CN1397647A - Process for transforming antibacterial peptide AD gene by yeast fermentation - Google Patents
Process for transforming antibacterial peptide AD gene by yeast fermentation Download PDFInfo
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- CN1397647A CN1397647A CN 02134570 CN02134570A CN1397647A CN 1397647 A CN1397647 A CN 1397647A CN 02134570 CN02134570 CN 02134570 CN 02134570 A CN02134570 A CN 02134570A CN 1397647 A CN1397647 A CN 1397647A
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Abstract
A process for preparing the AD gene transformed antibacterial peptide yeast by fermentation features that the antibacterial peptide contained engineering yeast obtained by transforming AD gene plasmid is fermented in proper culture medium and culture condition to express the antibacterial peptide gene and secrete the antibacterial peptide outside the thallus. The sterilizing activity of antibacterial peptide in the fermented liquid can reach 7000 unit/ml. Its advantages are high E/C ratio, and low cost.
Description
Technical field
The invention belongs to the biological fermentation field, more specifically say so about a kind of process for transforming antibacterial peptide AD gene by yeast fermentation.
Background technology
The antibacterial peptide AD gene is according to the aminoacid sequence design of tussah natural antibacterial peptide A and antibacterial peptide D gene and the gene of synthetic.Because the sterilization mechanism uniqueness of antibacterial peptide, pathogenic bacteria are difficult for it is developed immunity to drugs, and has a broad antifungal spectrum, therefore, antibacterial peptide is as a kind of novel environment friendly antiseptic feed additive, and is significant to the Sustainable development of livestock industry.And so far, the domestic also fermentation method for producing of this genetic engineering bacterium not.
Summary of the invention
The invention provides a kind of process for transforming antibacterial peptide AD gene by yeast fermentation.To contain gene engineering yeast that the antibacterial peptide AD gene plasmid transforms under suitable condition successful expression antibacterial peptide gene justacrine antibacterial peptide outside thalline.The technology of method mainly comprises shake-flask culture, fermentor cultivation, and specific embodiment is as follows:
(1) shake-flask culture: the yeast that will contain the conversion of antibacterial peptide AD gene plasmid inserts in the triangular flask as bacterial classification, at 28-32 ℃, under shaking speed 150-300 rev/min the condition, cultivates 24-72 hour;
(2) fermentor cultivation: with in (1) cultured seed insert fermentor tank, inoculum size is 2-20%, at 28-32 ℃, stirring velocity 180-400 rev/min, tank pressure 0.04-0.12 MPa, air quantity 1: 0.5-1: 1.8 volume/volume/minute, under the condition of pH 6.5-7.5, cultivated 24-96 hour.The gained antibacterial peptide that will ferment heated 8-20 minute down at 90-110 ℃.
Wherein used nutrient media components and weight percentage are: yeast powder 0.1-3%, peptone 0.5-4%, sugared 0.1-3%, pH 6.5-7.5.
When fermentative production bacterial classification consumption surpassed 5 liters, the technology of method mainly comprised shake-flask culture, seed tank culture, fermentor cultivation, and specific embodiment is as follows:
(1) shake-flask culture: the yeast that will contain the conversion of antibacterial peptide AD gene plasmid inserts in the triangular flask as bacterial classification, at 28-32 ℃, under shaking speed 150-300 rev/min the condition, cultivates 24-72 hour;
(2) seed tank culture: the shake-flask seed that shakes in (1) is inserted in the seeding tank, and inoculum size 1-10% is at 28-32 ℃, stirring velocity 200-600 rev/min, tank pressure 0.03-0.12 MPa, air quantity 1: 0.5-1: 2 volume/volume/minute condition under, cultivated 24-72 hour;
(3) fermentor cultivation: with in (2) cultured seed insert fermentor tank, inoculum size is 2-20%, at 28-32 ℃, stirring velocity 180-400 rev/min, tank pressure 0.04-0.12 MPa, air quantity 1: 0.5-1: 1.8 volume/volume/minute, under the condition of pH 6.5-7.5, cultivated 24-96 hour.The gained antibacterial peptide that will ferment heated 8-20 minute down at 90-110 ℃.
Wherein used nutrient media components and weight percentage are: yeast powder 0.1-3%, peptone 0.5-4%, sugared 0.1-3%, pH 6.5-7.5.
The engineering yeast that contains the conversion of antibacterial peptide AD gene plasmid that the present invention is used can obtain with existing genetic engineering means.
The used nutrient media components and the preferred version of weight percentage are among the present invention: yeast powder 0.5-2%, peptone 1-3%, glucose 0.5-2%, pH 6.5-7.5.
The antibacterial peptide yeast preparation sterilization that utilizes this fermentation culture method the to produce height of tiring, steady quality can be mass-produced, and is with short production cycle and cost is low.
Embodiment
Below in conjunction with embodiment method of the present invention is further described.
The production of the antibacterial peptide of 1 50 liters of jars of embodiment
1. shake-flask culture
50 liters of fermentor tanks, 30 liters of loading amounts, establishing inoculum size is 5%, the amount of then joining the shake-flask culture base is 30 a liters * 5%=1.5 liter, presses yeast powder 0.8%, peptone 2%, glucose 1%, the prescription of pH 6.8, join 1.5 liters of substratum, be divided in 500 milliliters of triangular flasks, every bottled amount is 100 milliliters, install the back sterilization, be cooled to 30 ℃ and insert bacterial classification (about 3 milliliters/bottle), shaking speed is 180 rev/mins, and controlled temperature is 30 ℃, after cultivating in 36 hours, insert 50 liters of fermentor cultivation.
2. fermentor cultivation
50 liters of actual loading amounts of jar are 30 liters, deduct 1.5 liters of shake-flask seeds, then real dosage is the 30-1.5=28.5 liter, the cultivation of joining 28.5 liters by present embodiment step 1 is based on (actual installation of water is 26.5 liters in 50 liters of jars, produce about 3 liters steam condensate during because of sterilization), sterilization when treating that temperature drops to 30 ℃ of left and right sides, inserts cultured shake-flask seed, the control culture temperature is 30 ℃, rotating speed is 380 rev/mins, tank pressure 0.1 MPa, air quantity be 1: 1.1 volume/volume/minute, cultivate after 24 hours, add 1% methyl alcohol (promptly 300 milliliters) and induce, cultivate after 48 hours, can produce antibacterial peptide, fermented liquid is through 95 ℃, after the deactivation in 15 minutes, be the antibacterial peptide finished product, fermentation titer is 7100 units per ml.
The production of the antibacterial peptide of 2 500 liters of jars of embodiment
1, shake-flask culture
50 liters of fermentor tanks, 30 liters of loading amounts, establishing inoculum size is 10%, the amount of then joining the shake-flask culture base is 30 a liters * 10%=3 liter, presses yeast powder 1%; Peptone 1.5%; Glucose 1.5%; The prescription of pH 7 is joined 3 liters of substratum, is divided in 500 milliliters of triangular flasks, and every bottled amount is 100 milliliters, install the back sterilization, be cooled to 28 ℃ and insert bacterial classification (about 3 milliliters/bottle), shaking speed is 180 rev/mins, controlled temperature is 28 ℃, after cultivating in 36 hours, inserts 50 liters of seed tank culture.
2, seed tank culture
50 liters of actual loading amounts of jar are 30 liters, deduct 3 liters of shake-flask seeds, then real dosage is the 30-3=27 liter, the cultivation of joining 27 liters by the prescription of present embodiment step 1 is based on (actual installation of water is 24 liters in 50 liters of jars, produce about 3 liters steam condensate during because of sterilization), sterilization, when treating that temperature drops to 30 ℃ of left and right sides, insert cultured shake-flask seed, the control culture temperature is 30 ℃, and rotating speed is 400 rev/mins, tank pressure 0.1 MPa, air quantity be 1: 1.5 volume/volume/minute, cultivate just can insert in the fermentor tank after 36 hours and cultivate.
3, fermentor cultivation
500 liters of actual loading amounts of jar are 300 liters, deduct 30 liters of seeding tank kind amounts (the kind amount is 10%), then real dosage is the 300-30=270 liter, the cultivation of joining 270 liters by present embodiment step 1 is based on (actual installation of water is 260 liters in 500 liters of jars, produce about 10 liters steam condensate during because of sterilization), sterilization, when treating that temperature drops to 30 ℃ of left and right sides, insert cultured seed jar seed, the control culture temperature is 30 ℃, rotating speed is 300 rev/mins, tank pressure 0.1 MPa, air quantity be 1: 1.4 volume/volume/minute, cultivate after 36 hours, adding 1% methyl alcohol (promptly 3000 milliliters) induces, cultivate after 48 hours, can produce antibacterial peptide, fermented liquid is through 95 ℃, after the deactivation in 15 minutes, be the antibacterial peptide finished product.Fermentation titer is 7000 units per ml.
Claims (4)
1, a kind of process for transforming antibacterial peptide AD gene by yeast fermentation comprises shake-flask culture, fermentor cultivation, it is characterized in that:
(1) shake-flask culture: the yeast that will contain the conversion of antibacterial peptide AD gene plasmid inserts in the triangular flask as bacterial classification, at 28-32 ℃, under shaking speed 150-300 rev/min the condition, cultivates 24-72 hour;
(2) fermentor cultivation: with in (1) cultured seed insert fermentor tank, inoculum size is 2-20%, at 28-32 ℃, stirring velocity 180-400 rev/min, tank pressure 0.04-0.12 MPa, air quantity 1: 0.5-1: 1.8 volume/volume/minute, under the condition of pH6.5-7.5, cultivated 24-96 hour.The gained antibacterial peptide that will ferment heated 8-20 minute down at 90-110 ℃;
Wherein used nutrient media components and weight percentage are: yeast powder 0.1-3%, peptone 0.5-4%, sugared 0.1-3%, pH6.5-7.5.
2, process for transforming antibacterial peptide AD gene by yeast fermentation according to claim 1 when the bacterial classification consumption surpasses 5 liters when it is characterized in that fermentative production, carries out seed tank culture behind shake-flask culture He before the fermentor cultivation.
3, process for transforming antibacterial peptide AD gene by yeast fermentation according to claim 2, it is characterized in that described seed tank culture, be that the shake-flask seed that will shake inserts in the seeding tank, inoculum size 1-10%, at 28-32 ℃, stirring velocity 200-600 rev/min, tank pressure 0.03-0.12 MPa, air quantity 1: 0.5-1: 2 volume/volume/minute condition under, cultivated 24-72 hour.
4,, it is characterized in that used nutrient media components and weight percentage are: yeast powder 0.5-2%, peptone 1-3%, glucose 0.5-2%, pH 6.5-7.5 according to claim 1,2 or 3 described process for transforming antibacterial peptide AD gene by yeast fermentation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021345708A CN1173030C (en) | 2002-08-16 | 2002-08-16 | Process for transforming antibacterial peptide AD gene by yeast fermentation |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021345708A CN1173030C (en) | 2002-08-16 | 2002-08-16 | Process for transforming antibacterial peptide AD gene by yeast fermentation |
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| CN1397647A true CN1397647A (en) | 2003-02-19 |
| CN1173030C CN1173030C (en) | 2004-10-27 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1309824C (en) * | 2004-08-27 | 2007-04-11 | 广州维观生物科技有限公司 | Antibiotic peptide gene converted viscons bacillus engineering bacterium and its preparing method and use |
| CN101381757B (en) * | 2008-03-27 | 2012-03-21 | 深圳市圣西马生物技术有限公司 | Solid fermentation preparation method of antibacterial peptide and application |
| CN104877924A (en) * | 2014-02-28 | 2015-09-02 | 雨润慕德生物科技（连云港）有限公司 | Fermentation method of interferon producing saccharomyces cerevisiae hansen strain at seed tank level |
-
2002
- 2002-08-16 CN CNB021345708A patent/CN1173030C/en not_active Expired - Lifetime
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1309824C (en) * | 2004-08-27 | 2007-04-11 | 广州维观生物科技有限公司 | Antibiotic peptide gene converted viscons bacillus engineering bacterium and its preparing method and use |
| CN101381757B (en) * | 2008-03-27 | 2012-03-21 | 深圳市圣西马生物技术有限公司 | Solid fermentation preparation method of antibacterial peptide and application |
| CN104877924A (en) * | 2014-02-28 | 2015-09-02 | 雨润慕德生物科技（连云港）有限公司 | Fermentation method of interferon producing saccharomyces cerevisiae hansen strain at seed tank level |
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| Publication number | Publication date |
|---|---|
| CN1173030C (en) | 2004-10-27 |
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Granted publication date: 20041027 |