CN114350638B - Method for producing high-temperature-resistant acidic beta-mannase - Google Patents

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a production method of high-yield high-temperature-resistant acid beta-mannase. The production strain adopted by the production method is Aspergillus niger (Aspergillus niger) HNG26-22, and the preservation number is CGMCC NO.23221. The fermentation enzyme activity level of industrial fermentation production by utilizing the strain can reach over 12000-13000U/mL, greatly reduces the production cost, is beneficial to large-scale application of beta-mannase, and saves energy and reduces emission. The produced beta-mannase has the characteristics of high temperature resistance, good pH stability, protease resistance and the like. Is more likely to play a role in the stomach and intestinal tracts of animals and poultry, and also shows great potential in feed applications.

Description

Method for producing high-temperature-resistant acidic beta-mannase

Technical field:

the invention belongs to the technical field of microorganisms, and particularly relates to an aspergillus niger strain for high-yield high-temperature-resistant acid beta-mannase and a production method thereof.

The background technology is as follows:

beta-mannanases (endo-1, 4-beta-D-mannanmanno hydroase EC, 3.2.1.78) are a class of hemicellulases that hydrolyze mannans containing beta-1, 4-D-mannosidic linkages, and are widely found in animals, plants and microorganisms. The substrate has a very wide range of actions, including plant polysaccharide such as mannans, glucomannans, galactomannans, galactoglucomannans and the like. The beta-mannase is a novel enzyme preparation, which has the functions of general non-starch polysaccharide enzymes, namely degradation of non-starch polysaccharide, reduction of intestinal viscosity and promotion of digestion and absorption of nutrient substances, and is a multifunctional accelerator, so that secretion of insulin growth factors, synthesis of proteins and growth promotion. Meanwhile, the growth of beneficial bacteria in intestines and stomach of animals can be promoted, the reproduction of harmful bacteria can be restrained, and the immunity and survival rate of the animals can be improved. In recent years, research on beta-mannase is increasingly paid attention to, and the beta-mannase has been widely applied to the fields of feed, food, petroleum exploitation and the like.

The reported microorganisms producing beta-mannanase include: bacillus, pseudomonas and vibrio in bacteria; aspergillus, trichoderma, and Penicillium among fungi; yeasts and Streptomyces among actinomycetes, and the like. Microorganisms are the main source of beta-mannanase, and the conditions for producing the mannanase by various microorganisms and the activities of the produced enzymes, the properties of the enzymes and the like are different. At present, research, production and application of beta-mannase from different strains at home and abroad are concentrated on alkaline and neutral enzymes, and mannase applied to feed needs to have higher activity under acidic conditions, so that research and development of the acidic beta-mannase have important significance in the application of feed industry.

In industrial applications, beta-mannanases are often subjected to high temperature treatment processes, resulting in enzyme inactivation. Therefore, to meet the requirements of various industrial applications, improving the high temperature resistance of beta-mannase is always an urgent problem to be solved. The mannanase applied to the feed needs to have higher activity under the acidic condition, so that the research and development of the high-temperature resistant acidic beta-mannanase have important significance in the feed industry application.

The invention comprises the following steps:

in order to solve the technical problems, the invention provides a high-yield high-temperature-resistant acid beta-mannase strain which is obtained by mutagenesis of a laboratory-preserved Aspergillus niger strain HQM-2, in particular Aspergillus niger (Aspergillus niger) HNG26-22, which is preserved in China general microbiological culture Collection center (address: national institute of Chinese sciences, national academy of sciences, no. 3, of Beijing, kogyo-area, north Chen West road No. 1, beijing) on day 25 of 2021, and has a preservation number of CGMCC No.23221.

The invention also provides an application of the aspergillus niger (Aspergillus niger) HNG26-22 in the production of beta-mannase, in particular to a liquid microorganism fermentation production method of the beta-mannase, which has high fermentation enzyme activity, high extraction yield and low manufacturing cost. The object of the invention can be achieved by the following measures:

(1) Production of beta-mannase by liquid fermentation

Fermentation process conditions of the fermentation tank: inoculating 1.5-2.5%, tank pressure 0.05-0.08MPa, culturing at 32-34 ℃ and rotating speed 200-800r/min, feeding when dissolved oxygen is reduced to the lowest and then increased to 30-35%, controlling dissolved oxygen to be 20-30% (initial feeding amount is 300g/h, reducing feeding amount when dissolved oxygen is lower than 20%, increasing feeding amount when dissolved oxygen is higher than 30%, properly adjusting feeding amount according to dissolved oxygen condition), culturing for about 110-130h, growing enzyme activity slowly, and discharging the tank when the thallus begins to be partially autolyzed;

when fermentation is finished, the enzyme activity of the fermentation liquor is 12000-13000U/mL;

(2) Extraction and refining of beta-mannase

After fermentation, adding 40-45% of water and 0.25-0.35% of calcium chloride according to the volume of fermentation liquid, adding 2.5-3.5% of perlite filter aid, and carrying out plate-frame filter pressing; carrying out ultrafiltration concentration on the clarified filter-press enzyme solution by using an ultrafiltration membrane with the aperture of 20 kDa; adding stabilizer (7-9% glucose, 8-10% sodium chloride) and antiseptic (0.15-0.2% potassium sorbate) into the concentrated solution, and filtering with diatomite for sterilization to obtain beta-mannanase final enzyme preparation ("%" represents mass/volume ratio).

Further, the fermenter medium composition is as follows: 1.5 to 2 percent of konjaku flour, 1.6 to 2 percent of corn steep liquor, 2 to 2.5 percent of dipotassium hydrogen phosphate, 0.2 to 0.3 percent of magnesium sulfate, 1.5 to 2.0 percent of ammonium sulfate, 0.25 to 0.3 percent of sodium nitrate, and the balance of water, wherein the pH value is 6.0 to 6.2 ("%" represents mass volume percent);

further, the fermentation tank sterilization process conditions: sterilizing for 30min at 121-124 deg.C and 0.11-0.12 MPa.

Further, the feed medium consisted of: 10-12% of konjak flour, 3-3.5% of corn steep liquor, 1.5-2.0% of ammonium sulfate, 1-2.5% of monopotassium phosphate and the balance of water, wherein the pH value is 6.0-6.2 ("%" represents mass and volume percentage).

The beta-mannase prepared by the invention has the following enzymatic characteristics:

(1) The optimal reaction temperature is 40 ℃, and the heat preservation is carried out for 2 hours at 90 ℃, so that the enzyme activity of more than 85% can be still maintained, and the thermal stability is good;

(2) The optimal reaction pH is 3.0, and the relative enzyme activity is still maintained above 80% after 2h of treatment under the condition of pH of 2.0-7.0.

The beneficial effects are that:

compared with the prior art, the aspergillus niger and the production method provided by the invention have outstanding substantive characteristics and remarkable progress, and can produce the following positive effects:

1. the invention firstly provides an Aspergillus niger mutant strain for high-yield beta-mannase, and the fermentation enzyme activity level of industrial fermentation production by utilizing the strain can reach over 12000-13000U/mL, so that the production cost is greatly reduced, the large-scale application of the beta-mannase is facilitated, and the energy conservation and the emission reduction are realized.

2. The beta-mannase has the characteristics of high temperature resistance, good pH stability, protease resistance and the like, the optimal reaction temperature is 40 ℃, and the beta-mannase can still keep more than 85% of enzyme activity under the condition of 90 ℃ for 2 hours, and has good thermal stability; the optimal reaction pH is 3.0, and the enzyme can maintain more than 80% of activity after being treated for 2 hours within the pH range of 2.0-7.0; has excellent pepsin and trypsin resisting capacity, and the relative enzyme activity is maintained over 95% after 2 hr pepsin treatment and over 90% after 2 hr trypsin treatment. The beta-mannanase of the invention is more likely to act in the stomach and intestinal tracts of animals and poultry.

In addition, the beta-mannase has better heat resistance and is suitable for heat treatment in the preparation process of the feed additive. Thus, the high temperature resistant acid beta-mannanases of the invention will show great potential in feed applications.

3. The invention provides a production method and a production strain for liquid biological fermentation of beta-mannase, which have higher fermentation activity, higher extraction yield and lower manufacturing cost.

Description of the drawings:

FIG. 1 is a graph of optimum reaction temperature;

FIG. 2 is a graph of thermal stability;

FIG. 3 is a graph of the optimum reaction pH;

FIG. 4pH stability graph;

FIG. 5 is a graph of the digestion resistance to pepsin and trypsin.

The specific embodiment is as follows:

in order to make the objects, technical solutions and advantages of the present patent more apparent, the present patent will be described in further detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the present invention.

EXAMPLE 1 mutagenesis seed selection of Strain

Collecting fresh culture slant of Aspergillus niger HQM-2, eluting thallus with sterile physiological saline, transferring into triangular flask with glass beads, shaking with shaking table for 30min to disperse thallus, filtering with sterile absorbent cotton, collecting monospore, and adjusting monospore concentration to 10 with physiological saline ⁶ About one/mL, and NTG mother liquor was added so that the final concentration was 0.2g/L. Then reacting for 30min at 34 ℃, after proper dilution, taking 100 mu L of the mixture to be coated on a screening plate, culturing for about 4 days at 34 ℃, picking up single colony with larger transparent ring on the screening plate, inoculating into a seed bottle for culturing, and inoculating into a fermentation shake flask after seeds grow well. Finally screening to obtain a strain of beta-mannaThe enzyme activity of the carbohydrase is improved by 4.6 times, and the beta-mannanase produced by the mutant strain has good heat resistance and pH stability through the research of the enzymatic characteristics.

Screening of plate medium: tryptone 1%; yeast powder 0.5%, sodium chloride 0.5%, locust bean gum 0.5%, congo red 0.3%, agar 2%, and water for the rest, with pH6.0;

fermentation shake flask medium: 2% of konjak fine powder; 2% of yeast powder, 1.6% of corn steep liquor, 0.2% of dipotassium hydrogen phosphate, 0.3% of magnesium sulfate, 0.5% of ammonium sulfate, 0.8% of sodium nitrate, 0.3% of sodium carbonate and the balance of water, and the pH value is 6.0.

(1) Stability passage experiment of beta-mannase high-producing strain HNG26-22

The beta-mannase high-yield strain Aspergillus niger HNG26-22 is cultured to be fresh inclined plane, an inoculating loop lawn is taken by a sterile iron shovel to be inoculated into a fermentation shake flask, and the fermentation shake flask is cultured for 4d at 34 ℃ and 200r/min, so that the enzyme activity of the beta-mannase is determined. The shake flask results of the strain were serially passaged 10 times and are shown in table 1:

TABLE 1 stability test results of strain HNG26-22

The strain HNG26-22 has the advantages of high growth speed, small spore yield and less mycelium pellet when cultured in a liquid shake flask. The mutant strain was subcultured for 10 generations, and the experimental results can be seen from Table 1, and the genetic stability of the mutant strain was good.

Example 2 method for determining the enzymatic Activity of beta-mannanase

(1) Definition of the enzyme Activity Unit of beta-mannanase of the invention

Under certain conditions (if not specified, the conditions are 40 ℃ C., pH 5.0), the amount of enzyme required to decompose beta-mannans in locust bean gum to 1. Mu. Mol of reducing sugar per minute is defined as 1 enzyme activity unit.

(2) Enzyme activity determination method

The activity of the beta-mannase is detected by adopting a DNS method, and the 3, 5-dinitrosalicylic acid solution and the reducing sugar solution are reduced into a reddish brown amino compound after being heated together, so that the quantity of the reducing sugar and the degree of the color depth of reddish brown substances are in a proportional relation within a certain range, and the beta-mannase can be used for colorimetric determination.

The specific method comprises the following steps: taking 900 mu L of 0.5% locust bean gum solution prepared by disodium hydrogen phosphate-citric acid buffer solution with pH of 5.0, adding 100 mu L of enzyme solution properly diluted by disodium hydrogen phosphate-citric acid buffer solution with pH of 5.0, accurately reacting for 10min at 40 ℃, adding 1.5mL of DNS to terminate the reaction, boiling water bath for 5min, and cooling to 540nm to measure OD value.

Example 3 liquid fermentation of Strain to produce beta-glucanase and extraction thereof

1. Seed culture

The culture medium comprises the following components in percentage by mass and volume:

(1) Plate separation medium

Tryptone 1%; yeast powder 0.5%, sodium chloride 0.5%, locust bean gum 0.5%, congo red 0.3%, agar 2%, and water for the rest, with pH6.0.

(2) Seed culture medium

2% of tryptone, 0.3% of beef extract, 1% of peptone, 0.5% of sodium chloride, 0.3% of ammonium sulfate and the balance of water, wherein the pH value is 6.0.

(3) Fermentation shake flask fermentation medium

2% of konjak fine powder; 2% of yeast powder, 1.6% of corn steep liquor, 0.2% of dipotassium hydrogen phosphate, 0.3% of magnesium sulfate, 0.5% of ammonium sulfate, 0.8% of sodium nitrate, 0.3% of sodium carbonate and the balance of water, and the pH value is 6.0.

(4) Culture conditions

Separation plate: culturing at 34 ℃ for 4 days;

liquid seed: taking a ring of strain from the flat plate, inoculating the strain to a seed culture medium, and culturing for 48 hours at 34 ℃ with the rotating speed of a shaking table of 200r/min;

fermenting and shaking: inoculating the seed solution into a fermentation shake flask culture medium according to the inoculum size of 2%, and culturing at 34 ℃ for 120h at the rotating speed of a shaking table of 200r/min.

2. Seed pot expansion culture

The culture medium comprises the following components in percentage by mass and volume:

(1) Seed tank culture medium

3% of corn starch, 1.5% of peptone, 2% of bean cake powder, 1% of sodium chloride, 0.5% of calcium chloride, 3% of ammonium sulfate, 1% of magnesium sulfate and the balance of water, wherein the pH value is 6.0;

(2) Seed tank sterilization process conditions: sterilizing at 121deg.C and 0.12MPa for 30min;

(3) Seed tank culture process conditions

The inoculation amount is 2 percent, the tank pressure is 0.05MPa, the culture temperature is 34 ℃, the stirring rotating speed is 200r/min, and the pH is controlled to be 6.0;

(4) Seed pot seed transfer conditions: the thalli are deeply and robustly dyed and have no bacteria.

3. Production of beta-mannase by liquid fermentation

The culture medium comprises the following components in percentage by mass and volume:

(1) Fermentation tank medium: 2% of konjaku flour, 1.8% of corn steep liquor, 2.2% of dipotassium hydrogen phosphate, 0.25% of magnesium sulfate, 1.8% of ammonium sulfate, 0.3% of sodium nitrate and the balance of water, wherein the pH value is 6.0;

(2) Fermentation tank sterilization process conditions: sterilizing at 121deg.C and 0.12MPa for 30min.

(3) Fermentation process conditions of the fermentation tank: the tank pressure is 0.05MPa, the culture temperature is 34 ℃, the rotating speed is 400r/min, the inoculation amount is 2%, the feeding is started when the dissolved oxygen is reduced to the lowest and then increased to 30%, the dissolved oxygen is controlled to be 20-30% (the initial feeding amount is 300g/h, the feeding amount is reduced when the dissolved oxygen is lower than 20%, the feeding amount is increased when the dissolved oxygen is higher than 30%, the feeding amount is properly regulated according to the dissolved oxygen condition), the fermentation is carried out until the bacterial autolysis is serious, and the tank is put when the enzyme activity is not obviously improved.

4. Supplementing material

(1) Feed medium: 10% of konjak flour, 3.5% of corn steep liquor, 1.8% of ammonium sulfate, 1.5% of monopotassium phosphate and the balance of water, wherein the pH value is 6.0.

(2) The material supplementing method comprises the following steps: when the dissolved oxygen is reduced to the lowest and then increased to 30%, feeding is started, and the dissolved oxygen is controlled to be 20-30%.

5. Tank for placing

Culturing for 110-120h, and slowly growing enzyme activity, and placing the thallus into a tank after partial autolysis.

6. Extraction and refining of beta-mannase

After fermentation, adding 40% of water and 0.3% of calcium chloride according to the volume of fermentation liquid, adding 3% of perlite filter aid, and carrying out plate-frame filter pressing; carrying out ultrafiltration concentration on the clarified filter-pressing enzyme solution by using a 20kDa ultrafiltration membrane; adding a stabilizing agent into the concentrated solution: 8% glucose, 10% sodium chloride, preservative: and (3) filtering and sterilizing the potassium sorbate with diatomite at 0.15% to obtain the beta-mannanase finished product enzyme preparation.

The Aspergillus niger mutant strain CGMCC No.23221 and the culture method are used for fermentation, and the fermentation period and the fermentation liquid enzyme activity of 6 batches of fermentation are shown in the table 2, wherein the fermentation liquid enzyme activity is 12000-13000U/mL.

TABLE 2 results of 50L jar fermentation experiments

As can be seen from Table 2, the fermentation level of the mutant strain HNG26-22 is relatively stable, and the fermentation enzyme activity reaches over 12000U/mL.

Example 4 optimal reaction temperature

Taking the beta-mannanase finished product prepared in the batch 1 of the example 3, determining the activity of the beta-mannanase under the normal condition of pH5.0 by adopting the method described in the example 2 at 30, 35, 40, 45, 50, 55 and 60 ℃ respectively, and calculating the relative enzyme activity by taking the enzyme activity at 40 ℃ as 100%. The results are shown in FIG. 1, and the optimum reaction temperature is 40 ℃.

Example 5 thermal stability

Taking the beta-mannase finished product prepared in the batch 1 in the example 3, respectively placing enzyme solutions at 60, 65, 70, 75, 80, 85, 90, 95 and 100 ℃ for heat preservation treatment for 2 hours, measuring the enzyme activity by adopting the method described in the example 2 after heat preservation, and calculating the relative enzyme activity by taking the untreated original enzyme activity as 100%. The experimental results are shown in fig. 2. The heat preservation is carried out for 2 hours at 90 ℃, more than 85% of enzyme activity can be still maintained, the heat stability is better, and the requirements of feeds and other industrial enzymes can be met.

Example 6 optimal reaction pH

Taking the beta-mannanase finished product prepared in the batch 1 of the example 3, according to the enzyme activity measuring method described in the example 2, the relative enzyme activities under the conditions of pH values of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0 are respectively measured at the temperature of 40 ℃, and the relative enzyme activities are calculated by taking the enzyme activities at the pH value of 3.0 as 100%. As a result of measurement, as shown in FIG. 3, the enzyme activity of the beta-mannanase was highest at a pH of around 3.0.

EXAMPLE 7 acid and alkali resistance

The beta-mannase finished product prepared in batch 1 of example 3 is respectively placed in disodium hydrogen phosphate-citric acid buffers with pH of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0, and the enzyme activity is measured by the method described in example 2 after standing for 2 hours at room temperature, and the relative enzyme activity is calculated by taking the untreated original enzyme activity as 100%. As shown in FIG. 4, the relative enzyme activity remained at 80% or more after 2 hours of treatment at pH 2.0-7.0.

EXAMPLE 8 beta-mannanase enzyme resistance to Trypsin and pepsin hydrolysis assay

Taking 0.1mL of the beta-mannanase finished enzyme solution prepared in the example 3 batch 1, respectively adding 0.5mL of pepsin (pH 2.0 concentration of 100U/mL) and 0.5mL of trypsin (pH 7.0 concentration of 150U/mL), treating at 37 ℃ for different time, and measuring the enzyme activity, wherein the relative enzyme activity is calculated by taking the untreated original enzyme activity as 100%. The results are shown in FIG. 5, wherein the relative enzyme activity is maintained at more than 95% after pepsin treatment for 2 hours, and at more than 90% after trypsin treatment for 2 hours, which indicates that the beta-mannase of the invention has very good pepsin and trypsin hydrolysis resistance.

The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the patent. It should be noted that, for a person skilled in the art, the above embodiments may also make several variations, combinations and improvements, without departing from the scope of the present patent. Therefore, the protection scope of the patent is subject to the claims.

Claims (5)

1. A method for producing beta-mannase by fermentation is characterized in that the production strain is aspergillus nigerAspergillus niger) HNG26-22 with preservation number of CGMCC No.23221.

2. The method for producing beta-mannanase by fermentation according to claim 1, wherein the fermentation process conditions in the fermenter are: the inoculation amount is 1.5-2.5%, the pot pressure is 0.05-0.08Mpa, the culture temperature is 32-34 ℃, the rotating speed is 200-800r/min, the material supplementing is started when the dissolved oxygen is reduced to the lowest and is increased to 30-35%, the dissolved oxygen is controlled to be 20-30%, the culture is carried out for about 110-130h, the enzyme activity is slowly increased, and the cell is put into the pot when the cell begins to be partially autolyzed.

3. A method for the fermentative production of β -mannanase according to claim 2, wherein the fermentation medium comprises, in mass volume percent: 1.5 to 2 percent of konjaku flour, 1.6 to 2 percent of corn steep liquor, 2 to 2.5 percent of dipotassium hydrogen phosphate, 0.2 to 0.3 percent of magnesium sulfate, 1.5 to 2.0 percent of ammonium sulfate, 0.25 to 0.3 percent of sodium nitrate, and the balance of water, wherein the pH value is 6.0 to 6.2.

4. A method for the fermentative production of β -mannanase according to claim 2, wherein the feed medium consists of, in mass volume percent: 10-12% of konjak flour, 3-3.5% of corn steep liquor, 1.5-2.0% of ammonium sulfate, 1-2.5% of monopotassium phosphate and the balance of water, and pH7.0-7.2.

5. The method for producing beta-mannanase by fermentation according to claim 2, wherein the method for extracting and refining beta-mannanase comprises the steps of: after fermentation, adding 40-45% of water and 0.25-0.35% of calcium chloride according to the volume of fermentation liquid, adding 2.5-3.5% of perlite filter aid, and carrying out plate-frame filter pressing; carrying out ultrafiltration concentration on the clarified filter-press enzyme solution by using an ultrafiltration membrane; adding a stabilizer and a preservative into the concentrated solution, and then filtering and sterilizing by using diatomite to obtain the beta-mannanase finished enzyme preparation.

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