High-yield high-temperature-resistant pectinase strain and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a high-yield high-temperature-resistant pectinase strain and application thereof. The invention provides an aspergillus niger mutant strain AR-11 for producing high-yield and high-temperature-resistant alkaline pectinase, the enzyme activity level of pectinase produced by utilizing the strain for industrial fermentation can reach 8000-8200U / mL or more, the production cost is greatly reduced, and large-scale application, energy conservation and emission reduction of the alkaline pectinase are facilitated. The produced pectinase has high enzyme activity under a high temperature condition, keeps the stability of the enzyme activity in a wide pH value range, can meet the requirements of a pectinase preparation in the aspects of ramie degumming and textile refining, and can be widely applied to the ramie degumming industry.

Description

Technical field: The invention belongs to the technical field of microorganisms, and particularly relates to a high-yield and high-temperature resistant pectinase strain and application thereof. Background technology: Pectinase is a general term for enzymes that can catalyze the decomposition of pectin in plants, mainly including pectinase, polygalacturonase, polymethylgalacturonase, polymethylgalacturonate lyase and polygalacturonate lyase. According to the application field and the optimal pH value, pectinase can usually be divided into two types: acidic pectinase and alkaline pectinase. Acidic pectinase generally refers to polygalacturonase, which is mainly used in clarifying juice and extracting pectin. Alkaline pectinase generally refers to polygalacturonate lyase, which can break the α-1,4 glycosidic bond of pectin molecules through trans-elimination and decompose polymer pectin into small molecular galacturonic acid. It is one of the most important industrial enzymes at this stage and has a wide range of applications in the textile industry, food industry, plant bast fiber degumming, cotton fiber biorefining, papermaking industry, feed additives, pectin-containing wastewater treatment and other fields.

[0003] At present, the optimum pH value of the developed pectinase is mostly in the neutral and acidic range, and the optimum action temperature is generally not high, which limits its scope of application in industry. In recent years, the research on high-temperature resistant alkaline pectinase has made up for these shortcomings. Alkaline pectinase is a general term for a class of enzymes that can efficiently decompose pectin in plant tissues under alkaline conditions. In recent years, it has attracted much attention as a degumming enzyme used in cotton and linen fibers.

[0004] Hemp textiles are known as green high-end textiles. This functional textile product is highly favored for its natural nature and unique functions. At the same time, hemp fiber is also a high-quality raw material for many industries, and can be used to make filter materials, non-wood paper, medical textiles, composite panels, etc.

[0005] my country has abundant types and output of hemp resources. Ramie ranks first in the world, accounting for more than 90% of the world's ramie output. It is mainly used in the hemp textile industry and is one of the main sources of hemp fiber in my country's hemp textile industry. Ramie fiber has the characteristics of long length and good toughness, and has better application prospects than other hemp fibers and cotton fibers. Hemp fiber is an ecological fiber, but hemp textile processing has not yet reached ecological standards. Problems such as wastewater pollution in hemp degumming are still common. To make hemp textiles truly ecological textiles, the problem of hemp degumming pollution must be solved.

[0006] Raw hemp is a strip-like material in which ramie fibers are bonded together by various gums. The content of gums in raw hemp is as high as 20-35%, mainly pectin and hemicellulose. Before spinning, the gums in the bast must be removed to separate the single hemp fibers from each other so that they can be spinnable. This process is called degumming. For decades, my country's hemp degumming has been using the chemical method of lye boiling. Although many improvements have been made in degumming technology and additives, it still does not deviate from the scope of chemical degumming. The traditional chemical degumming method uses the different stability of cellulose and gum components in raw hemp to acids, alkalis and oxidants to remove the gum components in raw hemp and retain the cellulose components. The chemical degumming method not only consumes a large amount of chemical raw materials and energy such as acids and alkalis, but also increases production costs. In addition, the intense chemical scouring process also seriously damages the fiber properties and structure of the hemp fiber, especially the fiber surface properties. The removal of colloid is uneven and thorough, and the yield of refined hemp is low, which greatly reduces the combing and spinning performance of the hemp fiber. The yarn quality is relatively poor, the fabric feels rough and hard, and cannot maintain the characteristics of natural plant fibers such as ramie.

[0007] Traditional chemical degumming method is to use high temperature and strong alkaline conditions to treat textile raw materials to achieve the purpose of degumming. This method has the disadvantages of large production energy consumption, long processing time, dangerous operating environment and wastewater pollution. Therefore, the current development trend of degumming treatment in the textile industry is to use bioenzyme method to degumming raw materials. Alkaline pectinase can be used for ramie degumming and the refining process of cotton fabric treatment. The fabric treated with alkaline pectinase under relatively mild pH and temperature conditions has a soft feel and high strength. It can replace the traditional hot alkali degumming process with large energy consumption and serious pollution, and has broad development prospects. In addition, alkaline pectinase has also become a new research hotspot in industries such as detergents, papermaking, and plant drug extraction, and has received more and more attention.

[0008] At present, there are many microorganisms that can produce pectinase, including bacteria, molds and actinomycetes, etc. However, most pectinase is used at room temperature or medium temperature, which cannot meet the high temperature and strong alkaline environment required for ramie degumming and cannot be widely used in industrial production. Therefore, it is of great significance to screen out alkaline pectinase with a comprehensive enzyme system, good specificity, high enzyme activity and high temperature resistance. Summary of the invention: In order to solve the above technical problems, one of the technical solutions provided by the present invention is a strain of high-yield thermostable alkaline pectinase, which is obtained by mutagenizing Aspergillus niger strain HQ-3 preserved in the laboratory by ultraviolet and lithium chloride composite mutagenesis, specifically Aspergillus niger ( Aspergillus niger)AR-11, which was deposited on November 8, 2022 at the General Microbiology Center of the China Culture Collection Administration (address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101), with the deposit number CGMCC No.40417.

[0010] The second technical solution provided by the present invention is Aspergillus niger ( Aspergillus niger )AR-11 application; Further, the application in the production of pectinase; Furthermore, the invention provides an application in ramie degumming, wherein the Aspergillus niger provided by the invention ( Aspergillus niger )AR-11 can produce not only highly active pectinase, but also hemicellulases such as xylanase and mannanase, but no cellulase, which is beneficial to the degradation and removal of hemicellulose in ramie gum and improves the degumming rate.

[0011] The third technical solution provided by the present invention is a liquid fermentation production method of pectinase with high fermentation enzyme activity and low manufacturing cost, which can be achieved by the following measures: Fermentation tank fermentation process conditions: inoculation amount 8-10%, tank pressure 0.05-0.08 MPa, culture temperature 29-31℃, rotation speed 200-800 rpm, control dissolved oxygen at 35-40%, start feeding when dissolved oxygen drops to the minimum and then rises to 40%, control dissolved oxygen at 30%-40%, culture for about 120h, release the tank when enzyme activity increases slowly and the bacteria begin to partially autolyze; At the end of fermentation, the enzyme activity of the fermentation broth was 8000-8200U / mL; Furthermore, the fermentation tank culture medium is composed of 2-2.5% orange peel powder, 2-3% corn starch, 3-4.5% bran, 1-2.5% corn steep liquor, 0.3-0.6% potassium dihydrogen phosphate, 0.3-0.6% dipotassium hydrogen phosphate, 0.1-0.2% calcium chloride, 0.15-0.2% magnesium sulfate, 1.5-2.0% ammonium sulfate, and the rest is water, pH 6.0-6.5 ("%" indicates mass volume percentage).

[0012] Furthermore, the fermentation tank sterilization process conditions are: 121°C-124°C, 0.11-0.12 MPa, sterilization for 20 minutes. Furthermore, the feed medium composition is as follows: 5-6% orange peel powder, 10-12% corn starch, 4-5% corn steep liquor, 3-4% ammonium sulfate, and the rest is water, pH 6.0-6.5 ("%" represents mass volume percentage).

[0013] The pectinase prepared by the present invention has the following enzymatic properties: (1) The optimum reaction temperature is 55°C. When kept at 70°C for 2 h, the enzyme activity can still be maintained at more than 85%, indicating good thermal stability. (2) The optimum reaction pH is 9.0, and it can remain stable in a wide pH range (pH 5.0-10.0). After being treated at pH 10.0 for 2 hours, the relative enzyme activity still remains above 85%, which meets the high temperature and strong alkaline environment required for hemp degumming. Beneficial effects: The Aspergillus niger mutant strain provided by the present invention has outstanding substantive characteristics and significant progress compared with the prior art, and can produce the following positive effects: 1. The present invention first provides a mutant strain of Aspergillus niger with high yield of high temperature resistant alkaline pectinase. The pectinase activity level produced by industrial fermentation using the strain can reach above 8000-8200 U / mL, which greatly reduces the production cost, is conducive to the large-scale application of alkaline pectinase, and saves energy and reduces emissions. 2. The pectinase has high enzyme activity under high temperature conditions and stability in a wide pH range. The optimum reaction temperature is 55°C. After being kept at 70°C for 2 hours, the enzyme activity can still be maintained at more than 85%. The thermal stability is good and can meet the high temperature environment required by pectinase preparations in ramie degumming and textile refining. The optimum reaction pH is 9.0, and it can remain stable in a wide pH range (pH5.0-10.0). After being treated at pH10.0 for 2 hours, the enzyme can maintain more than 85% of its activity.

[0014] 3. In addition to producing highly active pectinase, the mutant strain can also produce hemicellulases such as xylanase and mannanase, but does not produce cellulase, which is beneficial to the degradation and removal of hemicellulose in ramie colloid and improves the degumming rate. In addition, the cellulase activity in the fermentation liquid of the mutant strain is very low, which reduces the degree of damage to ramie fiber during the degumming process and can better maintain the strength and integrity of the fiber.

[0015] 4. The addition of some degumming aids commonly used in the fiber degumming process has no side effects on the pectinase produced by the mutant strain, and some aids even have an activating effect on the enzyme. Description of the drawings: Figure 1 This is the optimal reaction temperature curve.

[0017] Figure 2 Thermal stability curve.

[0018] Figure 3 This is the optimal reaction pH curve.

[0019] Figure 4 The pH stability curve is shown in FIG.

[0020] Figure 5 The effect of degumming aids on pectinase. Specific implementation method: In order to make the purpose, technical solution and advantages of this patent more clear, this patent is further described in detail in combination with specific embodiments. It should be understood that the specific embodiments described here are only used to explain this patent and are not used to limit the present invention.

[0022] Example 1 Mutation breeding of strains Take a fresh culture slant of the starting strain Aspergillus niger HQ-3, wash the cells with sterile saline, transfer to a triangular flask filled with glass beads, place it on a shaker for 30 minutes to disperse the cells, filter with sterile absorbent cotton, collect single spores, and adjust the single spore concentration to 10 with saline. 6 About 1000 spores / ml, take 1 mL of the single spore suspension prepared by the above method and place it in a sterile culture dish, place the culture dish vertically at 30 cm under a UV lamp, and irradiate it for 0 to 5 minutes under magnetic stirring. Dilute the irradiated mutagenic bacterial suspension appropriately and spread it on a screening plate medium containing 0.3% lithium chloride, place it in a 30°C incubator away from light for 5 days, add a small amount of 1% hexadecyltrimethylammonium bromide (CATB) to the plate, let it stand for 10 minutes, and observe whether there is a colorless transparent circle. The larger the transparent circle, the stronger the ability of the strain to utilize pectin. Select the mutant strain according to the size of the transparent circle diameter around the colony and the colony diameter, and pick the colony with a large ratio and store it on the slant. After fermentation and shake flask pectinase production, a mutant strain AR-11 with a shake flask pectinase activity of 1250U / mL was finally screened, among which the shake flask pectinase activity of the starting strain HQ-3 was 452U / mL, and the enzyme activity of AR-11 was 2.8 times higher than that of the starting strain. The mutant strain AR-11 was then preserved and the preservation number was CGMCC No.40417.

[0023] Some of the culture media and culture methods involved in the above screening process are as follows: (1) Plate screening culture medium Pectin 5%, glucose 1%, peptone 2%, sodium chloride 0.2%, malt powder 1.5%, ammonium sulfate 1.5%, agar powder 2%, and the rest is water, pH 6.0.

[0024] (2) Slant culture medium Glucose 2%, peptone 1%, agar powder 2%, 4 degree malt juice 95%, pH 6.0.

[0025] (3) Seed culture medium Bran 2%, peptone 1%, glucose 2%, calcium chloride 0.1%, corn steep liquor 0.5%, ammonium sulfate 1%, the rest is water, pH 6.0.

[0026] (4) Fermentation shake flask medium Orange peel powder 2%, glucose 1%, yeast powder 1.5%, corn steep liquor 2%, potassium dihydrogen phosphate 0.2%, calcium chloride 0.1%, ammonium sulfate 0.5%, and the rest is water, pH 7.2.

[0027] (5) Culture conditions Separation plate: culture at 30°C for 5 days; Liquid seeds: culture at 30°C for 48 h, shaker speed 200 rpm; Fermentation shake flask: culture at 30℃ for 120h, shaker speed of 200 rpm, inoculation amount of 10%.

[0028] 2. Stability test of high-yield pectinase strain AR-11 Pick a single colony of AR-11 with good growth on the screening plate and inoculate it on the slant culture medium. Culture it at 30℃ for 5-6 days. After the slant grows well, use a sterile iron shovel to take a piece of bacterial moss and inoculate it into the seed culture medium. Culture it at 30℃ and 200rpm for about 48h. Transfer it to the fermentation shake flask at 10% inoculation amount, culture it at 30℃ and 200rpm for about 120h, and measure the enzyme activity. The results of the shake flask after 10 consecutive passages of the strain are shown in Table 1 (the culture medium and culture method are the same as step 1): Table 1. Stability test results of strain AR-11

[0029] The mutant strain was subcultured for 10 generations. The experimental results are shown in Table 1. It can be seen that the mutant strain has high enzyme activity and good genetic stability.

[0030] Example 2 Alkaline Pectinase Activity Assay Method (1) Definition of alkaline pectinase activity used in the present invention Under certain conditions (45°C, pH 9.0 unless otherwise specified), the amount of enzyme required to degrade polygalacturonic acid to produce 1 μmol of unsaturated galacturonic acid per minute.

[0031] (2) Enzyme activity determination method Take 2mL of glycine-sodium hydroxide buffer (pH9.0, containing 0.44mol / L CaCl2) containing 0.2% (mass volume fraction, g / mL) polygalacturonic acid, preheat it in a 45℃ water bath for 5min, then draw 20μL of crude enzyme solution and add it to the above solution, mix thoroughly, react for 15min, terminate the reaction with 3mL of 0.03mol / L phosphoric acid, measure the absorbance at 235nm, and use the enzyme solution heated and boiled for 10min as a blank control in the same system.

[0032] U=OD 235 ×10 6 ×n×reaction mixture volume / 10 3 ×t×4600×b×volume of crude enzyme solution Where: U—enzyme activity of alkaline pectinase (U / mL); t (min)—enzyme reaction time (within the linear range of enzyme reaction); n—enzyme solution dilution multiple; b (cm) - cuvette thickness; 4600 (Lmol -1 cm -1 ) is the molar absorption coefficient of unsaturated polygalacturonic acid at 235 nm.

[0033] Example 3 Production of pectinase by liquid fermentation of strain AR-11 1. Seed tank expansion culture (1) Seed tank culture medium: 2% bran, 3% glucose, 1% soybean meal, 2% ammonium sulfate, 2% corn steep liquor, 0.5% potassium dihydrogen phosphate, 1.0% magnesium sulfate, and the rest water, pH 6.0.

[0034] (2) Seed tank sterilization process conditions: 121°C, 0.12MPa, sterilization for 30min; (3) Seed tank culture process conditions: tank pressure 0.05 MPa, culture temperature 30°C, stirring speed 400 rpm, inoculation amount 10%, pH control 6.0-6.2; (4) Seed tank transplanting conditions: The bacteria are darkly stained, strong, and free of foreign bacteria.

[0035] 2. Liquid fermentation to produce pectinase (1) Fermentation tank culture medium: orange peel powder 2.5%, corn starch 3%, bran 3.5%, corn steep liquor 2%, potassium dihydrogen phosphate 0.3%, dipotassium hydrogen phosphate 0.3%, calcium chloride 0.1%, magnesium sulfate 0.15%, ammonium sulfate 2.0%, and the rest is water, pH 6.0-6.2 ("%" indicates mass volume percentage).

[0036] (2) Fermentation tank sterilization process conditions: 121℃, 0.12 MPa, sterilization for 20 min.

[0037] (3) Fermentation tank process conditions: inoculation volume 10%, tank pressure 0.05MPa, culture temperature 30℃, rotation speed 200-800rpm. In the early stage, the dissolved oxygen is controlled at 40% by adjusting the rotation speed. When the dissolved oxygen drops to the minimum and then rises to 40%, feed is started. The dissolved oxygen is controlled at 30%-40%. The fermentation is carried out until the enzyme activity increases slowly and the bacteria begin to partially autolyze. 3. Feed (1) Feed medium: orange peel powder 6%, corn starch 12%, corn steep liquor 4%, ammonium sulfate 3%, and the remainder water, pH 6.0-6.2 (% indicates mass volume percentage).

[0038] (2) Feeding method: When the dissolved oxygen drops to the minimum and then rises to 40%, start feeding (the initial feeding amount is 15g / L / h), control the dissolved oxygen at 30%-40%, and adjust the feeding amount appropriately according to the dissolved oxygen situation.

[0039] 4. Place the can After 120-130 hours of cultivation, when the enzyme activity increases slowly and the bacteria begin to autolyze, the culture can be released from the tank.

[0040] 5. Extraction and purification of pectinase After the fermentation is completed, 40% water, 0.3% calcium chloride and 0.5% disodium hydrogen phosphate are added according to the volume of the fermentation liquid, and 3% perlite filter aid is added to perform plate and frame filtration; the clarified filtered enzyme liquid is ultrafiltered and concentrated using a 20KDa ultrafiltration membrane; stabilizers: 2.5% trehalose, 1.6% glycerol, preservatives: 0.15% potassium sorbate, 0.2% sodium metabisulfite are added to the concentrate, and then 6% diatomaceous earth is used for filtration and sterilization to obtain a finished pectinase enzyme preparation ("%" represents mass volume percentage).

[0041] Fermentation was carried out using the Aspergillus niger mutant strain AR-11 and culture medium. Table 2 shows the fermentation cycle and enzyme activity of the fermentation broth for 6 batches of fermentation. The average enzyme activity of the fermentation broth was 8116 U / mL.

[0042] Table 2.50L small tank fermentation test results

[0043] As can be seen from Table 2, the fermentation level of the induced strain AR-11 was relatively stable, and the fermentation enzyme activity reached more than 8000U / mL.

[0044] At the same time, the same method as above was used to ferment and produce pectinase using wild-type Aspergillus niger strain HQ-3 as the production strain and prepare a finished enzyme preparation for subsequent determination of enzymatic properties.

[0045] Example 4 Analysis of Enzymatic Properties (1) Optimum reaction temperature The pectinase product prepared in Example 3 was taken, and the pectinase activity was measured at pH 9.0 and different temperatures (30, 35, 40, 45, 50, 55, 60, 65, 70° C.) using the method described in Example 2. The relative enzyme activity was calculated with the highest enzyme activity as 100%.

[0046] The results are as follows Figure 1 As shown in the figure, the activity of pectinase produced by AR-11 gradually increased with the increase of temperature before 55℃, and the enzyme activity was highest at 55℃. At 60℃, the enzyme activity was still more than 85%. The optimum temperature of wild-type pectinase produced by HQ-3 was 45℃, and the enzyme activity was only about 70% at 60℃, indicating that the pectinase produced by the mutant strain AR-11 is more in line with the high temperature environment required for ramie degumming.

[0047] (2) Thermal stability The pectinase product prepared in Example 3 was taken, and the enzyme solution was placed at 50, 55, 60, 65, 70, 75, 80, 85, and 90° C. for 2 h, respectively. The enzyme activity was determined by the method described in Example 2, and the relative enzyme activity was calculated with the untreated original enzyme activity as 100%.

[0048] The experimental results are as follows Figure 2 As shown in the data, the pectinase produced by the mutant strain AR-11 has basically no loss of enzyme activity when kept at 50℃ for 2h, and can still maintain more than 85% of the enzyme activity when kept at 70℃ for 2h, while the relative enzyme activity of the wild-type pectinase produced by HQ-3 is only about 50%. The pectinase produced by AR-11 has better thermal stability than the wild-type, and can meet the high temperature environment required for pectinase preparations in ramie degumming and textile refining.

[0049] (3) Optimal reaction pH The finished pectinase product prepared in Example 3 was taken, and the enzyme activity of the pectinase at pH values ​​of 8.0, 9.0, 10.0, and 11.0 was measured at a temperature of 55° C. according to the enzyme activity determination method described in Example 2, and the relative enzyme activity was calculated with the highest enzyme activity as 100%.

[0050] The results of the test are as follows Figure 3As shown in the figure, the pectinase activity produced by the mutant strain AR-11 is the highest at pH 9.0, which is basically the same as the wild type. Under pH 10.0, the relative enzyme activity can still be maintained at more than 85%, while the wild type is only 70%. Ramie fiber is a cellulose fiber. Acidic conditions damage the strength of ramie fiber. Alkaline degumming environment can protect the fiber strength to the greatest extent. This requires pectinase to have a higher enzyme activity under alkaline conditions. The pectinase produced by the mutant strain AR-11 just meets this requirement.

[0051] (4) Acid and alkali resistance The finished pectinase prepared in Example 3 was placed in buffer solutions with pH values ​​of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, and 13.0, respectively (sodium hydrogen phosphate-citric acid buffer solution was used for pH 3.0-6.0, Tris-HCl buffer solution was used for pH 7.0-8.0, glycine-sodium hydroxide buffer solution was used for pH 9.0-11.0, and sodium hydrogen phosphate-sodium hydroxide buffer solution was used for pH 12-13), and the residual enzyme activity was determined according to the enzyme activity determination method described in Example 2 after standing at room temperature for 2 h, and the relative enzyme activity was calculated with the untreated original enzyme activity as 100%.

[0052] The results of the test are as follows Figure 4 As shown in the figure, the pectinase produced by the mutant strain AR-11 remains stable in a wide pH range (pH5.0-10.0). When treated at pH10.0 for 2 hours, the enzyme can maintain more than 85% of its activity. The process of pectinase catalyzing colloid will lead to the formation of acidic products and the decrease of pH of the reaction solution. Therefore, it is of great significance to develop pectinase with high stability in a wide pH range.

[0053] (5) Analysis of enzyme system in fermentation broth The pectinase product prepared in Example 3 was taken, and the xylanase (GB / T23874-2009), mannanase (GB / T36861-2018), and cellulase activity (GB / T23881-2009) thereof were measured respectively. The experimental results are shown in Table 3. The xylanase and mannanase activities produced by the mutant strain AR-11 were improved compared with the starting strain. Since 15% of the component in ramie colloid is cellulose, it is helpful for degumming to contain some hemicellulase in the degumming enzyme system. Therefore, the increase of xylanase and mannanase in the enzyme solution is more conducive to the application of the enzyme solution in ramie degumming.

[0054] At the same time, the mutant strain AR-11 maintains a high ability to degrade pectin, and the enzyme liquid it produces does not contain cellulase activity. Therefore, it can not only complete the traditional degumming work, but also reduce the damage of the enzyme liquid to the ramie fiber during the degumming process of ramie fiber, maintain the flexibility and integrity of the fiber, and has great research and development and promotion value.

[0055] Table 3. Enzyme activities in fermentation broth

[0056] (6) Effect of degumming aids on pectinase activity At present, the commonly used additives in fiber degumming are JFC, AES, OP-10, TX-10, EDTA, STPP, peregal O, sodium acetate, sodium pyrophosphate. The finished pectinase enzyme preparation prepared in Example 3 was added with the above degumming additives at a final concentration of 0.3%, and treated at 55°C for 2h. The residual enzyme activity was measured and the residual enzyme activity was calculated with the untreated original enzyme activity as 100%. The experimental results are as follows Figure 5 As shown, the degumming aids used above have no obvious side effects on the pectinase produced by AR-11, and some of the aids even have an activating effect on the enzyme.

[0057] (7) Evaluation of ramie degumming effect 100g of ramie was soaked in 1000g of glycine-sodium hydroxide buffer solution with pH 9.0 containing 1% penetrant JFC, and an appropriate amount of the finished pectinase prepared in Example 3 was added, and enzymatic degumming was performed in a water bath at 60°C for 20h, wherein the amount of pectinase added was 50U of alkaline pectinase per 1g of ramie for degumming. After the enzymolysis was completed, the treated ramie was washed with water, and dried to obtain degummed refined ramie, and the residual rubber rate, whiteness, doubling rate and bundle fiber strength of the degummed refined ramie were measured according to the following method.

[0058] (1) Referring to the national standard GBT5859-1986 "Quantitative Analysis Method for Chemical Composition of Ramie", the residual glue rate of the degummed ramie was determined, where the residual glue rate (%) = (dry weight of the sample - dry weight of the sample after residual glue extraction) / dry weight of the sample × 100%. The residual glue rate directly reflects the degumming effect of ramie fiber. The lower the residual glue rate, the better the degumming effect.

[0059] (2) With reference to GBT5885-1986 "Test Method for Whiteness of Ramie Fiber", the whiteness of the degummed ramie fiber was tested. Whiteness is an indicator for evaluating the whiteness of a substance surface. In the textile field, it generally refers to the degree of distance from the ideal white (with high-purity barium sulfate as the standard whiteness of 100 degrees). The higher the whiteness value, the more conducive it is to subsequent dyeing processing.

[0060] (3) With reference to GB / T5882-1986 "Test method for breaking strength of ramie bundle fiber", the breaking strength of degummed ramie fine hemp was tested. Bundle fiber strength refers to the maximum load that the fiber can withstand when it is broken. The greater the bundle fiber strength, the stronger the spinnability of the fiber, and the more favorable it is for subsequent processing.

[0061] (4) Take about 5.0 g of dry hemp from the sample to be tested, and accurately weigh its weight on an analytical balance after loosening it. Use tweezers to pinch out hard strips (i.e., strands) from the loosened hemp. Finally, accurately weigh the mass of the strands and calculate the strand rate.

[0062] The experimental results are shown in Table 4. The pectinase produced by the mutant strain AR-11 can significantly reduce the residual rubber rate of ramie fiber, improve the whiteness and bundle fiber breaking strength, and is more suitable for ramie degumming industry.

[0063] Table 4. Effects of different degumming methods on ramie fiber properties

[0064] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A strain of Aspergillus niger, characterized in that: Specifically, Aspergillus niger ( Aspergillus niger )AR-11, the deposit number is CGMCC No.40417.

2. The Aspergillus niger of claim 1 ( Aspergillus niger )Application of AR-11 in the production of pectinase.

3. The Aspergillus niger of claim 1 ( Aspergillus niger )Application of AR-11 in ramie degumming.

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