] Figure 1 A curve graph of the influence of pH on the activity of the thermophilic beta-1,4-endomannanase C4111;

[0028] Figure 2 A curve graph of the influence of temperature on the activity of the thermophilic beta-1,4-endomannanase C4111;

[0029] Figure 3 A column graph of the influence of metal ions on the activity of the thermophilic beta-1,4-endomannanase C4111. DETAILED DESCRIPTION

[0030] The present application will be further described by the following examples, which are not limited to the present embodiments. The experimental methods not specified in the examples are usually carried out according to the conventional conditions and the conditions described in the manual, or using the general equipment, materials, reagents, etc. suggested by the manufacturer, and if not otherwise specified, they can be obtained from commercial channels.

[0031] Example 1: Gene cloning, expression and preparation of crude enzyme solution of the thermophilic beta-1,4-endomannanase C4111

[0032] 1. Obtaining of the thermophilic beta-1,4-endomannanase C4111 gene c4111

[0033] The soil sample was collected from a high-temperature hot spring in Yuanjiang County, Yunnan Province, and the genomic DNA of the microbial community in the environment was extracted for second-generation sequencing of the metagenome. After quality control of the sequencing results, the data were subjected to binning, assembly and annotation to obtain open reading frames (ORFs) encoding amino acid sequences similar to known endomannanases (sequence identity > 20%, expectation value E-value < 1e-20). Primers were designed based on the base sequence information of the ORFs, and a candidate gene c4111 (as shown in SEQ ID No. 2) was cloned from the metagenomic DNA by PCR. The gene was codon-optimized for E. coli and synthesized by GenScript Biotech (Shanghai) Co., Ltd., and cloned into the pET-28a(+) vector to construct the recombinant plasmid pET28a-c4111.

[0034] 2. Plasmid extraction

[0035] The SanPrep column plasmid DNA mini-extraction kit (GenScript Biotech (Shanghai) Co., Ltd.) was used to extract the recombinant plasmid pET28a-c4111 according to the instructions.

[0036] 3. Transformation

[0037] The recombinant plasmid dry powder (about 4 μg) pET28a-c4111 after gene synthesis was placed in a centrifuge at 12000 r / min for 2 min, 40 μL of ddH2O was added, and the final concentration of the recombinant plasmid was 100 ng / μL. The prepared E. coli BL21(DE3) competent cells were transformed by heat shock method. When the E. coli competent cells were in an ice-water mixture, 20 μL of the recombinant plasmid was immediately added to the BL21(DE3) competent cells, and the mixture was gently mixed by a pipette gun and then incubated on ice for 30 min. The E. coli competent cells after ice incubation were placed in a 42°C water bath for 90 s (opening of the cell membrane, and the plasmid entered the cells), and then immediately taken out and placed on ice for 5 min (closing of the cell membrane). 500 μL of LB liquid medium without antibiotics was added to the centrifuge tube, and the medium was incubated in a constant-temperature shaker at 37°C, 220 r / min for 45 min. 200 μL of the above-mentioned medium was taken and spread on solid LB medium containing kanamycin sulfate (final concentration 50 μg / mL) using a sterile spreader. The inoculated medium was first placed in a 37°C incubator for 30 min, and then incubated upside down for 12-16 h to obtain the E. coli BL21(DE3) strain containing the recombinant plasmid.

[0038] 4. Induced expression of recombinant thermophilic β-1,4-endomannanase C4111

[0039] Monoclonal small volume (5 mL) culture: pick recombinant E. coli BL21 (DE3) monoclonal in 10 mL centrifuge tube, 5 mL LB resistance liquid medium (Kan 50 μg / mL), 37°C, 220 rpm, constant temperature shaker culture overnight 12-16 h to obtain E. coli BL21 (DE3) seed liquid containing recombinant plasmid.

[0040] 5. Expansion culture

[0041] Take 3 mL of bacterial liquid (1% inoculation amount) and inoculate 300 mL of fresh LB liquid medium (Kan 50 μg / mL), 37°C, 220 rpm, constant temperature shaker culture 4-6 h to OD 600 = 0.4-0.6, add 714 μL IPTG (final concentration 0.5 mM), constant temperature shaker 16°C, 220 rpm culture 20 h, centrifuge 8000 rpm / min, 5 min, discard the supernatant and collect the bacterial body, ensure that each centrifuge tube collects 4.0 g of bacterial body, and store at -20°C to obtain E. coli BL21 (DE3) bacterial body with complete natural thermophilic β-1,4-endomannanase C4111 expression.

[0042] 6. Crude enzyme acquisition

[0043] Add 4.0 g of bacterial body collected in a 50 mL centrifuge tube to 20 mL of non-denaturing lysis buffer, mix well with a pipette gun, centrifuge at 8000 r / min for 5 min, discard the supernatant, collect the bacterial body, add 20 mL of non-denaturing lysis buffer and 20 μL of lysozyme, mix well with a pipette gun, mix well with a vortex instrument, and incubate on ice for 30 min. Use ultrasonic crusher to perform ultrasonic crushing, power 40 w, amplitude rod 6, engineering 1, ultrasonic working 2 s, stop 8 s, whole process crushing on ice for 30 min, centrifuge at 8000 r / min for 15 min, take the supernatant, place it in a 70°C constant temperature water bath for preheating for 2 h, centrifuge at 8000 r / min for 15 min, take the supernatant to remove most of the E. coli impurities to obtain high-temperature impurity-removed natural thermophilic β-1,4-endomannanase C4111 crude enzyme liquid.

[0044] Example 2 Enzymatic properties and enzyme activity experiment of thermophilic β-1,4-endomannanase C4111

[0045] The activity of the thermophilic β-1,4-endomannanase C4111 was determined by the dinitrosalicylic acid method (DNS). The crude enzyme solution of the thermophilic β-1,4-endomannanase C4111 was pre-treated at 70°C for 2 h in a water bath, and then centrifuged at 8000 rpm / min for 15 min. After diluting the supernatant to an appropriate multiple, 50 μL of the solution was added to 50 μL of konjac mannan (0.3% in 50 mM citric acid-disodium hydrogen phosphate buffer at pH 5.5) in a 96-well PCR plate. After incubation at 95°C and pH 5.5 for 20 min in a water bath, 100 μL of DNS was added, and the mixture was incubated at 90°C for 10 min in a water bath. After cooling to room temperature in an ice box, 50 μL of the reaction solution was added to 150 μL of deionized water in a 96-well enzyme-labeled plate, and the absorbance of the sample was determined by an enzyme-labeled instrument. 540nm The determination of the following enzymatic properties was performed by this method.

[0046] The enzyme activity unit (U) was defined as the amount of enzyme required for hydrolyzing 1 μmol of reducing sugar per minute under the determination conditions.

[0047] 1. Optimum pH of the thermophilic β-1,4-endomannanase C4111

[0048] The determination of the optimum pH was performed at 95°C using different buffer systems, including disodium hydrogen phosphate-citric acid buffer (200 mM, pH 3.0-7.0) and glycine-NaOH buffer (20 mM, pH 8.0-12.0). The enzyme activity at the optimum pH was defined as 100% activity.

[0049] According to the experimental results, the optimum reaction pH of the thermophilic β-1,4-endomannanase C4111 was 5.5, as shown in Figure 1

[0050] 2. Optimum temperature of the thermophilic β-1,4-endomannanase C4111

[0051] The optimum temperature was determined by measuring the activity of the enzyme at a temperature ranging from 30 to 115°C. The activity of the enzyme at the optimum temperature was defined as 100%.

[0052] As shown in Figure 2 , the optimum reaction temperature of the enzyme was 95°C.

[0053] 3. Effect of metal ions on the enzyme activity

[0054] In order to observe the effect of different metal ions on the catalytic activity of the thermophilic β-1,4-endomannanase C4111, the metal salts (Na + , Al 3+ , K + ​Ni 2+ Fe 2+ Co 2+ Hg 2+ Mg 2+ Mn 2+ Cu 2 + and Zn 2+ The effect of metal ion deficiency on enzyme activity was investigated. Enzyme activity was determined under optimal reaction conditions of 95°C and pH 5.5. The absence of metal ions was defined as 100%, and relative enzyme activity was defined as activity relative to the control.

[0055] like Figure 3 As shown, Hg 2+ It can significantly inhibit the activity of thermophilic β-1,4-endomannanase C4111, 1 mM Hg 2+ In its presence, the remaining enzyme activity of C4111 is less than 25%; K + Ni 2+ Mg 2+ and Zn 2+ It promotes the enzyme activity of C4111.

[0056] Example 3: Validation of the effect of thermophilic β-1,4-endomannanase C4111

[0057] 1. Sample preparation and processing

[0058] On a pilot production line for tobacco processing, the same batch of K326 first-cured tobacco leaves were taken, and after standardized rehydration and shredding, they were divided into two groups and subjected to the exact same process, the only difference being the additives:

[0059] Experimental group: In the feeding process, C4111 enzyme solution was diluted with 50mM citrate-disodium hydrogen phosphate buffer (pH 5.5) to an activity of 50U / mL, and then sprayed evenly on the leaf fibers together with the feed solution.

[0060] Control group: During the feeding process, under the same process parameters, only an equal amount of 50mM citrate-disodium hydrogen phosphate blank buffer (pH 5.5) and feed solution were sprayed.

[0061] Subsequently, both sets of blades undergo the same subsequent processes:

[0062] Leaf storage: Store at room temperature (25±3℃) for 60±10 minutes to allow the liquid to penetrate evenly.

[0063] High-temperature and high-efficiency catalysis: Enter the pretreatment stage at 90-95℃ and treat for 30 minutes.

[0064] Synchronous inactivation: Enter the drying stage at >120℃ and dry and set for 8-12 minutes.

[0065] Collect the finished leaf fibers and equilibrate them for 48 hours under standard conditions (temperature 22±1℃, relative humidity 60±2%) for testing.

[0066] 2. Physical index analysis

[0067] Key physical parameters of the two groups of leaf filaments were measured, and the results are shown in the table below:

[0068] Table 1: Comparison of Main Physical Indicators of Tobacco Shreds (Mean ± Standard Deviation)

[0069]

[0070] After undergoing identical physicochemical conditions (temperature, humidity, and mechanical action), the experimental group showed significantly higher tobacco filling value and significantly lower draw resistance than the control group. This strongly suggests that the observed improvement is indeed due to the catalytic effect of the C4111 enzyme, rather than the pretreatment process itself.

[0071] 2. Sensory evaluation and absorption analysis

[0072] The two groups of tobacco leaves were rolled into cigarette samples with identical physical parameters. According to GB5606.4-2005 "Cigarettes Part 4: Sensory Technical Requirements", a professional panel of seven senior tasters conducted a double-blind tasting. Quantitative scoring (out of 9 points) was performed on six dimensions: gloss, aroma, harmony, off-flavors, irritation, and aftertaste, and qualitative evaluation opinions were recorded. The tasting results are shown in Table 2 below:

[0073] Table 2 Sensory evaluation scores of cigarette samples (average, n=7)

[0074]

[0075]

[0076] The evaluation results show that, after excluding interference from the process background, the enzyme-treated group has a significant advantage in sensory quality, confirming that the C4111 enzyme has a clear and positive contribution to improving the smoking quality of cigarettes.

[0077] This embodiment, by setting up a scientific control (replacing only with buffer solution), verifies that thermophilic β-mannanase C4111 is the fundamental reason for the significant synergistic improvement in the physical processing properties of tobacco and the sensory smoking quality of cigarettes in the integrated process of "online addition—low-temperature pre-catalysis—high-temperature high-efficiency catalysis—simultaneous inactivation". This design ensures the reliability of the experimental conclusions and provides strong evidence for the innovation and practicality of this invention.

[0078] The application has been described above by way of example only, and it should be appreciated that any simple modification, change or other equivalent replacement which does not depart from the core of the application falls within the scope of the application.

[0079]

[0080]

Claims

1. A novel thermostable β-1,4-endomannanase, characterized in that, The amino acid sequence of which is shown as SEQ ID No.

1.

2. The novel thermophilic β-1,4-endomannanase according to claim 1, characterized in that, The optimum reaction temperature thereof is 90-100°C.

3. A DNA molecule encoding the thermophilic β-1, 4-endomannanase of claim 1 or 2.

4. The DNA molecule of claim 3, wherein, The nucleotide sequence of which is optimized for expression in E. coli.

5. A recombinant expression vector containing the DNA molecule of claim 3 or 4.

6. The recombinant expression vector of claim 5, wherein, It is plasmid pET28a.

7. A host cell containing the recombinant expression vector of claim 5 or 6.

8. The host cell of claim 7, wherein, The host cell is E. coli BL21 (DE3).

9. A method of processing tobacco, characterized by, The method comprises applying the thermophilic β-1, 4-endomannanase of claim 1 or 2 to tobacco material, followed by subjecting the tobacco material to a treatment phase at a temperature of 90-100°C, followed by a curing phase at a temperature higher than 120°C.

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