CN-117511896-B - Method for preparing alkaline catalase by microbial fermentation method

CN117511896BCN 117511896 BCN117511896 BCN 117511896BCN-117511896-B

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 good state and are more suitable for practical production can be obtained, strain cells are quickly bred through a strain production culture medium, a culture solution containing a large amount of strain cells is finally formed at the stage, and by adding specific carbon sources, nitrogen sources, inorganic salts, growth factors and other nutrient substances into the culture solution, enough substances and energy can be provided for the cultured strain cells, favorable enzyme production conditions are created for producing strains, the yield of the alkaline catalase is improved, and the activity of the hydrogen catalase secreted by the strains is obviously improved through the carbon sources, the nitrogen sources, the inorganic salts and the growth factors.

Inventors

LIU HUANMIN
TIAN CHANGSHENG
ZHANG FENG
SONG YONG
WANG JIAJUN

Assignees

常州大学

Dates

Publication Date: 20260512
Application Date: 20231110

Claims (5)

1. A method for preparing alkaline catalase by using microbial fermentation method is characterized by comprising the specific steps of taking Arthrobacter sp.CGMCC No.8181 as a production strain, activating the production strain, culturing in a seed culture medium to obtain first-grade seeds, inoculating the first-grade seeds into a fermentation tank containing the seed culture medium according to an inoculum size of 2.0% -4.0% under aseptic operation condition, performing fermentation culture, adding 45g/L of tapioca starch, 60g/L of nitrogen source, 0.5g/L of potassium dihydrogen phosphate and 0.35g/L of nicotinic acid into the fermentation tank containing the seed culture medium, fully and uniformly stirring to form a fermentation culture medium, and fermenting bacterial cells in the fermentation tank to produce the alkaline catalase, wherein the nitrogen source consists of 10g/L of yeast extract, 20g/L of protein peptone, 2.5g/L of sodium chloride, and 2.5g/L of sodium nitrate according to a mass ratio of 3:1.
2. 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 steps of picking a loop of activated somatic cells by using an inoculating needle, inoculating the bacterial cells into a wide-mouth triangular shake flask containing 50ml of seed culture medium and having the capacity of 250ml, and shake-culturing alkaline catalase production strains in the shake flask at the temperature of 30-37 ℃.
3. 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.
4. 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 36-68 hours under the condition of 200-250 rpm.
5. The method for preparing alkaline catalase by using the microbial fermentation method according to claim 1, further comprising the steps of taking fermentation liquor and centrifuging under the condition of 10800 Xg after fermentation in a fermentation tank is finished, washing the obtained bacterial sludge with 50mmol/L Na 2 HPO 4 -NaH 2 PO 4 buffer with the pH value of 7.0, re-suspending the bacterial sludge with the buffer, then placing the bacterial sludge in ice water for precooling, crushing the bacterial sludge with an ultrasonic crusher, centrifuging under the condition of 12700 Xg, and taking supernatant, wherein the supernatant is the alkaline catalase crude enzyme liquid.

Description

Method for preparing alkaline catalase by microbial fermentation method 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) is an enzyme that catalyzes H 2O2 to produce water and oxygen, and is typically found in the peroxisome of animal and plant cells, and the marker enzyme of the peroxisome is catalase. The use of catalase is very widespread. (1) The medicine is commonly used for instrument disinfection because the H 2O2 has the effects of sterilization, cleaning, bleaching and disinfection. If H 2O2 in the CAT disinfectant is added during contact lens disinfection, it can be decomposed. (2) The catalase can keep food fresh and can be used as antioxidant to eliminate 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 protection industry in most developed countries, and this specific gravity is 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) The textile industry needs printing and dyeing, so that the removal of hydrogen peroxide is a necessary step, the traditional removal method has two different versions, one is to remove the hydrogen peroxide by alternately cleaning with cold and hot water after bleaching with hydrogen peroxide, and the second is to use a peroxide reducer, then cleaning and then dyeing. 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 has many applications in other fields, such as adding catalase and peroxidation at the same time in rubber molding, and can be used for molding quickly, and adding plastic as a binder, of course, in many industries requiring bleaching, adding hydrogen peroxide bleaching is a common operation, and then catalase becomes very necessary, and 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 production of catalase is mainly carried out from animal livers, but the process for producing catalase is easily limited by raw material supply, has very high cost, is low in cost, is easy to obtain by a microbial fermentation method, so that most of catalase in the market is produced by the microbial fermentation method, and not only is the influence of nutrients such as carbon sources, nitrogen sources, inorganic salts, growth factors (nicotinic acid) and the like on 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 quantity of catalase produced by microbial fermentation and the specific activity of enzyme are improved, and the fermentation conditions of microorganisms are required to be optimized. 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 stra