Method for preparing alkaline catalase by microbial fermentation

Abstract

The application discloses a method for preparing alkaline peroxidase by microbial fermentation, and belongs to the technical field of enzyme preparation by microbial fermentation. The fermentation process for alkaline peroxidase production can preliminarily cultivate original strains screened in a laboratory through strain culture, can obtain strain cell bacteria with high activity, excellent state and more suitable for actual production, and can rapidly breed the strain cell bacteria through a production strain culture medium, so that a culture solution containing a large number of bacterial cells is finally formed in this stage. Through adding specific carbon source, nitrogen source, inorganic salt, growth factor and other nutrients to the culture solution, sufficient material and energy can be provided for the cultured bacterial cells, meanwhile, favorable enzyme production conditions are created for the production strain, the yield of alkaline peroxidase is improved, and the carbon source, nitrogen source, inorganic salt and growth factor can significantly improve the activity of peroxidase secreted by the strain.

Description

Technical Field

[0001] This invention belongs to the field of enzyme preparation technology by microbial fermentation, specifically relating to a method for preparing alkaline catalase by microbial fermentation. Background Technology

[0002] Catalase (CAT) is an enzyme that catalyzes the production of water and oxygen from H2O2. It is generally found in peroxisomes in animal and plant cells, and catalase is the marker enzyme of peroxisomes.

[0003] Catalase has a wide range of applications. (1) Medicine: Because H2O2 has bactericidal, cleaning, bleaching and disinfecting effects, it is often used for instrument disinfection. If H2O2 in CAT disinfectant is added during contact lens disinfection, it can be decomposed. (2) Food processing: Catalase can preserve food and act as an antioxidant to eliminate molecular oxygen, reactive oxygen and free radicals in beer and beverages. It is used as an oxygen and glucose oxidase remover, milk sterilizer and cheese raw milk sterilizer. (3) Environmental protection: In most developed countries, hydrogen peroxide is generally used in the environmental protection industry, accounting for about one-tenth to one-fifth. The reason for using catalase for environmental protection is that it can replace other polluting chemical reagents, which generally have secondary pollution, while catalase does not. In addition, catalase can also have other functions, and it has a degradation effect on aromatic cyclic compounds and aliphatic compounds. The famous environmental catalase is horseradish peroxidase. (4) Textiles: In the textile industry, dyeing is necessary, so removing hydrogen peroxide is an essential step. There are two different versions of traditional removal methods: one is to bleach with hydrogen peroxide and then wash it with alternating hot and cold water to remove the hydrogen peroxide; the other is to use a peroxide reducing agent, then wash it, and then proceed with the dyeing step. Compared with the traditional process, the advantages of peroxide bleaching are evident. Using catalase to remove hydrogen peroxide is not only convenient and quick, but also allows for dyeing directly with just one wash or without washing. Catalase is environmentally friendly and economical, consumes less work time, provides a safe working environment, and ensures product quality. (5) Other industries: Catalase also has many other applications. For example, in rubber molding, adding catalase and peroxide at the same time can make it mold quickly; it is also added to plastics as an adhesive; of course, in many industries that require bleaching, adding hydrogen peroxide for bleaching is a common operation, at which point catalase becomes very necessary; in the cosmetics industry, catalase is added as an anti-aging agent. The popularity of hydrogen peroxide means that the market demand for catalase will only increase.

[0004] The primary source of catalase production is extraction from animal livers. However, this process is highly dependent on raw material supply, resulting in very high costs. In contrast, catalase production via microbial fermentation is much cheaper and uses readily available raw materials. Therefore, the vast majority of catalase on the market is produced through microbial fermentation. Not only do nutrients such as carbon sources, nitrogen sources, inorganic salts, and growth factors (nicotinic acid) significantly influence the process of catalase production via microbial fermentation, but the secretion of catalase by microorganisms is also subject to various fermentation conditions. Therefore, optimizing the fermentation conditions is necessary to improve the yield and specific activity of catalase produced by microbial fermentation.

[0005] Because catalase has such wide applications and significant industrial value, and because researchers in various countries are currently using different methods to produce it, optimizing the microbial fermentation conditions to increase the quantity and specific activity of catalase produced by specific microorganisms is a crucial technical approach. Against this backdrop, we have developed a new process for producing alkaline catalase through microbial fermentation. Summary of the Invention

[0006] To further improve the efficiency of the produced catalase, this invention provides a method for preparing alkaline catalase by microbial fermentation.

[0007] The production strain involved in this invention is Arthrobacter sp., with accession number CGMCCNO.8181, and has been published in Chinese Patent Publication No. CN103555620A.

[0008] This invention provides a novel process for producing alkaline catalase via microbial fermentation. The specific fermentation process steps for alkaline catalase production are as follows:

[0009] Using Arthrobacter sp. (CGMCC NO.8181) as the production strain, the production strain was cultured. The culture of the production strain included the preparation of solid and liquid culture media. The solid culture medium can efficiently prepare bacterial cells. Based on the components of the solid culture medium, a liquid seed culture medium for fermentation production was further obtained.

[0010] The optimization of the alkaline catalase fermentation process specifically includes adding specific carbon sources, nitrogen sources, inorganic salts, and growth factors (nicotinic acid) to the fermenter. Experimental data show that these measures significantly increase the amount of alkaline catalase produced by the strain and the specific activity of the enzyme.

[0011] The solid culture medium used in this invention consists of: 10 g / L yeast extract, 20 g / L peptone, 2.5 g / L sodium chloride, 25 g / L agar, 2.5 g / L sodium nitrate, and 20 g / L glucose. The specific preparation process is as follows: Preparation of Group A nutrients: The following components are added sequentially to 1000 mL of water: 10 g yeast extract, 20 g peptone, 2.5 g sodium chloride, and 25 g agar. The mixture is then thoroughly mixed and sterilized at 122.5°C for 20 minutes. Preparation of Group B nutrients: 2.5 g sodium nitrate and 20 g glucose are sterilized separately at 122.5°C for 20 minutes. On a clean bench, the sterilized Group B nutrients are added to the Group A nutrients. After sterilization, the mixture is allowed to cool naturally for later use.

[0012] Solid culture media are used for the selection of production strains. Specifically, the activated original starting strain is inoculated into a culture medium that has been thoroughly steam-sterilized and cooled, and cultured at 30℃-37℃ for 24 hours. During this stage, single colonies of the production strain capable of producing catalase through fermentation are selected.

[0013] The seed culture medium used in this invention differs from the solid culture medium in that it does not contain agar. Its specific composition is: yeast extract 10 g / L, peptone 20 g / L, sodium chloride 2.5 g / L, sodium nitrate 2.5 g / L, and glucose 20 g / L. This seed culture medium is used for the expansion of primary and secondary seed cultures.

[0014] The specific preparation process of the primary seed is as follows: a loop of bacterial cells that have been cultured on solid culture medium is picked up with an inoculation needle and then inoculated into a 250ml wide-mouth triangular shake flask containing 50ml of seed culture medium. Finally, the shake flask is fixed in a BS-2F shaking incubator and the alkaline catalase producing strain in the shake flask is shaken and cultured at a temperature of 30℃-37℃. Shaking culture can ensure that the strain and the culture medium are fully mixed, so that each bacterial cell can be fully nourished. At this stage, the primary seed for fermentation can be obtained.

[0015] Preferably, the specific preparation conditions for the primary seed are as follows: culture at 200-250 rpm and 30℃-37℃ for 8-12 hours, with a culture medium pH of 7.8. When the OD600 value of the primary seed culture reaches 0.9-1.5 and the pH of the culture medium is between 7.0 and 8.1, the secondary seed is then transferred for culture.

[0016] The specific preparation process of the secondary seed is as follows: the primary seed is transferred at an inoculation rate of 2.0%-4.0%, and the primary seed is transferred into a fermenter containing seed culture medium under aseptic conditions. The bacterial cells are then cultured in the fermenter to expand the secondary seed.

[0017] The specific process for fermenting alkaline catalase production is as follows: When the secondary seed cell concentration reaches a certain level (i.e., the OD600 value of the culture medium reaches 1.6-2.2), a certain amount of carbon source, nitrogen source, inorganic salt, and growth factor (nicotinic acid) are added to the fermenter in a specific order. After thorough mixing, a fermentation medium for alkaline catalase production is formed. The pH of the medium is 7.8. The cells ferment in the fermenter to produce alkaline catalase. The fermentation temperature in the fermenter is controlled at 30℃-37℃, and the fermentation speed is 200-250 rpm for continuous fermentation for 36-68 hours.

[0018] Specifically, 30-45 g / L of carbon source, 15-60 g / L of nitrogen source, 0.5-0.6 g / L of inorganic salt and 0.02-0.35 g / L of growth factor are added to the fermentation tank; wherein, the nitrogen source is composed of soybean meal and sodium nitrate in a mass ratio of 3:1.

[0019] In a specific embodiment of the present invention, a parallel control experiment was conducted to investigate the effect of different carbon sources on the fermentation of the strain. The carbon sources were glycerol, lactose, glucose, soluble starch, cassava starch, citric acid, sucrose, and cyclodextrin. The results showed that when sucrose, soluble starch, or cassava starch was used as the carbon source, the yield and activity of alkaline catalase were higher, with cassava starch being the most preferred.

[0020] In a specific embodiment of the present invention, a parallel control experiment was conducted to investigate the effect of different nitrogen sources on the fermentation of the strain. The nitrogen sources were corn steep liquor, soybean meal, beef extract, corn steep liquor powder, peptone, yeast extract, compound nitrogen source 1 (corn steep liquor: sodium nitrate mass ratio 3:1), compound nitrogen source 2 (yeast extract: sodium nitrate mass ratio 3:1), and compound nitrogen source 3 (soybean meal: sodium nitrate mass ratio 3:1). The results showed that the yield and activity of alkaline catalase were higher when the nitrogen source was compound nitrogen source 3.

[0021] In a specific embodiment of the present invention, parallel control experiments with inorganic salts were conducted to investigate the effects of different inorganic salts on the fermentation of the strain. The range of inorganic salts included: KCl, ZnCl2, FeCl2, CaCl2, MnCl2, NaCl, MgCl2, and KH2PO4. The results showed that fermentation media containing sodium chloride or potassium dihydrogen phosphate had higher yields and activities of alkaline catalase, with potassium dihydrogen phosphate being the most preferred.

[0022] Based on the screening of specific carbon sources, nitrogen sources, and inorganic salts mentioned above, this invention further adjusts the dosage of each component in the fermentation medium. The carbon source is cassava starch, with an addition amount of 45 g / L; the nitrogen source is 60 g / L, consisting of 45 g / L soybean meal and 15 g / L sodium nitrate; the inorganic salt is potassium dihydrogen phosphate, with an addition amount of 0.5 g / L; and the growth factor is nicotinic acid, with an addition amount of 0.35 g / L.

[0023] After fermentation in the fermenter is completed, the fermentation broth is centrifuged at 10800×g. The obtained sludge is washed once with 50mmol / L Na2HPO4-NaH2PO4 buffer (pH 7.0), then resuspended in the buffer, pre-cooled in ice water, and then crushed with an ultrasonic homogenizer. After centrifugation at 12700×g, the supernatant is collected. The supernatant is the crude alkaline catalase solution.

[0024] This fermentation process for producing alkaline catalase involves the initial cultivation of a laboratory-selected strain to obtain highly active and optimally functioning cells suitable for actual production. These cells are then rapidly multiplied using a seed culture medium, ultimately forming a fermentation seed solution rich in bacterial cells. Adding specific carbon sources, nitrogen sources, inorganic salts, and growth factors (nicotinic acid) to the culture medium provides sufficient matter and energy to the cultured cells, creating favorable conditions for enzyme production and increasing the yield of alkaline catalase. Furthermore, the carbon source, nitrogen source, inorganic salts, and growth factor (nicotinic acid) significantly enhance the activity of the catalase secreted by the strain. In other words, this fermentation process not only substantially increases the yield of alkaline catalase from the production strain but also produces alkaline catalase with even higher activity. Detailed Implementation

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] The production strain involved in this invention belongs to *Arthrobacter* sp., and was deposited on September 13, 2013, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCC NO. 8181. This strain has been published in patent publication number CN103555620A.

[0027] Example 1

[0028] The specific steps of the process for producing alkaline catalase by fermentation are as follows:

[0029] Starting with the cultivation of the production strain and the preparation of the seed culture, the cultivation of the production strain includes the preparation of solid and liquid culture media for the strain. Solid culture media can efficiently prepare bacterial cells. Based on the components of the solid culture media, a liquid seed culture medium for fermentation production is further obtained.

[0030] The specific composition and preparation process of the nutrient components in the culture medium for alkaline catalase production strain are as follows: Preparation of nutrient components A: Add the following components sequentially to 1000mL of water: 10g yeast extract, 20g peptone, 2.5g sodium chloride, and 25g agar. Mix thoroughly and sterilize at 122.5℃ for 20 minutes. Nutrient components B: Sterilize 2.5g sodium nitrate and 20g glucose separately at 122.5℃ for 20 minutes. On a clean bench, add the sterilized nutrients from group B to the nutrients from group A. The pH of the culture medium is 7.8.

[0031] The culture medium for the inoculum has been thoroughly steam-sterilized and cooled. The activated original starting strain is inoculated into a 250ml wide-mouth Erlenmeyer shake flask containing 50ml of culture medium and cultured at 32℃ for 24 hours. During this stage, single colonies capable of producing catalase through fermentation are selected.

[0032] The specific preparation process of the seed liquid for fermentation is as follows: The selected alkaline catalase producing strain is purified on a solid culture medium. Then, a loopful of the cultured bacterial cells is picked up with an inoculation needle and inoculated into a 250ml wide-mouth Erlenmeyer flask containing 50ml of bacterial culture medium (pH 7.8). Finally, the flask is fixed in a BS-2F shaking incubator and the alkaline catalase producing strain in the flask is shaken at 230rpm for 10 hours at 32℃. Shaking culture can ensure that the bacterial strain and the culture medium are thoroughly mixed, so that each bacterial cell can be fully nourished. At this stage, the seed liquid for fermentation can be obtained.

[0033] The specific preparation process of the fermentation medium for producing alkaline catalase is as follows: Based on the nutrient composition of the pre-prepared seed culture medium for alkaline catalase production strain, the carbon source (30 g / L), nitrogen source (15 g / L yeast extract), inorganic salt (NaCl, 0.6 g / L), and growth factor (nicotinic acid, 200 mg / L) are added sequentially to the pre-prepared seed culture medium. The mixture is then thoroughly stirred using a magnetic stirrer to form the fermentation medium for producing alkaline catalase. The pH value of the culture medium is 7.8.

[0034] The microbial fermentation process for producing alkaline catalase is characterized as follows: the seed liquid for fermentation is transferred at an inoculation rate of 3.5%. The calculated amount of seed liquid is transferred under aseptic conditions into a fermenter containing an appropriate amount of liquid fermentation medium. The bacterial cells are fermented at 32°C for 49 hours to produce alkaline catalase.

[0035] After fermentation in the fermenter, 15 ml of the fermentation broth was taken and centrifuged at 10800×g for 10 min. The obtained bacterial sludge was washed once with 50 mmol / L Na2HPO4-NaH2PO4 buffer (pH 7.0), resuspended in 5 mL of buffer, pre-cooled in ice water, and then disrupted by ultrasonic disruption for 10 min. It was then centrifuged at 12700×g for 15 min, and the supernatant was collected. This supernatant was the crude enzyme solution for alkaline catalase assay. Protein content determination: The total protein content in the crude enzyme solution was determined using the Coomassie brilliant blue method, with bovine serum albumin as the standard protein. Catalase activity determination: The decomposition rate of H2O2 was measured using a UV-Vis spectrophotometer at 30℃ and a wavelength of 240 nm. For ease of measurement, the total volume of the reaction system can be controlled at 3 mL; the reaction system contains H2O2 (120 mmol / L), Na2HPO4-NaH2PO4 buffer (pH 7.0, 50 mmol / L), and an appropriate amount of enzyme solution sample.

[0036] This example serves as a parallel control for carbon sources. The carbon sources included glycerol, lactose, glucose, soluble starch, tapioca starch, citric acid, sucrose, and cyclodextrin. The effects of different carbon sources on the fermentation of the strain were investigated, and the results are shown in Table 1. As can be seen from Table 1, tapioca starch was the preferred carbon source in this example, with an addition amount of 30 g / L.

[0037] Table 1 Enzyme yield and specific activity under different carbon sources

[0038]

[0039] Example 2

[0040] This example is a parallel control example of nitrogen source preparation. The specific preparation process of the fermentation medium for producing alkaline catalase is as follows: Based on the nutrient composition of the pre-prepared seed culture medium for the alkaline catalase production strain, the carbon source (cassava starch 30 g / L), nitrogen source (15 g / L), inorganic salt (NaCl, 0.6 g / L), and growth factor (nicotinic acid, 200 mg / L) are added sequentially to the pre-prepared seed culture medium. The mixture is thoroughly stirred using a magnetic stirrer to form the fermentation medium for producing alkaline catalase. The pH of the culture medium is 7.8. Other steps are the same as in Example 1.

[0041] The range of nitrogen sources available for fermentation production includes: corn steep liquor, soybean meal, beef extract, corn steep liquor powder, peptone, yeast extract, compound nitrogen source 1 (corn steep liquor:sodium nitrate mass ratio 3:1), compound nitrogen source 2 (yeast extract:sodium nitrate mass ratio 3:1), and compound nitrogen source 3 (soybean meal:sodium nitrate mass ratio 3:1). The effects of different nitrogen sources on the fermentation of the strains are shown in Table 2. Table 2 shows that adding different types of nitrogen sources helps the production strains secrete alkaline catalase. Adding an appropriate amount of suitable nitrogen source to the fermentation medium can increase the yield and specific activity of alkaline catalase in the production strains, and reduce the production cost of alkaline catalase. Among them, compound nitrogen source 3 showed the highest enzyme yield and specific activity.

[0042] Table 2 Enzyme yield and specific activity under different nitrogen sources

[0043]

[0044] Example 3

[0045] This example is a parallel control example using inorganic salts. The specific preparation process of the fermentation medium for producing alkaline catalase is as follows: Based on the nutrient composition of the pre-prepared seed culture medium for the alkaline catalase production strain, the following components were directly modified: carbon source (glucose 30 g / L), nitrogen source (yeast extract, 15 g / L), inorganic salts (0.6 g / L), and growth factor (nicotinic acid, 200 mg / L) were added sequentially to the pre-prepared seed culture medium. The mixture was then thoroughly stirred using a magnetic stirrer to form the fermentation medium for producing alkaline catalase. The pH of the medium was 7.8. Other steps were the same as in Example 1.

[0046] The range of inorganic salts available for fermentation production includes: KCl, ZnCl2, FeCl2, CaCl2, MnCl2, NaCl, MgCl2, and KH2PO4. The effects of different inorganic salts on the fermentation of the strains are shown in Table 3. Table 3 shows that adding different types of inorganic salts helps the production strains secrete alkaline catalase. Adding appropriate amounts of suitable inorganic salts to the fermentation medium can increase the yield and specific activity of alkaline catalase in the production strains, and reduce the production cost of alkaline catalase. Among these, KH2PO4 is the optimal choice.

[0047] Table 3 Enzyme yield and specific activity after culture with different inorganic salts

[0048]

[0049] Example 4

[0050] The fermentation medium used in this example consisted of 10g yeast extract, 20g peptone, 2.5g sodium chloride, 2.5g sodium nitrate, and 20g glucose. Then, 45g cassava starch and a compound nitrogen source (soybean meal + sodium nitrate) were added: 45g soybean meal + 15g sodium nitrate, 0.5g KH2PO4, and 350mg niacin. After fermentation for 49 hours, the final catalase yield was 61443U / mL, and the specific activity of the enzyme was 74636U / mg.

[0051] Comparative Example 1

[0052] The difference between this comparative example and Example 4 is that nicotinic acid was not added to the culture medium, and the final catalase yield was 56527 U / mL, with a specific activity of 67918 U / mg.

[0053] The experimental results of the above examples and comparative examples demonstrate that adding nicotinic acid can significantly increase the yield of alkaline catalase in the producing strain. This is probably because nicotinic acid is a precursor to NADH. The increased concentration of nicotinic acid in the fermentation broth enhances the synthesis of NADH in the strain cells, increases the reducing power concentration, and thus enhances the yield of alkaline catalase in the strain cells.

[0054] The above are merely some specific embodiments of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only some examples of the present invention and are not intended to limit the present invention. Various changes and modifications can be made to the present invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A method for preparing alkaline catalase by microbial fermentation, characterized in that, The specific steps are as follows: Using *Arthrobacter sp.* CGMCC NO.8181 as the production strain, the strain is activated and cultured in a seed culture medium to obtain primary seed culture. The primary seed culture is then inoculated into a fermenter containing seed culture medium at an inoculation rate of 2.0%-4.0% under aseptic conditions for expansion culture. Fermentation is carried out when the OD600 value of the bacterial culture reaches 1.6-2.

2. In the fermenter containing seed culture medium, 45 g / L cassava starch, 60 g / L nitrogen source, 0.5 g / L potassium dihydrogen phosphate, and 0.35 g / L nicotinic acid are added and thoroughly mixed to form a fermentation medium. The bacterial cells ferment in the fermenter to produce alkaline catalase. The nitrogen source consists of soybean meal and sodium nitrate in a 3:1 mass ratio. The seed culture medium consists of: 10 g / L yeast extract, 20 g / L peptone, 2.5 g / L sodium chloride, 2.5 g / L sodium nitrate, and 20 g / L glucose.

2. The method for preparing alkaline catalase by microbial fermentation according to claim 1, characterized in that, The specific cultivation process of the primary seed: Use an inoculation needle to pick up a loop of activated bacterial cells and inoculate them into a 250ml wide-mouth triangular shake flask containing 50ml of seed culture medium. The alkaline catalase producing strain in the shake flask is then cultured under shaking conditions at 30℃-37℃.

3. The method for preparing alkaline catalase by microbial fermentation according to claim 1, characterized in that, The concentration of the primary seed culture medium has an OD600 value of 0.9-1.5 and a pH value of 7.0-8.

1.

4. The method for preparing alkaline catalase by microbial fermentation according to claim 1, characterized in that, Alkaline catalase is produced by fermentation. The fermentation temperature in the fermenter is controlled at 30℃-37℃, and the rotation speed is 200-250rpm for continuous fermentation for 36-68 hours.

5. The method for preparing alkaline catalase by microbial fermentation according to claim 1, characterized in that, The preparation method further includes, after fermentation in the fermenter is completed, centrifuging the fermentation broth at 10800×g, washing the obtained sludge with a 50mmol / L Na2HPO4-NaH2PO4 buffer solution with a pH of 7.0, resuspending it with the buffer solution, pre-cooling it in ice water, crushing it with an ultrasonic disruptor, centrifuging it at 12700×g, and taking the supernatant, which is the crude alkaline catalase solution.

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