CN117511896A - Method for preparing alkaline catalase by microbial fermentation method - Google Patents
Method for preparing alkaline catalase by microbial fermentation method Download PDFInfo
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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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
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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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01006—Catalase (1.11.1.6)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/06—Arthrobacter
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Abstract
The invention discloses a method for preparing alkaline catalase by a microbial fermentation method, and belongs to the technical field of processes for preparing enzyme by the microbial fermentation method. According to the fermentation process for producing alkaline catalase, primary culture is carried out on original strains which can be screened in a laboratory in a strain culture process, strain cell bacteria which have high activity and excellent state and are more suitable for actual production can be obtained, strain cells are quickly bred through a strain production culture medium, and a culture solution containing a large number of thallus cells is finally formed at the stage; by adding specific carbon source, nitrogen source, inorganic salt, growth factor and other nutrient substances into the culture solution, enough substances and energy can be provided for the cultured bacterial cells, favorable enzyme production conditions are created for producing strains, the yield of alkaline catalase is improved, and the activity of the carbon source, nitrogen source, inorganic salt and growth factor on catalase secreted by the strains is obviously improved.
Description
Technical Field
The invention belongs to the technical field of processes for preparing enzymes by a microbial fermentation method, and particularly relates to a method for preparing alkaline catalase by microbial fermentation.
Background
Catalase (CAT), a type of enzyme capable of converting H 2 O 2 Enzymes that catalyze the production of water and oxygen are typically found in peroxisomes in animal and plant cells, and the marker enzyme of peroxisomes is catalase.
The use of catalase is very widespread. (1) medicine: due to H 2 O 2 Has the effects of sterilization, cleaning, bleaching and disinfection, and is therefore commonly used for instrument disinfection. If H in CAT disinfectants is added during contact lens disinfection 2 O 2 Can be decomposed. (2) food processing: the catalase can keep food fresh, and can be used as antioxidant for eliminating molecular oxygen, active oxygen and free radical in beer and beverage. It is used as oxygen and glucose oxidase remover for sterilizing cow milk and cheese material milk. (3) environmental protection: hydrogen peroxide is commonly used in the environmental industry in most developed countries, with a specific gravity of about one tenth to one fifth. The reason for environmental protection with catalase is that it can replace other polluting chemical reagents, and these chemical reagents capable of degrading hydrogen peroxide generally have secondary pollution, but catalase is not, and catalase has other functions, and has degradation function on aromatic cyclic compounds and aliphatic compounds. A well-known environmentally friendly catalase is horseradish peroxidase. (4) spinning: in the textile industry, printing and dyeing are required, so that the removal of hydrogen peroxide is a necessary step, the traditional removal method has two different versions, and one is to remove the hydrogen peroxide by alternately cleaning with cold and hot water after bleaching with the hydrogen peroxide; the second is to use peroxide reducing agent, then wash, and then perform the dyeing step. Compared with the traditional process, the peroxide bleaching has the advantages that the hydrogen peroxide can be removed by using the catalase conveniently and quickly, and the dyeing can be directly performed by only one time or no cleaning. The catalase is environment-friendly, saves time, is safe in operation environment and ensures the quality of products. (5) other industries: catalase is otherwise identical toThere are many applications, such as the simultaneous addition of catalase and peroxidation in rubber molding, which allow rapid molding; plastic is also added as an adhesive; of course, in many industries requiring bleaching, the addition of hydrogen peroxide bleaching is a common practice, where catalase becomes a very essential necessity; catalase is added as an anti-aging agent in the cosmetic industry. The popularity of hydrogen peroxide has led to the market of increasing only the need for catalase.
The main source of the catalase is extracted from animal livers, but the process for producing the catalase is easily limited by raw material supply, has very high cost, and has low cost when producing the catalase by a microbial fermentation method, and the raw materials are also easily obtained, so most of the catalase in the market is produced by the microbial fermentation method; not only are the nutrition components such as carbon source, nitrogen source, inorganic salt, growth factor (nicotinic acid) and the like greatly affected in the process of producing catalase by microbial fermentation, but also the process of producing catalase by microbial secretion is limited by various fermentation conditions, so that the fermentation conditions of the microorganisms are required to be optimized in order to improve the quantity of catalase produced by microbial fermentation and the specific activity of the enzymes.
As the catalase has such wide application and important industrial value, the catalase production process carried out by scientific researchers in various countries is different, and the method improves the quantity and specific activity of the catalase produced by strains through optimizing the fermentation conditions of the catalase microorganisms, thereby being a very important technical route. Under such background conditions, we have developed a novel process for producing alkaline catalase by microbial fermentation.
Disclosure of Invention
In order to further improve the efficiency of the produced catalase, the invention provides a method for preparing alkaline catalase by a microbial fermentation method.
The production strain related to the invention is Arthrobacter sp, the preservation number is CGMCC NO.8181, and the production strain is disclosed in China patent publication No. CN 103555620A.
The invention provides a novel process for preparing alkaline catalase by a microbial fermentation method, which comprises the following specific steps:
the Arthrobacter sp (with the preservation number of CGMCC No. 8181) is used for producing a bacterial strain, the bacterial strain is cultured, the solid and liquid culture mediums of the bacterial strain are prepared, the solid culture mediums can be used for efficiently preparing bacterial cells, and the liquid seed culture medium for fermentation production is further obtained on the basis of all components of the solid culture mediums.
Optimization of alkaline catalase fermentation process, which specifically comprises adding specific carbon source, nitrogen source, inorganic salt and growth factor (nicotinic acid) nutrient substances into a fermentation tank, wherein experimental data show that the measures remarkably improve the quantity of alkaline catalase produced by the strain and the specific activity of enzyme.
The solid culture medium used in the invention comprises the following components: yeast extract 10g/L, peptone 20g/L, sodium chloride 2.5g/L, agar 25g/L, sodium nitrate 2.5g/L, and glucose 20g/L. The specific preparation process comprises the following steps: the preparation process of the nutrient components of the group A comprises the following steps: the following components were added sequentially to 1000mL of water: 10g of yeast extract, 20g of peptone, 2.5g of sodium chloride and 25g of agar, and then thoroughly mixing, and sterilizing at 122.5 ℃ for 20 minutes; the nutrition components of the group B: 2.5g of sodium nitrate and 20g of glucose were sterilized separately for 20 minutes at 122.5 ℃; and adding the sterilized nutrient substances of the group B into the nutrient components of the group A on an ultra-clean workbench. And after the sterilization process is finished, naturally cooling for standby.
The solid culture medium is used for breeding production strains. Specifically, the activated original strain is inoculated into a strain culture medium which is fully subjected to steam sterilization and cooling, and is cultured for 24 hours at the temperature of 30-37 ℃, and single bacterial colony of a production strain capable of being used for producing catalase through fermentation is obtained through breeding at the stage.
The seed culture medium used in the invention has the composition which is different from that of the solid culture medium in that the seed culture medium does not contain agar, and the specific composition is as follows: yeast extract 10g/L, peptone 20g/L, sodium chloride 2.5g/L, sodium nitrate 2.5g/L, and glucose 20g/L. The seed culture medium is used for the expansion culture of primary seeds and secondary seeds.
The specific preparation process of the first-stage seeds comprises the following steps: picking a loop of somatic cells which have been cultured in a solid culture medium by using an inoculating needle, then inoculating the somatic cells into a wide-mouth triangular shake flask containing 50ml of seed culture medium and having the capacity of 250ml, finally fixing the shake flask in a BS-2F shaking incubator, and carrying out shaking culture on alkaline catalase production strains in the shake flask at the temperature of 30-37 ℃, wherein the shaking culture can ensure that the strains are fully mixed with the culture medium, each somatic cell can be fully nourished, and finally obtaining first-stage seeds for fermentation at the stage.
Preferably, the specific preparation process conditions of the first-stage seeds are as follows: culturing at 200-250rpm and 30-37 deg.c for 8-12 hr to pH 7.8. When the concentration OD600 value of the bacterial culture solution of the first-stage seeds reaches 0.9-1.5 and the pH value of the bacterial culture solution is 7.0-8.1, the second-stage seeds are transferred and cultured.
The specific preparation process of the secondary seeds comprises the following steps: the first-level seeds are transferred according to the inoculation amount of 2.0% -4.0%, the first-level seeds are transferred into a fermentation tank containing a seed culture medium under the aseptic operation condition, and strain cells are used for expanding culture of the second-level seeds in the fermentation tank.
The specific process engineering for producing alkaline catalase by fermentation comprises the following steps: when the secondary seed bacterial cells reach a certain concentration (namely the bacterial concentration OD600 value of the culture solution reaches 1.6-2.2), adding a certain amount of carbon source, nitrogen source, inorganic salt and growth factor (nicotinic acid) into a fermentation tank according to a certain sequence, fully stirring and uniformly mixing to finally form a fermentation culture medium for producing alkaline catalase, wherein the pH value of the culture medium is 7.8, and fermenting bacterial cells in the fermentation tank to produce the alkaline catalase. Controlling the fermentation temperature in the fermentation tank, wherein the temperature of the fermentation tank is set to be 30-37 ℃ and the fermentation is continuously carried out for 36-68h under the condition of the rotating speed of 200-250 rpm.
Specifically, adding 30-45g/L of carbon source, 15-60g/L of nitrogen source, 0.5-0.6g/L of inorganic salt and 0.02-0.35g/L of growth factor into a fermentation tank; wherein the nitrogen source consists of cypress and sodium nitrate according to the mass ratio of 3:1.
In the specific embodiment of the invention, carbon source parallel control experiments are carried out to examine the influence of different carbon sources on the fermentation of the strain, wherein the carbon sources are respectively glycerol, lactose, glucose, soluble starch, tapioca starch, citric acid, sucrose, cyclodextrin, and the results show that when the sucrose, the soluble starch or the tapioca starch are used as the carbon sources, the yield and the activity of alkaline catalase are higher, and the tapioca starch is the most preferable.
In the specific embodiment of the invention, the influence of different nitrogen sources on the fermentation of the bacterial strain is examined by carrying out a nitrogen source parallel control experiment, wherein the nitrogen sources are respectively corn steep liquor, bean pulp, beef extract, corn steep liquor powder, peptone, yeast extract, composite nitrogen source 1 (corn steep liquor: sodium nitrate mass ratio 3:1), composite nitrogen source 2 (yeast extract: sodium nitrate mass ratio 3:1) and composite nitrogen source 3 (bean pulp: sodium nitrate mass ratio 3:1), and the results show that the yield and activity of alkaline catalase are higher when the nitrogen source is the composite nitrogen source 3.
In the specific embodiment of the invention, the influence of different inorganic salts on the fermentation of the strain is examined by performing an inorganic salt parallel control experiment, and the types of the inorganic salts are as follows: KCl, znCl 2 ，FeCl 2 ，CaCl 2 ，MnCl 2 ，NaCl，MgCl 2 ，KH 2 PO 4 The results show that the fermentation medium containing sodium chloride or potassium dihydrogen phosphate has higher yield and activity of alkaline catalase, and potassium dihydrogen phosphate is most preferable.
On the basis of the screening of the specific carbon source, the nitrogen source and the inorganic salt, the invention further adjusts the dosage of each component in the fermentation culture medium, wherein the carbon source is tapioca starch, and the dosage is 45g/L; the adding amount of the nitrogen source is 60g/L, and the nitrogen source is prepared from 45g/L of soybean meal and 15g/L of sodium nitrate; the inorganic salt is potassium dihydrogen phosphate, and the addition amount of the inorganic salt is 0.5g/L; the growth factor is nicotinic acid, and the addition amount of the growth factor is 0.35g/L.
After the fermentation in the fermenter was completed, the fermentation broth was centrifuged at 10800 Xg to obtain bacterial sludge containing 50mmol/LNa 2 HPO 4 -NaH 2 PO 4 Buffer (pH value is)7.0 And (3) washing once, re-suspending by using a buffer solution, pre-cooling by placing the buffer solution in ice water, crushing by using an ultrasonic crusher, centrifuging under the condition of 12700 Xg, and taking supernatant, wherein the supernatant is alkaline catalase crude enzyme solution.
According to the fermentation process for producing alkaline catalase, primary culture can be carried out on original strains screened in a laboratory in a strain culture process, strain cells which have high activity and excellent state and are more suitable for actual production can be obtained, the strain cells are quickly bred through a seed culture medium for producing the strain, and fermentation seed liquid containing a large amount of thallus cells is finally formed at the stage; by adding specific carbon source, nitrogen source, inorganic salt, growth factor (nicotinic acid) and other nutrient substances into the culture solution, enough substances and energy can be provided for the cultured bacterial cells, favorable enzyme production conditions are created for the production strains, the yield of alkaline catalase is improved, and the activity of catalase secreted by the strains can be obviously improved by the carbon source, the nitrogen source, the inorganic salt and the growth factor (nicotinic acid), namely, the fermentation process can not only greatly improve the yield of the alkaline catalase of the production strains, but also can use the alkaline catalase produced by the production strains to have higher activity.
Detailed Description
The following description of the embodiments of the present invention will clearly and fully describe the technical solutions of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The production strain related by the invention belongs to Arthrobacter sp, and is preserved in China general microbiological culture Collection center (CGMCC, address: north Chen West Lu No. 1, 3 of the area of the Korean of Beijing, and the post code 100101 of the institute of microorganisms of the national academy of sciences of China) in 9 and 13 days of 2013, and the preservation number is CGMCC No.8181. This strain is disclosed in patent publication No. CN 103555620A.
Example 1
The process for producing alkaline catalase by fermentation comprises the following specific steps:
the method comprises the steps of starting production strain culture and seed liquid preparation, wherein the production strain culture comprises preparation of a strain solid and liquid culture medium, the solid culture medium can be used for efficiently preparing bacterial cells, and the liquid seed culture medium for fermentation production is further obtained on the basis of all components of the solid culture medium.
The specific composition of nutrient components of the culture medium for producing the strain by alkaline catalase and the specific preparation process thereof are as follows: the preparation process of the nutrient components of the group A comprises the following steps: the following components were added sequentially to 1000mL of water: 10g of yeast extract, 20g of peptone, 2.5g of sodium chloride and 25g of agar, and then thoroughly mixing, and sterilizing at 122.5 ℃ for 20 minutes; the nutrition components of the group B: 2.5g of sodium nitrate and 20g of glucose were sterilized separately for 20 minutes at 122.5 ℃; and adding the sterilized nutrient substances of the group B into the nutrient components of the group A on an ultra-clean workbench, wherein the pH value of the culture medium is 7.8.
The strain culture medium is fully steam sterilized and cooled, the activated original strain is inoculated into a wide-mouth triangular shake flask containing 50ml of strain culture medium and having the capacity of 250ml, the strain culture medium is cultured for 24 hours at the temperature of 32 ℃, and single bacterial colony capable of being used for producing catalase through fermentation is obtained through breeding at the stage.
The specific preparation process of the seed liquid for fermentation comprises the following steps: pure culturing the bred alkaline catalase production strain on a solid medium, then picking a ring of cultured bacterial cells by an inoculating needle, then inoculating into a wide-mouth triangular shake flask containing 50ml of strain culture medium (pH 7.8) and having the capacity of 250ml, finally fixing the shake flask in a BS-2F shake incubator, shake culturing the alkaline catalase production strain in the shake flask at 230rpm for 10h under the temperature condition of 32 ℃, and fully mixing the strain with the culture medium by shake culturing so that each bacterial cell can be fully nourished, thereby finally obtaining the seed liquid for fermentation at the stage.
The specific preparation process of the fermentation medium for producing alkaline catalase comprises the following steps: directly modifying the prepared alkaline hydrogen peroxidase producing strain seed culture medium based on the nutrition components contained in the seed culture medium, sequentially adding a carbon source (30 g/L), a nitrogen source (15 g/L yeast extract), inorganic salt (NaCl, 0.6 g/L) and growth factors (nicotinic acid, 200 mg/L) into the prepared seed culture medium, and fully stirring and uniformly mixing in a magnetic stirrer to finally form a fermentation culture medium for producing alkaline hydrogen peroxidase, wherein the pH value of the culture medium is 7.8.
The microbial fermentation process for producing alkaline catalase is characterized by comprising the following steps: the seed liquid for fermentation is transferred according to the inoculation amount of 3.5 percent, the calculated amount of the seed liquid is transferred into a fermentation tank containing a proper amount of liquid fermentation medium under the aseptic operation condition, and strain cells are fermented for 49 hours under the condition of 32 ℃ to produce alkaline catalase.
After the fermentation in the fermenter was completed, 15ml of the fermentation broth was centrifuged at 10800 Xg for 10min. The obtained bacterial sludge is treated with 50mmol/LNa 2 HPO 4 -NaH 2 PO 4 Washing with buffer solution (pH 7.0), re-suspending with 5mL buffer solution, pre-cooling in ice water, crushing with ultrasonic crusher for 10min, centrifuging for 15min under 12700 Xg condition, collecting supernatant, and measuring with alkaline catalase to obtain crude enzyme solution. Determination of protein content: the total protein content in the crude enzyme solution is determined by adopting a Coomassie Brilliant method, and bovine serum albumin is used as a standard protein. Determination of catalase Activity: at 30deg.C and wavelength of 240nm, using ultraviolet-visible spectrophotometer for H 2 O 2 Is measured. For the convenience of measurement, the total volume of the reaction system can be controlled to be 3mL; the reaction system comprises H 2 O 2 (120mmol/L),Na 2 HPO 4 -NaH 2 PO 4 Buffer (pH 7.0,50 mmol/L), and an appropriate amount of enzyme solution sample.
The example is a parallel control of carbon sources, which are respectively glycerol, lactose, glucose, soluble starch, tapioca starch, citric acid, sucrose and cyclodextrin, and the influence of different carbon sources on the fermentation of the strain is examined, and the results are shown in Table 1. As can be seen from Table 1, the carbon source in this example is preferably tapioca starch, and the amount added is 30g/L.
TABLE 1 enzyme yields and specific Activity in different carbon source cultures
Example 2
The example is a nitrogen source parallel comparison example, and the specific preparation process of the fermentation medium for producing alkaline catalase comprises the following steps: the method is characterized in that the method is directly modified on the basis of nutrition contained in the prepared seed culture medium of the alkaline catalase production strain, a carbon source (30 g/L of tapioca starch), a nitrogen source (15 g/L), inorganic salt (NaCl, 0.6 g/L) and growth factors (nicotinic acid, 200 mg/L) are sequentially added into the prepared seed culture medium, and the prepared seed culture medium is fully stirred and uniformly mixed by a magnetic stirrer to finally form a fermentation culture medium for producing the alkaline catalase, wherein the pH value of the culture medium is 7.8. The other steps are the same as in example 1.
The variety range of nitrogen sources available for fermentation production is: corn steep liquor, bean pulp, beef extract, corn steep liquor powder, peptone, yeast extract, a composite nitrogen source 1 (corn steep liquor: sodium nitrate mass ratio 3:1), a composite nitrogen source 2 (yeast extract: sodium nitrate mass ratio 3:1), and a composite nitrogen source 3 (bean pulp: sodium nitrate mass ratio 3:1). The effect of different nitrogen sources on strain fermentation is shown in Table 2. As can be seen from Table 2, the addition of different kinds of nitrogen sources is helpful for the cells of the production strain to secrete alkaline catalase, and the addition of a proper amount of proper nitrogen sources to the fermentation medium can improve the yield of the alkaline catalase of the production strain and the specific activity of the enzyme, so that the production cost of the alkaline catalase can be reduced. Wherein, the enzyme yield and specific activity of the compound nitrogen source 3 are highest.
TABLE 2 enzyme yields and specific Activity in different Nitrogen Source cultures
Example 3
The embodiment is an inorganic salt parallel comparison example, and the specific preparation process of the fermentation medium for producing alkaline catalase comprises the following steps: directly modifying the prepared alkaline catalase production strain seed culture medium on the basis of nutritional ingredients contained in the seed culture medium, sequentially adding a carbon source (glucose 30 g/L), a nitrogen source (yeast extract, 15 g/L), inorganic salts (0.6 g/L) and growth factors (nicotinic acid, 200 mg/L) into the prepared seed culture medium, fully stirring and uniformly mixing in a magnetic stirrer to finally form a fermentation culture medium for producing alkaline catalase, wherein the pH value of the culture medium is 7.8. The other steps are the same as in example 1.
The variety range of inorganic salt for fermentation production is as follows: KCl, znCl 2 ，FeCl 2 ，CaCl 2 ，MnCl 2 ，NaCl，MgCl 2 ，KH 2 PO 4 . The effect of different inorganic salts on strain fermentation is shown in Table 3. As can be seen from Table 3, the addition of different inorganic salts helps the cells of the production strain to secrete alkaline catalase, and the addition of appropriate amount of suitable inorganic salts to the fermentation medium can improve the yield of alkaline catalase and the specific activity of the enzyme of the production strain, and can reduce the production cost of alkaline catalase. Wherein KH 2 PO 4 Most preferably.
TABLE 3 enzyme yields and specific Activity of different inorganic salt cultures
Example 4
The fermentation medium used in this example was: 10g of yeast extract, 20g of peptone, 2.5g of sodium chloride, 2.5g of sodium nitrate and 20g of glucose; 45g of tapioca starch and a compound nitrogen source (soybean meal + sodium nitrate) were then added: 45g of soybean meal plus 15g of sodium nitrate, KH 2 PO40.5g, nicotinic acid addition amount is 350mg, fermentation is carried out for 49h, final catalase yield is 61443U/mL, and specific activity of enzyme is 74636U/mg.
Comparative example 1
The difference between this comparative example and example 4 is that no nicotinic acid was added to the medium, the final hydrogenase yield was 56527U/mL and the specific activity of the enzyme was 67918U/mg.
The experimental results of the above examples and comparative examples prove that the addition of nicotinic acid can greatly improve the yield of alkaline catalase of the production strain, presumably because the nicotinic acid is a precursor substance of NADH, and the concentration of the nicotinic acid in the fermentation broth is improved, thereby enhancing the synthesis of NADH in cells of the strain, improving the concentration of reducing power, and further enhancing the yield of alkaline catalase of cells of the strain.
The foregoing is only illustrative of some of the specific embodiments of the invention. It will be understood by those skilled in the art that the present invention is not limited to the foregoing embodiments, which have been described in the foregoing embodiments and description as examples only, and not intended to limit the invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as claimed.
Claims (9)
1. A method for preparing alkaline catalase by a microbial fermentation method, which is characterized by comprising the following specific steps: taking Arthrobacter sp.CGMCC No.8181 as a production strain, and culturing the production strain in a seed culture medium after activating the production strain to obtain first-stage seeds; inoculating the first-stage seeds into a fermentation tank containing a seed culture medium according to the inoculum size of 2.0% -4.0% under aseptic operation condition for expanded culture, wherein the OD600 value of a bacterial culture solution reaches 1.6-2.2, adding 30-45g/L of a carbon source, 15-60g/L of a nitrogen source, 0.5-0.6g/L of inorganic salt and 0.02-0.35g/L of growth factor into the fermentation tank, and fermenting bacterial cells in the fermentation tank to produce alkaline catalase; wherein the nitrogen source consists of cypress and sodium nitrate according to the mass ratio of 3:1.
2. The method for preparing alkaline catalase by microbial fermentation according to claim 1, wherein the carbon source comprises one or more of sucrose, soluble starch and tapioca starch.
3. The method for producing alkaline catalase by microbial fermentation according to claim 1, wherein the inorganic salt comprises one or more of sodium chloride and potassium dihydrogen phosphate.
4. The method for preparing alkaline catalase by using the microbial fermentation method according to claim 1, wherein the carbon source is tapioca starch, and the addition amount of the tapioca starch is 45g/L; the adding amount of the nitrogen source is 60g/L; the inorganic salt is potassium dihydrogen phosphate, and the addition amount of the inorganic salt is 0.5g/L; the growth factor is nicotinic acid, and the addition amount of the growth factor is 0.35g/L.
5. The method for preparing alkaline catalase by using a microbial fermentation method according to claim 1, wherein the composition of the seed culture medium is as follows: yeast extract 10g/L, peptone 20g/L, sodium chloride 2.5g/L, sodium nitrate 2.5g/L, and glucose 20g/L.
6. The method for preparing alkaline catalase by using the microbial fermentation method according to claim 1, wherein the specific culture process of the primary seeds comprises the following steps: a loop of activated somatic cells is selected by an inoculating needle, and is inoculated into a wide-mouth triangular shake flask containing 50ml of seed culture medium and having the capacity of 250ml, and alkaline catalase production strains in the shake flask are subjected to shake culture at the temperature of 30-37 ℃.
7. The method for preparing alkaline catalase by using the microbial fermentation method according to claim 1, wherein the concentration OD600 value of the bacterial culture solution of the primary seeds is 0.9-1.5, and the pH value of the bacterial culture solution is 7.0-8.1.
8. The method for preparing alkaline catalase by using the microbial fermentation method according to claim 1, wherein the alkaline catalase is produced by fermentation, the fermentation temperature in the fermentation tank is controlled to be 30-37 ℃, and the fermentation is continuously carried out for 36-68h under the condition of 200-250 rpm.
9. The microorganism according to claim 1A method for preparing alkaline catalase by fermentation, characterized in that the preparation method further comprises centrifuging the fermentation broth under 10800 Xg after fermentation in the fermentation tank, and obtaining bacterial sludge with concentration of 50mmol/LNa 2 HPO 4 -NaH 2 PO 4 Washing with buffer solution (pH 7.0), re-suspending with buffer solution, pre-cooling in ice water, crushing with ultrasonic crusher, centrifuging at 12700 Xg, and collecting supernatant which is alkaline catalase crude enzyme solution.
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