CN114196573A - Bacillus for producing alkaline mannase and application thereof - Google Patents
Bacillus for producing alkaline mannase and application thereof Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
- C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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Abstract
The invention discloses a Bacillus mutant strain for high yield of alkaline mannase and a liquid fermentation method thereof, wherein the Bacillus mutant strain is (Bacillus sp.) GL-21372 with the preservation number of CGMCC No. 23194. The strain is cultured for 120h on a 100L fermentation tank by feeding fermentation through liquid submerged fermentation, and the fermentation enzyme activity reaches between 24300 and 25600U/mL. The alkaline mannan produced by the invention has the optimum reaction pH value of 10.0 and the optimum reaction temperature of 75 ℃. The residual enzyme activity is more than 70% after the heat preservation is carried out for 4h at the temperature of 80 ℃, and the residual enzyme activity is more than 85% after the enzyme is preserved for 24h at the pH value of 10.5, so that the enzyme has good alkali resistance and heat resistance, and can be applied to industries such as washing, spinning, biological bleaching and the like.
Description
The technical field is as follows:
the invention belongs to the technical field of microorganisms, and particularly relates to a bacillus mutant strain for producing alkaline mannanase and a liquid fermentation technology thereof.
Background art:
the beta-mannase can be used for producing water-soluble mannose and mannooligosaccharides by catalyzing and hydrolyzing mannan, glucomannan, galactomannan and galactoglucomannan, is an important industrial enzyme preparation, and is widely applied to the fields of feed, food processing, textile, biological bleaching, washing industry and the like. At present, most of mannase used in the fields of feed, food processing and the like is acidic mannase, and the mannase can not fully play a role in the fields of biological bleaching, washing and the like; harmony between human beings and the environment, the need to reduce the use and discharge of chemical waste; the price of raw materials rises, the fermentation level of the alkaline mannase is generally low, the competitiveness of the product is insufficient, and the product use amplification is influenced. Therefore, the production strain of the high-yield alkaline mannase is bred, and the produced alkaline mannase has good alkali resistance and heat resistance, and has great significance for promoting the conversion of new and old kinetic energy and upgrading the industrial structure.
The invention content is as follows:
in order to solve the technical problems, the invention selects and breeds a bacillus capable of highly producing the alkaline mannase, and produces and prepares the high-activity mannase with good heat resistance and alkali resistance by a liquid fermentation method.
One of the technical schemes provided by the invention is a Bacillus mutant strain, in particular to Bacillus (Bacillus sp.) GL-21372, wherein the strain GL-21372 is obtained by joint mutagenesis and breeding of nitrosoguanidine and diethyl sulfate, and is preserved in China general microbiological culture Collection center at 25.8.8.2021, the preservation address is as follows: the microbial research institute of Chinese academy of sciences, No. 3 Xilu-Beijing, Chaozhou, Chao code No. 1, facing Yang, has a preservation number of CGMCC No. 23194.
The second technical scheme provided by the invention is the application of Bacillus (Bacillus sp.) GL-21372 in the production of mannase, in particular the application in the production of alkaline mannase by a fermentation method;
specifically, the fermentation method is as follows:
inoculating the seed liquid into a fermentation culture medium according to the proportion of 10-12% of the inoculation amount, wherein the culture conditions are as follows: at the temperature of 36-38 ℃, the tank pressure of 0.07-0.08MPa, the air volume of 0-4vvm, the rotation speed of 300-;
after fermentation for 100-120h, the fermentation enzyme activity reaches 24300U/mL-25600U/mL;
further, the fermentation medium consists of (m: v): corn flour 4-5%, corn starch 1.5-2%, cottonseed protein powder 5-6%, ammonium chloride 1-1.2%, KH2PO42-2.2 percent of corn steep liquor, 1.8-2 percent of corn steep liquor, 2-2.5 percent of palm meal and CaCl20.8-1 percent of water, and the balance of water, adjusting the pH value to 6.5, and sterilizing for 30-40min at the temperature of 121-;
further, the feed medium composition was (m: v): dextrin 30-35%, ammonium chloride 5-5.5%, KH2PO4 0.1-0.12％，NaH2PO42-2.2%, corn steep liquor 5-6%, CaCl20.2-0.3 percent of water and the balance of water, adjusting the pH value to 7.5, and sterilizing at the temperature of 121-.
Has the advantages that:
1. the invention breeds a mutagenic strain GL-21372 for high-yield alkaline mannanase by mutagenizing nitrosoguanidine and diethyl sulfate of bacillus GL-X167, and optimizes the fermentation process of the mutagenic strain GL-21372 so that the enzyme activity of the mutagenic strain can reach 24300U/mL to 25600U/mL.
2. Alkaline mannase obtained by fermentation of mutagenic strain GL-21372, the effective pH range of the enzyme is 6.5-11.0, and the optimum pH range of the enzyme is 8.0-10.5; the optimum reaction pH value of the enzyme is 10.0. The effective temperature range of the enzyme is 40-85 ℃, the optimal temperature range of the enzyme is 60-80 ℃, and the optimal reaction temperature of the enzyme is 75 ℃. The residual enzyme activity is more than 70% after the heat preservation is carried out for 4h at the temperature of 80 ℃, and the residual enzyme activity is more than 85% after the enzyme is preserved for 24h at the pH value of 10.5, so that the enzyme has good alkali resistance and heat resistance, and can be applied to industries such as washing, spinning, biological bleaching and the like.
Description of the drawings:
FIG. 1 relative enzyme activity at different pH;
FIG. 2 relative enzyme activities at different temperatures;
FIG. 380 ℃ relative enzyme activity after incubation for 0-6 h.
FIG. 4 shows the relative enzyme activity of the enzyme treated under alkaline conditions for 24 hours.
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.
The enzyme activity determination method of the alkaline mannase adopted by the invention comprises the following steps:
(1) definition of enzyme activity unit: 1mL of liquid enzyme was subjected to hydrolysis of mannan under conditions (not specifically mentioned, the conditions were 40 ℃ C., pH8.0), and the amount of enzyme producing 1. mu.g of mannose in 1min was defined as 1 enzyme activity unit (U) in U/mL.
(2) Apparatus and device
A constant-temperature water bath kettle: 40 + -0.2 deg.C
A spectrophotometer: in accordance with the provisions of GB9721
(3) DNS reagent
3.15g of 3, 5-dinitrosalicylic acid is accurately weighed and poured into 500mL of the solution, and the solution is stirred in a water bath to 45 ℃. Then 100mL of sodium hydroxide solution was added stepwise) with constant stirring until the solution was clear and transparent (note: the solution temperature should not exceed 48 c during the addition of sodium hydroxide. 91.0g of potassium sodium tartrate tetrahydrate, 2.5g of phenol and 2.5g of anhydrous sodium sulfite are gradually added. The 45 ℃ water bath heating was continued while adding 300mL of water, with constant stirring, until the added material was completely dissolved. The heating was stopped, the mixture was cooled to room temperature, and then the volume of the mixture was adjusted to 1000mL with water. Filtering with sintered glass filter. Taking the filtrate, storing in a brown bottle and keeping away from light. Can be used after being stored for 7 days at room temperature, and the effective period is 6 months.
(4) Drawing of mannose standard curve
Adding 1mg/mL standard mannose solution 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL into a 25mL test tube with a plug respectively, supplementing to 2mL with distilled water, adding 3mL DNS reagent, shaking uniformly, boiling in boiling water for 5min, taking out, rapidly cooling to room temperature with tap water, diluting to 25mL with distilled water, and fully mixing. The absorbance was measured at 540nm and a standard curve was drawn with mannose content (mg) as the abscissa and absorbance as the ordinate.
(5) Measurement of
Taking 2mL of 0.6g/L mannan solution to a 25mL test tube with a plug, carrying out water bath at 40 ℃ for 30min, adding 2mL of enzyme solution to be detected and 5mL of DNS reagent which are balanced at 40 ℃, immediately heating in the boiling water bath for 5min after oscillation, taking out, rapidly cooling to room temperature by using water, adding water to fix the volume to 25mL, and uniformly mixing. Zeroing with distilled water and measuring the absorbance at 540nm with a 10mm cuvette (A)0)。
Adding 2mL of 0.6g/L mannan solution into a 25mL test tube with a plug, balancing in a 40 ℃ water bath for 10min, adding 2mL of appropriately diluted enzyme solution (balanced in a 40 ℃ water bath), shaking, accurately preserving heat for 30min, adding 5mL of DNS reagent, shaking, heating in a boiling water bath for 5min, taking out, rapidly cooling with water to room temperature, adding water to fix the volume to 25mL, and uniformly mixing. And (4) adjusting to zero by using distilled water, measuring the absorbance (A) of the sample at the wavelength of 540nm, and calculating the activity of the beta-mannase.
(6) Computing
Beta mannanase activity is expressed as X in units of enzyme activity per milliliter (U/mL), calculated according to formula (I):
in the formula:
a- - -absorbance of the sample tube;
A0-absorbance of sample blank tube;
k- -the slope of the standard curve;
b- -intercept of the standard curve;
m- - - -the molar mass of mannose in grams per mole (g/mol);
30- -enzymolysis reaction time, unit is min;
1000-unit conversion factor;
n- - -dilution multiple of sample to be measured.
EXAMPLE 1 guanidine hydrochloride mutagenesis of strains
Activating and culturing a wild type bacillus GL-X167 preserved in the laboratory of the applicant, collecting thalli growing to the logarithmic phase, centrifugally washing the thalli for 2 to 3 times by using sterile physiological saline, and suspending the thalli in a proper amount of physiological saline. Placing the prepared bacterial suspension into a triangular flask with glass beads, and oscillating in a shaking table at 200r/min for 10min to disperse thallus sufficiently and make thallus concentration reach 108～l09one/mL.
Respectively sucking 20mL of bacterial suspension into a triangular flask, adding nitrosoguanidine into the triangular flask to make the final concentration of nitrosoguanidine in the triangular flask to be 10-30 mu g/mL, placing the triangular flask in a 36 ℃ shaking table at 200rpm, and performing mutagenesis for 30-40min to make the lethality rate in 70-90%.
After mutagenesis, putting the mutagenic bacteria suspension into a 50mL centrifuge tube, using a high-speed refrigerated centrifuge, setting the temperature to be 4 ℃, 10000rpm, centrifuging for 3min, discarding the supernatant, immediately cleaning precipitated bacteria by using a seed culture medium preheated to 36 ℃ for 4 times, finally suspending all liquid seed culture mediums for mutagenesis again, fixing the volume to 10-30mL, taking 100 mu L of the suspension to be coated on a solid culture medium for screening, culturing for 48h at 36 ℃, and picking out a bacterial colony with a high HC value (HC value is the diameter of a hydrolysis ring/the diameter of a bacterial colony) after a single bacterial colony grows out.
Solid slant culture medium for mutagenesis: 20g of glucose, 5g of beef extract, 2g of peptone and NaH2PO4 2g， MgSO40.5g，CaCl2 0.5g，KH2PO44g of konjak powder and 15g of agar, dissolving the components in 1000mL of water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 20 min;
liquid seed culture medium for mutagenesis: 2g of konjac flour, 3g of beef extract, 8g of peptone, 2g of glucose and NaH2PO4 2g，MgSO4 0.5g，CaCl20.5g, dissolving the components in 1000mL of water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 20 min;
solid medium for screening: 10g of konjac flour, 3g of beef extract, 8g of peptone, 2g of glucose and NaH2PO4 2g，MgSO40.5g，CaCl20.5g of agar and 2.3 percent of agar, dissolving the components in 1000mL of water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 20 min;
and (3) shaking a flask for re-screening: selecting a bacterial colony with a larger ratio of the diameter of the transparent circle of the konjac flour to the diameter of the bacterial colony to carry out shake flask re-screening, inoculating the mutagenic bacterial strain with a high HC value into a shake flask screening culture medium to carry out fermentation, carrying out culture at 36 ℃ and 300rpm for 80h, and then measuring the fermentation enzyme activity of each mutagenic strain;
shake flask screening medium: corn flour 5%, corn starch 4%, cottonseed protein 8%, ammonium chloride 1%, K2HPO43％，KH2PO45 percent of konjak powder and 5 percent of CaCl 21 percent of water, and the balance of water, adjusting the pH value to 6.5, and sterilizing for 30-40min at the temperature of 121-;
the list of 1 mutagenic strain with higher enzyme activity screened by repeated shake flask screenings is as follows:
| bacterial strains | Original bacteria | GL-21163 |
| Enzyme activity (U/mL) | 5208 | 9230 |
The shake flask enzyme activity level of the high-producing strain GL21163 after 5 serial passages is as follows:
| number of passages | Shake flask enzyme activity level (U/mL) | Relative enzyme activity level (%) |
| Y1 | 9238 | 100.09％ |
| Y2 | 9263 | 100.36％ |
| Y3 | 9182 | 99.48％ |
| Y4 | 9103 | 98.62％ |
| Y5 | 9290 | 100.65％ |
The verification proves that the high-yield strain GL21163 obtained by mutagenesis has good genetic stability.
EXAMPLE 2 guanidine hydrochloride mutagenesis of strains
Then the mutant strain GL21163 is activated and cultured, the thallus growing to the logarithmic phase is collected, and is centrifugally washed for 2-3 times by using sterile normal saline, and is suspended in a proper amount of normal saline. Placing the prepared bacterial suspension into a triangular flask with glass beads, and oscillating in a shaking table at 200r/min for 10min to disperse thallus sufficiently and make thallus concentration reach 107～l08one/mL.
Taking 3mL of bacterial suspension into a sterile centrifuge tube, adding 3mL of phosphate buffer solution (pH 7.0), adding 0.05mL of DES solution, mixing uniformly, placing on a shaker at 30 ℃ and 180r/min respectively, treating for 15-20 minutes, controlling the lethality at 70-90%, and adding 3mL of 25% Na2S2O3The reaction was terminated.
Centrifuging the centrifuge tube for 3min at 4 deg.C and 10000rpm for 3min, discarding the supernatant, immediately cleaning the precipitated thallus with seed culture medium preheated to 36 deg.C for 4 times, suspending all liquid seed culture medium for mutagenesis again, metering to 10-30mL, coating 100 μ L of the suspension on solid culture medium for screening, culturing at 36 deg.C for 48h, and picking out bacterial colony with high HC value (HC value is hydrolysis ring diameter/colony diameter) after single thallus grows out.
Solid slant culture medium for mutagenesis: 20g of glucose, 5g of beef extract, 2g of peptone and NaH2PO4 2g， MgSO40.5g，CaCl2 0.5g，KH2PO44g of konjak powder and 15g of agar, dissolving the components in 1000mL of water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 20 min;
liquid seed culture medium for mutagenesis: 2g of konjac flour, 3g of beef extract, 8g of peptone, 2g of glucose and NaH2PO4 2g，MgSO4 0.5g，CaCl20.5g, dissolving the components in 1000mL of water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 20 min;
solid medium for screening: 10g of konjac flour, 3g of beef extract, 8g of peptone, 2g of glucose and NaH2PO4 2g，MgSO40.5g，CaCl20.5g of agar 2.3%, dissolving the above components in 1000mL of water, and adjusting pH to6.5, sterilizing at 121 ℃ for 20 min;
and (3) shaking a flask for re-screening: and (3) carrying out shake flask re-screening on the bacterial colony with the larger ratio of the diameter of the transparent circle of the konjac flour to the diameter of the bacterial colony, inoculating the mutagenic strain into a shake flask screening culture medium for fermentation, culturing at 36 ℃ at 300rpm for 80h, and then determining the fermentation enzyme activity of each mutagenic strain.
Shake flask screening medium: corn flour 5%, corn starch 4%, cottonseed protein 8%, ammonium chloride 1%, K2HPO4 3％，KH2PO45 percent of konjak powder and 5 percent of CaCl 21 percent of water, and the balance of water, adjusting the pH value to 6.5, and sterilizing for 30-40min at the temperature of 121-;
the list of 1 mutagenic strain with higher enzyme activity screened by repeated shake flask screenings is as follows:
| bacterial strains | GL-21163 | GL-21372 |
| Enzyme activity (U/mL) | 9230 | 11590 |
The shake flask enzyme activity level of the high-producing strain GL-21372 after 5 serial passages is as follows:
the verification proves that the high-yield strain GL-21372 obtained by mutagenesis has good genetic stability and can be used for amplification production.
EXAMPLE 3 production of alkaline mannanase by liquid fermentation
1) Solid slant culture medium: 5g of beef extract, 25g of peptone, 20g of glucose, 5.0g of sodium chloride and 20g of agar, dissolving the components in 1000mL of water, naturally adjusting the pH, and sterilizing at 121 ℃ for 20min for later use;
2) liquid seed culture medium: 30g of corn protein, 3g of beef extract, 8g of peptone, 2g of glucose and NaH2PO4 2g，MgSO4 0.5g，CaCl20.5g, adding water to a constant volume of 1000mL, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 20 min;
3) seeding tank medium (m: v): 3% of corn starch, 2% of corn protein, 2.5% of peptone, 0.5% of beef extract and KH2PO4 0.15％，CaCl20.005 percent of water and the balance of water, adjusting the pH value to 6.5, and sterilizing for 30-40min at the temperature of 121-;
4) fermenter Medium (m: v): corn flour 4%, corn starch 2%, cottonseed protein powder 6%, ammonium chloride 1%, KH2PO42 percent of corn steep liquor, 2 percent of palm meal and CaCl 21 percent of water, and the balance of water, adjusting the pH value to 6.5, and sterilizing for 30-40min at the temperature of 121-;
5) feed flask medium (m: v): dextrin 30%, ammonium chloride 5%, KH2PO4 0.1％，NaH2PO42% of corn steep liquor 6% of CaCl20.3 percent of water and the balance of water, adjusting the pH value to 7.5, and sterilizing for 30-40min at the temperature of 121-;
solid slant seed culture: inoculating a strain of bacillus mutagenic strain GL-21372 to a solid inclined plane, and culturing at a constant temperature of 36 ℃ for 60 hours;
liquid seed culture: inoculating one strain of the bacterial strain obtained by solid slant culture into a seed culture medium, and culturing for 36h under the conditions of initial pH6.5, 36 deg.C and shaking table rotation speed of 200 rpm;
seed tank culture: inoculating the seed liquid fermented in the shake flask into a seed tank according to the proportion of 15% of the inoculum size, controlling the dissolved oxygen to be 20% -30% by continuously increasing the rotating speed and the rotating speed to be 0.07-0.08MPa, controlling the air volume to be 0-4vvm and the rotating speed to be 300-800rpm, controlling the pH to be 6.5 initially by supplementing ammonia when the pH is lower than 6.0, and transferring the seed liquid to a fermentation tank when the pH begins to rise and is higher than 7.5;
culturing in a fermentation tank: inoculating the seed liquid in the seed tank into the fermentation tank according to the proportion of 10 percent of the inoculation amount, and culturing under the conditions that: at the temperature of 36 ℃, the tank pressure of 0.08MPa, the air volume of 0-4vvm and the rotation speed of 300-800rpm, controlling the dissolved oxygen to be 10-20% by continuously increasing the rotation speed and the air volume, controlling the initial pH to be 6.5, supplementing ammonia when the pH is lower than 6.0, controlling the pH to be 6.5, starting material supplement when the pH is higher than 7.5 during the fermentation period, controlling the pH to be 6.5-6.8 by material supplement, ending the fermentation when the enzyme activity is slowly increased and the cell autolysis is serious, wherein the fermentation period is about 100-120 h.
The following table shows the enzyme production of the fermentation of 5 batches of 100L fermentor, with the average enzyme production level of the fermentation broth of 24986U/mL.
| Batches of | Fermentation period (h) | Fermentation vigor (U/mL) |
| 1 | 105 | 25100 |
| 2 | 100 | 24300 |
| 3 | 115 | 24830 |
| 4 | 120 | 25600 |
| 5 | 115 | 25100 |
Example 4 pH Range of action of enzymes
Alkaline mannase with the enzyme activity of 20000U/mL prepared by centrifuging the clear liquid of the fermentation liquid is used as a uniform sample, the enzyme activity is measured under different pH values (6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5 and 12.0) respectively at the temperature of 40 ℃, the measured relative enzyme activity change curve is shown in figure 1, the effective pH range of the enzyme is 6.5-11.5, the optimal pH range of the enzyme is 8.0-10.5, and the optimal reaction pH value of the enzyme is 10.0.
Example 5 temperature Range of action of enzymes
Alkaline mannase with the enzyme activity of 20000U/mL is used as a uniform sample, the enzyme activity is measured under the condition that the pH value is 8.0 and under different temperatures (40, 45, 50, 55, 60, 65, 70, 75, 80, 85 and 90), the variation curve of the measured relative enzyme activity is shown in figure 2, the effective temperature range of the enzyme is 40-85 ℃, the optimal temperature range of the enzyme is 60-80 ℃, and the optimal reaction temperature of the enzyme is 75 ℃.
Example 6 thermostability of enzymes
The alkaline mannase with the enzyme activity of 20000U/mL is used as a uniform sample, the temperature is kept for 0-6h at 80 ℃ under the condition that the pH value is 8.0, and the residual enzyme activity is determined, as shown in figure 3, after the temperature is kept for 4h at 80 ℃, the residual enzyme activity is more than 70%, and the heat-resistant preservation activity is good.
Example 7 alkali-resistant stability of the enzyme
Alkaline mannase with the enzyme activity of 20000U/mL is used as a uniform sample, and the sample is stored at room temperature for 24 hours under the conditions of pH values of 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0 and 11.5 to determine the residual enzyme activity, as shown in figure 4, the residual enzyme activity is more than 85% at the pH value of 10.5, and the alkaline mannase has good alkali resistance.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.
Claims (5)
1. A Bacillus mutant strain for high yield of alkaline mannase is characterized in that the strain is Bacillus (Bacillus sp.) GL-21372, and the collection number of the strain is CGMCC No. 23194.
2. Use of the bacillus GL-21372 of claim 1 in the production of a mannanase.
3. The use of claim 2, wherein the mannanase is produced by fermentation using bacillus GL-21372 as follows:
inoculating the seed liquid into a fermentation culture medium according to the proportion of 10-12% of the inoculation amount, wherein the culture conditions are as follows: at the temperature of 36-38 ℃, the tank pressure of 0.07-0.08MPa, the air volume of 0-4vvm, the rotation speed of 300-.
4. The use according to claim 3, wherein the fermentation medium consists of: corn flour 4-5%, corn starch 1.5-2%, cottonseed protein powder 5-6%, ammonium chloride 1-1.2%, KH2PO42-2.2 percent of corn steep liquor, 1.8-2 percent of corn steep liquor, 2-2.5 percent of palm meal and CaCl20.8-1% and the balance of water, and adjusting the pH value to 6.5.
5. The use according to claim 3, wherein the feed medium consists of: dextrin 30-35%, ammonium chloride 5-5.5%, KH2PO4 0.1-0.12％，NaH2PO42-2.2%, corn steep liquor 5-6%, CaCl20.2-0.3 percent of the total weight of the mixture and the balance of water, and the pH value is adjusted to 7.5.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101157903A (en) * | 2007-09-17 | 2008-04-09 | 云南师范大学 | Producing Strain for beta- mannose and preparation method thereof |
| CN112522243A (en) * | 2020-12-09 | 2021-03-19 | 山东隆科特酶制剂有限公司 | Acid protease and production method thereof |
| CN113502249A (en) * | 2021-07-28 | 2021-10-15 | 河南省科学院生物研究所有限责任公司 | Bacillus amyloliquefaciens HTGC-10 and fermentation application method thereof for producing beta-mannase |
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2021
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101157903A (en) * | 2007-09-17 | 2008-04-09 | 云南师范大学 | Producing Strain for beta- mannose and preparation method thereof |
| CN112522243A (en) * | 2020-12-09 | 2021-03-19 | 山东隆科特酶制剂有限公司 | Acid protease and production method thereof |
| CN113502249A (en) * | 2021-07-28 | 2021-10-15 | 河南省科学院生物研究所有限责任公司 | Bacillus amyloliquefaciens HTGC-10 and fermentation application method thereof for producing beta-mannase |
Non-Patent Citations (1)
| Title |
|---|
| 张婕等: "产β-甘露聚糖酶细菌的筛选及产酶条件的优化" * |
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| CN114196573B (en) | 2023-08-01 |
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