CN112522243B - Acid protease and production method thereof - Google Patents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
- C12N9/62—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from Aspergillus
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/14—Pretreatment of feeding-stuffs with enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/06—Enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to acid-producing protease and a production method thereof. The acid protease is produced by Aspergillus niger FSM-208063, the strain is preserved in China general microbiological culture Collection center (CGMCC NO. 20724) at 9-18 th of 2020. Is obtained by mutagenizing and breeding a strain of Aspergillus niger by nitrosoguanidine. The acid protease produced by the strain has the residual enzyme activity of more than 70 percent after heat preservation for 4 hours at the temperature of 80 ℃, has good acid resistance and heat resistance, and can be applied to industries of wine brewing, food, feed, leather processing and the like.
Description
The technical field is as follows:
the invention belongs to the technical field of bioengineering, and particularly relates to acid-producing protease and a production method thereof.
Background art:
the acid protease is an enzyme capable of hydrolyzing protein under the environment of pH value of 2.5-5.0. This requirement for pH of the acid protease may be related to the carboxyl group contained in its active center. Since acid protease has good acid resistance, it is widely used in food, medicine, light industry, leather process, and feed processing industries.
At present, most of commercialized acid protease is derived from microorganisms, and microbial strains capable of producing acid protease comprise: aspergillus saitoi, Aspergillus awamori, Mucor miehei, Penicillium, Rhizopus, Aspergillus usamii, Aspergillus niger, Aspergillus oryzae, Penicillium, Saccharomyces cerevisiae, Lactobacillus, Bacillus subtilis, etc. The commercial acid protease producing strain is mainly Aspergillus oryzae, Aspergillus niger and Aspergillus usamii.
Patent CN 201810409771.4 discloses a method for improving enzyme activity by changing the traditional yeast structure and liquid fermentation process and adopting a solid fermentation mode, wherein the enzyme activity of the fermented material reaches 62314.0U/g.
The invention discloses an Aspergillus niger mutant strain for high yield of acid protease and a liquid fermentation method thereof, wherein the Aspergillus niger mutant strain is Aspergillus niger TP-235, the Aspergillus niger mutant strain is cultured for 120h by feeding fermentation on a 50L fermentation tank through liquid submerged fermentation, and the enzyme production level is 21852U/mL. The optimum pH range of the produced acidic protease is 2.5-3.5, the optimum action temperature range is 55-65 ℃, the residual enzyme activity is 78% after heat preservation is carried out for 1h at 60 ℃, and the residual enzyme activity is 58.4% after heat preservation is carried out for 2h at 80 ℃.
Patent CN201810014018.5 discloses an acid protease, its coding gene and application, the optimum pH of the protease is 3.5, in the range of pH 2.5-pH 3.5, the enzyme can maintain its 70% above enzyme activity; the optimum temperature is 60 ℃, and the enzyme activity is still more than 60% at 65 ℃.
However, due to the rising of the price of raw materials and the continuous improvement of the enzyme activity level in the same industry at home and abroad, the fermentation level of the acid protease in the prior art at home is generally lower and the competitiveness of the product is insufficient, so that a production strain of high-yield acid protease is bred, and the produced acid protease has good acid 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:
the invention aims to provide an Aspergillus niger mutant strain with high yield of acid protease and a liquid fermentation enzyme production method thereof.
The Aspergillus niger FSM-208063 strain of high-yield acidic protease is deposited in China general microbiological culture Collection center (CGMCC) at 9/18 of 2020, and the preservation number is CGMCC NO. 20724. The strain is obtained by mutation breeding of nitrosoguanidine in Aspergillus niger with the preservation number of CGMCC NO. 10789.
The liquid fermentation technology mainly comprises the following steps:
1) the solid slant medium was as follows: 5g/L of yeast powder, 2g/L of peptone, 40g/L of sucrose and NaH2PO4 2g/L，MgSO4 0.5g/L，CaCl2 0.5g/L，KH2PO44g/L, 2g/L casein, 15g/L agar and the balance of water, adjusting the pH to 4.0, and sterilizing at 121 ℃ for 20min for later use;
2) the liquid seed culture medium is prepared as follows: 30g/L of soybean protein concentrate, 3g/L of yeast powder, 8g/L of peptone, 2g/L of sucrose and NaH2PO4 2g/L，MgSO4 0.5g/L，CaCl20.5g/L, and the balance of water, adjusting the pH to 4.0, and sterilizing at 121 ℃ for 20 min;
3) seeding tank culture medium: 30g/L of corn starch, 30g/L of soybean protein concentrate, 20g/L of peptone, 10g/L of yeast powder and KH2PO4 0.15g/L，CaCl20.005g/L and the balance of water, adjusting the pH value to 4.0, and sterilizing at the temperature of 121-;
4) fermentation tank culture medium: 40g/L of corn flour, 20g/L of corn starch, 60g/L of cottonseed protein powder and NH4Cl 10g/L，KH2PO422g/L, 18g/L corn steep liquor and CaCl210g/L, the balance being water, adjusting the pH to 4.0, and sterilizing at the temperature of 121-;
5) and (3) feeding bottle culture medium: 300g/L of maltose syrup, 60g/L of ammonium chloride and KH2PO4 1.0g/L，NaH2PO420g/L, 8.0g/L sodium citrate, 60g/L corn steep liquor and CaCl23.0g/L and the balance of water, adjusting the pH value to 3.5, and sterilizing at the temperature of 121-;
6) the culture method comprises the following steps:
solid slant seed culture: inoculating one strain of the aspergillus niger mutagenic strain to a solid inclined plane, and culturing at a constant temperature of 34 ℃ 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 55h under the conditions of initial pH4.0, 34 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 12 percent of the inoculation amount, controlling the pressure of the seed tank to be 0.08MPa, the air volume to be 0-3vvm and the rotation speed to be 200-800rpm, controlling the dissolved oxygen to be 20-30 percent by continuously increasing the rotation speed and the air volume, controlling the initial pH to be 4.0 by supplementing ammonia when the pH is lower than 4.0, transferring the seed liquid into a fermentation tank when the pH begins to rise and is higher than 4.5, and culturing for about 50 hours;
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: the fermentation process comprises the steps of controlling dissolved oxygen to be 10-20% by continuously increasing the rotating speed and the wind quantity at the temperature of 34 ℃, controlling the air quantity to be 0.08MPa, controlling the air quantity to be 0-3vvm and the rotating speed to be 200-800rpm, controlling the dissolved oxygen to be 10-20% by continuously increasing the rotating speed and the wind quantity, controlling the pH to be 4.0 by supplementing ammonia when the initial pH is lower than 4.0, starting material supplementing when the pH is higher than 4.5 during the fermentation period, controlling the pH to be 4.5-4.7 by supplementing material, ending the fermentation when the enzyme activity is slowly increased and the thalli are seriously autolyzed, wherein the fermentation period is about 90-110h, and the activity of the fermentation enzyme reaches 82800U/mL-84600U/mL.
Has the advantages that:
1. the invention breeds a mutagenic strain FSM-208063 of high-yield acid protease by carrying out nitrosoguanidine mutagenesis on an aspergillus niger strain with the preservation number of CGMCC10789, and optimizes the fermentation process of the mutagenic strain FSM-208063 so that the enzyme activity of the mutagenic strain FSM-208063 reaches 82800U/mL to 84600U/mL.
2. The acid protease obtained by fermentation of the mutagenic strain FSM-208063 has an effective pH range of 1.0-5.5, an optimal pH range of 1.5-4.5 and an optimal reaction pH value of 2.5. The effective temperature range of the enzyme is 46-78 ℃, the optimal temperature range of the enzyme is 50-74 ℃, and the optimal reaction temperature of the enzyme is 66 ℃. The residual enzyme activity is more than 70% after the heat preservation is carried out for 4 hours at the temperature of 80 ℃, the acid resistance and the heat resistance are good, and the method can be applied to industries such as wine making, food, feed and leather processing.
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.
The specific implementation mode is as follows:
the present invention will be described in more detail with reference to the following specific examples, which are given by way of illustration only and are not intended to limit the scope of the present invention.
EXAMPLE 1 mutagenic Breeding of strains
In each mutagenesis batch, 100 fresh inclined planes of the mutagenesis strain are taken, the thalli are eluted into a large shake flask by using about 300mL of sterilized normal saline, glass beads are added into a shaking table for 200rmp for 30min to disperse the thalli, and the thalli are centrifugally collected to be used as mutagenesis bacterial suspension for spawn running.
20mL of the bacterial suspension is respectively sucked into 1 triangular flask with 50mL, nitrosoguanidine is added into the triangular flask to make the final concentration of the nitrosoguanidine in the triangular flask to be 10-30 mu g/mL, the triangular flask is placed in a shaking table with the temperature of 34 ℃ and the rpm is 200, the time for mutagenesis is 30-40min from the time of adding the nitrosoguanidine, and the lethality is 70-95%.
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 for 4 times by using about 1200mL seed culture medium preheated to 34 ℃, finally suspending all mutagenic liquid seed culture medium again, fixing the volume to 10-30mL, taking 100 mu L of the culture medium to be coated on the mutagenic seed screening culture medium, culturing for 60h at 34 ℃, and picking out bacterial colonies with high HC value (HC value is hydrolysis ring diameter/bacterial colony diameter) after single bacterial colony grows out.
Solid slant culture medium for mutagenesis: 40g of sucrose, 5g of yeast powder, 2g of peptone and NaH2PO4 2g，MgSO4 0.5g，CaCl2 0.5g，KH2PO44g of casein, 2g of agar and 15g of agar, dissolving the components in 1000mL of water, adjusting the pH value to 4.0, and sterilizing at 121 ℃ for 20 min;
liquid seed culture medium for mutagenesis: 10g of casein, 3g of yeast powder, 8g of peptone, 2g of sucrose and NaH2PO4 2g，MgSO4 0.5g，CaCl20.5g, dissolving the above components in 1000mL water, adjusting pH to 4.0, sterilizing at 121 deg.C for 20min；
Mutagenic seed screening culture medium: 30g of casein, 3g of yeast powder, 8g of peptone, 2g of sucrose 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 4.0, and sterilizing for 20min at 121 ℃;
primary screening by a flat plate:
the primary screening method comprises the following steps: and (3) dibbling the colonies grown after mutagenesis on a screening plate, culturing at 34 ℃ for 60h, observing the size of a transparent ring around the colonies, and selecting the colonies with a larger ratio of the diameter of the casein transparent ring to the diameter of the colonies for shake flask rescreening.
And (3) shaking a flask for re-screening:
the secondary screening method comprises the following steps: inoculating the mutagenic strain into a fermentation shake flask for fermentation, culturing at 34 ℃ and 300rpm for 80h, and measuring the fermentation enzyme activity of each mutagenic strain by a spectrophotometry.
Shake flask screening medium: corn flour 5%, corn starch 4%, soybean protein concentrate 8%, ammonium chloride 1%, K2HPO43％，KH2PO45% of corn steep liquor, 5% of CaCl 21 percent of water, and the balance of water, adjusting the pH value to 4.0, 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 | FSM-208063 |
| Enzyme activity (U/mL) | 19208 | 35800 |
The shake flask enzyme activity level of the high-producing strain FSM-208063 after 5 continuous passages is as follows:
the verification proves that the high-yield strain FSM-208063 obtained by mutagenesis has good genetic stability and can be used for large-scale production and amplification production.
EXAMPLE 2 production of acid protease by liquid fermentation
Solid slant seed culture: inoculating an inoculation of the aspergillus niger mutagenic strain FSM-208063 on a solid inclined plane, and culturing at a constant temperature of 34 ℃ 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 55h under the conditions of initial pH4.0, 34 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 12 percent of the inoculation amount, controlling the pressure of the seed tank to be 0.08MPa, the air volume to be 0-3vvm and the rotation speed to be 200-800rpm, controlling the dissolved oxygen to be 20-30 percent by continuously increasing the rotation speed and the air volume, controlling the initial pH to be 4.0 by supplementing ammonia when the pH is lower than 4.0, transferring the seed liquid into a fermentation tank when the pH begins to rise and is higher than 4.5, and culturing for about 50 hours;
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 34 ℃, the pressure of the tank is 0.08MPa, the air volume is 0-3vvm, the rotating speed is 200-800rpm, the dissolved oxygen is controlled to be 10-20% by continuously increasing the rotating speed and the air volume, the initial pH value is 4.0, the pH value is controlled to be 4.0 by ammonia supplementation when the pH value is lower than 4.0, the material supplementation is started when the pH value is higher than 4.5 during the fermentation period, the pH value is controlled to be 4.5-4.7 by the material supplementation, the fermentation is finished when the enzyme activity is slowly increased and the cell autolysis is serious, and the fermentation period is about 90-110 h.
The following table shows the enzyme production of the fermentation of 5 batches of a 100L fermentor with an average enzyme production level of 83540U/mL.
EXAMPLE 3 method for measuring enzyme Activity of acid protease
(1) Definition of enzyme activity unit: 1mL of liquid enzyme hydrolyzes casein at 40 ℃ and pH value of 3.0 for 1min to generate 1 mu g of tyrosine as an enzyme activity unit in U/mL.
(2) The principle is as follows: proteases hydrolyze casein (a protein that mammals provide a source of nitrogen for juvenile growth) substrates under conditions of temperature and pH to produce amino acids containing phenolic groups [ e.g.: tyrosine (α -amino- β -p-hydroxyphenylpropionic acid), tryptophan (α -amino- β -indolylpropanoic acid), etc. ], the molybdenum blue and tungsten blue are produced by reducing the green reagent under alkaline conditions, and the enzyme activity is calculated by measuring the absorbance with a spectrophotometer.
(3) Reagents and solutions
Preparation of a forrine reagent: adding sodium tungstate (Na) into 2000mL ground reflux device2WO4·2H2O)100g, sodium molybdate (Na)2MoO4·2H2O)25g, water 700mL, 85% phosphoric acid 50mL, concentrated hydrochloric acid 100mL, boiling for 10h with soft fire, taking off a reflux cooler, adding lithium sulfate (LiSO4)50g, water 50mL and several drops of concentrated bromine water (99%), slightly boiling for 15min to remove redundant bromine (the bromine is still green after cooling, then adding bromine water again, slightly boiling to remove redundant bromine), cooling, adding water to a constant volume of 1000mL, uniformly mixing, and filtering. The reagent should be golden yellow and stored in a brown bottle.
The use solution: one part of the formalin reagent was mixed with the two parts of water and shaken up.
Sodium carbonate solution [ c (Na)2CO3)＝0.4mol/L]: 42.4g of anhydrous sodium carbonate is taken, dissolved in water and metered to 1000 mL.
Trichloroacetic acid [ c (CCL)3·COOH)]0.4 mol/L: 65.4g of trichloroacetic acid is taken, dissolved in water and metered to 1000 mL.
Sodium hydroxide solution [ c ═ 0.5mol/L ]: prepared according to GB 601.
Hydrochloric acid solution [ c 1mol/L and 0.1mol/L ]
GB 601: measuring 90mL of hydrochloric acid, injecting into 1000mL of water, and shaking up to obtain 1mol/L hydrochloric acid.
Weighing 9mL of hydrochloric acid, injecting into 1000mL of water, and shaking up to obtain 0.1mol/L hydrochloric acid.
Phosphate buffer (pH 7.5): disodium hydrogen phosphate (Na) was weighed2HPO4·12H2O)6.02g and sodium dihydrogen phosphate (NaH)2PO4·2H2O)0.5g, dissolved in water and made to volume of 1000mL, which is corrected by a pH meter.
10g/L casein (casein) solution: weighing 1.000g of casein, accurately weighing to 0.001g, wetting with 2-3 drops of concentrated lactic acid, adding about 80mL of buffer solution with pH7.5, heating in a boiling water bath while stirring until the solution is completely dissolved, cooling, transferring into a 100mL volumetric flask, and diluting to a scale with the buffer solution. The solution was stored in a refrigerator for a 3 day pot life.
L-tyrosine standard solution:
a. weighing 0.1000g of L-tyrosine which is pre-dried to constant weight at 105 ℃ to an accuracy of 0.0002g, and selecting 1 mol/ml
And dissolving 60mL of L-hydrochloric acid, and metering to 100mL to obtain a 1mg/mL tyrosine standard solution.
b. Sucking 10.00mL of 1mg/mL tyrosine standard solution, and diluting to 100mL with 0.1mol/L hydrochloric acid to obtain 100 ug/mL-tyrosine standard solution.
(4) Apparatus and device
A constant-temperature water bath kettle: 40 + -0.2 deg.C
A spectrophotometer: should comply with the provisions of GB9721
(5) Analytical procedure
Drawing of standard curve
Respectively sucking 0, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0ml of the L-tyrosine standard solution of 100mg/ml into a 10ml volumetric flask, and fixing the volume to the scale by using distilled water to prepare the dilute standard solution containing 0, 10, 20, 30, 40, 50 and 60mg of L-tyrosine in each ml. Taking 1.00ml of the solution (needing to be subjected to parallel test) into a test tube, adding 5.00ml of 0.4mol/L sodium carbonate solution and 1.00ml of dilute formalin reagent, placing the test tube in a water bath at 40 ℃ for color development for 20min, taking out the test tube, using a 10mm cuvette, and respectively measuring the absorbance of the test tube at the wavelength of 660nm by using a spectrophotometer, wherein the tube without tyrosine is a blank control. And drawing a standard curve or calculating a regression equation by taking the absorbance as a vertical coordinate and the corresponding tyrosine concentration as a horizontal coordinate. The amount of tyrosine (mg) when the absorbance is 1 is the colorimetric constant K value.
(6) Preparation of enzyme solution to be tested
And (3) colorimetric determination: accurately sucking 1.0ml of enzyme solution to be detected (3 parallel test tubes per sample), and preheating in water bath at 40 ℃ for 2 min. Meanwhile, taking a proper amount of 1% casein solution, preheating in 40 ℃ water bath for 3-5 min, then taking 1.0ml of the solution, adding the solution into the preheated enzyme solution, immediately starting timing, carrying out accurate reaction in 40 ℃ water bath for 10min, immediately adding 2.0ml of 0.4mol/L trichloroacetic acid, shaking up, taking out, standing for 10min, filtering, and taking 1.0ml of filtrate. 5.0ml of 0.4mol/L sodium carbonate solution is added, and then the absorbance of the sample is measured according to the standard curve drawing step.
Meanwhile, blank control determination is carried out, which comprises the steps of absorbing 1.0ml of enzyme solution to be determined, adding 2.0ml of 0.4mol/L trichloroacetic acid after water bath at 40 ℃ for 2min, adding 1.0ml of 1% casein solution after water bath at 40 ℃ for 10min, taking out, standing for 10min, filtering, taking 1.0ml of filtrate, and carrying out the same steps as sample determination.
(7) Computing
X＝(A×K×4×n)/(1×10)
In the formula: x-enzyme activity of the sample, U/ml;
a-average absorbance of the sample parallel test;
k-colorimetric constant;
4-volume of reaction reagents (ml);
n-sample dilution factor;
1-amount of enzyme solution participating in the reaction, ml;
10-reaction time, min
Example 4 pH Range of action of enzymes
The acidic protease with the enzyme activity of 80000U/mL produced by the invention is used as a uniform sample, the enzyme activity is measured under different pH values (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.5) at 40 ℃, the measured relative enzyme activity change curve is shown in figure 1, the effective pH range of the enzyme is 1.0-5.5, the optimal pH range of the enzyme is 1.5-4.5, and the optimal reaction pH value of the enzyme is 2.5.
Example 5 temperature Range of action of enzymes
Acid protease with the enzyme activity of 80000U/mL is used as a uniform sample, the enzyme activity is measured under the condition that the pH value is 4.0 and under different temperatures (38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82 and 86) respectively, the change curve of the measured relative enzyme activity is shown in figure 2, the effective temperature range of the enzyme is 46-78 ℃, the optimal temperature range of the enzyme is 50-74 ℃, and the optimal reaction temperature of the enzyme is 66 ℃.
Example 6 thermostability of enzymes
Acid protease with the enzyme activity of 80000U/mL is used as a uniform sample, the enzyme activity is measured by keeping the temperature at 80 ℃ for 0-6h under the condition that the pH value is 4.0, as shown in figure 3, the enzyme activity is still more than 70% (the initial activity is 100%) after keeping the temperature at 80 ℃ for 4h, and the heat-resistant preservation activity is good.
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 (1)
1. The method for producing the acid protease is characterized in that a production strain is Aspergillus niger FSM-208063 with the preservation number of CGMCC NO.20724, and the production method specifically comprises the following steps:
inoculating the seed liquid into a fermentation culture medium according to the proportion of 10 percent of the inoculation amount, wherein the culture conditions are as follows: at 34 ℃, the tank pressure is 0.08MPa, the air volume is 0-3vvm, the rotating speed is 200-800rpm, the dissolved oxygen is controlled to be 10-20% by continuously increasing the rotating speed and the air volume, the initial pH is 4.0, the pH is controlled to be 4.0 by ammonia supplementation when the pH is lower than 4.0, the material supplementation is started when the pH is higher than 4.5 during the fermentation period, the pH is controlled to be 4.5-4.7 by the material supplementation, the fermentation is finished when the enzyme activity is slowly increased and the thallus autolysis is serious, and the fermentation period is 90-110 h;
the fermentation medium consisted of: 40g/L of corn flour, 20g/L of corn starch, 60g/L of cottonseed protein powder and NH4Cl10g/L，KH2PO422g/L, 18g/L corn steep liquor and CaCl210g/L, the balance being water, adjusting the pH to 4.0, and sterilizing at the temperature of 121-;
the feed medium consisted of: 300g/L of maltose syrup, 60g/L of ammonium chloride and KH2PO4 1.0g/L，NaH2PO420g/L, 8.0g/L sodium citrate, 60g/L corn steep liquor and CaCl23.0g/L and the balance of water, adjusting the pH value to 3.5, and sterilizing at the temperature of 121-123 ℃ for 30-40 min.
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