CN113957110B - Method for improving streptomyces aureochromogenes mitochondrial complex enzyme system - Google Patents
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Abstract
The invention relates to a method for improving the enzyme system of a mitochondrial complex of streptomyces aureochromogenes (S.aurea chromogemes), which effectively improves the biological potency of streptomyces aureochromogenes for producing polyoxin and further improves the tolerance of streptomyces aureochromogenes for 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 aureochromogenes mitochondrial complex enzyme system.
Background
Polyoxin, also called polyoxin, is known in japan from the species streptomyces cacao, associn, a variety of species @S .cacaoi var .asoensis) Separated into a nucleoside antibiotic which is Streptomyces aureochromogenes in ChinaS .aurea chromogemes,4896 A secondary metabolite of (a) a protein. As for the metabolite of Streptomyces aureochromogenes, A-N has been reported at home and abroad. The polyoxin belongs to broad-spectrum biological bactericides, has better systemic conduction effect, and has the action mechanism of inhibiting the biosynthesis of the chitin on the cell wall of the thallusThe cell walls of the bacteria cannot carry out biological synthesis to cause death, and the bacterial spore production and the disease spot expansion can be inhibited. The polyoxin is mainly used for preventing and controlling various 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 polyoxin produced by the streptomyces aureofaciens, the polyoxin synthesis gene cluster, the polyoxin nucleoside skeleton and the secondary metabolites produced by the hybridization of the streptomyces aureofaciens and other streptomyces genes, but the genome, regulatory proteins, transcription and translation factors and the like of the secondary metabolites of the streptomyces aureofaciens are not clarified yet, and especially the streptomyces aureofaciens is not clarified yetS .aurea chromogemes,4896 The change of the respiratory chain-related complex enzyme system has not been reported yet. Experiments show that if the dissolved oxygen is insufficient, the secondary metabolite content of the streptomyces aureochromogenes is often lower, even the mitochondria of the streptomyces aureochromogenes are swollen, the mitochondrial complex enzyme system of the streptomyces aureochromogenes is further influenced to lose activity, and finally the mitochondrial inner membrane electrons of the streptomyces aureochromogenes are promoted to be accumulated, and the electron transfer chain is blocked, so that Na is caused + -k + -ATP or Ca 2+ (Mg 2+ ATP) enzyme activity is reduced, and how to alleviate the situation, thereby solving the technical problem that the content of secondary metabolites, namely components A-N, of the streptomyces aureochromogenes is not further influenced by the rapid consumption of dissolved oxygen in the fermentation process, and further improving the potency of polyoxin produced by fermentation of the streptomyces aureochromogenes, which is to be solved in the art.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides a method for improving the enzyme system of the mitochondrial complex of streptomyces aureochromogenes, which comprises the steps of sequentially adding a certain amount of marine chlorella filter membrane treatment liquid, rubidium chloride and sodium citrate into a polyoxin fermentation medium, thereby effectively improving the biological potency of streptomyces aureochromogenes for producing polyoxin and further improving the tolerance of streptomyces aureochromogenes to low dissolved oxygen, and the invention adopts the following technical scheme:
a method for improving a streptomyces aureochromogenes mitochondrial complex enzyme system, comprising the following steps:
(1) Preparing a culture solution of marine chlorella to culture the marine chlorella
The formula of the marine chlorella culture medium is as follows: the mass concentration of the ammonia nitrate is 0.10-0.30g/L, the mass concentration of the 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 1% ferric trichloride solution, adding a proper amount of distilled water, stirring for dissolution under the heating condition, taking out after the ferric trichloride solution is completely dissolved, cooling the mixture in the room temperature environment, adjusting the pH of the solution to 7.0, sterilizing the solution, and storing the solution in the environment of 4 ℃ for later use;
(2) Culture conditions: inoculating Chlorella species with low-temperature preserved culture medium, culturing at room temperature with 2800Lx illumination intensity for 10-14 hr each day in the culture process, and culturing for 6-8d with chlorella maximum density of 1.8-3.0X10 8 cell/m L；
(3) When the density of the cultured chlorella is 2.4X10 8 collecting chlorella at cell/mL, ultrasonically crushing chlorella, performing ultrasonic frequency of 20-40KHz, power of 1-4.0Kw, ultrasonic time of 30-60min, and filtering with 0.2 μm microporous filter membrane to obtain marine chlorella filter membrane treatment solution;
(4) The polyoxin fermentation medium formula is as follows: corn flour 0.2-0.4%, glucose 0.20-0.50%, yeast extract powder 0.20-0.50%, KH 2 PO 4 0.1-0.3%，CaCO 3 0.30 to 0.60 percent, 0.2 to 0.7 percent of bean pulp, 0.10 to 0.16 percent of fish meal, 0.1 to 0.2 percent of NaCl, 0.01 to 0.02 percent of soybean oil (NH) 4 ) 2 SO 4 0.05-0.015%, GPE defoamer 0.004-0.008%, amylase 0.0004-0.0009%, balance sterilized water, pH6.5 before sterilization, sterilizing at 121 ℃ for 30min to obtain polyoxin fermentation medium;
(5) 10-30mL of the marine chlorella filter membrane treatment liquid in the step (3) is added into the polyoxin fermentation medium in the step (4) to be used as a streptomyces aureochromogenes fermentation medium for standby.
In a preferred embodiment of the present invention, in step (1), the marine chlorella medium formulation is: the mass concentration of ammonia nitrate is 0.20g/L, the mass concentration of 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 step (3), the ultrasonic frequency is 30KHz, the power is 2.0Kw, and the ultrasonic time is 45min.
In a preferred embodiment of the invention, the volume ratio of the marine chlorella filter membrane treatment liquid of step (3) to the polyoxin fermentation medium of step (4) is 1:100 to 100:1, preferably 1:25.
In a preferred embodiment of the present invention, in step (5), rubidium chloride and sodium citrate are added to the prepared solution, wherein the addition amount of rubidium chloride is 20-40mg/L, and the addition amount of sodium citrate is 0.15-0.25g/L; more preferably, the adding amount of rubidium chloride is 30mg/L, the adding 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 method has the advantages that a certain amount of marine chlorella ultrasonic filtrate, rubidium chloride and sodium citrate are sequentially added into the polyoxin fermentation medium, so that the biological titer of the streptomyces aureochromogenes for producing polyoxin is effectively improved, the tolerance of the streptomyces aureochromogenes to low-dissolved oxygen is further improved, and meanwhile, the activities of mitochondrial complex I, II and enzyme III are also improved.
Detailed Description
The present invention will be described in further detail with reference to examples, which are not intended to limit the scope of the present invention.
The method for improving the mitochondrial complex enzyme system of the streptomyces aureochromogenes comprises the following steps:
(1) Preparing a culture solution of marine chlorella to culture the marine chlorella
The formula of the marine chlorella culture medium is as follows: the mass concentration of ammonia nitrate is 0.20g/L, the mass concentration of 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 1% ferric trichloride solution, adding a proper amount of distilled water, stirring for dissolution under the heating condition, taking out after the ferric trichloride solution is completely dissolved, cooling the mixture in the room temperature environment, adjusting the pH of the solution to 7.0, sterilizing the solution, and storing the solution in the environment of 4 ℃ for later use;
(2) Culture conditions: inoculating Chlorella species with low-temperature preserved culture medium, culturing at room temperature under 2800Lx illumination intensity for 12 hr each in daily illumination and dark environment for 7d, wherein the maximum density of Chlorella is 2.4X10 8 cell/m L；
(3) When the density of the cultured chlorella is 2.4X10 8 collecting chlorella at cell/mL, crushing chlorella by ultrasonic, wherein the ultrasonic frequency is 30KHz, the power is 2Kw, the ultrasonic time is 45min, and then filtering by a 0.2 mu m microporous filter membrane;
(4) The polyoxin fermentation medium formula is as follows: corn flour 0.3%, glucose 0.35%, yeast extract 0.35%, KH 2 PO 4 0.15%，CaCO 3 0.45%, soybean meal 0.45%, fish meal 0.13%, naCl0.15%, soybean oil 0.015%, (NH) 4 ) 2 SO 4 0.1 percent of GPE defoamer 0.006 percent, amylase 0.0005 percent and the balance of sterilized water, sterilizing for 30 minutes at 121 ℃ and pH6.5 before sterilization to obtain a polyoxin fermentation medium;
(5) 10-30mL of the marine chlorella filter membrane treatment liquid in the step (3) is added into the polyoxin fermentation medium in the step (4) to be used as a streptomyces aureochromogenes fermentation medium, wherein the volume ratio of the filtrate in the step (3) to the polyoxin fermentation medium in the step (4) is 1:100-100:1.
By the first to fourth examples, the influence of chlorella ultrasonic filtrate, rubidium chloride and sodium citrate on the mitochondrial complex enzyme system and production titer during fermentation of Streptomyces aureochromogenes and the influence of chlorella, sodium citrate and rubidium chloride on the mitochondrial complex enzyme system and production titer during fermentation of Streptomyces aureochromogenes are respectively examined. Wherein, the marine chlorella is purchased from Zhanjiang sea mao aquatic products company.
Embodiment one:
in order to examine the influence of chlorella ultrasonic filtrate on the mitochondrial complex enzyme system and production titer during fermentation of Streptomyces aureochromogenes, the embodiment adopts a three-stage fermentation process for Streptomyces aureochromogenes, the three-stage fermentation equipment of which is purchased by Nanjing Tianhui biotechnology Co., ltd, the total volume of a zero-stage fermentation tank is 5L, the volume of a primary fermentation tank is 30L, the volume of the three-stage fermentation tank is 50L, a sterilized air conduit is installed in each fermentation tank, sterilized air is produced by a small air compressor and is input into the fermentation tank (provided with an air flowmeter) by the conduit, steam of a sterilizing tank body is produced 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, feed supplement, defoaming and acid-base supplement in real time. Fermentation medium of each stage of fermentation tank: corn flour 0.3%, glucose 0.35%, yeast extract 0.35%, KH 2 PO 4 0.15%，CaCO 3 0.45%, soybean meal 0.45%, fish meal 0.13%, naCl0.15%, soybean oil 0.015%, (NH) 4 ) 2 SO4 0.1%, GPE defoamer 0.006%, amylase 0.0005%, balance of sterilized water. When the volume ratio of the fermentation medium of the streptomyces aureochromogenes to the ultrasonic filtrate of the marine chlorella is 1:100, 1:50 and 1:25 and 12.5:1, examining the influence of chlorella ultrasonic filtrate on the mitochondrial complex enzyme system and production titer during fermentation of the streptomyces aureofaciens, wherein the batch inoculation amount is 10-15% of the fermentation volume, the zero-order fermentation time is 30h, the primary fermentation time is 24h and the secondary fermentation time is 120h. Filtering the supernatant of the fermentation broth after fermenting in a secondary tank for 120 hr with ceramic membrane (membrane average pore size 0.1 μm, transmembrane pressure 0.2-0.3 mPa) sequentially by ultracentrifugation at 12000rmp for 30min, and collecting filtrateFiltering with a filter membrane (nanofiltration membrane with molecular weight cutoff of more than 500), and preparing polyoxin coarse powder from the trapped liquid by a spray drying tower. Pretreatment is carried out on mitochondrial complex enzyme system determination of zero-order, first-order and second-order fermentation liquor: taking 1-2mL of fermentation liquor in different fermentation periods, centrifuging at 15000-20000rmp for 15-20min, and taking supernatant as a sample for testing the streptomyces aureochromogenes mitochondrial complex enzyme system; the methods for determining mitochondrial complex enzyme I from the zero-order tank fermentation sample (fermented in the zero-order tank for 24 hours), the first-order tank fermentation sample (fermented in the first-order tank for 12 hours) and the second-order tank fermentation sample (fermented in the second-order tank for 96 hours) are as follows: the method for measuring S.aureochromogenes mitochondrial complex enzyme II comprises adding 100 μl of the above-mentioned pretreated test sample to 900 μl of buffer (0.03 mol/L iron potassium hydride 0.2mmol/LPBS,1% bovine serum albumin and 0.6mol/L succinic acid) for 2min, measuring OD value at 420nm of ultraviolet spectrometer, adding 900 μl of buffer (0.3 mM/L cytochrome C) for 2min, and measuring OD value at 550nm of ultraviolet spectrometer.
TABLE 1 Effect of Chlorella filtrate on mitochondrial Complex enzyme System and production titers of Streptomyces aureochromogenes
From Table 1, it is clear that the chlorella filtrate can improve the mitochondrial complex enzymes I and II of Streptomyces aureofaciens and can also improve the production titer of Streptomyces aureofaciens, but has no significant effect on the mitochondrial complex enzyme III of Streptomyces aureofaciens.
Embodiment two:
to examine fermentation of rubidium chloride on Streptomyces aureofaciensThe three-stage fermentation process is adopted for streptomyces aureochromogenes, three-stage fermentation equipment of the three-stage fermentation process is purchased by Nanjing Tianhui biotechnology limited company, the total volume of a zero-stage fermentation tank is 5L, the volume of a first-stage fermentation tank is 30L, the volume of the three-stage fermentation tank is 50L, each fermentation tank is provided with a sterilized air duct, sterilized air is produced by a small air compressor and is input into the fermentation tank (provided with an air flowmeter) through the duct, water vapor of the sterilization tank body is produced 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 supplementing in real time. Fermentation medium of each stage of fermentation tank: corn flour 0.3%, glucose 0.35%, yeast extract 0.35%, KH 2 PO 4 0.15%，CaCO 3 0.45 percent, 0.45 percent of bean pulp, 0.13 percent of fish meal, 0.15 percent of NaCl, 0.015 percent of soybean oil (NH 4) 2 SO4 0.1%, GPE defoamer 0.006%, amylase 0.0005%, balance of sterilized water. When the adding amount of the fermentation medium of the streptomyces aureochromogenes and the rubidium chloride is 10mg/L, 20mg/L, 30mg/L and 40mg/L, the influence of the rubidium chloride on the mitochondrial complex enzyme system and the production titer during fermentation of the streptomyces aureochromogenes is examined, the batch inoculation amount is 10-15% of the fermentation volume, the zero-order fermentation time is 30h, the primary fermentation time is 24h and the secondary fermentation time is 120h. The fermentation liquor after fermentation in the secondary tank for 120 hours is subjected to ultracentrifugation for 30 minutes at 12000rmp, the supernatant is sequentially filtered by a ceramic membrane (the average pore diameter of the membrane is 0.1 mu m, the transmembrane pressure is 0.2-0.3 mPa), the filtrate is filtered by a nanofiltration membrane (the nanofiltration membrane with the molecular weight cutoff of more than 500), and the cutoff is subjected to spray drying to obtain polyoxin coarse powder. Pretreatment is carried out on mitochondrial complex enzyme system determination of zero-order, first-order and second-order fermentation liquor: taking 1-2mL of fermentation liquor in different fermentation periods, centrifuging at 15000-20000rmp for 15-20min, and taking supernatant as a sample for testing the streptomyces aureochromogenes mitochondrial complex enzyme system; the methods for determining mitochondrial complex enzyme I from the zero-order tank fermentation sample (fermented in the zero-order tank for 24 hours), the first-order tank fermentation sample (fermented in the first-order tank for 12 hours) and the second-order tank fermentation sample (fermented in the second-order tank for 96 hours) are as follows: taking experiments of zero level, first level and second level after the pretreatmentThe sample 900. Mu.L was added with 100. Mu.L of a glycylglycine buffer (6 mmol/L NADH 0.2mmol/L, glycylglycine 0.5mmol, cytochrome C, pH8.5, and the reaction was carried out for 2 minutes, and the absorbance was measured at 550nm by a UV spectrometer, and the method for measuring S.aureochromogenes mitochondrial complex enzyme II was carried out by adding 100. Mu.L of the above-mentioned pretreated experimental sample to a buffer (0.03 mol/L of potassium iron hydride 0.2mmol/LPBS,1% bovine serum albumin and 0.6mol/L of succinic acid) and measuring the OD value at 420nm by a UV spectrometer, and the method for measuring S.aureochromogenes mitochondrial complex enzyme III was carried out by adding 100. Mu.L of the above-mentioned pretreated experimental sample to 900. Mu.L of buffer (0.3 mM/L cytochrome C) and reacting at 75mol/L of phosphate buffer for 2 minutes, and measuring the OD value at 550nm by a UV spectrometer.
TABLE 2 influence of rubidium chloride on mitochondrial Complex enzyme System and production titers of Streptomyces aureofaciens
As can be seen from Table 2, rubidium chloride can increase the mitochondrial complex enzyme I, II and enzyme III of Streptomyces aureofaciens and can also increase the production titer of Streptomyces aureofaciens.
Embodiment III:
in order to examine the influence of sodium citrate on the mitochondrial complex enzyme system and production titer during fermentation of Streptomyces aureochromogenes, the embodiment adopts a three-stage fermentation process for Streptomyces aureochromogenes, the three-stage fermentation equipment of which is purchased by Nanjing Tianhui biotechnology limited company, the total volume of a zero-stage fermentation tank is 5L, the volume of a primary fermentation tank is 30L, the volume of the three-stage fermentation tank is 50L, a sterilized air conduit and sterilized air are installed in each fermentation tank, the sterilized air is generated by a small air compressor and is input into the fermentation tank (provided with an air flowmeter) by the conduit, the water vapor of the sterilization 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, material supplementing, defoaming and acid-base supplementing in real time. Fermentation culture in each stage of fermentation tankA base: corn flour 0.3%, glucose 0.35%, yeast extract 0.35%, KH 2 PO 4 0.15%，CaCO 3 0.45 percent, 0.45 percent of bean pulp, 0.13 percent of fish meal, 0.15 percent of NaCl, 0.015 percent of soybean oil (NH 4) 2 SO 4 0.1%, GPE defoamer 0.006%, amylase 0.0005%, balance of sterilized water. When the adding amount of the fermentation medium of the streptomyces aureochromogenes and the citric acid is 0.1g/L, 0.15g/L, 0.25g/L and 0.3/L, the influence of sodium citrate on the mitochondrial complex enzyme system and the production titer during fermentation of the streptomyces aureochromogenes is examined, the batch inoculation amount is 10-15% of the fermentation volume, the zero-order fermentation time is 30h, the primary fermentation time is 24h and the secondary fermentation time is 120h. The fermentation liquor after fermentation in the secondary tank for 120 hours is subjected to ultracentrifugation for 30 minutes at 12000rmp, the supernatant is sequentially filtered by a ceramic membrane (the average pore diameter of the membrane is 0.1 mu m, the transmembrane pressure is 0.2-0.3 mPa), the filtrate is filtered by a nanofiltration membrane (the nanofiltration membrane with the molecular weight cutoff of more than 500), and the cutoff is subjected to spray drying to obtain polyoxin coarse powder. Pretreatment is carried out on mitochondrial complex enzyme system determination of zero-order, first-order and second-order fermentation liquor: taking 1-2mL of fermentation liquor in different fermentation periods, centrifuging at 15000-20000rmp for 15-20min, and taking supernatant as a sample for testing the streptomyces aureochromogenes mitochondrial complex enzyme system; the methods for determining mitochondrial complex enzyme I from the zero-order tank fermentation sample (fermented in the zero-order tank for 24 hours), the first-order tank fermentation sample (fermented in the first-order tank for 12 hours) and the second-order tank fermentation sample (fermented in the second-order tank for 96 hours) are as follows: the method for measuring S.aureochromogenes mitochondrial complex enzyme II comprises adding 100 μl of the above-mentioned pretreated test sample to 900 μl of buffer (0.03 mol/L iron potassium hydride 0.2mmol/LPBS,1% bovine serum albumin and 0.6mol/L succinic acid) for reaction for 2min, measuring OD value at 420nm of ultraviolet spectrophotometer, measuring absorbance at 550nm of S.aureochromogenes mitochondrial complex enzyme III by adding 100 μl of the above-mentioned pretreated test sample to 900 μl of buffer (0.03 mol/L iron potassium hydride 0.2mmol/LPBS,1% bovine serum albumin and 0.6mol/L succinic acid), reacting for 2min, and measuring OD value at 75mol/L phosphateThe reaction was carried out for 2min, and 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
From Table 3, it is known that sodium citrate can increase mitochondrial complex enzyme I of Streptomyces aureochromogenes, but has no significant effect on mitochondrial complexes II and III, and although sodium citrate addition has no significant effect on the production titer of Streptomyces aureochromogenes, acid-base balance can also assist in increasing the titer of Streptomyces aureochromogenes when rubidium chloride and marine chlorella filtrate are added, and specific results are shown in example IV.
Embodiment four:
in order to comprehensively examine the influence of chlorella, sodium citrate and rubidium chloride on the mitochondrial complex enzyme system and production titer during fermentation of Streptomyces aureochromogenes, the embodiment adopts a three-stage fermentation process for Streptomyces aureochromogenes, the three-stage fermentation equipment of which is purchased by Nanjing Tianhui biotechnology limited company, the total volume of a zero-stage fermentation tank is 5L, the volume of a primary fermentation tank is 30L, the volume of the three-stage fermentation tank is 50L, a sterilized air conduit is arranged in each fermentation tank, sterilized air is produced by a small air compressor and is input into the fermentation tank (provided with an air flowmeter) by the conduit, steam of a sterilizing tank body is produced 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, material supplementing, defoaming and acid-base supplementing in real time. Fermentation medium of each stage of fermentation tank: corn flour 0.3%, glucose 0.35%, yeast extract 0.35%, KH 2 PO 4 0.15%，CaCO 3 0.45%, soybean meal 0.45%, fish meal 0.13%, naCl0.15%, soybean oil 0.015%, (NH) 4 ) 2 SO 4 0.1%, GPE defoamer 0.006%, amylase 0.0005%, balance of sterilized water. The influence of sodium citrate on mitochondrial complex enzyme system and production titer during fermentation of streptomyces aureofaciens is examined, and the inoculation amount of batch is fermentation10-15% of the volume, the zero-order fermentation time is 30h, the primary fermentation time is 24h and the secondary fermentation time is 120h. The fermentation liquor after fermentation in the secondary tank for 120 hours is subjected to ultracentrifugation for 30 minutes at 12000rmp, the supernatant is sequentially filtered by a ceramic membrane (the average pore diameter of the membrane is 0.1 mu m, the transmembrane pressure is 0.2-0.3 mPa), the filtrate is filtered by a nanofiltration membrane (the nanofiltration membrane with the molecular weight cutoff of more than 500), and the cutoff is subjected to spray drying to obtain polyoxin coarse powder. Pretreatment is carried out on mitochondrial complex enzyme system determination of zero-order, first-order and second-order fermentation liquor: taking 1-2mL of fermentation liquor in different fermentation periods, centrifuging at 15000-20000rmp for 15-20min, and taking supernatant as a sample for testing the streptomyces aureochromogenes mitochondrial complex enzyme system; the methods for determining mitochondrial complex enzyme I from the zero-order tank fermentation sample (fermented in the zero-order tank for 24 hours), the first-order tank fermentation sample (fermented in the first-order tank for 12 hours) and the second-order tank fermentation sample (fermented in the second-order tank for 96 hours) are as follows: the method for measuring S.aureochromogenes mitochondrial complex enzyme II comprises adding 100 μl of the above-mentioned pretreated test sample to 900 μl of buffer (0.03 mol/L iron potassium hydride 0.2mmol/LPBS,1% bovine serum albumin and 0.6mol/L succinic acid) for 2min, measuring OD value at 420nm of ultraviolet spectrometer, adding 900 μl of buffer (0.3 mM/L cytochrome C) for 2min, and measuring OD value at 550nm of ultraviolet spectrometer.
TABLE 4 Effect of three substances on mitochondrial Complex enzyme System and production titers of Streptomyces aureochromogenes
As can be seen from Table 4, the optimal combination of the amounts of sodium citrate, rubidium chloride and chlorella filtrate is 25:1 of the volume ratio of the fermentation medium of Streptomyces aureofaciens to the chlorella filtrate, and the addition of rubidium chloride is 30mg/L and the addition of citric acid is 0.02g/L, thereby improving the mitochondrial complex enzymes I, II and III of Streptomyces aureofaciens and also improving the production titer of Streptomyces aureofaciens.
While the invention has been described in detail in terms of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, it is intended to claim all such modifications and improvements as may be made without departing from the scope of the invention.
Claims (5)
1. A method for increasing the enzyme system of the mitochondrial complex of streptomyces aureochromogenes (s. aurea chromogemes), comprising the steps of:
(1) Preparing a culture solution of marine chlorella to culture the marine chlorella
The formula of the marine chlorella culture medium is as follows: the mass concentration of the ammonia nitrate is 0.10-0.30g/L, the mass concentration of the 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 1% ferric trichloride solution, adding a proper amount of distilled water, stirring for dissolution under the heating condition, taking out after the ferric trichloride solution is completely dissolved, cooling the mixture in the room temperature environment, adjusting the pH of the solution to 7.0, sterilizing the solution, and storing the solution in the environment of 4 ℃ for later use;
(2) Culture conditions: inoculating Chlorella species with low-temperature preserved culture medium, culturing at room temperature with 2800Lx illumination intensity for 10-14 hr each day in the culture process, and culturing for 6-8d with chlorella maximum density of 1.8-3.0X10 8 cell/m L；
(3) When the density of the cultured chlorella is 2.4X10 8 cell/mL, receiveCollecting chlorella, crushing chlorella by ultrasonic, wherein the ultrasonic frequency is 20-40KHz, the power is 1-4.0Kw, the ultrasonic time is 30-60min, and then filtering by a 0.2 mu m microporous filter membrane to obtain a marine chlorella filter membrane treatment solution;
(4) The polyoxin fermentation medium formula is as follows: corn flour 0.2-0.4%, glucose 0.20-0.50%, yeast extract powder 0.20-0.50%, KH 2 PO 4 0.1%-0.3%，CaCO 3 0.30 to 0.60 percent, 0.2 to 0.7 percent of bean pulp, 0.10 to 0.16 percent of fish meal, 0.1 to 0.2 percent of NaCl, 0.01 to 0.02 percent of soybean oil (NH) 4 ) 2 SO 4 0.05 to 0.015 percent, 0.004 to 0.008 percent of GPE defoamer, 0.0004 to 0.0009 percent of amylase and the balance of sterilized water, wherein the pH value before sterilization is 6.5, and the fermentation medium of polyoxin is obtained after sterilization for 30 minutes at 121 ℃;
(5) 10-30mL of the marine chlorella filter membrane treatment solution in the step (3) is added into the polyoxin fermentation medium in the step (4) to be used as a streptomyces aureochromogenes fermentation medium for standby;
the volume ratio of the marine chlorella filter membrane treatment liquid in the step (3) to the polyoxin fermentation medium in the step (4) is 1:100-100:1;
in the step (5), adding rubidium chloride and sodium citrate into the prepared solution, wherein the adding amount of the rubidium chloride is 20-40mg/L, the adding amount of the sodium citrate is 0.15-0.25g/L, and regulating the pH of the solution to 6.5-7.0.
2. The method of claim 1, wherein in step (1), the marine chlorella medium formulation is: the mass concentration of ammonia nitrate is 0.20g/L, the mass concentration of 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.
3. The method of claim 1, wherein in step (3), the ultrasonic frequency is 30KHz, the power is 2.0Kw, and the ultrasonic time is 45min.
4. The method of claim 1, wherein the volume ratio of the marine chlorella filter membrane treatment fluid of step (3) to the polyoxin fermentation medium of step (4) is 1:25.
5. The method according to claim 1, wherein in step (5), rubidium chloride and sodium citrate are added to the prepared solution, wherein the amount of rubidium chloride added is 30mg/L, the amount of sodium citrate added is 0.2 g/L, and the pH of the solution is adjusted to 6.5-7.0.
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