CN103981114A - L-phenylalanine high-yield bacterial strain and applications thereof in fermentation production of L-phenylalanine - Google Patents
L-phenylalanine high-yield bacterial strain and applications thereof in fermentation production of L-phenylalanine Download PDFInfo
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Abstract
The invention discloses a L-phenylalanine high-yield bacterial strain, an induced mutation method thereof, and applications of the L-phenylalanine high-yield bacterial strain in fermentation production of L-phenylalanine. The L-phenylalanine high-yield bacterial strain is named as C. glutamicum BT-N508, and is preserved in China Center for Type Culture Collection (CCTCC); preservation number is CCTCC No.M2013484. According to the induced mutation method, nitrosoguanidine (NMMG) is injected for induced mutation; a bacterial strain with high L-phenylalanine high-yield is selected as an original strain of a next cycle of induced mutation via shake flask fermentation; and at last the target bacterial strain C. glutamicum BT-N508 is obtained via selection. The C. glutamicum BT-N508 is capable of increasing L-phenylalanine in fermentation products greatly and reducing other ingredients, and possesses excellent hereditary stability. Corn flour is taken as a carbon source by the L-phenylalanine high-yield bacterial strain; L-phenylalanine fermentation yield can reach 56g/L in a 5L fermentation tank, and the L-phenylalanine fermentation yield is increased 27.9% compared with an original strain BT-008; the L-phenylalanine high-yield bacterial strain can be used for industrialized production, is capable of increasing fermentation titer unit greatly, and possesses significant economic application values.
Description
Technical field
The present invention relates to microorganism fermentation field, be specifically related to a kind of L-Phe superior strain and mutafacient system thereof and its application in fermentative production L-Phe.
Background technology
L-Phe is the die aromatischen Aminosaeuren with physiologically active, is one of human body and animal necessary amino acid that can not synthesize, and is also the important component part of high sugariness novel sweetener aspartame low in calories.As a kind of important biochemical product, L-Phe has a wide range of applications in food, fodder additives and medicine and other fields.
The preparation method of L-Phe mainly contains microbe fermentation method and microbial enzyme method forwards method.Microbe fermentation method is to have self amino acid needed ability by biology, by processing such as the mutagenesis to bacterial strain, select various auxotroph and Amino acid analogue resistance dissociant, to remove the feedback inhibition in metabolism adjusting and to check factor, reach the object of excess accumulation L-Phe.For example: the long victory of Fudan University model waits from utilizing, UV-LiCl (UV-LiCl) and ultraviolet ray-nitrosoguanidine (UV-NTG) carry out multiple mutated to it and obtained the relative starting strain content of a strain and improve the superior strain of 50% left and right.The Monsanto company of the U.S. relies on makes its fermentation produce L-Phe to the improvement of bacterial classification, and acid production rate is up to 45g/L.But domestic to prepare aspect research work for phenylalanine relatively backward.It is the genetic engineering bacterium of carrier that existing fermentation process adopts intestinal bacteria mostly, and complicated operation and bacterial classification are easily degenerated.In actual production process, cause producing unstable.
Summary of the invention
One of the technical problem to be solved in the present invention is to provide a strain can increase substantially L-Phe superior strain in tunning.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is as follows: a strain L-Phe superior strain, its Classification And Nomenclature is Corynebacterium glutamicum C.glutamicum BT-N5086, Chinese Typical Representative culture collection center C CTCC, deposit number: CCTCC NO.M2013484 have been preserved on October 21st, 2013.
It is as follows that the morphology of Corynebacterium glutamicum C.glutamicum BT-N5086 and Physiology and biochemistry are learned feature:
Colony colour: rice white.
Aerobic mode: aerobic growth.
Bacterium colony size: 3～6mm.
Suitable growth temperature: 31～33 DEG C.
Suitable growth pH:7.0～7.2.
Thalli morphology: circular bacterium colony, tarnish.
Gramstaining: the positive.
Two of the technical problem to be solved in the present invention is to provide a kind of simple, efficient, safe L-Phe Producing Strain mutafacient system, can obtain aforementioned L-Phe superior strain by mutagenesis screening.
The mutagenesis screening method of L-Phe superior strain Corynebacterium glutamicum C.glutamicum BT-N5086 of the present invention is: by L-Phe starting strain, after nitrosoguanidine (NMMG) mutagenesis, screen and obtain bacterial strain that L-Phe output the is high starting strain as next round mutagenesis by shake flask fermentation, repeat above-mentioned mutagenic processes, finally filter out the aimed strain that L-Phe output is high.Concrete steps are as follows:
(a), sample suspension liquid preparation: obtain cell suspension to be mutagenic from the Corynebacterium glutamicum C.glutamicum culture solid medium: Corynebacterium glutamicum starting strain is made to the acellular overlapping cell suspension of microscopy.
(b), in order to obtain phenylalanine superior strain, through the multiple mutafacient system of contrast, select the method for nitrosoguanidine (NMMG) mutagenesis Corynebacterium glutamicum C.glutamicum bacterial strain.Under 20 DEG C and 100rpm, by 10
8the cell suspension of cell/ml step (a) joins the 0.1mol/L sodium citrate buffer solution that contains 300～500mg/ml nitrosoguanidine MNNG solution, the lower temperature of environment at pH5.0 is bathed 60～120min, then by mutagenized cell liquid medium within, 26～30 DEG C, the lower cultivation of environment of 100rpm 10 hours, obtain the bacterium liquid after mutagenesis.
(c), the screening of mutagenic strain:
Screening: bacterium liquid after step (b) mutagenesis is coated on solid medium and is separated, then by the single bacterium colony access seed culture medium on solid medium, 31～33 DEG C of culture temperature, under 240～320rpm shaking speed, after enlarged culturing 12～24h, access fermention medium by the inoculum size of 5～15% (v/v).After 31～33 DEG C, 240～320rpm shaking speed bottom fermentation, 24～44h, lower shaking table, gets 3mL fermented liquid in centrifuge tube, and 3500rpm high speed centrifugation 10min, abandons supernatant liquor, adds methyl alcohol to 9mL, is shaking 30min fast on vortex mixer; 3500rpm high speed centrifugation 10min, gets supernatant liquor and measures L-Phe content in fermented liquid.Get bacterial strain that fermented liquid L-Phe content the is the highest starting strain as next mutagenesis, repeating step (a)～(c), until filter out the aimed strain Corynebacterium glutamicum C.glutamicum BT-N5086 that L-Phe output is high.
In step (b), the preferred mutagenesis dosage of nitrosoguanidine NMMG is 400mg/ml; Liquid nutrient medium used in step (b) is: carbon source 2.0%～4.0%, nitrogenous source 0.5%～1.5%, all the other are water, pH7.0～7.2; Wherein said carbon source is one or both the mixing in amylum hydrolysate of the sugar and molasses; Described nitrogenous source is one or both the mixing in extractum carnis and yeast powder.
Solid medium used in step (c) is: carbon source 2.0%～4.0%, nitrogenous source 0.5%～1.5%, all the other are water, pH7.0～7.2; Wherein said carbon source is one or both the mixing in amylum hydrolysate of the sugar and molasses; Described nitrogenous source is one or both the mixing in extractum carnis and yeast powder; Seed culture medium used in step (c) is: carbon source 3.0%～5.0%, nitrogenous source 1.0%～3.0%, inorganic salt 0.1%～0.4%, and all the other are water, pH7.0～7.2; Wherein said carbon source is one or both the mixing in amylum hydrolysate of the sugar and molasses; Described nitrogenous source is one or more the mixing in yeast powder, soybean cake powder; Described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts; Fermention medium used in step (c) is: carbon source 1.0%～3.0%, nitrogenous source 2.0%～6.0%, inorganic salt 0.1%～0.4%, trace element 0.0001%～0.0004%, and all the other are water, pH7.0～7.2; Wherein said carbon source is one or both the mixing in amylum hydrolysate of the sugar, molasses; Described nitrogenous source is one or more the mixing in urea, soybean cake powder; Described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts; Described trace element is VITMAIN B1.
Step (b) and (c) in solid medium used be: amylum hydrolysate of the sugar 2.0%, Fructus Hordei Germinatus soaks powder 1.0%, agar powder 1.5%, pH7.0～7.2, distilled water configuration.
Seed culture medium used in step (c) is: amylum hydrolysate of the sugar 3.0%, soybean cake powder 2.0%, potassium primary phosphate 0.2%, magnesium sulfate 0.05%, pH7.0～7.2, tap water configuration; Fermention medium used in step (c) is: amylum hydrolysate of the sugar 10%, soybean cake powder 2.0%, potassium primary phosphate 0.2%, magnesium sulfate 0.05%, vitaminB10 .0002%, pH7.0～7.2, tap water configuration.
Three of the technical problem to be solved in the present invention is to provide the application in fermentative production L-Phe of above-mentioned L-Phe superior strain.Concrete steps are as follows:
(1), solid culture: Corynebacterium glutamicum C.glutamicum BT-N5086 is inoculated on solid medium, and culture temperature is 31～33 DEG C, and incubation time is 24～44h;
(2), seed culture: the Corynebacterium glutamicum C.glutamicum BT-N5086 of step (1) solid culture is inoculated in seed culture medium, in 250mL culturing bottle, liquid amount is 20～50mL, culture temperature is 31～33 DEG C, under 240～320rpm shaking speed, cultivates 24～44h;
(3), fermentation culture: the seed culture fluid in step (2) is inoculated in fermention medium, inoculum size is 5～15% (v/v), in 500mL culturing bottle, liquid amount is 35～65mL, 31～33 DEG C is 31～33 DEG C, 240～320rpm shaking speed bottom fermentation, 24～44h in culture temperature.
Wherein, the component that described solid medium comprises following mass percent: carbon source 2.0%～4.0%, nitrogenous source 0.5%～1.5%, agar 1.2%～1.8%, all the other are water, pH7.0～7.2; Wherein said carbon source is one or both the mixing in amylum hydrolysate of the sugar and molasses; Described nitrogenous source is one or both the mixing in extractum carnis and yeast powder.
The component that described seed culture medium comprises following mass percent: carbon source 3.0%～5.0%, nitrogenous source 1.0%～3.0%, inorganic salt 0.1%～0.4%, all the other are water, pH7.0～7.2; Wherein said carbon source is one or both the mixing in amylum hydrolysate of the sugar and molasses; Described nitrogenous source is one or more the mixing in yeast powder, soybean cake powder; Described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts.
The component that described fermention medium comprises following mass percent: carbon source 1.0%～3.0%, nitrogenous source 2.0%～6.0%, inorganic salt 0.1%～0.4%, trace element 0.0001%～0.0004%, all the other are water, pH7.0～7.2; Wherein said carbon source is one or both the mixing in amylum hydrolysate of the sugar, molasses; Described nitrogenous source is one or more the mixing in urea, soybean cake powder; Described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts; Described trace element is VITMAIN B1.
Adopt after technique scheme, the present invention has following beneficial effect: (1)) the present invention adopts (NMMG) mutagenesis of nitrosoguanidine (NMMG), final screening obtains a strain L-Phe superior strain Corynebacterium glutamicum C.glutamicum BT-N5086, can improve by a relatively large margin in tunning L-Phe component and genetic stability good, after bacterial strain continuous passage 5 times, tire and there is no considerable change.
(2) successful in fermentative production L-Phe of L-Phe superior strain of the present invention, in 5L fermentor tank, taking cheap molasses as carbon source, fermentation is produced L-Phe output and is reached 56g/L, compares original starting strain and has improved 27.9%.Technology provided by the present invention meets industrialization L-Phe completely produces the mutagenesis of bacterial strain and screens needs, plays a role in energy saving, and reduces costs.And can be applicable to industrial fermentation production, there is great social effect and economic worth.
(3) the present invention adopts Corynebacterium glutamicum direct fermentation, has avoided using the uncertain factors such as the bacterial classification running in the middle of the genetic engineering bacterium productions such as intestinal bacteria is easily degenerated, and productive rate is unstable.Having great economy at suitability for industrialized production L-Phe is worth.
Brief description of the drawings
Fig. 1: be the positive and negative mutation rate of bacterial strain under different MNNG dosage, as can be seen from the figure 300mg/ml positive mutation rate is the highest.Can determine that 300mg/ml is the best mutagenesis dosage of bacterial classification, easily filters out the increase rate of tiring and exceedes 10% bacterial strain;
L-Phe color atlas in Fig. 2: embodiment 3 fermented liquids.
Embodiment
According to following embodiment, the present invention may be better understood.But, those skilled in the art will readily understand, the described concrete material proportion of embodiment, processing condition and result thereof be only for the present invention is described, and should also can not limit the present invention described in detail in claims.
(embodiment 1):
The method that Corynebacterium glutamicum starting strain is carried out mutagenesis by the present embodiment explanation.
Embodiment 1: the method that Corynebacterium glutamicum starting strain is carried out mutagenesis by the present embodiment explanation.
The Corynebacterium glutamicum BT-008 preserving taking laboratory, as starting strain, carries out mutagenesis, and concrete steps are as follows:
(a), bacterium suspension preparation: Corynebacterium glutamicum starting strain is made to bacteria suspension, and it is overlapping that microscopy is acellular.
(b), in order to obtain phenylalanine superior strain, through the multiple mutafacient system of contrast, select the method for nitrosoguanidine (NMMG) mutagenesis Corynebacterium glutamicum C.glutamicum bacterial strain.Under 20 DEG C and 100rpm, by 10
8the cell suspension of cell/ml step (a) joins the 0.1mol/L sodium citrate buffer solution that contains 300～500mg/ml nitrosoguanidine MNNG solution, the lower temperature of environment at pH5.0 is bathed 60～120min, then by mutagenized cell liquid medium within, 26～30 DEG C, the lower cultivation of environment of 100rpm 10 hours, obtain the bacterium liquid after mutagenesis.
(c), the screening of mutagenic strain:
Screening: bacterium liquid after step (b) mutagenesis is coated on solid medium and is separated, then by the single bacterium colony access seed culture medium on solid medium, 31～33 DEG C of culture temperature, under 240～320rpm shaking speed, after enlarged culturing 12～24h, access fermention medium by the inoculum size of 5～15% (v/v).After 31～33 DEG C, 240～320rpm shaking speed bottom fermentation, 24～44h, lower shaking table, gets 3mL fermented liquid in centrifuge tube, and 3500rpm high speed centrifugation 10min, abandons supernatant liquor, adds methyl alcohol to 9mL, is shaking 30min fast on vortex mixer; 3500rpm high speed centrifugation 10min, gets supernatant liquor and measures L-Phe content in fermented liquid.Get bacterial strain that fermented liquid L-Phe content the is the highest starting strain as next mutagenesis, repeating step (a)～(c), until filter out the aimed strain Corynebacterium glutamicum C.glutamicum BT-N5086 that L-Phe output is high.
Solid medium used in step (b) is: amylum hydrolysate of the sugar 2.0%, Fructus Hordei Germinatus soaks powder 1.0%, agar powder 1.5%, pH7.0～7.2, distilled water configuration.
Seed culture medium used in step (c) is: amylum hydrolysate of the sugar 3.0%, soybean cake powder 2.0%, potassium primary phosphate 0.2%, magnesium sulfate 0.05%, pH7.0～7.2, tap water configuration; Fermention medium used in step (c) is: amylum hydrolysate of the sugar 10%, soybean cake powder 2.0%, potassium primary phosphate 0.2%, magnesium sulfate 0.05%, vitaminB10 .0002%, pH7.0～7.2, tap water configuration.
(embodiment 2):
The mitotic stability of the present embodiment explanation mutant strain.
In the fermention medium taking Semen Maydis powder as carbon source, detect the mitotic stability of mutant strain Corynebacterium glutamicum C.glutamicum BT-N5086, strain passage fermentation test result is as shown in table 1:
Table 1 enhanced variant Corynebacterium glutamicum C.glutamicum BT-N5086 stability test
Can find out from experimental result, go down to posterity through 5 times, the L-Phe output of mutant strain Corynebacterium glutamicum C.glutamicum BT-N5086 is more stable, has good mitotic stability, can be used as the production bacterial strain of further research and development.
(embodiment 3):
The technique of the present embodiment explanation superior strain Corynebacterium glutamicum C.glutamicum BT-N5086 fermentative production L-Phe.
The culture medium prescription that the present embodiment is used:
Solid medium is: glucose 2.0%, Fructus Hordei Germinatus soaks powder 1.0%, agar powder 1.5%, pH7.0～7.2, distilled water configuration.
Seed culture medium is: amylum hydrolysate of the sugar 3.0%, soybean cake powder 2.0%, potassium primary phosphate 0.2%, magnesium sulfate 0.05%, pH7.0～7.2, tap water configuration.
Fermention medium is: amylum hydrolysate of the sugar 10%, soybean cake powder 2.0%, potassium primary phosphate 0.2%, magnesium sulfate 0.05%, vitaminB10 .0002%, pH7.0～7.2, tap water configuration.
Corynebacterium glutamicum C.glutamicum BT-N5086 is inoculated on solid medium, and culture temperature is 31 DEG C, and incubation time is 24h; The Corynebacterium glutamicum C.glutamicum BT-N5086 culture of solid culture is seeded in seed culture medium, and in 250mL culturing bottle, liquid amount is 30mL, and culture temperature is 31 DEG C, under 280rpm rotating speed, cultivates 24h; Seed culture fluid is inoculated in fermention medium, and initial pH is 7.2, and inoculum size is 10% (v/v), and in 500mL culturing bottle, liquid amount is 50mL, and culture temperature is 31 DEG C, 220rpm shaking speed bottom fermentation 44h.It is 56g/L that L-Phe output is produced in fermentation, has improved 27.9% with respect to original starting strain.
Wherein, the detection method of L-Phe output in fermented liquid.
Plant and instrument: high performance liquid chromatograph, chromatographic working station, whizzer, ultrasonic cleaning machine, eddy mixer, microsyringe, millipore filtration, chromatographic column are the stainless steel column of Agilent (in-built filler PLRS-S, 10nm, long 150nm, internal diameter 4.6mm) etc.
Reagent: 40mM Na
2hPO
4pH7.8 (5.5g Na
2hPO
4+ 1L water), with NaOH solution tune pH to 7.8
Testing conditions: moving phase is ACN:MeOH: water (45:45:10); Flow velocity is 2mLmin
-1; Detection wavelength is 338nm, sample size 5 μ L.
Operation steps
(1) preparation of standard specimen
Accurately take L-Phe standard substance 0.05g (being accurate to 0.0002g) and be dissolved in 50mL volumetric flask, use Na
2hPO
4dissolve and be settled to scale, shaking up.Get 1mL to 10mL volumetric flask, Na with transfer pipet again
2hPO
4dissolve, and be settled to scale, for subsequent use.
(2) sample preparation
Get 3mL fermented liquid in centrifuge tube, 3500rpm high speed centrifugation 10min, abandons supernatant liquor, adds methyl alcohol to 9mL, is shaking 30min fast on vortex mixer; 3500rpm high speed centrifugation 10min, get supernatant liquor after certain dilution with carrying out HPLC detection after filtering with microporous membrane.
Under these conditions, after injecting the continuous two pin mark sample L-Phe peak areas of standard specimen solution relatively change and be less than 1.5% after instrument stabilizer, then the sample feeding preparing is detected.Fig. 2 is L-Phe color atlas in standard specimen and fermented liquid.
(3) method of calculation
L-Phe output (g/L)=(sample peak area/standard substance peak area) × standard substance concentration × extension rate.
(embodiment 4):
The technique of the present embodiment explanation superior strain Corynebacterium glutamicum C.glutamicum BT-N5086 fermentative production L-Phe.
The culture medium prescription that the present embodiment is used:
Solid medium is: amylum hydrolysate of the sugar 2.0%, Fructus Hordei Germinatus soaks powder 1.0%, agar powder 1.5%, pH7.0～7.2, distilled water configuration.
Seed culture medium is: amylum hydrolysate of the sugar 3.0%, soybean cake powder 2.0%, potassium primary phosphate 0.2%, magnesium sulfate 0.05%, pH7.0～7.2, tap water configuration.
Fermention medium is: amylum hydrolysate of the sugar 10%, soybean cake powder 2.0%, potassium primary phosphate 0.2%, magnesium sulfate 0.05%, vitamins B
10.0002%, pH7.0～7.2, tap water configuration.
Corynebacterium glutamicum C.glutamicum BT-N5086 is inoculated on solid medium, and culture temperature is 31 DEG C, and incubation time is 24h; The Corynebacterium glutamicum C.glutamicum BT-N5086 culture of solid culture is seeded in seed culture medium, and in 250mL culturing bottle, liquid amount is 30mL, and culture temperature is 31 DEG C, under 280rpm rotating speed, cultivates 24h; Seed culture fluid is inoculated in fermention medium, and initial pH is 7.2, and inoculum size is 10% (v/v), and in 500mL culturing bottle, liquid amount is 50mL, and culture temperature is 31 DEG C, 220rpm shaking speed bottom fermentation 44h.It is 53g/L that L-Phe output is produced in fermentation, has improved 21.1% with respect to original starting strain.
Above-described specific embodiment; object, technical scheme and beneficial effect to invention further describe; institute is understood that; the foregoing is only specific embodiments of the invention; be not limited to the present invention; within the spirit and principles in the present invention all, any amendment of making, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (9)
1. a L-Phe superior strain, its Classification And Nomenclature is Corynebacterium glutamicum (C.glutamicum) BT-N0086, has been preserved in Chinese Typical Representative culture collection center C CTCC, deposit number: CCTCC NO.M2013484.
2. the mutafacient system of L-Phe superior strain according to claim 1, is characterized in that concrete steps are as follows:
(a), sample suspension preparation: obtain cell suspension to be mutagenic from the Corynebacterium glutamicum C.glutamicum culture solid medium: Corynebacterium glutamicum starting strain is made to the acellular overlapping cell suspension of microscopy;
(b), nitrosoguanidine NMMG mutagenesis: under 20 DEG C and 100rpm, by 10
8the cell suspension of cell/ml step (a) joins the 0.1mol/L sodium citrate buffer solution that contains 300～500mg/ml nitrosoguanidine MNNG solution, the lower temperature of environment at pH5.0 is bathed 60～120min, then by mutagenized cell liquid medium within, 26～30 DEG C, the lower cultivation of environment of 100rpm 10 hours, obtain the bacterium liquid after mutagenesis;
(c), the screening of mutagenic strain: bacterium liquid after step (b) mutagenesis is coated on solid medium and is separated, then by the single bacterium colony access seed culture medium on solid medium, 31～33 DEG C of culture temperature, under 240～320rpm shaking speed, after enlarged culturing 12～24h, access fermention medium by the inoculum size of 5～15%v/v; Then lower shaking table after 31～33 DEG C, 240～320rpm shaking speed bottom fermentation, 24～44h, gets 3mL fermented liquid in centrifuge tube, and 3500rpm high speed centrifugation 10min, abandons supernatant liquor, adds methyl alcohol to 9mL, is shaking 30min fast on vortex mixer; 3500rpm high speed centrifugation 10min, gets supernatant liquor and measures L-Phe content in fermented liquid; Get bacterial strain that fermented liquid L-Phe content the is the highest starting strain as next mutagenesis, repeating step (a)～(c), until filter out the aimed strain Corynebacterium glutamicum C.glutamicum BT-N5086 that L-Phe output is high.
3. the mutafacient system of L-Phe superior strain according to claim 2, is characterized in that: in step (b), the preferred mutagenesis dosage of nitrosoguanidine NMMG is 400mg/ml; Solid medium used in step (b) is: carbon source 2.0%～4.0%, nitrogenous source 0.5%～1.5%, all the other are water, pH7.0～7.2; Wherein said carbon source is one or both the mixing in amylum hydrolysate of the sugar and molasses; Described nitrogenous source is one or both the mixing in extractum carnis and yeast powder.
4. the mutafacient system of L-Phe superior strain according to claim 2, is characterized in that: solid medium used in step (c) is: carbon source 2.0%～4.0%, nitrogenous source 0.5%～1.5%, all the other are water, pH7.0～7.2; Wherein said carbon source is one or both the mixing in amylum hydrolysate of the sugar and molasses; Described nitrogenous source is one or both the mixing in extractum carnis and yeast powder; Seed culture medium used in step (c) is: carbon source 3.0%～5.0%, nitrogenous source 1.0%～3.0%, inorganic salt 0.1%～0.4%, and all the other are water, pH7.0～7.2; Wherein said carbon source is one or both the mixing in amylum hydrolysate of the sugar and molasses; Described nitrogenous source is one or more the mixing in yeast powder, soybean cake powder; Described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts; Fermention medium used in step (c) is: carbon source 1.0%～3.0%, nitrogenous source 2.0%～6.0%, inorganic salt 0.1%～0.4%, trace element 0.0001%～0.0004%, and all the other are water, pH7.0～7.2; Wherein said carbon source is one or both the mixing in amylum hydrolysate of the sugar, molasses; Described nitrogenous source is one or more the mixing in urea, soybean cake powder; Described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts; Described trace element is VITMAIN B1.
5. the application of L-Phe superior strain claimed in claim 1 in fermentative production L-Phe.
6. the application of L-Phe superior strain according to claim 5 in fermentative production L-Phe, is characterized in that: the method for described fermentative production L-Phe comprises following steps:
(1), solid culture: Corynebacterium glutamicum (C.glutamicum) BT-N5086 is inoculated on solid medium, and culture temperature is 31～33 DEG C, and incubation time is 12～24h;
(2), seed culture: Corynebacterium glutamicum (C.glutamicum) BT-N5086 of step (1) solid culture is inoculated in seed culture medium, in 250mL culturing bottle, liquid amount is 20～50mL, culture temperature is 31～33 DEG C, under 240～320rpm shaking speed, cultivates 24～44h;
(3), fermentation culture: the seed culture fluid in step (2) is inoculated in fermention medium, inoculum size is 5～15% (v/v), in 500mL culturing bottle, liquid amount is 35～65mL, culture temperature is 31～33 DEG C, under 240～320rpm shaking speed, cultivates 24～44h.
7. the application of L-Phe superior strain according to claim 6 in fermentative production L-Phe, it is characterized in that the component that the solid medium of described step (1) comprises following mass percent: carbon source 2.0%～4.0%, nitrogenous source 0.5%～1.5%, agar 1.2%～1.8%, all the other are water, pH7.0～7.2; Wherein said carbon source is one or both the mixing in amylum hydrolysate of the sugar and molasses; Described nitrogenous source is one or both the mixing in extractum carnis and yeast powder.
8. the application of L-Phe superior strain according to claim 6 in fermentative production L-Phe, it is characterized in that the component that the seed culture medium of described step (2) comprises following mass percent: carbon source 3.0%～5.0%, nitrogenous source 1.0%～3.0%, inorganic salt 0.1%～0.4%, all the other are water, pH7.0～7.2; Wherein said carbon source is one or both the mixing in amylum hydrolysate of the sugar and molasses; Described nitrogenous source is one or more the mixing in yeast powder, soybean cake powder; Described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts.
9. the application of L-Phe superior strain according to claim 6 in fermentative production L-Phe, it is characterized in that the component that the fermention medium of described step (3) comprises following mass percent: carbon source 1.0%～3.0%, nitrogenous source 2.0%～6.0%, inorganic salt 0.1%～0.4%, trace element 0.0001%～0.0004%, all the other are water, pH7.0～7.2; Wherein said carbon source is one or both the mixing in amylum hydrolysate of the sugar, molasses; Described nitrogenous source is one or more the mixing in urea, soybean cake powder; Described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts; Described trace element is VITMAIN B1.
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| CN103074292A (en) * | 2013-01-22 | 2013-05-01 | 江南大学 | Recombinant corynebacterium glutamicum capable of being used for highly yielding L-phenylalanine and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110484483A (en) * | 2019-08-09 | 2019-11-22 | 山东汇冠康博生物科技有限公司 | A kind of preparation method of coli strain |
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