CN102382787A - Corynebacterium pekinense and application thereof - Google Patents
Corynebacterium pekinense and application thereof Download PDFInfo
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- CN102382787A CN102382787A CN201110414623XA CN201110414623A CN102382787A CN 102382787 A CN102382787 A CN 102382787A CN 201110414623X A CN201110414623X A CN 201110414623XA CN 201110414623 A CN201110414623 A CN 201110414623A CN 102382787 A CN102382787 A CN 102382787A
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- rod bacillus
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Abstract
The invention relates to the field of microorganisms and discloses a corynebacterium pekinense, wherein the collection number thereof is CGMCC No.5361. The corynebacterium pekinense with the collection number of CGMCC No.5361 takes a wild corynebacterium pekinense with the collection number of CICC No.21721 as a parent strain, and is prepared by performing chemical mutagenesis and screening through a culture medium containing neomycin; and the capability of generating L-lysine through fermentation is obviously higher than that of the wild corynebacterium pekinense, and the yield of the L-lysine is increased by 14.7 percent, so that the corynebacterium pekinense with the collection number of CGMCC No.5361 is widely applied to the fermentation of the L-lysine.
Description
Technical field
The present invention relates to microorganism field, relate to a kind of Beijing rod bacillus and application thereof specifically.
Background technology
L-Methionin, formal name used at school are 2, and the 6-diaminocaproic acid is a kind of basic aminoacids in common 20 seed amino acids of constitutive protein matter, is one of essential amino acid.
Protein is the major ingredient that constitutes human body cell; Protein in the food resolves into amino acid earlier through digestion after getting into human body; Human body utilizes the new human body protein of these amino acid resynthesis again then, all is the human body protein after synthetic like immune antibody, digestive ferment, plasma proteins, tethelin etc.In each seed amino acid of synthetic protein, L-Methionin is most important a kind of, has lacked it, and other amino acid just is restricted or is not used, and scientist claims that it is human body first indispensable amino acid.In recent years, scientist also finds, L-Methionin be control people bulk-growth important substance-pressing down most important in the long element also is the most essential composition, people's nervus centralis and peripheral nervous system all played an important role.Human body can not self synthesize L-Methionin; Must from food, draw Methionin; It is the key substance that helps other nutritive substance fully to be absorbed and utilize by human body; Human body has only and has replenished absorption and the utilization that enough L-Methionin could improve food proteins, reaches balanced nutritious, promotes to grow.
Prior art mainly is that raw material produces through microbial fermentation with the saccharic, adopts " iso-electric point extraction " to add that " ion exchange resin " separates acquisition then.L-lysine production height is inseparable with the microbe species that is adopted, and Beijing rod bacillus is one of bacterial classification commonly used in the L-fermenting lysine, but its L-lysine production is not high.Therefore, a kind of L-Methionin superior strain is provided, significant for the fermentation efficiency that improves L-Methionin.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of Beijing rod bacillus and application thereof, make said Beijing rod bacillus can improve the output of L-Methionin.
For realizing the foregoing invention purpose; The present invention is a starting strain with wild-type Beijing rod bacillus CICC 21721 (available from Chinese industrial microbial strains preservation administrative center); With N-methyl-N ' nitro-N-nitrosoguanidine is that mutagenic compound carry out chemomorphosis to it; To be coated on the substratum that contains Xin Meisu through the suspension bacteria liquid of the Beijing after mutagenesis rod bacillus CICC 21721 then, filter out bacterial strain from containing the Xin Meisu substratum, called after bacterial strain MHZ-0910 with neomycin resistance.
The present invention carries out physio-biochemical characteristics according to the method for the elegant pearl in east, Cai Miaoying chief editor " common bacteria system identification handbook " to it with the above-mentioned bacterial strain MHZ-0910 that filters out and detects, and detected result is consistent with wild-type Beijing rod bacillus.In addition, analyze through the 16S DNA detection, bacterial strain MHZ-0910 and wild-type Beijing rod bacillus homology in conjunction with physiological and biochemical property, identify that it is Beijing rod bacillus up to 99%.
Beijing rod bacillus MHZ-0910 according to the invention has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on October 20th, 2011; The address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.5361.
The L-fermenting lysine simultaneous test of Beijing rod bacillus MHZ-0910 according to the invention and wild-type Beijing rod bacillus CICC 21721 shows; Beijing rod bacillus MHZ-0910 fermentation synthetic L-lysine content according to the invention is 29.6g/L; And rod bacillus CICC 21721 fermentation synthetic L-lysine contents in wild-type Beijing are 25.8g/L, and the L-lysine production has improved 14.7%.Therefore, Beijing rod bacillus MHZ-0910 according to the invention can be applied to produce in the L-Methionin.
Xin Meisu is a kind of aminoglycoside antibiotics; Through being incorporated into interfere with bacterial protein synthesis on the intracellular rrna; Make it the protein of resulting anomaly, and the bacterial cell membrane permeability is increased, cause in the cell important physiological material to leak outside and present germicidal action; Stronger to bacterium germicidal action stationary phase, be sterilization stationary phase class microbiotic.And the resistance that Beijing rod bacillus MHZ-0910 according to the invention has Xin Meisu also can improve L-Methionin synthesis capability.After wild strain obtains neomycin resistance; Maybe be because the protein opposing Xin Meisu of Beijing according to the invention rod bacillus MHZ-0910 resulting anomaly; These unusual protein cause L-Methionin synthetase series to remove the feedback inhibition of some product; Thereby cause the L-lysine production of mutant strain to be improved; Perhaps the Methionin translocator function on the MHZ-0910 cytolemma changes and has promoted the speed that Methionin is gone out from intracellular transport, thereby causes the L-lysine production of mutant strain to be improved.
In addition; The present invention also provides a kind of L-Methionin working method; With deposit number is that Beijing rod bacillus of CGMCCNo.5361 is inoculated in seed culture medium and expands numerously, and the culture that will expand then after numerous changes the fermention medium fermentation over to, and keeping the pH value between yeast phase is 6-9.
Wherein, as preferably, the pH value between yeast phase is 6.5-7.4.
Working method according to the invention can be performed until and no longer produce till the L-Methionin, can separate L-Methionin according to this area ordinary method then.
Fermention medium for seed culture medium that expands numerous use and fermentation use is known in this field, and those skilled in the art all can adopt suitable medium to carry out the fermentation of L-Methionin.As preferably, seed culture medium according to the invention is made up of 20g/L glucose, 5g/L ammonium sulfate, 1g/L potassium primary phosphate, 30g/L steeping water, 3g/L soybean meal hydrolysate, 100 μ g/L vitamin Hs, 200 μ g/L VitB1s, and the pH value is 7.2-7.4.Said fermention medium is made up of 95g/L molasses, 1g/L potassium primary phosphate, 25g/L steeping water, 3g/L soybean meal hydrolysate, 40g/L ammonium sulfate, 50g/L glucose, 30g/L lime carbonate, 100 μ g/L vitamin Hs, 200 μ g/L VitB1s, and the pH value is 7.2-7.4.
Can know by above technical scheme; Deposit number according to the invention is that Beijing rod bacillus MHZ-0910 of CGMCC No.5361 is a starting strain with wild-type Beijing rod bacillus CICC 21721; After chemomorphosis, obtain through containing the Xin Meisu screening of medium; The ability that its fermentation generates L-Methionin significantly improves than the excellent bacillus in wild-type Beijing, and the L-lysine production has improved 14.7%.Therefore, deposit number according to the invention is that Beijing rod bacillus of CGMCC No.5361 can be widely used in the fermentation of L-Methionin.
The explanation of biological preservation information
Classification name: Beijing rod bacillus; Corynebacterium pekinense. is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on October 20th, 2011; The address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica, deposit number is CGMCCNo.5361.
Embodiment
The embodiment of the invention discloses a kind of Beijing rod bacillus and application thereof.Those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included in the present invention.Bacterial strain of the present invention is described through preferred embodiment, and the related personnel obviously can change or suitably change and combination product as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use technology of the present invention.
In order further to understand the present invention, the present invention is elaborated below in conjunction with embodiment.
Embodiment 1: the screening of Beijing rod bacillus MHZ-0910 according to the invention
Starting strain-wild-type Beijing rod bacillus CICC 21721 is seeded in the LB liquid nutrient medium 30 ℃ cultivates the bacteria suspension that obtained to be in logarithmic phase in 16 hours; Then this bacteria suspension is coated on the LB solid plate; An amount of N-methyl-N ' nitro-N-nitrosoguanidine solid particulate is put to LB solid plate central authorities; Placing 30 ℃ of incubators to cultivate to scrape after 16 hours has the lawn of faint growth to be suspended in an amount of SPSS around getting N-methyl-N ' nitro-N-nitrosoguanidine particle; The saline water that is suspended with lawn is coated on the LB solid plate that contains 0.5mg/L; 30 ℃ are continued to cultivate 2 days, according to characteristic picking list bacterium colonies such as colonial morphology, colors, and called after MHZ-0910.
LB liquid nutrient medium/LB solid plate prescription is following: Carnis Bovis seu Bubali cream 5g, peptone 10g, NaCl 5g, vitamin H 50ug, VitB1 100ug, leucine 100mg, homoserine 100mg, Threonine 100mg, methionine(Met) 100mg, agar 18g are settled to 1000ml with dissolved in distilled water and after regulating pH to 6.8-7.4.
Wherein, the difference of LB liquid nutrient medium and LB solid plate is whether agar adds.Vitamin H, VitB1, leucine, homoserine, Threonine, the nonessential interpolation of methionine(Met) can be added according to actual needs.
The above-mentioned bacterial strain MHZ-0910 that filters out is carried out physio-biochemical characteristics according to the method for the elegant pearl in east, Cai Miaoying chief editor " common bacteria system identification handbook " to it detect, detected result is consistent with wild-type Beijing rod bacillus.In addition, analyze through the 16S DNA detection, bacterial strain MHZ-0910 and wild-type Beijing rod bacillus homology in conjunction with physiological and biochemical property, identify that it is Beijing rod bacillus up to 99%.
Embodiment 2: L-Methionin working method according to the invention and L-fermenting lysine simultaneous test
1, fermentation process
Beijing according to the invention rod bacillus MHZ-0910 is seeded in expand in the seed culture medium of the following stated numerous; In 30 ℃ of shaking culture 16 hours; The gained culture is inoculated in the fermention medium of the following stated; The volume ratio of culture and fermention medium is 1: 10 (those skilled in the art can suitably regulate according to the amount of the fermention medium in the actual production, and this ratio is a preferred proportion), and 30 ℃ of shaking culture are to no longer producing (being generally about 72 hours) till the L-Methionin.In fermention medium during shaking culture, keep pH value in the fermention medium between 6.5-7.4.
Seed culture medium:
20g/L glucose, 5g/L ammonium sulfate, 1g/L potassium primary phosphate, 30g/L steeping water, 3g/L soybean meal hydrolysate, 100 μ g/L vitamin Hs, 200 μ g/L VitB1s are formed, and the pH value is 7.2-7.4.
Fermention medium: 95g/L molasses, 1g/L potassium primary phosphate, 25g/L steeping water, 3g/L soybean meal hydrolysate, 40g/L ammonium sulfate, 50g/L glucose, 30g/L lime carbonate, 100 μ g/L vitamin Hs, 200 μ g/L VitB1s are formed, and the pH value is 7.2-7.4.
2, L-fermenting lysine simultaneous test
The excellent bacillus CICC in Beijing according to the invention rod bacillus MHZ-0910 and wild-type Beijing 21721 is fermented according to aforementioned production method simultaneously; The yeasting of two kinds of bacterial strains, inoculum size are all identical, stop the fermentation back and measure the two kinds of synthetic L-of bacterial strain institute lysine contents through the bio-sensing appearance.
The result is in L lysine HCL, and wild-type Beijing rod bacillus CICC 21721 output are 25.8g/L, and Beijing according to the invention rod bacillus MHZ-0910 output is 29.6g/L, and mutant strain L-lysine production has improved 14.7% than starting strain CICC 21721.
The explanation of above embodiment just is used for helping to understand method of the present invention and core concept thereof.Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of claim of the present invention.
Claims (6)
1. Beijing rod bacillus is characterized in that deposit number is CGMCC No.5361.
2. deposit number is the application of Beijing rod bacillus in producing L-Methionin of CGMCC No.5361.
3. a L-Methionin working method is characterized in that, is that Beijing rod bacillus of CGMCC No.5361 is inoculated in seed culture medium and expands numerously with deposit number, and the culture that will expand then after numerous changes the fermention medium fermentation over to, and keeping the pH value between yeast phase is 6-9.
4. according to the said working method of claim 3, it is characterized in that the pH value between said yeast phase is 6.5-7.4.
5. according to the said working method of claim 3; It is characterized in that; Said seed culture medium is made up of 20g/L glucose, 5g/L ammonium sulfate, 1g/L potassium primary phosphate, 30g/L steeping water, 3g/L soybean meal hydrolysate, 100 μ g/L vitamin Hs, 200 μ g/L VitB1s, and the pH value is 7.2-7.4.
6. according to the said working method of claim 3; It is characterized in that; Said fermention medium is made up of 95g/L molasses, 1g/L potassium primary phosphate, 25g/L steeping water, 3g/L soybean meal hydrolysate, 40g/L ammonium sulfate, 50g/L glucose, 30g/L lime carbonate, 100 μ g/L vitamin Hs, 200 μ g/L VitB1s, and the pH value is 7.2-7.4.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660483A (en) * | 2012-05-17 | 2012-09-12 | 通辽梅花生物科技有限公司 | Corynebacterium pekinense and application thereof |
| CN105112323A (en) * | 2015-08-14 | 2015-12-02 | 广东省微生物研究所 | Corynebacterium guangdongensis producing various amino acids and application of Corynebacterium guangdongensis |
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2011
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| 张久丽等: "一株赖氨酸产生菌的菌种鉴定及发酵条件优化", 《CNKI-中国优秀硕士学位论文全文数据库》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660483A (en) * | 2012-05-17 | 2012-09-12 | 通辽梅花生物科技有限公司 | Corynebacterium pekinense and application thereof |
| CN105112323A (en) * | 2015-08-14 | 2015-12-02 | 广东省微生物研究所 | Corynebacterium guangdongensis producing various amino acids and application of Corynebacterium guangdongensis |
| CN105112323B (en) * | 2015-08-14 | 2018-02-23 | 广东省微生物研究所 | A kind of Guangdong bar bacterium for producing several amino acids and its application |
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