Method and application for producing manooligosaccharide by mannanase ManG3

Abstract

The present invention belongs to the field of bioengineering technology, and particularly relates to a method and application of producing mannan oligosaccharides by mannanase ManG3. The present invention provides an application of mannanase ManG3 in the production of mannan oligosaccharides, and the amino acid sequence of the mannanase ManG3 is as shown in SEQ ID NO.1. The mannanase ManG3 of the present invention has good pH stability and temperature stability, and has a wide range of uses, solving the problems of complex environment, uneven pH and temperature distribution during the industrial application of mannanase; and the mannanase ManG3 can effectively degrade various substrates such as locust bean gum, konjac mannan, guar gum, etc., especially hydrolyze locust bean gum to produce mannan oligosaccharides with a degree of polymerization of 2 to 5, and has potential application value in the production of composite mannan oligosaccharides.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a method and application for producing manno-oligosaccharides by mannanase ManG3. Background Art

[0002] Mannanase (β-mannanase, EC 3.2.1.78) is a hemicellulose hydrolase widely present in bacteria, fungi, plants and animals. According to its catalytic mechanism and sequence homology, mannanases are mainly divided into GH5, GH26, GH76 and GH120 families. Among them, mannanases of the GH5 family have relatively high catalytic activity and substrate specificity.

[0003] Mannanase has currently been widely used in multiple fields such as food, medicine and chemical industry. However, mannanase is easily inactivated at higher temperatures, which limits its application in some high-temperature industrial processes. The stability of mannanase under extreme pH conditions is also poor, which restricts its application in acidic or alkaline environments. For example, the optimal temperature of the mannanase mutant disclosed in Chinese Patent CN109251913B is 45°C, and the optimal pH is 6.0. There are still some disadvantages in aspects such as production cost, stability, substrate specificity and application effect of mannanase, and further research and improvement are needed to overcome these problems to improve its application value in various fields. Summary of the Invention

[0004] The purpose of the present invention is to provide a method and application for producing manno-oligosaccharides by mannanase ManG3. The mannanase ManG3 of the present invention has good pH stability and temperature stability, and has a wide range of uses, and can be used for producing manno-oligosaccharides.

[0005] The present invention provides an application of mannanase ManG3 in the production of manno-oligosaccharides, and the amino acid sequence of the mannanase ManG3 is as shown in SEQ ID NO.1.

[0006] As a preferred embodiment, the nucleotide sequence of the coding gene of the mannanase ManG3 is as shown in SEQ ID NO.2.

[0007] As a preferred embodiment, the production method of the mannanase ManG3 includes: culturing recombinant bacteria and inducing the expression of mannanase ManG3; the recombinant bacteria include the coding gene of the mannanase ManG3.

[0008] As a preferred embodiment, the basic bacteria of the recombinant bacteria include Escherichia coli.

[0009] The present invention also provides a method for producing mannan oligosaccharides, which uses mannanase ManG3 to degrade a substrate; the substrate includes at least one of locust bean gum, konjac mannan, and guar gum; the amino acid sequence of the mannanase ManG3 is as shown in SEQ ID NO.1.

[0010] The present invention also provides an application of mannanase ManG3 in breaking the gel of oilfield fracturing fluid, and the amino acid sequence of the mannanase ManG3 is as shown in SEQ ID NO.1; the fracturing fluid includes guar gum fracturing fluid.

[0011] Beneficial effects:

[0012] The present invention provides an application of mannanase ManG3 in the production of mannan oligosaccharides, and the amino acid sequence of the mannanase ManG3 is as shown in SEQ ID NO.1. The mannanase ManG3 of the present invention is suitable for use in the range of pH 5.0 - 9.0 and temperature 40 - 70 °C, has good pH stability and temperature stability, has a wide range of use, and solves the problems of complex environment, uneven pH and temperature distribution during the industrial application of mannanase; and the mannanase ManG3 can effectively degrade various substrates such as locust bean gum, konjac mannan, and guar gum, especially hydrolyzes locust bean gum to produce mannan oligosaccharides with a degree of polymerization of 2 - 5, and has potential application value in the production of composite mannan oligosaccharides. Description of the drawings

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0014] Figure 1 It is the SDS-PAGE result diagram after purification of mannanase ManG3 in Example 2;

[0015] Figure 2 It is the thin layer chromatography result diagram of the product of mannanase ManG3 hydrolyzing locust bean gum in Example 3;

[0016] Figure 3 It is the quantitative result diagram of mannanase ManG3 hydrolyzing locust bean gum in Example 3;

[0017] Figure 4 It is the standard curve diagram of D-mannose in Example 4;

[0018] Figure 5 It is the enzyme activity detection result diagram under different temperature conditions in Example 4;

[0019] Figure 6 It is the enzyme activity detection result diagram under different pH value conditions in Example 4;

[0020] Figure 7 It is the viscosity change diagram of the guar gum fracturing fluid in Example 6. Detailed implementation manners

[0021] The present invention provides an application of mannanase ManG3 in the production of manooligosaccharides. The amino acid sequence of the mannanase ManG3 is shown as SEQ ID NO.1: MKKKWIAAISSVVLGCSTVMMPFASSFTVNAADTRSGFVTTNGTQFMLDGSTFYYAGTNNYYLNFKPKESVDAVLEDAAEMGLKVVRTWGNLDAGVKTDKVSDKGYTVFTDNVDGSGEKEGVYYQYFDADLGRPVVNEGEDGLQKLDYALYKAEQEGIKLLITFTNNWEAFGGMGQYVQWAKLAGENVSGHDDFYTNETIKGWYKDYIKTLLNHENVYTGVKYKDDPTIFSWELANEPRCESDAGCENNTVVKWATEMSAYVKSIDNNHMLAVGDEGFYNYGYNDFPEGDHKYVYHGSSGMDYLQLTSIPDIDFGTLHVYCDQWGLTKEQGEFWFKKHGEDTAAMNKPLIAEEFGWKDQGERADVYTDWFNIFEGNTYEGVEFAGTNYWMLASMDGDSLYQDYDGYTVYFRDYPKTNDARDVIMAHAERMNARNAQNSVSPKKADFDIVNPKDVTLQATIKLGSISGLQLDDTTLTADTDYSISGTTVTISKNALQSLELGNHKVTLLTTEGAQPTALIRVYDSTAEEQSRSVIDDFESYADSKAVAAAYQQNSSGDSLTLSLDTEHVKNGKAALKYDYSVTDGGAGYCGATKNLGSADWTGFDGIRFWILSDGSNRETTFQFVDGAGAYWESVQKVTAEEGWTEVKIPFSDFYVQQWGTAAETPTLSGVKEFSLYTGQNGNPGTGVWYFDDIGLYHDGTVTVPDAEATETEATFDPQDPTDIIFTIITNGHVLTGITEDGNALQQGTDYAVNGSQFRFNRSYLETLTEGKHTLHVTFATCPDVVLTIQVLGQSGTTETTVTTEETTTTETETTSSAADTTSETASSESTTETVASTTETTAQTTASSSEVLGQVLYGDVNLDGRVDITDAVLLNKATAGVVTLNDAARQNADCDANGMTDTNDATVLLRFLVQIINNLPEMAE。The mannanase ManG3 of the present invention comprises five domains, which are sequentially annotated as endo-1,4-β-mannosidase, carbohydrate-binding module X2 (CBM_X2), carbohydrate-binding module 11 (CBM_11), carbohydrate-binding module X2 (CBM_X2), and dockerin domain from the N-terminus to the C-terminus.

[0022] The mannanase ManG3 of the present invention is suitable for use in the range of pH 5.0 to 9.0 and temperature 40 to 70 °C, has good pH stability and temperature stability, has a wide range of use, and the mannanase ManG3 can produce manno-oligosaccharides using a variety of substrates, and the substrates may include at least one of locust bean gum, konjac mannan, and guar gum.

[0023]

[0024] As an implementation manner, the production method of the mannanase ManG3 includes culturing a recombinant bacterium and inducing the expression of the mannanase ManG3; the recombinant bacterium includes the coding gene of the mannanase ManG3. As an implementation manner, the basic bacterium of the recombinant bacterium is Escherichia coli. As an implementation manner, the preparation of the recombinant bacterium includes: transferring a recombinant vector containing the coding gene of the mannanase ManG3 into Escherichia coli competent cells, resuscitating on a shaker at 37 °C and 220 rpm for 60 min, coating on an LB plate containing kanamycin and culturing overnight, picking several single colonies with a sterile inoculation loop and using primers for verification by bacterial liquid PCR and DNA sequencing verification, obtaining positive transformants with correct sequencing, which are the recombinant bacteria. The present invention cultures the recombinant bacterium until the OD 600 value is in the range of 0.6 to 0.8, adding isopropyl-β-D-thiogalactoside for induced expression to produce the mannanase ManG3. As an implementation manner, the temperature of the induced expression is 16 °C; the time of the induced expression is 20 to 22 h. As an implementation manner, the final concentration of isopropyl-β-D-thiogalactoside is 0.5 mmol / L.

[0025] As an implementation manner, after the induced expression ends in the present invention, it further includes collecting the fermentation broth and centrifuging to collect the bacterial cells; after resuspending and centrifuging the bacterial cells, collecting the washed bacterial cells; after ultrasonically disrupting the washed bacterial cells, centrifuging and collecting the supernatant to obtain the crude enzyme solution of the mannanase ManG3. As an implementation manner, the working conditions of the ultrasonic disruption are: 300 W, working for 4 s, intermittent for 6 s, and the total working time is 15 min.

[0026] The present invention also provides a method for producing manooligosaccharides, using the mannanase ManG3 to degrade a substrate; the substrate includes at least one of locust bean gum, konjac mannan, and guar gum; the amino acid sequence of the mannanase ManG3 is as shown in SEQ ID NO.1. In the present invention, the mannanase ManG3 can degrade locust bean gum into mannobiose, mannotriose, mannotetraose, and mannopentaose.

[0027] The present invention also provides an application of mannanase ManG3 in breaking the gel of oilfield fracturing fluid. The amino acid sequence of the mannanase ManG3 is as shown in SEQ ID NO.1; the fracturing fluid includes guar gum fracturing fluid. The guar gum in the guar gum fracturing fluid can form a colloidal solution with high viscosity, enabling the fracturing fluid to have sufficient viscosity to carry proppants into the formation fractures. After the fracturing operation is completed, it is necessary to break the gel of the fracturing fluid. At present, chemical degradation method is generally used for breaking the gel of fracturing fluid in oilfield sites, that is, using strong acids or oxidant chemical reagents to degrade in the fracturing fluid. The operation is convenient, but there will be problems of pipeline corrosion and damage to the formation, which has a negative impact on the ecosystem. Applying mannanase ManG3 to break the gel of guar gum fracturing fluid has the characteristics of mild reaction conditions and fast degradation rate, overcoming the disadvantages of uneven molecular weight distribution of the products obtained by chemical degradation method and incomplete degradation by physical degradation method. In addition, compared with chemical gel breakers, the products after breaking the gel by mannanase ManG3 are environmentally friendly, will not produce harmful chemical substances, and will not pollute the oil reservoir. Guar gum is a natural galactomannan, and its main chain is composed of mannose units connected by β-1,4-glycosidic bonds; mannanase ManG3 can catalyze the hydrolysis of the mannose backbone connected by β-1,4-glycosidic bonds, specifically acting on the β-1,4-glycosidic bonds in the main chain of guar gum and breaking the connection between mannose units. With the breakage of β-1,4-glycosidic bonds, the guar gum macromolecules are decomposed into shorter oligosaccharide products, and their molecular weight will be significantly reduced, and the viscosity of the guar gum solution will also be significantly reduced accordingly, thus realizing the rapid gel breaking of the fracturing fluid.

[0028] In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the drawings and embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0029] Example 1

[0030] Entrusted Ascenda Biotechnology Co., Ltd. to synthesize ManG3 The gene (SEQ ID NO.2) sequence, after inserting it behind the T7 promoter of the expression vector pET-28a(+), construct the mannanase expression vector pET-28a-ManG3, and the total length of pET-28a-ManG3 is 8141 bp.

[0031] Take out the Escherichia coli BL21 competent cells from the -80 °C refrigerator and immediately place them on ice for 5 min. Measure the concentration of the pET-28a-ManG3 expression vector using a micro-spectrophotometer to make the total transformation amount within the range of 50 - 100 ng. In a clean bench sterilized by ultraviolet light, gently add pET-28a-ManG3 to the competent cells, flick the tube wall with your finger to mix evenly, place the mixture on ice for incubation for 30 min, put the incubated mixture into a 42 °C water bath for heat shock for 90 s, and then immediately place it back on ice for 2 - 3 min. Add 900 μL of LB liquid medium without antibiotics to the centrifuge tube, gently mix, and culture and resuscitate it on a shaker at 37 °C and 220 rpm for 60 min. Centrifuge at 5000 rpm for 1 min, discard the supernatant, and collect the bacterial cells. Retain about 100 μL of the supernatant, gently pipette to resuspend the bacterial pellet, and then spread the resuspended solution onto an LB plate containing kanamycin with a final concentration of kanamycin of 50 μg / mL. Place the plate in an incubator at 37 °C for overnight culture. The next day, uniform and dense single colonies grow on the plate. Use a sterile inoculation loop to pick several single colonies and inoculate them into an LB liquid medium containing kanamycin, and culture them on a shaker at 37 °C and 220 rpm for 5 - 6 h, and then use primers for bacterial liquid PCR verification.

[0032] Among them, the composition of the LB liquid medium is: using distilled water as the solvent, 5 g / L of yeast extract, 10 g / L of peptone, and 10 g / L of NaCl, and the pH value is 7.4 - 7.6.

[0033] The composition of the LB solid medium is: using distilled water as the solvent, 5 g / L of yeast extract, 10 g / L of peptone, 10 g / L of NaCl, and 15 - 20 g / L of agar, and the pH value is 7.4 - 7.6.

[0034] The primer sequences for bacterial liquid PCR verification are:

[0035] G3-F (SEQ ID NO.3): 5′-GGATCTTCCAGAGATATGAAAAAGAAATGGATTGCGG-3′;

[0036] G3-R (SEQ ID NO.4): 5′-CTGCCGTTCGACGATTTATTCCGCCATTTCCGGC-3′.

[0037] The steps of PCR amplification are: (1) pre-denaturation at 95 °C for 5 min; (2) denaturation at 94 °C for 1 min; (3) annealing at 55 °C for 30 s; (4) extension at 72 °C for 90 s; steps (2) - (4) are repeated 30 times; (5) continue to extend at 72 °C for 10 min and cool to 4 °C.

[0038] The PCR amplification system was as follows: 12.5 μL of rTaq enzyme, 1.0 μL of template DNA, 1.0 μL of each of the upstream and downstream primers, and supplemented with ddH2O to 25 μL. The PCR product was purified by agarose gel electrophoresis to obtain the target band, and DNA sequencing was performed by Ascent Biotechnology Co., Ltd. The full length was 2772 bp, and the sequence was as shown in SEQ ID NO.2.

[0039] The single colonies verified to be correct by sequencing were separately purified and preserved as glycerol bacteria. A 50% glycerol solution was prepared, which could be obtained by mixing equal volumes of distilled water and glycerol. Under sterile conditions, the cultured bacterial solution was mixed with 50% glycerol at a ratio of 1:1 to make the final glycerol concentration reach 25% - 30%. The mixed solution was dispensed into pre-sterilized preservation tubes, 1 - 2 mL per tube, and then the preservation tubes were placed in a -80°C refrigerator for storage, labeled as E.coli -pET-28a-ManG3. The glycerol bacteria stored at -80°C need to be activated before use.

[0040] Add E.coli -pET-28a-ManG3 was inoculated into LB liquid medium containing kanamycin at a final concentration of 50 μg / mL and cultured overnight to obtain a seed solution. Then the seed solution was inoculated into fresh LB liquid medium containing kanamycin and cultured on a shaker at 37°C and 220 rpm for 3 - 4 h. The OD value at 600 nm was measured using an ultraviolet spectrophotometer and was in the range of 0.6 - 0.8. At this time, isopropyl-β-D-thiogalactoside could be added for induction expression. The final concentration of isopropyl-β-D-thiogalactoside was 0.5 mmol / L, the induction temperature was 16°C, and the time was 20 - 22 h.

[0041] After the induction expression was completed, 100 mL of the fermentation broth was collected and centrifuged at 9000 rpm and 4°C for 20 min using a pre-cooled high-speed centrifuge. The supernatant was discarded and the bacterial cells were collected. After the bacterial cells were resuspended with buffer, they were centrifuged again at 9000 rpm and 4°C for 20 min, and the supernatant was discarded. The operation of washing and resuspending with buffer was repeated twice to wash the bacterial cells to avoid the influence of culture medium components on subsequent experiments. The obtained bacterial cells were resuspended with 10 mL of buffer and placed in an ice-water mixture. The bacterial cells were ultrasonically disrupted using a cell ultrasonic disruptor. The working conditions of the cell ultrasonic disruptor were: 300 W, working for 4 s, intermittent for 6 s, and the total working time was 15 min. The ultrasonically disrupted bacterial solution changed from opaque milky white to clear and transparent. The disrupted bacterial solution was centrifuged at 9000 rpm / 4°C for 20 min, and the supernatant was retained, which was the crude enzyme solution. The crude enzyme solution was diluted 10 times with Tris-HCl buffer (pH 7.0) to obtain the crude enzyme solution of mannanase ManG3 and stored in a 4°C refrigerator for later use.

[0042] Example 2 Purification of Mannanase

[0043] Use a 1 mL pre-packed gravity column of HyPur T Ni-NTA 6FF (His-Tag) from Sangon Biotech (Shanghai) Co., Ltd. to purify the crude mannanase ManG3 solution obtained in Example 1 by gravity method. The specific method is as follows:

[0044] The loaded gravity column is equilibrated with 5 column volumes of Tris-HCl Buffer to bring the packing into the same buffer system as the target protein, and this is repeated 2 - 3 times. Add the sample to the equilibrated gravity column, and let the sample stay for at least 2 min to ensure sufficient contact between the sample and the packing. Collect the effluent, and the sample can be loaded repeatedly to increase the binding efficiency. Wash with 10 - 15 column volumes of Wash Buffer to remove non-specifically adsorbed impurity proteins, and collect the wash solution. Among them, the Wash Buffer is a 20 - 50 mM imidazole solution, and the concentration of imidazole can be adjusted for washing. Elute with 5 - 10 column volumes of Elution Buffer, collect in fractions, collect one tube for each column volume, and detect them separately, which can not only ensure that all bound target proteins are eluted, but also obtain proteins with high purity and high concentration. The Elution Buffer is a 200 mM imidazole solution. Subsequently, wash the packing with 3 column volumes of Tris-HCl Buffer and 5 column volumes of deionized water in sequence, equilibrate the packing with 5 column volumes of 20% ethanol, and finally store the packing in 1×PBS containing 20% ethanol at 4°C.

[0045] Use a pipette to aspirate 20 μL of the purified mannanase ManG3 protein obtained by nickel column purification, and mix it well by pipetting with 5 μL of 6× protein loading buffer, and heat it in a boiling water bath for 5 min to fully denature the protein and bind it to SDS. After removing the insulating rubber strip at the bottom of the precast SDS-PAGE gel, install it into the electrophoresis tank, add 1× electrophoresis buffer to submerge the bottom of the gel, let it stand for 5 min, and check whether there is any liquid leakage in the gel tank. Use a micropipette to aspirate the sample and carefully add the sample into each sample well, taking care to avoid sample overflow or mixing. Perform separation electrophoresis at a voltage of 120V until the front of the bromophenol blue indicator approaches the bottom of the gel. Take out the electrophoresed gel, put it into Coomassie Brilliant Blue R-250 staining solution, and stain it overnight on a staining shaker at room temperature. Transfer the stained gel to the destaining solution, gently shake it for destaining, and change the destaining solution every half hour to one hour until the background is clear and the protein bands appear. Place the destained gel under a gel imaging system to observe and take pictures for recording. The theoretical size of mannanase ManG3 is 98.7 kDa. The result diagram after destaining of the SDS-PAGE electrophoresis experiment is as Figure 1As shown. According to Figure 1 it can be seen that a band of the expected size is obtained, indicating that the purification of mannanase ManG3 is successful.

[0046] Example 3 Qualitative and Quantitative Detection of Mannanase Hydrolysis Products

[0047] (1) Hydrolysis of locust bean gum by mannanase ManG3: Weigh 0.0010 g each of mannose, mannotriose, mannotetraose, mannopentaose standard pure products for preparing the stock solution of standard products. Dissolve them in 1 mL of ultrapure water respectively, and fully dissolve them by shaking to prepare stock solutions with respective concentrations of 1 mg / mL. Take 200 μL from each of the above-prepared stock solutions of standard products, mix them together to prepare a mixed standard product, and finally obtain 1 mL of the mixed standard.

[0048] Mix 50 μL of the crude mannanase ManG3 solution prepared in Example 1 with 450 μL of 0.5% (w / v) locust bean gum, react in a water bath at 55 °C for 10 min, after the reaction, centrifuge the product at 12000 rpm for 30 min, then take 80 μL of the supernatant and store it at 4 °C for subsequent use.

[0049] (2) Qualitative detection: Prepare the developing agent by mixing 100 mL of n-butanol, 50 mL of glacial acetic acid, and 50 mL of water in a ratio of 2:1:1. Weigh 0.4 g of diphenylamine and 0.4 mL of aniline, then add 2 mL of 85% concentrated phosphoric acid, and finally dissolve all of them in 20 mL of acetone to prepare the color-developing agent. The preparation processes of the developing agent and the color-developing agent should be carried out in a fume hood to ensure experimental safety and reagent stability.

[0050] Put the thin-layer plate into an oven at 60 °C and dry it for 30 min. The purpose is to make it completely dry and free of moisture, providing good conditions for subsequent experimental operations. Add the pre-prepared developing agent into the developing tank and let it saturate in the tank for 30 min. At the same time, pay attention to controlling the height of the developing agent liquid level, ensuring that it is less than 0.5 cm. First, draw a line on the thin-layer plate with a pencil. The line is about 0.5 cm from the bottom of the thin-layer plate, and the distance between each spotting position should be 1 cm to clearly distinguish the development of different samples subsequently. Use a capillary to suck the sample for spotting. After spotting the sample each time, use a fan to dry it. Repeat this operation 10 times to complete the sample loading, ensuring that the sample adheres stably on the thin-layer plate and does not interfere with each other. With the help of tweezers, carefully place the silica gel plate that has completed the spotting operation into the developing tank, and let the developing solution develop upward along the silica gel plate until the developing solution reaches 0.5 cm from the top of the silica gel plate. Then take out the silica gel plate and place it in a ventilated place to make it dry thoroughly. After the silica gel plate is dry, evenly smear the developing solution on its surface. Then put the silica gel plate into an oven at 105 °C for color development for 15 min. After the color development is completed, take it out and take a photo. The result diagram of the hydrolysis product of locust bean gum hydrolyzed by mannanase ManG3 is as Figure 2 shown. The Mn lane is a standard mixture of mannose-mannobiose-mannotriose-mannotetraose-mannopentaose, which are represented by M1, M2, M3, M4, and M5 respectively. The ManG3 lane is the hydrolysis product of locust bean gum hydrolyzed by mannanase ManG3. By comparing with the standard, it can be seen that the hydrolysis products of locust bean gum include mannobiose, mannotriose, mannotetraose, and mannotetraose.

[0051] (3)Quantitative detection: Entrust Jiangsu Sanshu Biotechnology Co., Ltd. to detect the manooligosaccharides in the supernatant in step (1). Since the entrusted company does not have a standard product of mannotetraose, the content of mannotetraose was not detected.

[0052] Specific operation: The mannooligosaccharide distribution of locust bean gum hydrolyzed by ManG3 was detected by high performance anion exchange chromatography - pulsed amperometry (HPAEC-PAD). The chromatographic system used was a Thermo ICS5000 ion chromatography system (ICS5000+, Thermo Fisher Scientific), and an electrochemical detector was used for analysis and detection. The liquid chromatography column was a Dionex™ CarboPac™ PA200 (250*4.0 mm, 10 μm) column, and the injection volume was 5 μL. Mobile phase A: 0.2 M NaOH; Mobile phase B: 0.2 M NaOH / 0.2 M NaAC. The column temperature was 30 °C, and an electrochemical detector was used to analyze and detect the components. The flow rate was 0.4 mL / min; Elution gradient: 0 min A / B (90:10 V / V), 10 min A / B (90:10 V / V), 30 min A / B (40:60 V / V), 50 min A / B (40:60 V / V); 50.1 min A / B (90:10 V / V); 60 min A / B (90:10 V / V). The detection results are as Figure 3 shown, Figure 3 where the abscissa represents the types of mannooligosaccharides, and the ordinate represents their content (μg / mL).

[0053] It can be seen from Figure 3 the figure that the content of mannohexaose is the highest, reaching 4.6888 μg / mL, indicating that locust bean gum has not been completely degraded within 10 min. Therefore, pure mannohexaose can be obtained by controlling the reaction time and then separation and purification. Mannohexaose can be used as a carrier for certain drugs and vaccines, which helps to improve the stability and delivery efficiency of drugs and has important significance in drug delivery and efficacy improvement. Followed by mannobiose with a content of 1.9809 μg / mL and mannotetraose with a content of 1.7148 μg / mL. As high-quality prebiotics, mannobiose and mannotetraose can selectively promote the proliferation of beneficial bacteria such as Bifidobacterium and Lactobacillus, regulate the intestinal microecological balance, and can replace traditional sweeteners in low-sugar or sugar-free foods. They have the function of dietary fiber and are suitable for the development of sugar substitute products for diabetic patients. The contents of mannose and mannotriose are relatively low, 0.6828 μg / mL and 0.5353 μg / mL respectively. Mannotriose can activate the surface receptors of macrophages, induce the release of anti-inflammatory factors, and relieve intestinal inflammation. Numerous studies have shown that mannooligosaccharides have advantages such as environmental protection, safety, non-toxicity, and low pollution. These characteristics make mannooligosaccharides show broad application prospects in the fields of biomedicine, feed industry, and functional foods.

[0054] Example 4 Determination of the enzyme activity of mannanase

[0055] (1) Draw a standard curve

[0056] Mannanase activity is determined by quantifying the amount of reducing sugars released from locust bean gum using the 3,5-dinitrosalicylic acid (DNS) method and D-mannose as a standard. One unit (U / mL) of enzyme activity is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under defined conditions.

[0057] First, a standard curve was drawn using D-mannose at concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mg / mL. The specific method was as follows: 500 μL of D-mannose standard solution of each concentration was incubated in a water bath at 55°C for 10 min, 500 μL of DNS reagent was added to each standard solution, mixed evenly, and incubated in a boiling water bath for 5 min. After cooling in an ice bath, the absorbance of the sample at 540 nm was measured by a microplate reader to determine the amount of reducing sugar released. The sample volume was 200 μL.

[0058] The method for determining the enzyme activity of mannanase hydrolyzate is as follows:

[0059] Blank group: 450 μL of 0.5% (w / v) locust bean gum solution was incubated at 55°C in a water bath for 10 min. 50 μL of the crude mannanase ManG3 enzyme solution prepared in Example 1 and 500 μL of DNS reagent were added to the sample, mixed thoroughly, and reacted in a boiling water bath for 5 min to complete the reaction. After cooling in an ice bath, the sample absorbance at 540 nm was measured with a microplate reader to determine the amount of reducing sugars released.

[0060] Experimental Group: A reaction mixture of 50 μL of the crude mannanase ManG3 enzyme solution prepared in Example 1 and 450 μL of 0.5% (w / v) locust bean gum solution (Tris-HCl buffer, pH 7.0) was shaken and reacted in a water bath at 55°C for 10 min. 500 μL of DNS reagent was added to the sample, mixed thoroughly, and incubated in a boiling water bath for 5 min to complete the reaction. After cooling in an ice bath, the sample absorbance at 540 nm was measured with a microplate reader to determine the amount of reducing sugars released. The sample volume was 200 μL.

[0061] DNS is a reagent that can terminate the experiment. A blank group and an experimental group were set up. The difference was whether the mannanase ManG3 was reacted with the locust bean gum solution in a water bath for 10 minutes. The standard curve of D-mannose is shown in the figure below. Figure 4 As shown in the figure, after drawing the standard curve based on the measured data, the calculation formula of enzyme activity is as follows:

[0062]

[0063] Where: U: enzyme activity value, unit U / mL

[0064] A E : OD of the experimental group 540nm value

[0065] A B : OD of the blank group 540nm value

[0066] 1000: Unit conversion coefficient (mg - μg)

[0067] T: Reaction time, 10 min

[0068] N: Dilution factor of the enzyme solution

[0069] 180.16: Molecular weight of mannose

[0070] (2) Determination of the enzyme activity of mannanase ManG3 under different temperature conditions

[0071] The determination method is as follows:

[0072] Blank group: 450 μL of 0.5% (w / v) locust bean gum solution was water-bathed at 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C for 10 min respectively. 50 μL of the crude mannanase ManG3 enzyme solution prepared in Example 1 and 500 μL of DNS reagent were added to the sample, mixed evenly, and reacted in a boiling water bath for 5 min to complete the reaction. After cooling in an ice bath, the absorbance of the sample at 540 nm was measured by an enzyme-labeling instrument to determine the amount of released reducing sugar.

[0073] Experimental group: The reaction mixture of 50 μL of the crude mannanase ManG3 enzyme solution prepared in Example 1 and 450 μL of 0.5% (w / v) locust bean gum solution (Tris-HCl buffer, pH 7.0) was shaken evenly and water-bathed at 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C for 10 min respectively. 500 μL of DNS reagent was added to the sample, mixed evenly, and reacted in a boiling water bath for 5 min to complete the reaction. After cooling in an ice bath, the absorbance of the sample at 540 nm was measured by an enzyme-labeling instrument to determine the amount of released reducing sugar, and the sample addition volume was 200 μL. The obtained data was sorted out, and the enzyme activity calculation formula was used to calculate the enzyme activity at each temperature. The relative enzyme activity detection results of mannanase ManG3 under different temperature conditions are shown in Table 1 and Figure 5 as follows.

[0074] Table 1 Relative enzyme activity of mannanase ManG3 at different temperatures

[0075]

[0076] From the results of the optimum temperature experiment, it can be seen that the optimum temperature of mannanase ManG3 is 60 °C, and more than 50% of the enzyme activity can be retained in the range of 45 °C to 70 °C, and the applicable temperature range is relatively wide (Table 1 and Figure 5 ).

[0077] (3) Determination of the enzyme activity of mannanase ManG3 under different pH conditions

[0078] The determination method is as follows:

[0079] First, prepare buffer solutions with different pH values. Citrate-disodium hydrogen phosphate buffer is used in the range of pH 3.0 - 6.0, Tris-HCl buffer is used in the range of pH 7.0 - 8.0, and glycine-sodium hydroxide buffer is used in the range of pH 9.0 - 10.0. The final concentration of the buffer is 50 mM, and the pH value is detected using a pH meter. Prepare a 0.5% (w / v) locust bean gum solution with the above buffer solutions of different pH values.

[0080] Blank group: 450 μL of 0.5% (w / v) locust bean gum solution with different pH values is water-bathed at 55 °C for 10 min. Add 50 μL of the crude enzyme solution of mannanase ManG3 prepared in Example 1 and 500 μL of DNS reagent to the sample, mix well, react in a boiling water bath for 5 min to complete the reaction, and after cooling in an ice bath, measure the absorbance of the sample at 540 nm using a microplate reader to determine the amount of released reducing sugar.

[0081] Experimental group: Shake the reaction mixture of 50 μL of the crude enzyme solution of mannanase ManG3 prepared in Example 1 and 450 μL of 0.5% (w / v) locust bean gum solution with different pH values evenly, and react in a water bath at 55 °C for 10 min. Add 500 μL of DNS reagent to the sample, mix well, react in a boiling water bath for 5 min to complete the reaction, and after cooling in an ice bath, measure the absorbance of the sample at 540 nm using a microplate reader to determine the amount of released reducing sugar. The sample addition volume is 200 μL. Organize the obtained data and calculate the enzyme activity at each temperature using the enzyme activity calculation formula. The relative enzyme activity detection results of mannanase ManG3 under different pH conditions are shown in Table 2 and Figure 6 as follows.

[0082] Table 2 Relative enzyme activity of mannanase ManG3 at different pH values

[0083]

[0084] From the results of the optimum pH experiment, it can be seen that the optimum pH of mannanase ManG3 is 8.0, and the applicable range is within 5.0 - 9.0 (Table 2 andFigure 6 ).

[0085] Example 5 Substrate Specificity of Mannanase

[0086] Prepare 0.5% (w / v) xanthan gum, locust bean gum, konjac mannan, guar gum, and carboxymethyl cellulose using Tris-HCl (pH = 7.0). The above polysaccharides are used as substrates to react with the mannanase ManG3 obtained by the purification method of Example 2, and the enzyme activity measurement method is as described in Example 4. The substrate specificity results are shown in Table 3.

[0087] Table 3 Substrate Specificity of Mannanase ManG3

[0088]

[0089] As can be seen from Table 3, the mannanase ManG3 has the ability to hydrolyze locust bean gum, konjac mannan, and guar gum, among which the hydrolysis ability for locust bean gum is the strongest.

[0090] Example 6 Application of Guar Gum Fracturing Fluid in Gel Breaking

[0091] (1) Group Setting:

[0092] A. Blank Control Group: Place 490 mL of water in a Wuyin mixer, turn on the rotation speed of 1500 rpm, weigh 1.75 g of guar gum, slowly add it, stir for 2 min, then adjust the rotation speed to 1000 rpm, add NaOH solution to adjust its pH to 10, and then add 10 mL of saturated borax solution as a crosslinking agent, stir for 3 min, as the blank control group.

[0093] B. Ammonium Persulfate Group: In oilfield sites, 0.05% ammonium persulfate is commonly used as a chemical gel breaker for gel breaking. Therefore, prepare an ammonium persulfate mother liquor (0.25 g / mL). Place 490 mL of water in a Wuyin mixer, turn on the rotation speed of 1500 rpm, weigh 1.75 g of guar gum, slowly add it, stir for 2 min, then adjust the rotation speed to 1000 rpm, add NaOH solution to adjust its pH to 10, add 1.0 mL of ammonium persulfate mother liquor (0.05% addition amount), and then add 10 mL of saturated borax solution as a crosslinking agent, stir for 3 min, as the ammonium persulfate control group.

[0094] C, Mannanase group: Measure the protein concentration of ManG3 in Example 1 and dilute it to 20 mg / mL. Take 490 mL of water and place it in a Wu Yin mixer. Turn on the rotation speed of 1500 rpm, weigh 1.75 g of guar gum, slowly add it, and stir for 2 min. Then adjust the rotation speed to 1000 rpm, add NaOH solution to adjust the pH to 10, add 500 μL of the diluted enzyme solution (20 mg / L addition amount), and then add 10 mL of saturated borax solution as a cross-linking agent and stir for 3 min to obtain the mannanase experimental group.

[0095] (2) Experimental setup: Place the fracturing fluids of groups A, B, and C in a water bath at 55 °C. Take out one portion every 0.5 h and measure the viscosity change using a viscometer. The results are as Figure 7 shown.

[0096] According to Figure 7 it can be seen that the viscosity of the blank control group remains basically stable within the time range of 3 h, and the viscosity value is approximately around 12000 mPa·s, with slight fluctuations, but the overall change is not significant. This indicates that without adding any breaker, the viscosity of the guar gum fracturing fluid has a certain stability and can maintain a relatively high viscosity level for a long time. The initial viscosity of the ammonium persulfate group is similar to that of the blank control group, also around 12000 mPa·s. As time goes by, the viscosity starts to decrease rapidly, dropping to approximately 6500 mPa·s at 0.5 h, further decreasing to close to 0 at 1 h, and remaining at a viscosity level close to 0 within the subsequent 1.5 - 3 h, indicating that ammonium persulfate, as a breaker, can effectively reduce the viscosity of the guar gum fracturing fluid and cause it to completely break. The viscosity of the mannanase group shows a characteristic of rapid decrease. Between 0 and 0.5 h, the viscosity drops sharply from the initial 12000 mPa·s to close to 500 mPa·s, and after 0.5 h until 3 h, the viscosity remains at a level close to 0. This shows that mannanase has a better breaking effect and can quickly destroy the viscosity structure of the guar gum fracturing fluid, reducing its viscosity to a very low level in a short time.

[0097] In summary, the mannanase ManG3 of the present invention is suitable for use in the range of pH 5.0 - 9.0 and temperature 40 - 70 °C, has good pH stability and temperature stability, and has a wide range of applications, solving the problems of complex environment, uneven pH and temperature distribution during the industrial application of mannanase; it can effectively degrade various substrates such as locust bean gum, konjac mannan and guar gum, especially hydrolyze locust bean gum to produce manno-oligosaccharides with a degree of polymerization of 2 - 5, and has potential application value in the production of composite manno-oligosaccharides; and the mannanase ManG3 can be applied to break the gel in guar gum fracturing fluid, and the products after gel breaking are environmentally friendly, will not produce harmful chemical substances, and will not cause pollution to the oil reservoir.

[0098] Although the above embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. Use of a mannanase ManG3 in the production of mannan oligosaccharides, characterized in that, The amino acid sequence of the mannanase ManG3 is shown in SEQ ID NO.

1.

2. The application according to claim 1, wherein The nucleotide sequence of the coding gene of the mannanase ManG3 is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that, The production method of the mannanase ManG3 includes: culturing a recombinant bacterium to induce the expression of the mannanase ManG3; the recombinant bacterium includes the coding gene of the mannanase ManG3.

4. The application according to claim 3, wherein The basic bacterium of the recombinant bacterium is Escherichia coli.

5. A method for producing mannan oligosaccharide, characterized in that, Using the mannanase ManG3 to degrade a substrate; the substrate is selected from at least one of locust bean gum, konjac mannan, and guar gum; the amino acid sequence of the mannanase ManG3 is shown in SEQ ID NO.

1.

6. Use of a mannanase ManG3 in breaking the gel of oilfield fracturing fluid, characterized in that, The amino acid sequence of the mannanase ManG3 is shown in SEQ ID NO.1; the fracturing fluid is a guar gum fracturing fluid.

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