CN106148220B - A kind of method preparing Nahsi peptide and its production bacterial strain - Google Patents
A kind of method preparing Nahsi peptide and its production bacterial strain Download PDFInfo
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- CN106148220B CN106148220B CN201510204706.4A CN201510204706A CN106148220B CN 106148220 B CN106148220 B CN 106148220B CN 201510204706 A CN201510204706 A CN 201510204706A CN 106148220 B CN106148220 B CN 106148220B
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Abstract
The present invention provides one plant of S.actuosus (streptomyces actuosus) BJX004, and deposit number is CGMCC NO.8732.The ability that the strain fermentation produces Nahsi peptide is high, can achieve 5000 μ g/mL fermentation liquids, and bacterial strain its ability for producing Nahsi peptide after continuous passage 5 times still keeps previous level, shows that the bacterial strain genetic stability is good.Using the method for bacterial strain BJX004 fermentation preparation Nahsi peptide, it can be improved the production efficiency of Nahsi peptide, and method is simple and easy, low in cost, be suitable for the large-scale industrial production of Nahsi peptide.
Description
Technical field
The present invention relates to field of microbial fermentation, specifically, being related to a kind of method for preparing Nahsi peptide and its production bacterium
Strain.
Background technique
Nahsi peptide be it is a kind of containing multiple thiazole rings and be rich in the peptide antibiotics of element sulphur, be a kind of novel non-absorbing
Property animal feed additive, be suitable for inhibiting the growth of gram-positive bacterium in enteron aisle, when intestinal canal administration, it has dosage
It is low, scope of restraining fungi is wide, the features such as not remaining in animal body, the growth of the animals such as chicken, pig, fish can be obviously promoted.
The mechanism of action of Nahsi peptide are as follows: Nahsi peptide (multhiomycin) can inhibit the synthesis of protein in vivo and in vitro,
Synthesis without inhibiting DNA and RNA, this effect is similar to tetracycline, is by forming with 23rRNA and ribosomal protein L ll
Complex combine closely, to inhibit the hydrolysis of the activity of elongation factor Tu and G, GTP, the final synthesis for inhibiting protein,
It is suppressed the growth of bacterium.
Since research of the country to Nahsi peptide is also immature, production level far lags behind developed country, therefore to Nahsi peptide
Production bacterial strain studied, have important economic value and vast market prospect.
Summary of the invention
The object of the present invention is to provide a kind of methods of fermentation preparation Nahsi peptide.
It is a further object of the present invention to provide the production bacterial strains for the preparation Nahsi peptide that ferments.
In order to achieve the object of the present invention, the present invention provides one plant of S.actuosus (streptomyces actuosus)
BJX004 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC), address north
The institute 3 of the Chaoyang District Jing Shi North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode 100101, deposit number CGMCC
NO.8732, preservation date on January 17th, 2014.
S.actuosus BJX004 can produce Nahsi peptide in the fermentation medium, and it is raw without spiral that aerial hyphae belongs to single-wheel
Shape, end form growing straight spore chain, and spore is cylindrical or elliptical cylinder-shape, size (0.6-0.8 μm) × (1.0-1.5 μm), table
Face is smooth, and aerial hyphae is light gray to yellowish grey, and the color of substrate mycelium and aerial hyphae is without significant difference (yellow ash to Huang
Brown), a small amount of soluble pigment (yellow ash to yellowish-brown) is generated in synthetic media or some organic culture mediums；In tyrosine
Furvous pigment is generated in agar medium.
Growthform of the S.actuosus BJX004 in different culture medium is as follows:
Glucose asparagus fern element agar:, cortex cinnamomi brown cotton-shaped for gas silk, light yellow to the shallow carmetta of base silk.
Glycerol asparagus fern element agar (ISP), inorganic salts starch agar (ISP): gas silk is canescence.Base silk reverse side yellow or green
Yellow: adding 0.05N HCl, becomes orange from yellow, can lysochrome yellow or yellowish, add 0.05N HCl, from yellow become orange
Color.
Starch agar: base silk is light yellow.
Calcium malate agar: base silk is light yellow to isabelline.Solvable pigment is isabelline.
Yeastex malt extract agar (ISP), oatmeal agar (ISP): gas silk ash color system, base silk reverse side yellow or greenish-yellow
Color, can lysochrome it is yellowish, uranidin meet acid become orange.
S.actuosus BJX004 gelatin liquefaction is limited, dun pigment.Milk does not solidify, and peptonizes weak.Starch Hydrolysis is limited.
The present invention also provides a kind of methods using S.actuosus BJX004 preparation Nahsi peptide, comprising the following steps:
1) fermented and cultured: the seed liquor of the bacterial strain is inoculated in fermented and cultured in fermentation medium and obtains fermentation liquid；
2) Nahsi peptide is extracted from fermentation liquid.
Wherein, the condition of fermented and cultured are as follows: 35-38 DEG C, 200-230rpm (radius of turn 50mm), shaken cultivation 65-
75 hours, preferably 37 DEG C, 220rpm, shaken cultivation 72 hours.The relative humidity for keeping yeasting is 50-60%.
The medium component that fermented and cultured uses are as follows: KNO30.5-1g/L、(NH4)2S040.8-1.5g/L、NaCl 3-5g/
L, soybean powder 35-45g/L, yeast powder 1-2.5g/L, glucose 5-6g/L, amylase 0.01-0.02g/L, starch 40-50g/L
With precipitated calcium carbonate 3.5-5g/L, prepared with water (preferably distilled water).115 DEG C of sterilizing 25min.
Medium component is preferred are as follows: KNO30.8g/L、(NH4)2S041g/L, NaCl 4g/L, soybean powder 40g/L, yeast powder
2g/L, glucose 5.5g/L, amylase 0.015g/L, starch 48g/L and precipitated calcium carbonate 4.5g/L.
The inoculum concentration of seed liquor is preferably 5-10% (percent by volume) in fermentation process, and the seed liquor is to cultivate to right
The seed liquor in number growth period.
Be used to prepare the medium component of seed liquor are as follows: soybean cake powder 20g/L, glucose 50g/L, corn pulp 10g/L,
MgSO43g/L、CaCO34g/L, pH6.8, tap water are prepared.
The condition of culture of seed liquor are as follows: 35 DEG C, envionmental humidity 50-60%, revolving speed 180-200rpm (radius of turn
50mm), it cultivates 30 hours.
The present invention also provides the specific primer pair for PCR detection S.actuosus BJX004, the primer pairs are as follows:
Forward primer: 5 '-AGAGTTTGATCCTGGCTCAG-3 '
Reverse primer: 5 '-AAGTCGTAACAAGGTAGCCGTA-3 '.
The ability of S.actuosus BJX004 fermenting and producing Nahsi provided by the invention peptide is high, can achieve 5000 μ g/mL
Fermentation liquid, bacterial strain its ability for producing Nahsi peptide after continuous passage 5 times still keep previous level, show the bacterial strain inheritance stability
Property is good.Using the method for bacterial strain BJX004 fermentation preparation Nahsi peptide, the production efficiency of Nahsi peptide can be improved, and method is simply easy
It is capable, low in cost, it is suitable for the large-scale industrial production of Nahsi peptide.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
The acquisition of 1 bacterial strain of embodiment
The high-throughput prescreening method used in the present embodiment is as follows:
Fermentation stage: 96 hole deep-well plates are used, fill culture medium 0.3mL in every hole.Strain, 35 DEG C of stationary cultures are accessed in every hole
7 days.Slant medium composition: soluble starch 15g/L, beef extract 3g/L, albumen freeze 4g/L, K2HPO40.4g/L, NaCl
0.4g/L, MgSO41g/L, KNO31g/L, agar powder 15g/L, pH7.0, distilled water are prepared.
The extraction stage: filtering is detected after obtaining filtrate.
The measurement stage: spectrophotometry is used.Determination step is as follows:
1) drafting of standard curve
Precision weighs Nahsi peptide mark product 5mg, and 0.lmL DMF is added, is prepared into lmg/mL's with boric acid mixed liquor after dissolution
Titer, accurate measuring Nahsi peptide 100-800 μ g (0.1-0.8mL titer) is mended in 8 test tubes with boric acid mixed liquor respectively
It is charged to 2mL, every pipe adds 10mL borate buffer and 8mL water-saturated n-butanol, shakes up, and 37 DEG C of water-baths stand 20min, takes
Clear liquid 4mL adds dehydrated alcohol 4mL, the colorimetric at wavelength 408nm, reads OD value, draws standard curve, and acquire curvilinear equation.
2) measurement of fermentation liquid potency
It taking fermentation liquid 5mL in test tube, 5mL dehydrated alcohol is added, jumps a queue and shakes up, 37 DEG C of water-baths keep the temperature 45min, it filters,
Filtrate 2mL is taken, 10mL borate buffer and 8mL water-saturated n-butanol solution is added, sufficiently shakes up, 37 DEG C of standing 20min take
Clear liquid 4mL adds dehydrated alcohol 4mL, the colorimetric at wavelength 408nm, read OD value, by OD value substitute into calibration curve equation to get
Calculated result, unit are μ g/ml.
1, the isolated strains from soil
Mudanjiang City, Heilongjiang Province Jingpo Lake soil nearby is acquired, suspension is made in soil, Soil Slurry is inoculated with
It is cultivated in isolation medium, picking single colonie is diluted scribing line and isolates and purifies, and obtains pure culture bacterial strain.
Isolation medium is by soybean cake powder 20g/L, glucose 50g/L, corn pulp 10g/L, MgSO43g/L, CaCO34g/L,
PH6.8, tap water are prepared.
Primary dcreening operation: the pure culture bacterial strain of acquisition is carried out shake flask fermentation culture and is surveyed after fermentation using spectrophotometry
Fixed, the bacterial strain that will generate Nahsi peptide carries out following serial mutagenesis respectively.
2, mutagenesis
(1) first round ultraviolet mutagenesis
Bacteria suspension 10mL is taken, is added in sterile petri dish, is irradiated with the ultraviolet irradiation device of 30W in 30cm eminence.If
Determining irradiation time is respectively 50s, 80s.It prepares isolation medium to be cultivated, after picking single colonie, 35 DEG C of cultures are imitated for 7 days
The superior strain of valence measurement, acquisition carries out next step mutagenesis.
(2) second wheel ultraviolet mutagenesis
Ultraviolet mutagenesis is carried out again using same method to the superior strain that first round ultraviolet mutagenesis obtains.It obtains
Superior strain continue chemical mutagenesis.
(3) 6-chloropurine mutagenesis
By bacterial strain inclined plane inoculating in the starvation media of no organic nitrogen source, overnight incubation, by strain inoculated in containing not
With in the 6-chloropurine plate of concentration, the final concentration of 6-chloropurine is respectively in the plate of 50 μ g/mL, 100 μ g/mL.35 DEG C of trainings
It supports 7 days.Picking single colonie culture, carries out titration, and the superior strain of acquisition continues mutagenesis.
(4) ethylmethane sulfonate (EMS) mutagenesis
With 0.2mol/L, the phosphate buffer of pH6.8 prepares spore suspension, be added EMS that concentration is 0.5mg/mL in
After 35 DEG C of oscillation 30min, with brine spore 3 times, 35 DEG C of coated plate picking single colonie are cultivated 7 days, and picking single bacterium is dropped into
Row high throughput primary dcreening operation, shaking flask retrial, screening obtain Nikemycin superior strain.
(5) NTG mutagenesis
Appropriate NTG concentrated solution is added in the monospore suspension of certain volume, its activity is made to reach 1.0mg/mL,
After a certain period of time, spore is collected by centrifugation, and will embrace son again after twice with brine and suspend in 30 DEG C of constant temperature shaking processing
In a small amount of physiological saline, dilution apply 28 DEG C of plate cultivate 4-5 days (used vessel with the NaOH of 1M immersion 1 day or more or
Massive laundering).
6) Co60 irradiates
Mutagenic treatment is carried out to spore suspension using the ray that Co60 is generated, exposure dose is by irradiation distance and irradiation time
It is controlled.
It is final to obtain Nahsi peptide high-yield strains BJX004 by above-mentioned series methods.
3, the identification of bacterial strain
Nahsi peptide can be produced in the fermentation medium by producing Nahsi peptide bacterial strain BJX004, and it is raw without spiral that aerial hyphae belongs to single-wheel
Shape, end form growing straight spore chain, and spore is cylindrical or elliptical cylinder-shape, size (0.6-0.8 μm) × (1.0-1.5 μm), table
Face is smooth, and aerial hyphae is light gray to yellowish grey, and the color of substrate mycelium and aerial hyphae is without significant difference (yellow ash to Huang
Brown), a small amount of soluble pigment (yellow ash to yellowish-brown) is generated in synthetic media or some organic culture mediums；In tyrosine
Furvous pigment is generated in agar medium.
Specific primer pair used in 16S rDNA, PCR by the PCR amplification bacterium are as follows:
Forward primer: 5 '-AGAGTTTGATCCTGGCTCAG-3 ' (SEQ ID No.2)
Reverse primer: 5 '-AAGTCGTAACAAGGTAGCCGTA-3 ' (SEQ ID No.3)；
PCR product is subjected to detected through gel electrophoresis, stripe size 1084bp surveys purpose band after gel extraction
Sequence, sequence is as shown in SEQ ID No.1.
The above physiological and biochemical property and sequencing result show that the mutant strain obtained in the present embodiment is S.actuosus
(streptomyces actuosus), is named as BJX004.
The fermentation of embodiment 2 S.actuosus BJX004 produces Nahsi peptide
Fermentation medium I composition: KNO30.5g/L、(NH4)2S040.8g/L, NaCl 3g/L, soybean powder 35g/L, yeast
Powder 1g/L, glucose 5g/L, amylase 0.01g/L, starch 40g/L, precipitated calcium carbonate 3.5g/L, are prepared with distilled water.115℃
Sterilize 25min.
Fermentation medium II forms: KNO31g/L、(NH4)2S041.5g/L, NaCl 5g/L, soybean powder 45g/L, yeast powder
2.5g/L, glucose 6g/L, amylase 0.02g/L, starch 50g/L, precipitated calcium carbonate 5g/L, are prepared with distilled water.115 DEG C go out
Bacterium 25min.
Fermentation medium III forms: KNO30.8g/L、(NH4)2S041g/L, NaCl 4g/L, soybean powder 40g/L, yeast powder
2g/L, glucose 5.5g/L, amylase 0.015g/L, starch 48g/L, precipitated calcium carbonate 4.5g/L, are prepared with distilled water.115
DEG C sterilizing 25min.
1, Nahsi peptide is produced using the fermentation of fermentation medium I S.actuosus BJX004
(1) inclined-plane culture
S.actuosus BJX004 is inoculated on slant medium, is 50-60% condition in 35 DEG C, envionmental humidity
Lower culture 7-8 days.
Wherein, inclined-plane culture based formulas are as follows: soluble starch 15g/L, beef extract 3g/L, albumen knee 4g/L,
K2HPO40.4g/L、NaCL 0.4g/L、MgSO41g/L、KNO31g/L, agar powder 15g/L, pH7.0, distilled water are prepared.
(2) seed culture
Take inclined-plane lawn 1cm obtained in step (1)2, accessed the kind of the seed culture medium equipped with 30mL sterilizing
In sub- bottle, 30 hours: 35 DEG C, envionmental humidity 50-60%, revolving speed 180-200rpm, rotation are cultivated under the following conditions
Radius 50mm, obtains seed culture fluid.
Wherein, the formula of seed culture medium are as follows: soybean cake powder 20g/L, glucose 50g/L, corn pulp 10g/L,
MgSO43g/L、CaCO34g/L, pH6.8, tap water are prepared.
(3) fermented and cultured
Fermentation culture method: above-mentioned seed culture fluid is taken to be inoculated in by the inoculum concentration of 10% (percent by volume) equipped with 30mL
In the triangular flask of the fermentation medium I of sterilizing, 65 hours: 35 DEG C of fermented and cultured under the following conditions, envionmental humidity 50%,
Revolving speed 200rpm (radius of turn 50mm), obtains fermentation liquid.
(4) Nahsi peptide bioactivity
Experiment sets 4 repetitions, is as a result averaged.Show that fermentation liquid potency is 4980 μ g/mL.
2, Nahsi peptide is produced using the fermentation of fermentation medium II S.actuosus BJX004
(1) inclined-plane culture
Same above step (1)
(2) seed culture
Same above step (1)
(3) fermented and cultured
Fermentation culture method: above-mentioned seed culture fluid is taken to be inoculated in by the inoculum concentration of 5% (percent by volume) equipped with 30mL
In the bottle of the fermentation medium II of sterilizing, 65 hours: 38 DEG C of fermented and cultured, envionmental humidity 60% turn under the following conditions
Fast 200rpm (radius of turn 50mm), obtains fermentation liquid.
(4) Nahsi peptide bioactivity
Experiment sets 4 repetitions, is as a result averaged.Show that fermentation liquid potency is 5100 μ g/mL.
3, Nahsi peptide is produced using the fermentation of fermentation medium III S.actuosus BJX004
(1) inclined-plane culture
Same above step (1)
(2) seed culture
Same above step (1)
(3) fermented and cultured
Fermentation culture method: above-mentioned seed culture fluid is taken to be inoculated in by the inoculum concentration of 8% (percent by volume) equipped with 30mL
In the bottle of the fermentation medium III of sterilizing, 72 hours: 37 DEG C of fermented and cultured, envionmental humidity 55% turn under the following conditions
Fast 220rpm (radius of turn 50mm), obtains fermentation liquid.
(4) Nahsi peptide bioactivity
Experiment sets 4 repetitions, is as a result averaged.Show that fermentation liquid potency is 5040 μ g/mL.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (7)
- S.actuosus 1. (streptomyces actuosus) BJX004, deposit number is CGMCC NO.8732.
- 2. using the method for the preparation Nahsi peptide of bacterial strain described in claim 1, which comprises the following steps:1) fermented and cultured: the seed liquor of the bacterial strain is inoculated in fermented and cultured in fermentation medium and obtains fermentation liquid；2) Nahsi peptide is extracted from fermentation liquid.
- 3. according to the method described in claim 2, it is characterized in that, in step 1) fermented and cultured condition are as follows: 35-38 DEG C, 200-230rpm, shaken cultivation 65-75 hours.
- 4. according to the method described in claim 3, it is characterized in that, in step 1) fermented and cultured condition are as follows: 37 DEG C, 220rpm, shaken cultivation 72 hours.
- 5. according to the method described in claim 2, it is characterized in that, the medium component that fermented and cultured uses in step 1) are as follows: KNO3 0.5-1g/L、(NH4) 2S040.8-1.5g/L, NaCl 3-5g/L, soybean powder 35-45g/L, yeast powder 1-2.5g/L, Glucose 5-6g/L, amylase 0.01-0.02g/L, starch 40-50g/L and precipitated calcium carbonate 3.5-5 g/L, are prepared with water.
- 6. according to the method described in claim 5, it is characterized in that, the medium component are as follows: KNO3 0.8g/L、(NH4)2S04 1g/L, NaCl 4g/L, soybean powder 40g/L, yeast powder 2g/L, glucose 5.5g/L, amylase 0.015g/L, starch 48g/L and Precipitated calcium carbonate 4.5g/L.
- 7. according to the described in any item methods of claim 2-6, which is characterized in that the envionmental humidity to ferment in step 1) is 50-60%。
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| 诺西肽产生菌—活跃链霉菌的研究;马舒;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20071115(第05期);E079-15 * |
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