CN107815426B - Special strain for fermentation production of kasugamycin and application thereof - Google Patents
Special strain for fermentation production of kasugamycin and application thereof Download PDFInfo
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Abstract
The invention relates to a special strain for producing kasugamycin by fermentation and application thereof. The invention separates an aerobic actinomycete from soil of a certain vegetable field in the Yanglin City of the peony river, and finally obtains a bacterial strain BJX007 with high yield of kasugamycin by carrying out ultraviolet and chemical mutagenesis treatment on the actinomycete, wherein the preservation number of the bacterial strain is CGMCC No. 11622. Through identification, the strain BJX007 is Streptomyces aureofaciens, has high capacity of producing kasugamycin through fermentation, the yield can reach 25000 mu g/mL of fermentation liquor, and the capacity of producing kasugamycin of the strain BJX007 after continuous passage for 5 times is still maintained at the original level, which shows that the strain BJX007 has good genetic stability and can be used for the production of industrial large-scale kasugamycin. The invention provides a valuable microorganism resource for producing kasugamycin by utilizing a biological fermentation technology.
Description
Technical Field
The invention relates to the field of microbial fermentation, in particular to a special strain for producing kasugamycin by fermentation and application thereof.
Background
Kasugamycin (Kasugamycin), also known as Kasugamycin, is an aminoglycoside antibiotic produced by actinomycetes, belongs to a systemic antibiotic, has both treatment and prevention effects, is a white needle-shaped crystalline solid, is stable at normal temperature, is stable under acidic and neutral conditions, and is easy to decompose under alkaline conditions. The aqueous solution is dark green liquid, can be stored for more than 2 years at normal temperature, is environment-friendly, is an ideal agricultural bactericide, is widely applied to agricultural production, has excellent prevention and treatment effects on rice blast on rice, and has special effects on preventing and treating diseases such as bacterial angular leaf spot of watermelons, gummosis of peach trees, scab, perforation disease and the like.
Disclosure of Invention
The invention aims to provide a special strain-streptomyces aureofaciens BJX007 for producing kasugamycin by fermentation.
Another purpose of the invention is to provide application of the strain BJX007 in fermentation production of kasugamycin.
In order to realize the purpose of the invention, the invention separates a strain of actinomycetes from a vegetable field near a cross river in the Yanglin city of the peony river, and the specific method comprises the following steps: preparing soil into suspension, inoculating the soil suspension into a separation culture medium for culture, selecting a single colony, diluting, streaking, separating and purifying to obtain a pure culture strain. The strain morphology identification is carried out according to the index table of the fungal identification manual.
The strain is identified to be aerobic actinomycetes, spore filaments are spiral, spore spheres are in an oval shape, the middle part is convex, the surface is smooth, the aerial hyphae on a synthetic culture medium are grey white, and the hyphae in the medium are earthy yellow.
Wherein the formula of the separation culture medium is as follows: 3g/L of tryptone, 10g/L of bean cake powder, 10mL/L of glycerin, 3g/L of sodium chloride, 2g/L of calcium carbonate and 25g/L of agar powder, and the pH value is natural. The formula of the synthetic medium is as follows: 20g/L of corn starch, 45g/L of bean cake powder, 2.5g/L of sodium chloride, 0.2g/L of monopotassium phosphate, 12mL/L of soybean oil, 0.1g/L of amylase and natural pH value.
The actinomycete strain is subjected to the following mutagenesis treatment:
(1) ultraviolet mutagenesis twice: 5ml of the bacterial suspension is taken, added into a sterile culture dish and vertically irradiated at a height of 30cm by using a 30W ultraviolet irradiator. The irradiation time was set to 60s and 90s, respectively. After the isolation medium was sterilized, streptomycin was added to the medium at a final concentration of 0.1. mu.g/ml and 0.2. mu.g/ml, which was sterilized by filtration, plated on a plate, and cultured at 28 ℃ for 10 days. Single colonies were picked for high throughput screening and shake flask retesting according to the methods described above. The obtained high-yield kasugamycin strain is subjected to next step mutagenesis.
(2) Sodium Nitrite (NIT) mutagenesis
Preparing bacterial suspension of the bacterial strain by using the bacterial strain screened by ultraviolet mutagenesis, respectively adding 10mL of the bacterial suspension into 250mL conical flasks, respectively filling 90mL of silicate modified culture medium containing NIT with different concentrations (0, 5, 10, 20, 40, 60 and 80mg/L) into the flasks, treating for 40min at the temperature of 30 ℃ and at the speed of 200r/min, then centrifugally separating thalli precipitates, washing for 3 times by using sterile water, removing NIT, and terminating mutagenesis. Then 1mL of each mutagenic bacterium liquid is diluted and coated on a modified medium agar plate, the mutagenic bacterium liquid is cultured for 7d at 30 ℃, then colony counting is carried out, the optimal mutagenic dose is determined, and the colony under the mutagenic dose is selected for primary screening and secondary screening. The obtained bacterial strain with high yield of kasugamycin is continuously subjected to chemical mutagenesis treatment.
(3) Lithium chloride treatment
Preparing spore suspension of the strain from the strain screened by NIT mutagenesis, and taking 10mL of spore suspension (the number of spores is 10)8Each mL) of the culture medium is added with LiCl according to the concentration of 1%, the mixture is shaken on a shaking table for 10 hours, 12 hours, 15 hours and 20 hours, mutagenesis is stopped, 1mL of each mutagenic bacterium liquid is taken to be diluted and coated on an agar medium plate, colony counting is carried out after the culture is carried out for 10 days at the temperature of 28 ℃, the optimal mutagenic dose is determined, colonies under the mutagenic dose are selected to be primarily screened and re-screened, and finally, a strain BJX007 with high kasugamycin yield is obtained.
Through identification, the strain BJX007 is aerobic actinomycetes, spore filaments are spiral, spore spheres are in an oval shape, the middle part is convex, the surface is smooth, aerial hyphae on a synthetic culture medium are grey white, and hyphae in the medium are earthy yellow.
The 16SrDNA sequence of the strain BJX007 is shown as SEQ ID NO:1, the 16SrDNA sequence is compared in GenBank, and a phylogenetic tree of the strain BJX007 is constructed. Wherein, the primers used for PCR amplification of the BJX 00716 SrDNA sequence are as follows:
a forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3'
Reverse primer: 5'-AAGTCGTAACAAGGTAGCCGTA-3'
By integrating the morphological characteristics of the strain and the evolutionary analysis result of a 16SrDNA gene sequence system, the strain BJX007 is identified to be Streptomyces aureofaciens (Streptomyces microaureus), which is deposited in the common microorganism center of China Committee for culture Collection of microorganisms, No. 3 of Xilu 1 of Beijing area north Chengyang, institute of microbiology of Chinese academy of sciences, zip code 100101, preservation number CGMCC No.11622, and preservation date of 2015, 11 months and 5 days.
The invention also provides application of the streptomyces aureofaciens BJX007 in fermentation production of kasugamycin.
The method comprises the following steps:
s1, preparing a seed solution: digging 1cm from slant culture medium of strain BJX0072Inoculating a bacterium-carrying culture medium into a 250mL seed bottle filled with a fermentation culture medium, wherein the liquid loading amount is 30mL, performing shake culture at 28 ℃ and 220r/min for 48h, inoculating the obtained bacterium liquid into a fermentation bottle filled with the fermentation culture medium according to the mass percentage of 10%, wherein the volume of the fermentation bottle is 250mL, the liquid loading amount is 35mL, and performing shake culture at 28 ℃ and 220r/min for 24h to obtain a seed liquid;
s2, fermentation culture: inoculating the seed liquid into a fermentation bottle filled with a fermentation culture medium according to the mass percentage of 10%, wherein the volume of the fermentation bottle is 250mL, the liquid loading amount is 35mL, performing shake culture on a shaking table at 220r/min at 28 ℃ for 165-170h (preferably 7d), and after the fermentation is finished, separating the kasugamycin from the fermentation liquid.
Wherein the formula of the fermentation medium is as follows: 15-20g/L of corn starch, 45-50g/L of bean cake powder, 2.5g-3/L of sodium chloride, 0.2g-0.5/L of monopotassium phosphate, 10-12mL/L of soybean oil, 0.05-0.1g/L of amylase and natural pH value.
Preferably, the fermentation medium formulation is as follows: 20g/L of corn starch, 45g/L of bean cake powder, 2.5g/L of sodium chloride, 0.2g/L of monopotassium phosphate, 12mL/L of soybean oil and 0.1g/L of amylase.
The Streptomyces aureofaciens BJX007 provided by the invention has high capacity of producing kasugamycin by fermentation, the yield can reach 25000 mu g/mL fermentation liquor, and the capacity of producing kasugamycin of the strain BJX007 after continuous passage for 5 times is still maintained at the original level, which shows that the strain BJX007 has good genetic stability and can be used for the production of industrial large-scale kasugamycin. The invention provides a valuable microorganism resource for producing kasugamycin by utilizing a biological fermentation technology.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.
The amylase used in the invention is high-temperature amylase, and the enzyme activity is 10000U/g.
Example 1 isolation and identification of Strain BJX007
1. Isolation and identification of original strains
An actinomycete is separated from a vegetable field near a cross river in the Yangtze Shanlin city of peony, and the strain morphology is identified according to an index table of 'fungus identification handbook'.
The strain is identified to be aerobic actinomycetes, spore filaments are spiral, spore spheres are in an oval shape, the middle part is convex, the surface is smooth, the aerial hyphae on a synthetic culture medium are grey white, and the hyphae in the medium are earthy yellow.
2. Mutagenesis of the original Strain
(1) Ultraviolet mutagenesis twice: 5ml of the bacterial suspension is taken, added into a sterile culture dish and vertically irradiated at a height of 30cm by using a 30W ultraviolet irradiator. The irradiation time was set to 60s and 90s, respectively. After the isolation medium was sterilized, streptomycin was added to the medium at a final concentration of 0.1. mu.g/ml and 0.2. mu.g/ml, which was sterilized by filtration, plated on a plate, and cultured at 28 ℃ for 10 days. Single colonies were picked for high throughput screening and shake flask retesting according to the methods described above. The obtained high-yield kasugamycin strain is subjected to next step mutagenesis.
(2) Sodium Nitrite (NIT) mutagenesis
Preparing bacterial suspension of the bacterial strain by using the bacterial strain screened by ultraviolet mutagenesis, respectively adding 10mL of the bacterial suspension into 250mL conical flasks, respectively filling 90mL of silicate modified culture medium containing NIT with different concentrations (0, 5, 10, 20, 40, 60 and 80mg/L) into the flasks, treating for 40min at the temperature of 30 ℃ and at the speed of 200r/min, then centrifugally separating thalli precipitates, washing for 3 times by using sterile water, removing NIT, and terminating mutagenesis. Then 1mL of each mutagenic bacterium liquid is diluted and coated on a modified medium agar plate, the mutagenic bacterium liquid is cultured for 7d at 30 ℃, then colony counting is carried out, the optimal mutagenic dose is determined, and the colony under the mutagenic dose is selected for primary screening and secondary screening. The obtained bacterial strain with high yield of kasugamycin is continuously subjected to chemical mutagenesis treatment.
(3) Lithium chloride treatment
Preparing spore suspension of the strain from the strain screened by NIT mutagenesis, and collecting10mL spore suspension (spore count 10)8Each mL) of the culture medium is added with LiCl according to the concentration of 1%, the mixture is shaken on a shaking table for 10 hours, 12 hours, 15 hours and 20 hours, mutagenesis is stopped, 1mL of each mutagenic bacterium liquid is taken to be diluted and coated on an agar medium plate, colony counting is carried out after the culture is carried out for 10 days at the temperature of 28 ℃, the optimal mutagenic dose is determined, colonies under the mutagenic dose are selected to be primarily screened and re-screened, and finally, a strain BJX007 with high kasugamycin yield is obtained.
3. Identification of Strain BJX007
Through identification, the strain BJX007 is aerobic actinomycetes, spore filaments are spiral, spore spheres are in an oval shape, the middle part is convex, the surface is smooth, aerial hyphae on a synthetic culture medium are grey white, and hyphae in the medium are earthy yellow.
The 16SrDNA sequence of the strain BJX007 is shown as SEQ ID NO:1, the 16SrDNA sequence is compared in GenBank, and a phylogenetic tree of the strain BJX007 is constructed. Wherein, the primers used for PCR amplification of the BJX 00716 SrDNA sequence are as follows:
a forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3'
Reverse primer: 5'-AAGTCGTAACAAGGTAGCCGTA-3'
And (3) identifying the strain BJX007 as Streptomyces minor aureofaciens by integrating the morphological characteristics of the strain and the phylogenetic analysis result of the 16SrDNA gene sequence.
Example 2 application of the Strain BJX007 to fermentation production of kasugamycin
1. Preparing a seed solution: digging 1cm from slant culture medium of strain BJX0072Inoculating a bacterium-carrying culture medium into a 250mL seed bottle filled with a fermentation culture medium, wherein the liquid loading amount is 30mL, performing shake culture at 28 ℃ and 220r/min for 48h, inoculating the obtained bacterium liquid into a fermentation bottle filled with the fermentation culture medium according to the mass percentage of 10%, wherein the volume of the fermentation bottle is 250mL, the liquid loading amount is 35mL, and performing shake culture at 28 ℃ and 220r/min for 24h to obtain the seed liquid.
2. Fermentation culture: inoculating the seed solution into a fermentation bottle filled with a fermentation culture medium according to the mass percentage of 10%, wherein the volume of the fermentation bottle is 250mL, the liquid loading amount is 35mL, performing shake culture on a shaking table at the temperature of 28 ℃ and the speed of 220r/min for 7d, after the fermentation is finished, the content of kasugamycin in the obtained fermentation liquid is up to 25000 mu g/mL, and then separating the kasugamycin from the fermentation liquid to obtain the kasugamycin.
Wherein the formula of the fermentation medium is as follows: 20g/L of corn starch, 45g/L of bean cake powder, 2.5g/L of sodium chloride, 0.2g/L of monopotassium phosphate, 12mL/L of soybean oil, 0.1g/L of amylase and natural pH value. And (3) sterilizing conditions of the culture medium: sterilizing at 121 deg.C for 30 min.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (4)
1. Streptomyces microaureus (BJX 007) with the preservation number of CGMCC No. 11622.
2. The use of Streptomyces aureofaciens BJX007 as claimed in claim 1 for the fermentative production of kasugamycin.
3. Use according to claim 2, characterized in that it comprises the following steps:
s1, preparing a seed solution: digging 1cm from slant culture medium of strain BJX0072Inoculating a bacterium-carrying culture medium into a 250mL seed bottle filled with a fermentation culture medium, wherein the liquid loading amount is 30mL, performing shake culture at 28 ℃ and 220r/min for 48h, inoculating the obtained bacterium liquid into a fermentation bottle filled with the fermentation culture medium according to the mass percentage of 10%, wherein the volume of the fermentation bottle is 250mL, the liquid loading amount is 35mL, and performing shake culture at 28 ℃ and 220r/min for 24h to obtain a seed liquid;
s2, fermentation culture: inoculating the seed liquid into a fermentation bottle filled with a fermentation culture medium according to the mass percentage of 10%, wherein the volume of the fermentation bottle is 250mL, the liquid loading amount is 35mL, performing shake culture at the temperature of 28 ℃ of 220r/min for 165-170h, and separating the kasugamycin from the fermentation liquid after the fermentation is finished;
wherein the formula of the fermentation medium is as follows: 15-20g/L of corn starch, 45-50g/L of bean cake powder, 2.5g-3/L of sodium chloride, 0.2g-0.5/L of monopotassium phosphate, 10-12mL/L of soybean oil, 0.05-0.1g/L of amylase and natural pH value; the enzyme activity of the amylase is 10000U/g.
4. The use according to claim 3, wherein the fermentation medium is formulated as follows: 20g/L of corn starch, 45g/L of bean cake powder, 2.5g/L of sodium chloride, 0.2g/L of monopotassium phosphate, 12mL/L of soybean oil and 0.1g/L of amylase.
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