CN113957110A - Method for improving streptomyces aureofaciens mitochondrial complex enzyme system - Google Patents
Method for improving streptomyces aureofaciens mitochondrial complex enzyme system Download PDFInfo
- Publication number
- CN113957110A CN113957110A CN202111369303.7A CN202111369303A CN113957110A CN 113957110 A CN113957110 A CN 113957110A CN 202111369303 A CN202111369303 A CN 202111369303A CN 113957110 A CN113957110 A CN 113957110A
- Authority
- CN
- China
- Prior art keywords
- mass concentration
- chlorella
- fermentation
- streptomyces
- level
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/005—Glycopeptides, glycoproteins
-
- 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
-
- 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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to a method for improving a streptomyces aureofaciens mitochondrial complex enzyme system, which effectively improves the biological value of the streptomyces aureofaciens for producing polyoxin and further improves the tolerance of the streptomyces aureofaciens to low dissolved oxygen by sequentially adding a certain amount of marine chlorella filter membrane treatment liquid, rubidium chloride and sodium citrate into a polyoxin fermentation medium.
Description
Technical Field
The invention belongs to the field of pesticides, and particularly relates to a method for improving a streptomyces aureofaciens mitochondrial complex enzyme system.
Background
Polyoxin, also called polyoxin, is derived from the family of cacao-streptogramin var axoloides (A. cacao, B. cacao)S .cacaoi var .asoensis) A nucleoside antibiotic is separated from the extract, and in China, the extract is streptomyces aureochromogenes ((S. aureochromogenes))S .aurea chromogemes, 4896) The secondary metabolite of (4). A-N is reported at home and abroad about metabolites of Streptomyces aureochromogenes. Polyoxin belongs to a broad-spectrum biological bactericide, has a good systemic conduction effect, and has an action mechanism of inhibiting biosynthesis of thallus cell wall chitin, so that the thallus cell wall can not be subjected to biosynthesis to cause death, and the sporulation and the enlargement of disease spots of pathogenic bacteria can be inhibited. The polyoxin is mainly used for preventing and controlling a plurality of plant diseases such as apple alternaria leaf spot, pear scab, grape gray mold, cucumber downy mildew, tomato late blight, ginseng black spot and the like. A great deal of reports are made at home and abroad on the synthesis mechanism of the streptomyces aureochromogenes for producing polyoxin, a polyoxin synthesis gene cluster, a polyoxin nucleoside skeleton and secondary metabolites heterozygously produced by the streptomyces aureochromogenes and other streptomyces genes, but the genomes, the regulatory proteins, the transcription and translation factors and the like of the secondary metabolites of the streptomyces aureochromogenes are not clarified, particularly the streptomyces aureochromogenes ((S. aureogenes)S .aurea chromogemes, 4896) Changes in respiratory chain-associated complex enzyme systems have not been reported. Experiments show that if the dissolved oxygen is insufficient, the content of secondary metabolites of the streptomyces aureochromogenes is often low, even mitochondria of the streptomyces aureochromogenes is swollen, the inactivation of a streptomyces aureochromogenes mitochondria complex enzyme system is further influenced, and finally, the electronic accumulation of mitochondria endomembrane of the streptomyces aureochromogenes and the blocking of an electron transfer chain are promoted, so that Na is used for preventing the loss of activity of the streptomyces aureochromogenes mitochondria complex enzyme system+-k+-ATP or Ca2+(Mg2+ATP) enzyme activity is reduced, and the situation is relieved, so that the problem that the streptomyces aureochromogenes does not cause dissolved oxygen to be fast in the fermentation process is solvedConsumption further influences the content of secondary metabolites, namely components A-N, and further improves the titer of the polyoxin produced by fermentation of the streptomyces aureochromogenes, which is a technical problem to be solved in the field.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides a method for improving a streptomyces aureofaciens mitochondrial complex enzyme system, which effectively improves the biological value of the streptomyces aureofaciens for producing polyoxin and further improves the tolerance of the streptomyces aureofaciens to low dissolved oxygen by sequentially adding a certain amount of marine chlorella filter membrane treatment solution, rubidium chloride and sodium citrate into a polyoxin fermentation medium, and adopts the following technical scheme:
a method for improving a streptomyces aureofaciens mitochondrial complex enzyme system comprises the following steps:
(1) preparation of culture solution for marine chlorella
The marine chlorella culture medium comprises the following components in percentage by weight: the mass concentration of the ammonium nitrate is 0.10-0.30g/L, the mass concentration of the calcium superphosphate is 0.02-0.04 g/L, the mass concentration of the magnesium chloride is 0.07-0.09g/L, the mass concentration of the sodium bicarbonate is 0.01-0.03 g/L, the mass concentration of the potassium chloride is 0.01-0.03 g/L, the mass concentration of the sea salt is 3.00-5.00 g/L, the mass concentration of the disodium hydrogen phosphate is 0.10-0.20 g/L, and 1000mL of sterilized distilled water is added; the configuration method comprises the following steps: weighing the required raw materials according to the proportion, adding a ferric trichloride solution with the concentration of 1%, then adding a proper amount of distilled water, stirring and dissolving under a heating condition, taking out after the ferric trichloride solution is completely dissolved, cooling at room temperature, adjusting the pH value of the solution to 7.0, sterilizing, and storing at 4 ℃ for later use;
(2) the culture conditions are as follows: inoculating Chlorella strain with low-temperature culture medium, culturing at room temperature under illumination intensity of 2800Lx for 6-8 days in light environment and dark environment each day, wherein the maximum density of Chlorella is 1.8-3.0 × 108 cell/m L；
(3) When the density of cultured chlorella is 2.4 × 108cell/mL, collecting chlorella, crushing chlorella by ultrasound with the ultrasound frequency of20-40KHz, power of 1-4.0Kw, ultrasonic time of 30-60min, and filtering with 0.2 μm microporous membrane to obtain marine chlorella membrane treatment liquid;
(4) the formula of the polyoxin fermentation medium is as follows: corn flour 0.2-0.4%, glucose 0.20-0.50%, yeast extract powder 0.20-0.50%, KH2PO4 0.1-0.3%，CaCO30.30-0.60%, soybean meal 0.2-0.7%, fish meal 0.10-0.16%, NaCl0.1-0.2%, soybean oil 0.01-0.02%, (NH)4)2SO40.05-0.015 percent, 0.004-0.008 percent of GPE defoaming agent, 0.0004-0.0009 percent of amylase and the balance of sterilized water, wherein the pH value is 6.5 before sterilization, and the sterilization is carried out for 30min at 121 ℃ to obtain a polyoxin fermentation medium;
(5) and (4) adding 10-30mL of the marine chlorella filter membrane treatment solution obtained in the step (3) into the polyoxin fermentation medium obtained in the step (4) to serve as a streptomyces aureochromogenes fermentation medium for later use.
In a preferred embodiment of the invention, in step (1), the marine chlorella culture medium formula is as follows: the mass concentration of ammonium nitrate is 0.20g/L, the mass concentration of calcium superphosphate is 0.03 g/L, the mass concentration of magnesium chloride is 0.08 g/L, the mass concentration of sodium bicarbonate is 0.02g/L, the mass concentration of potassium chloride is 0.02g/L, the mass concentration of sea salt is 4.00 g/L, the mass concentration of disodium hydrogen phosphate is 0.15g/L, and 1000mL of sterilized distilled water is added.
In a preferred embodiment of the present invention, in the step (3), the ultrasonic frequency is 30KHz, the power is 2.0Kw, and the ultrasonic time is 45 min.
In a preferred embodiment of the invention, the volume ratio of the marine chlorella filter membrane treatment solution in the step (3) to the polyoxin fermentation medium in the step (4) is 1: 100-100: 1, preferably 1: 25.
In a preferred embodiment of the invention, in the step (5), rubidium chloride and sodium citrate are added into the prepared solution, wherein the addition amount of the rubidium chloride is 20-40mg/L, and the addition amount of the sodium citrate is 0.15-0.25 g/L; more preferably, the addition amount of rubidium chloride is 30mg/L, the addition amount of sodium citrate is 0.2 g/L, and the pH of the solution is adjusted to 6.5-7.0.
Compared with the prior art, the invention effectively improves the biological value of the streptomyces aureochromogenes for producing the polyoxin, further improves the tolerance of the streptomyces aureochromogenes to low dissolved oxygen and simultaneously improves the activity of mitochondrial compound I, II and enzyme III by sequentially adding a certain amount of marine chlorella ultrasonic filtrate, rubidium chloride and sodium citrate into a polyoxin fermentation medium.
Detailed Description
The present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited thereto.
The method for improving the streptomyces aureofaciens mitochondrial complex enzyme system comprises the following steps:
(1) preparation of culture solution for marine chlorella
The marine chlorella culture medium comprises the following components in percentage by weight: the mass concentration of ammonium nitrate is 0.20g/L, the mass concentration of calcium superphosphate is 0.03 g/L, the mass concentration of magnesium chloride is 0.08 g/L, the mass concentration of sodium bicarbonate is 0.02g/L, the mass concentration of potassium chloride is 0.02g/L, the mass concentration of sea salt is 4.00 g/L, the mass concentration of disodium hydrogen phosphate is 0.15g/L, and 1000mL of sterilized distilled water is added; the configuration method comprises the following steps: weighing the required raw materials according to the proportion, adding a ferric trichloride solution with the concentration of 1%, then adding a proper amount of distilled water, stirring and dissolving under a heating condition, taking out after the ferric trichloride solution is completely dissolved, cooling at room temperature, adjusting the pH value of the solution to 7.0, sterilizing, and storing at 4 ℃ for later use;
(2) the culture conditions are as follows: inoculating Chlorella strain with low-temperature culture medium, culturing at room temperature under 2800Lx light intensity for 12 hr each day in light environment and dark environment, and culturing for 7d with maximum density of 2.4 × 108 cell/m L；
(3) When the density of cultured chlorella is 2.4 × 108collecting chlorella per mL, carrying out ultrasonic crushing on the chlorella, carrying out ultrasonic frequency of 30KHz, power of 2Kw and ultrasonic time of 45min, and then filtering by using a 0.2-micron microporous filter membrane;
(4) the formula of the polyoxin fermentation medium is as follows: corn flour 0.3%, glucose 0.35%, yeast extract powder0.35% of KH2PO4 0.15%，CaCO3 0.45%, soybean meal 0.45%, fish meal 0.13%, NaCl0.15%, soybean oil 0.015%, (NH)4)2SO40.1 percent of GPE defoaming agent, 0.006 percent of amylase, and the balance of sterilized water, wherein the pH value is 6.5 before sterilization, and the sterilization is carried out for 30min at 121 ℃ to obtain polyoxin fermentation medium;
(5) and (3) adding 10-30mL of the marine chlorella filter membrane treatment solution obtained in the step (3) into the polyoxin fermentation medium obtained in the step (4) to serve as a streptomyces aureochromogenes fermentation medium for later use, wherein the volume ratio of the filtrate obtained in the step (3) to the polyoxin fermentation medium obtained in the step (4) is 1: 100-100: 1.
Through the first to the fourth examples, the influence of chlorella ultrasonic filtrate, rubidium chloride and sodium citrate on the mitochondrial complex enzyme system and the production titer of the streptomyces aureochromogenes during fermentation is respectively considered, and the influence of chlorella, sodium citrate and rubidium chloride on the mitochondrial complex enzyme system and the production titer of the streptomyces aureochromogenes during fermentation is comprehensively considered. Among them, marine chlorella is purchased from Zhanjiang Haimaoyao aquatic products Co.
The first embodiment is as follows:
in order to examine the influence of chlorella ultrasonic filtrate on a mitochondrial complex enzyme system and production titer of streptomyces aureochromogenes during fermentation, a three-stage fermentation process is adopted for the streptomyces aureogenes, a three-stage fermentation device of the three-stage fermentation process is purchased from Nanjing Tianhui Biotech limited, the total volume of a zero-stage fermentation tank is 5L, the volume of a first-stage fermentation tank is 30L, the volume of a third-stage fermentation tank is 50L, each fermentation tank is provided with a sterilized air conduit, sterilized air is generated by a small air compressor and is input into the fermentation tank (provided with an air flow meter) through a conduit, water vapor in a sterilized tank body is generated by a steam generator and is input into the fermentation tank (provided with an electromagnetic valve and a steam meter in the middle), and each fermentation tank is provided with a system for monitoring dissolved oxygen, pH, temperature, rotating speed, feeding, defoaming and acid and alkali supplementation in real time. Fermentation medium of each stage of fermentation tank: corn flour 0.3%, glucose 0.35%, yeast extract powder 0.35%, KH2PO4 0.15%，CaCO3 0.45%, soybean meal 0.45%, fish meal 0.13%, NaCl0.15%, soybean oil 0.015%, (NH)4)240.1% of SO, 0.006% of GPE defoaming agent, 0.0005% of amylase and the balance of sterilized water. When the volume ratio of the golden streptomyces colorogenes fermentation medium to the addition amount of the marine chlorella ultrasonic filtrate is 1:100, 1:50 and 1: and 25 and 12.5:1, investigating the influence of the chlorella ultrasonic filtrate on a mitochondrial complex enzyme system and production titer during fermentation of the streptomyces aureochromogenes, wherein the batch inoculation amount is 10-15% of the fermentation volume, the zero-level fermentation time is 30h, and the first-level fermentation time is 24h and the second-level fermentation time is 120 h. And (3) performing ultracentrifugation on the fermentation liquor fermented for 120h in the secondary tank for 30min at 12000rmp, sequentially filtering the supernatant by using a ceramic membrane (the average pore diameter of the membrane is 0.1 mu m, the transmembrane pressure is 0.2-0.3mPa), filtering the filtrate by using a nanofiltration membrane (the nanofiltration membrane with the molecular weight cutoff of more than 500), and preparing polyoxin coarse powder by using the retentate through a spray drying tower. Pre-treating the mitochondria complex enzyme system determination of zero-level, first-level and second-level fermentation liquor: taking 1-2mL of fermentation liquid in different fermentation periods, centrifuging at the rotating speed of 15000-; the method for measuring the mitochondrial complex enzyme I of the zero-level tank fermentation sample (fermented for 24h in the zero-level tank), the first-level tank fermentation sample (fermented for 12h in the first-level tank) and the second-level tank fermentation sample (fermented for 96h in the second-level tank) is as follows: taking 900 mu L of the zero-level, first-level and second-level pretreated experimental samples, adding 100 mu L of glycylglycine buffer solution (6mmol/L NADH 0.2mmol/L, glycylglycine 0.5mmol, cytochrome C, pH8.5, reacting for 2min, measuring the absorbance at 550nm of an ultraviolet spectrophotometer, measuring the complex enzyme II of the streptomyces aureochromogenes mitochondria, taking 100 mu L of the zero-level, first-level and second-level pretreated experimental samples, adding 900 mu L of buffer solution (0.03mol/L potassium iron hydride 0.2mmol/LPBS, 1% bovine serum albumin and 0.6mol/L succinic acid), reacting for 2min, measuring the OD value at 420nm of the ultraviolet spectrophotometer, measuring the complex enzyme III of the streptomyces aureochromogenes mitochondria by taking 100 mu L of the zero-level, first-level and second-level pretreated experimental samples, adding 900 mu L of buffer solution (0.3mM/L cytochrome C) to react for 2min at 75mol/L of phosphate buffer solution, the OD value was measured at 550nm in an ultraviolet spectrophotometer. The specific results are shown in table 1 below.
TABLE 1 influence of Chlorella filtrate on the mitochondrial Complex enzyme System and production titer of Streptomyces aureochromogenes
As can be seen from Table 1, the chlorella filtrate can improve mitochondrial complex enzymes I and II of Streptomyces aureochromogenes and also can improve the production titer of Streptomyces aureochromogenes, but has no significant effect on mitochondrial complex enzyme III of Streptomyces aureochromogenes.
Example two:
in order to investigate the influence of rubidium chloride on a mitochondrial complex enzyme system and production titer during fermentation of streptomyces aureochromogenes, the three-stage fermentation process is adopted for the streptomyces aureogenes, the three-stage fermentation equipment is purchased from Nanjing Tianhui Biotechnology Limited, the total volume of a zero-stage fermentation tank is 5L, the volume of a first-stage fermentation tank is 30L, the volume of a third-stage fermentation tank is 50L, each fermentation tank is provided with a sterilized air conduit, sterilized air is generated by a small-sized air compressor and is input into the fermentation tank (provided with an air flow meter) through a conduit, water vapor in a sterilizing tank body is generated by a steam generator and is input into the fermentation tank (provided with an electromagnetic valve and a steam meter in the middle), and each fermentation tank is provided with a system for monitoring dissolved oxygen, pH, temperature, rotating speed, feeding, defoaming and acid and alkali supplementation in real time. Fermentation medium of each stage of fermentation tank: corn flour 0.3%, glucose 0.35%, yeast extract powder 0.35%, KH2PO40.15%，CaCO3 0.45%, soybean meal 0.45%, fish meal 0.13%, NaCl0.15%, soybean oil 0.015%, (NH4)240.1% of SO, 0.006% of GPE defoaming agent, 0.0005% of amylase and the balance of sterilized water. When the addition amounts of the fermentation medium of the streptomyces aureochromogenes and rubidium chloride are 10mg/L, 20mg/L, 30mg/L and 40mg/L, the influence of the rubidium chloride on a mitochondrial complex enzyme system and the production titer during fermentation of the streptomyces aureochromogenes is considered, the batch inoculation amount is 10-15% of the fermentation volume, the zero-level fermentation time is 30h, and the first-level fermentation time is 24h and the second-level fermentation time is 120 h. The supernatant fluid of 120h fermentation broth after the secondary tank fermentation is ultracentrifuged for 30min at 12000rmp is sequentially filtered by ceramic membrane (the average membrane pore diameter is 0.1 μm, the transmembrane pressure is 0.2-0.3)mPa), filtering the filtrate with a nanofiltration membrane (the nanofiltration membrane with the molecular weight cutoff is more than 500), and preparing polyoxin coarse powder from the cutoff liquid through a spray drying tower. Pre-treating the mitochondria complex enzyme system determination of zero-level, first-level and second-level fermentation liquor: taking 1-2mL of fermentation liquid in different fermentation periods, centrifuging at the rotating speed of 15000-; the method for measuring the mitochondrial complex enzyme I of the zero-level tank fermentation sample (fermented for 24h in the zero-level tank), the first-level tank fermentation sample (fermented for 12h in the first-level tank) and the second-level tank fermentation sample (fermented for 96h in the second-level tank) is as follows: taking 900 mu L of the zero-level, first-level and second-level pretreated experimental samples, adding 100 mu L of glycylglycine buffer solution (6mmol/L NADH 0.2mmol/L, glycylglycine 0.5mmol, cytochrome C, pH8.5, reacting for 2min, measuring the absorbance at 550nm of an ultraviolet spectrophotometer, measuring the complex enzyme II of the streptomyces aureochromogenes mitochondria, taking 100 mu L of the zero-level, first-level and second-level pretreated experimental samples, adding 900 mu L of buffer solution (0.03mol/L potassium iron hydride 0.2mmol/LPBS, 1% bovine serum albumin and 0.6mol/L succinic acid), reacting for 2min, measuring the OD value at 420nm of the ultraviolet spectrophotometer, measuring the complex enzyme III of the streptomyces aureochromogenes mitochondria by taking 100 mu L of the zero-level, first-level and second-level pretreated experimental samples, adding 900 mu L of buffer solution (0.3mM/L cytochrome C) to react for 2min at 75mol/L of phosphate buffer solution, the OD value was measured at 550nm in an ultraviolet spectrophotometer. The specific results are shown in Table 2 below.
TABLE 2 influence of rubidium chloride on the mitochondrial Complex enzyme System and production titer of Streptomyces aureochromogenes
As can be seen from Table 2, rubidium chloride can improve mitochondrial complex enzyme I, II and enzyme III of Streptomyces aureochromogenes, and can also improve the production titer of Streptomyces aureochromogenes.
Example three:
in order to examine the mitochondrial complex enzyme system and the production titer of sodium citrate during the fermentation of Streptomyces aureochromogenesInfluence, this embodiment adopts tertiary fermentation technology to golden streptomyces chromogenes, south Beijing Tianhui biotechnology limited is purchased to its tertiary fermentation equipment, zero order fermentation cylinder total volume is 5L, first order fermentation cylinder volume is 30L, third order fermentation cylinder volume is 50L, sterile air pipe is installed to every fermentation cylinder, sterile air is produced by small-size air compressor and by pipe input to fermentation cylinder (being equipped with air flow meter), the vapor of the disinfection jar body is produced by steam generator and by pipe input to fermentation cylinder (middle solenoid valve that is equipped with, the steam meter), every fermentation cylinder is equipped with real time monitoring dissolved oxygen, pH, temperature, the rotational speed, the feed supplement, defoaming and acid and alkali system of benefit. Fermentation medium of each stage of fermentation tank: corn flour 0.3%, glucose 0.35%, yeast extract powder 0.35%, KH2PO40.15%，CaCO3 0.45%, soybean meal 0.45%, fish meal 0.13%, NaCl0.15%, soybean oil 0.015%, (NH4)2SO40.1 percent, 0.006 percent of GPE defoaming agent, 0.0005 percent of amylase and the balance of sterilized water. When the addition amounts of the streptomyces aureochromogenes fermentation medium and citric acid are 0.1g/L, 0.15g/L, 0.25g/L and 0.3/L, the influence of sodium citrate on a mitochondrial complex enzyme system and production titer during fermentation of the streptomyces aureochromogenes is considered, the batch inoculation amount is 10-15% of the fermentation volume, the zero-order fermentation time is 30h, and the first-order fermentation time is 24h and the second-order fermentation time is 120 h. And (3) performing ultracentrifugation on the fermentation liquor fermented for 120h in the secondary tank for 30min at 12000rmp, sequentially filtering the supernatant by using a ceramic membrane (the average pore diameter of the membrane is 0.1 mu m, the transmembrane pressure is 0.2-0.3mPa), filtering the filtrate by using a nanofiltration membrane (the nanofiltration membrane with the molecular weight cutoff of more than 500), and preparing polyoxin coarse powder by using the retentate through a spray drying tower. Pre-treating the mitochondria complex enzyme system determination of zero-level, first-level and second-level fermentation liquor: taking 1-2mL of fermentation liquid in different fermentation periods, centrifuging at the rotating speed of 15000-; the method for measuring the mitochondrial complex enzyme I of the zero-level tank fermentation sample (fermented for 24h in the zero-level tank), the first-level tank fermentation sample (fermented for 12h in the first-level tank) and the second-level tank fermentation sample (fermented for 96h in the second-level tank) is as follows: taking 900 mu L of experimental sample subjected to zero-level, first-level and second-level pretreatment, and adding 100 mu L of experimental sampleL glycylglycine buffer solution (6mmol/L NADH 0.2mmol/L glycylglycine 0.5mmol, cytochrome C, pH8.5, reaction for 2min, measured at 550nm absorbance of ultraviolet spectrophotometer; the determination method of Streptomyces aureochromogenes mitochondrial complex enzyme II comprises taking 100. mu.L of the above-mentioned pretreated experimental sample, adding buffer solution (0.03mol/L potassium iron hydride 0.2mmol/LPBS, 1% bovine serum albumin and 0.6mol/L succinic acid) 900. mu.L, reacting for 2min, measuring its OD value at 420nm of ultraviolet spectrophotometer, and the determination method of Streptomyces aureochromogenes mitochondrial complex enzyme III comprises taking 100. mu.L of the above-mentioned pretreated experimental sample, adding 900. mu.L buffer solution (0.3mM/L cytochrome C) to 75mol/L phosphate buffer solution, reacting for 2min, the OD value was measured at 550nm in an ultraviolet spectrophotometer. The specific results are shown in Table 3 below.
TABLE 3 Effect of sodium citrate on mitochondrial Complex enzyme System and production Titers of Streptomyces aureochromogenes
As can be seen from Table 3, sodium citrate can improve mitochondrial complex enzyme I of Streptomyces aureochromogenes, but has no significant effect on mitochondrial complex II and III, and although the addition of sodium citrate has no significant effect on the production titer of Streptomyces aureochromogenes, the addition of acid and base can be balanced to assist the increase of the titer of Streptomyces aureochromogenes when rubidium chloride and marine chlorella filtrate are added, and specific results are shown in example IV.
Example four:
in order to comprehensively investigate the influence of chlorella, sodium citrate and rubidium chloride on a mitochondria complex enzyme system and production titer during fermentation of streptomyces aureochromogenes, the three-stage fermentation process is adopted for the streptomyces aureochromogenes, the three-stage fermentation equipment is purchased from Nanjing Tianhui Biotech limited, the total volume of a zero-stage fermentation tank is 5L, the volume of a first-stage fermentation tank is 30L, the volume of a third-stage fermentation tank is 50L, each fermentation tank is provided with a sterilized air conduit, sterilized air is generated by a small-sized air compressor and is input into the fermentation tank (provided with an air flow meter) through a conduit, and water vapor in a sterilization tank body is generated by steamThe device is produced and input into fermentation tanks (electromagnetic valves and steam meters are arranged in the middle), and each fermentation tank is provided with a system for monitoring dissolved oxygen, pH, temperature, rotating speed, feeding, defoaming and acid and alkali feeding in real time. Fermentation medium of each stage of fermentation tank: corn flour 0.3%, glucose 0.35%, yeast extract powder 0.35%, KH2PO4 0.15%，CaCO3 0.45%, soybean meal 0.45%, fish meal 0.13%, NaCl0.15%, soybean oil 0.015%, (NH)4)2SO40.1 percent, 0.006 percent of GPE defoaming agent, 0.0005 percent of amylase and the balance of sterilized water. And (3) investigating the influence of the sodium citrate on a mitochondrial complex enzyme system and the production titer during fermentation of the streptomyces aureochromogenes, wherein the batch inoculation amount is 10-15% of the fermentation volume, the zero-level fermentation time is 30h, and the first-level fermentation time is 24h and the second-level fermentation time is 120 h. And (3) performing ultracentrifugation on the fermentation liquor fermented for 120h in the secondary tank for 30min at 12000rmp, sequentially filtering the supernatant by using a ceramic membrane (the average pore diameter of the membrane is 0.1 mu m, the transmembrane pressure is 0.2-0.3mPa), filtering the filtrate by using a nanofiltration membrane (the nanofiltration membrane with the molecular weight cutoff of more than 500), and preparing polyoxin coarse powder by using the retentate through a spray drying tower. Pre-treating the mitochondria complex enzyme system determination of zero-level, first-level and second-level fermentation liquor: taking 1-2mL of fermentation liquid in different fermentation periods, centrifuging at the rotating speed of 15000-; the method for measuring the mitochondrial complex enzyme I of the zero-level tank fermentation sample (fermented for 24h in the zero-level tank), the first-level tank fermentation sample (fermented for 12h in the first-level tank) and the second-level tank fermentation sample (fermented for 96h in the second-level tank) is as follows: taking 900 mu L of the zero-level, first-level and second-level pretreated experimental samples, adding 100 mu L of glycylglycine buffer solution (6mmol/L NADH 0.2mmol/L, glycylglycine 0.5mmol, cytochrome C, pH8.5, reacting for 2min, measuring absorbance at 550nm of an ultraviolet spectrophotometer, and measuring the compound enzyme II of the streptomyces aureochromogenes mitochondria by taking 100 mu L of the zero-level, first-level and second-level pretreated experimental samples, adding buffer solution (0.03mol/L potassium iron hydride 0.2mmol/LPBS, 1% bovine serum albumin and 0.6mol/L succinic acid) 900 mu L of buffer solution (0.03mol/L potassium iron hydride 0.2mmol/LPBS, reacting for 2min, measuring the OD value at 420nm of the ultraviolet spectrophotometer, and measuring the compound enzyme III of the streptomyces aureochromogenes mitochondria: taking 100 μ L of the test sample subjected to the above pre-treatment in the zero, first and second stages, adding 900 μ L of buffer solution (0.3mM/L cytochrome C) to 75mol/L of phosphate buffer solution, reacting for 2min, and measuring OD value at 550nm of an ultraviolet spectrophotometer. The specific results are shown in Table 4 below.
TABLE 4 Effect of the three substances on the mitochondrial Complex enzyme System and the production titer of Streptomyces aureochromogenes
As can be seen from Table 4, the optimal combination of the sodium citrate, the rubidium chloride and the chlorella filtrate is that the volume ratio of the streptomyces aureochromogenes fermentation culture medium to the chlorella filtrate is 25:1, the addition amount of the rubidium chloride is 30mg/L, and the addition amount of the citric acid is 0.02g/L, so that mitochondrial complex enzymes I, II and III of the streptomyces aureochromogenes can be improved, and the production titer of the streptomyces aureochromogenes can also be improved.
Although the invention has been described in detail in the foregoing with reference to general description, specific embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto based on the invention. Therefore, it is intended that the present invention cover the modifications and improvements made without departing from the spirit and scope of the present invention.
Claims (6)
1. A method for improving a streptomyces aureofaciens mitochondrial complex enzyme system is characterized in that a certain amount of marine chlorella filter membrane treatment solution, rubidium chloride and sodium citrate are sequentially added into a polyoxin fermentation medium.
2. The method of claim 1, comprising the steps of:
(1) preparation of culture solution for marine chlorella
The marine chlorella culture medium comprises the following components in percentage by weight: the mass concentration of the ammonium nitrate is 0.10-0.30g/L, the mass concentration of the calcium superphosphate is 0.02-0.04 g/L, the mass concentration of the magnesium chloride is 0.07-0.09g/L, the mass concentration of the sodium bicarbonate is 0.01-0.03 g/L, the mass concentration of the potassium chloride is 0.01-0.03 g/L, the mass concentration of the sea salt is 3.00-5.00 g/L, the mass concentration of the disodium hydrogen phosphate is 0.10-0.20 g/L, and 1000mL of sterilized distilled water is added; the configuration method comprises the following steps: weighing the required raw materials according to the proportion, adding a ferric trichloride solution with the concentration of 1%, then adding a proper amount of distilled water, stirring and dissolving under a heating condition, taking out after the ferric trichloride solution is completely dissolved, cooling at room temperature, adjusting the pH value of the solution to 7.0, sterilizing, and storing at 4 ℃ for later use;
(2) the culture conditions are as follows: inoculating Chlorella strain with low-temperature culture medium, culturing at room temperature under illumination intensity of 2800Lx for 6-8 days in light environment and dark environment each day, wherein the maximum density of Chlorella is 1.8-3.0 × 108 cell/m L；
(3) When the density of cultured chlorella is 2.4 × 108collecting chlorella per mL, carrying out ultrasonic crushing on the chlorella, carrying out ultrasonic frequency of 20-40KHz, power of 1-4.0Kw and ultrasonic time of 30-60min, and then filtering by using a 0.2-micron microporous filter membrane to obtain marine chlorella filter membrane treatment liquid;
(4) the formula of the polyoxin fermentation medium is as follows: corn flour 0.2-0.4%, glucose 0.20-0.50%, yeast extract powder 0.20-0.50%, KH2PO4 0.1-0.3%，CaCO30.30-0.60%, soybean meal 0.2-0.7%, fish meal 0.10-0.16%, NaCl0.1-0.2%, soybean oil 0.01-0.02%, (NH)4)2SO40.05-0.015 percent, 0.004-0.008 percent of GPE defoaming agent, 0.0004-0.0009 percent of amylase and the balance of sterilized water, wherein the pH value is 6.5 before sterilization, and the sterilization is carried out for 30min at 121 ℃ to obtain a polyoxin fermentation medium;
(5) and (4) adding 10-30mL of the marine chlorella filter membrane treatment solution obtained in the step (3) into the polyoxin fermentation medium obtained in the step (4) to serve as a streptomyces aureochromogenes fermentation medium for later use.
3. The method according to claim 2, wherein in the step (1), the marine chlorella culture medium formula is as follows: the mass concentration of ammonium nitrate is 0.20g/L, the mass concentration of calcium superphosphate is 0.03 g/L, the mass concentration of magnesium chloride is 0.08 g/L, the mass concentration of sodium bicarbonate is 0.02g/L, the mass concentration of potassium chloride is 0.02g/L, the mass concentration of sea salt is 4.00 g/L, the mass concentration of disodium hydrogen phosphate is 0.15g/L, and 1000mL of sterilized distilled water is added.
4. The method according to claim 2, wherein in the step (3), the ultrasonic frequency is 30KHz, the power is 2.0Kw, and the ultrasonic time is 45 min.
5. The method according to claim 2, wherein the volume ratio of the marine chlorella filter membrane treatment solution in the step (3) to the polyoxin fermentation medium in the step (4) is 1: 100-100: 1, preferably 1: 25.
6. The method according to claim 2, wherein in the step (5), rubidium chloride and sodium citrate are added into the prepared solution, wherein the addition amount of the rubidium chloride is 20-40mg/L, and the addition amount of the sodium citrate is 0.15-0.25 g/L; more preferably, the addition amount of rubidium chloride is 30mg/L, the addition amount of sodium citrate is 0.2 g/L, and the pH of the solution is adjusted to 6.5-7.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111369303.7A CN113957110B (en) | 2021-11-18 | 2021-11-18 | Method for improving streptomyces aureochromogenes mitochondrial complex enzyme system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111369303.7A CN113957110B (en) | 2021-11-18 | 2021-11-18 | Method for improving streptomyces aureochromogenes mitochondrial complex enzyme system |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113957110A true CN113957110A (en) | 2022-01-21 |
| CN113957110B CN113957110B (en) | 2023-08-08 |
Family
ID=79471286
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111369303.7A Active CN113957110B (en) | 2021-11-18 | 2021-11-18 | Method for improving streptomyces aureochromogenes mitochondrial complex enzyme system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113957110B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5084389A (en) * | 1989-08-01 | 1992-01-28 | Her Majesty In Right Of Canada As Represented By The Minister Of Energy, Mines And Resources Canada | Bioadsorption composition and process for production thereof |
| CN102325868A (en) * | 2008-12-23 | 2012-01-18 | Xtu公司 | Device for cultivating algae and/or microorganisms for treating an effluent, and biological frontage |
| CN102643764A (en) * | 2012-03-06 | 2012-08-22 | 南京工业大学 | Streptomyces and multi-antibiotics metabolic regulation fermentation process |
| CN104513805A (en) * | 2013-10-07 | 2015-04-15 | 鲁南制药集团股份有限公司 | Method for producing anti CD20 antibody |
| US20170218328A1 (en) * | 2016-01-28 | 2017-08-03 | Nanogen Pharmaceutical Biotechnology Co., Ltd | Universal, glycosylation enhancer, completely chemically defined medium formulation |
-
2021
- 2021-11-18 CN CN202111369303.7A patent/CN113957110B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5084389A (en) * | 1989-08-01 | 1992-01-28 | Her Majesty In Right Of Canada As Represented By The Minister Of Energy, Mines And Resources Canada | Bioadsorption composition and process for production thereof |
| CN102325868A (en) * | 2008-12-23 | 2012-01-18 | Xtu公司 | Device for cultivating algae and/or microorganisms for treating an effluent, and biological frontage |
| CN102643764A (en) * | 2012-03-06 | 2012-08-22 | 南京工业大学 | Streptomyces and multi-antibiotics metabolic regulation fermentation process |
| CN104513805A (en) * | 2013-10-07 | 2015-04-15 | 鲁南制药集团股份有限公司 | Method for producing anti CD20 antibody |
| US20170218328A1 (en) * | 2016-01-28 | 2017-08-03 | Nanogen Pharmaceutical Biotechnology Co., Ltd | Universal, glycosylation enhancer, completely chemically defined medium formulation |
Non-Patent Citations (2)
| Title |
|---|
| M. LAKSHMIKANDAN 等: "Co-cultivation of Streptomyces and microalgal cells as an efficient system for biodiesel production and bioflocculation formation", BIORESOURCE TECHNOLOGY, vol. 332, no. 1, pages 125118 * |
| 黄文福;牛春;孙金刚;张瑞敏;张萍;: "微量元素和发酵增效剂影响金色链霉菌发酵研究", 饲料工业, no. 06, pages 44 - 48 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113957110B (en) | 2023-08-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109439701B (en) | Method for preparing ergothioneine by biosynthesis and fermentation medium | |
| CN104726510B (en) | A kind of method of preparing lysine through fermentation | |
| CN112375755A (en) | Breeding method of aspergillus niger capable of producing beta-glucosidase at high yield | |
| CN102154168A (en) | Abamectin producing bacterium and preparation method thereof | |
| CN102690857A (en) | Method for preparing medium-low molecular weight dextran by using biological method | |
| CN108866128B (en) | Method for improving biological value of kasugamycin | |
| CN104277989B (en) | One plant of Saccharomyces cerevisiae and its application in fermenting and producing DPN | |
| CN109234334A (en) | A kind of method of fermenting and producing tremella polysaccharides and fermentation medium used | |
| CN113957110A (en) | Method for improving streptomyces aureofaciens mitochondrial complex enzyme system | |
| CN112592860A (en) | Bifidobacterium infantis fermentation medium without animal-derived nitrogen source and application thereof | |
| CN104878059B (en) | A kind of method for preparing s-adenosylmethionine | |
| CN107603884A (en) | One plant height produces the trichoderma reesei mutant strain of neutral cellulase | |
| CN106591401B (en) | Fermentation promoter for increasing yield of gentamicin C1a and addition method thereof | |
| CN107384879B (en) | A kind of fermentation manufacturing technique of recombinant yeast pichia pastoris bacterium production liver detoxification enzyme | |
| CN102559814A (en) | Method for preparing acarbose | |
| CN109504725A (en) | A kind of method and fermentation medium of fermentation Hericium erinaceus preparation high-purity Hericium erinaceus Polysaccharides | |
| CN102399845B (en) | Based on CO in tail gas 2the vitamin B12 fermentative production Controlling Technology of concentration | |
| CN108531418A (en) | A kind of preparation method of chitosan enzyme | |
| CN107988086B (en) | A kind of bacterial strain of high yield rhamnosan galacturonic hydrolase | |
| CN105505787A (en) | Aspergillus niger mutant strain for producing transglucosidase | |
| CN102260716A (en) | Fermentation broth for citric acid fermentation and fermentation method using same | |
| CN110699409A (en) | Method for producing pingyangmycin by fermentation | |
| TWI792803B (en) | Method of producing natural type n-acetylglucosamine | |
| CN109486877A (en) | The method of fed-batch fermentation technique production rapamycin | |
| CN108424944B (en) | Optimized production method and culture medium of candicidin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |