CN105112323A - Corynebacterium guangdongensis producing various amino acids and application of Corynebacterium guangdongensis - Google Patents
Corynebacterium guangdongensis producing various amino acids and application of Corynebacterium guangdongensis Download PDFInfo
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- CN105112323A CN105112323A CN201510497963.1A CN201510497963A CN105112323A CN 105112323 A CN105112323 A CN 105112323A CN 201510497963 A CN201510497963 A CN 201510497963A CN 105112323 A CN105112323 A CN 105112323A
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- corynebacterium
- guangdongensis
- guangdong
- coryneform bacteria
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- 241000186216 Corynebacterium Species 0.000 title abstract description 10
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Abstract
The invention discloses Corynebacterium guangdongensis and application of the Corynebacterium guangdongensis. Corynebacterium guangdongensis is collected at China General Microbiological Culture Collection Center (CGMCC), in the Institute of Microbiology of the Chinese Academy of Sciences in Chaoyang District of Beijing, China and under number of CGMCC No:11162. Corynebacterium guangdongensis S01 can produce 15 amino acids including aspartic acid, threonine, glutamic acid, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine and arginine, thereby having important application value in developing amino acid products.
Description
Technical field:
The invention belongs to microbial technology field, be specifically related to a kind of bacterium new species-Guangdong coryneform bacteria and the application thereof that can produce multiple amino acids.
Background technology:
Amino acid is the fundamental unit forming bioprotein, has irreplaceable biological function, has been widely used in the fields such as medicine, health care, food, feed, fertilizer, makeup.Sodium Glutamate is first amino acid that the mankind apply, and is also the seed amino acid that range of application is the widest, volume of production and marketing is maximum in the world.The industry development such as medicine, food, feed is rapid in the world in recent years, increasing to amino acid whose demand.Current amino acids production enterprise of China has reached nearly various schools of thinkers, and ultimate production is more than 3,000,000 tons, and wherein, the annual production of large amino acid products L-glutamic acid and its esters reaches 2,200,000 tons, becomes amino acid products genuine " world's factory ".Fermentable produces amino acid whose important channel.Within 1940, start to adopt fermentative Production amino acid, nineteen fifty-seven Japan's fermentation using bacteria carry out commercial production L-glutamic acid (monosodium glutamate), kind more than 20 can be had with the amino acid of fermentative Production in the world at present.Along with the development of modern biotechnology, utilize DNA recombinant bacterial strain to produce amino acid and increasingly increase, benefit to increase.China amino acids production technology aggregate level is comparatively large apart from international most advanced level, and especially show that bacterial classification acid production rate is low, productivity effect is poor, and the amino acid of high quality, high value such as to lack at the aspect.Therefore, actively excavate and produce amino acid whose microorganism high-quality new resources, the amino acids production bacterial classification of the special premium properties of restructuring tool can play very important effect for the development promoting whole amino acid Application Areas.
Summary of the invention:
First object of the present invention is to provide a kind of bacterium new species that can produce multiple amino acids---Guangdong coryneform bacteria (Corynebacteriumguangdongensis) S01, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on July 31st, 2015, address: No. 3 Institute of Microorganism, Academia Sinica of No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute, deposit number is CGMCCNo:11162.
Guangdong of the present invention coryneform bacteria (Corynebacteriumguangdongensis) S01 can produce 15 seed amino acids such as aspartic acid, Threonine, L-glutamic acid, glycine, L-Ala, Gelucystine, α-amino-isovaleric acid, methionine(Met), Isoleucine, leucine, tyrosine, phenylalanine, Methionin, Histidine and arginine.
Therefore, second object of the present invention is to provide Guangdong coryneform bacteria (Corynebacteriumguangdongensis) S01 and is preparing the application in aspartic acid, Threonine, L-glutamic acid, glycine, L-Ala, Gelucystine, α-amino-isovaleric acid, methionine(Met), Isoleucine, leucine, tyrosine, phenylalanine, Methionin, Histidine and/or arginine.
Guangdong of the present invention coryneform bacteria (Corynebacteriumguangdongensis) S01 can produce 15 seed amino acids such as aspartic acid, Threonine, L-glutamic acid, glycine, L-Ala, Gelucystine, α-amino-isovaleric acid, methionine(Met), Isoleucine, leucine, tyrosine, phenylalanine, Methionin, Histidine and arginine.Therefore, Guangdong coryneform bacteria S01 of the present invention has important using value in exploitation amino acids product.
Guangdong of the present invention coryneform bacteria (Corynebacteriumguangdongensis) S01 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on July 31st, 2015, address: No. 3 Institute of Microorganism, Academia Sinica of No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute, deposit number is CGMCCNo:11162.
Accompanying drawing illustrates:
Fig. 1 is the transmission electron microscope photo of Guangdong coryneform bacteria S01 of the present invention;
Fig. 2 is the NJ phylogenetic tree that the 16SrRNA gene order of Guangdong coryneform bacteria S01 of the present invention builds.
Embodiment:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1:
One, the separation of Guangdong coryneform bacteria S01.
Guangdong of the present invention coryneform bacteria (Corynebacteriumguangdongensis) S01 is separated on the NA flat board of the laboratory pollution of laboratory, Guangdong Microbes Inst super clean bench, there is the light yellow bacterium colony polluted in NA flat board, obtain Guangdong coryneform bacteria S01 of the present invention in picking culture & identification light yellow bacterium colony process in put procedure.
Described NA culture medium prescription is: peptone 10.0g, extractum carnis 3.0g, sodium-chlor 5.0g, agar 15.0g, distilled water 1000mL, pH7.3, and after mixing, sterilizing is for subsequent use.
Two, the qualification of Guangdong coryneform bacteria S01.
1. phenotypic characteristic: adopt three rides to be inoculated on NA substratum Guangdong coryneform bacteria S01, is placed in 30 DEG C of incubators and cultivates 2 days, takes out and observes colony characteristics.The fermentation etc. of cellular form observation (Fig. 1), gramstaining, mobility experiment, catalase, oxydase, urase, nitrate reduction, hydrolysis polychrom, carbohydrate measures see " common bacteria identification handbook ".Guangdong coryneform bacteria S01 then have employed BiologGN III to the utilization of different carbon nitrogen source, and operation is carried out to specifications.
Substratum growth test adopts the single bacterium colony three ride mode of inoculation, have employed NA, LB, R2A, TSA, 2216E substratum respectively.
NA substratum: peptone 10.0g, extractum carnis 3.0g, sodium-chlor 5.0g, agar 15.0g, distilled water 1000mL, pH7.3, after mixing, sterilizing is for subsequent use.
LB culture medium prescription: trypsinase 10.0g, yeast extract 5.0g, sodium-chlor 10.0g, agar 15.0g, distilled water 1000mL, pH7.0, after mixing, sterilizing is for subsequent use.
R2A culture medium prescription: yeast extract 0.5g, peptone 0.5g, acid hydrolysis casein food grade 0.5g, glucose 0.5g, Zulkovsky starch 0.5g, K
2hPO
40.3g, MgSO
47H
2o0.05g, Sodium.alpha.-ketopropionate 0.3g, agar powder 15g, distilled water 1000mL, pH7.0 ~ 7.4, after mixing, sterilizing is for subsequent use.
TSA culture medium prescription: Tryptones 15.0g, soy peptone 5.0g, sodium-chlor 5.0g, agar powder 15.0g, distilled water 1000mL, pH value 7.3, after mixing, sterilizing is for subsequent use.
2216E culture medium prescription adopts the rich product (article No.: HB0132) in sea, Qingdao, and after mixing, sterilizing is for subsequent use.
Morphological specificity: 30 DEG C are cultivated bacterium colony after 48h to Guangdong coryneform bacteria S01 on NA flat board be faint yellow, circle, regular edges, smooth surface is translucent.Cell is shaft-like, and atrichia does not move, without gemma, and facultative aerobic growth (Fig. 1).
Physiological and biochemical property: Gram-positive, oxidase negative, catalase, nitrate reduction and urase are positive, and the fermentation and acid of glucose, ribose, maltose, lactose and raffinose is feminine gender.Can not gelatin hydrolysate and polychrom, can grow on NA, LB, R2A, TSA and 2216E substratum.
Type strain of delivering the highest with its homology for Guangdong coryneform bacteria S01 of the present invention has been carried out comparing of physiological and biochemical property, and result is as shown in table 1.
Coryneform bacteria S01 relative type strain Microbiological Characteristics in table 1 Guangdong compares
Note: "+" represents positive; "-" represents negative; " w " represents the weak positive.
2. genetic development analysis: adopt bacterial universal primers 27F (5 '-AGAGTTTGATCMTGGCTCAG-3 ') and 1492R (5 '-GGYTACCTTGTTACGACTT-3 ') to carry out the pcr amplification of the 16SrRNA gene of Guangdong coryneform bacteria S01, checked order by Shanghai Ying Jun Bioisystech Co., Ltd, obtain its sequence information (its nucleotide sequence is as shown in SEQIDNO.1), sequence information and ncbi database are carried out Blast comparison, obtains the highlyest with its similarity delivering type species information.Biological software MEGA5 is adopted to build the NJ phylogenetic tree of Guangdong coryneform bacteria S01, as shown in Figure 2.
16SrRNA gene amplification and sequence alignment find that Guangdong of the present invention coryneform bacteria (Corynebacteriumguangdongensis) S01 maximum similarity type strain is CorynebacteriumhumireducensMFC-5
t(95.9%) the novel species threshold value 97% of generally acknowledging in the world, is less than.Further genetic development analysis finds that this bacterial strain belongs to corynebacterium (Corynebacterium), with CorynebacteriummarisJCM17018 on evolutionary tree
tand CorynebacteriumdoosanenseJCM17317 (94.8%)
t(95.0%) form stable branch, there is nearest sibship (Fig. 2).These evidences show that Guangdong coryneform bacteria S01 may be a new species of corynebacterium.
3. chemical classification feature: lipid acid abstraction and quantification adopts the Standard operation procedure SOP (Sasser, 1990) of MIDI company.The measuring method of DNAG+C content is with reference to Mesbahetal. (1989).
Type species of delivering nearest with its sibship for Guangdong coryneform bacteria S01 of the present invention have been carried out comparing of lipid acid moiety, and result is as shown in table 2.Guangdong coryneform bacteria S01 is (C on main fatty acid content
18:1ω 9c, C
16:0and 10-methylC
18:0) there is obvious difference to relevant type strain, showing that Guangdong coryneform bacteria S01 is different from the associative mode bacterial classification delivered, may be a new species of corynebacterium.
Table 2 Guangdong coryneform bacteria S01 and relevant type strain lipid acid ratio of components thereof are comparatively (%)
| Fatty acid | C.guangdongensis S01 | C.maris JCM 17018 T | C.doosanense JCM 17317 T |
| C 14:0 | 1.8 | 0.4 | 0.4 |
| C 16:1ω9c | 1.7 | 1.5 | 1.9 |
| C 16:0 | 27.8 | 22.5 | 17.2 |
| C 17:0 | / | / | 12.4 |
| C 18:1ω9c | 49.8 | 55.7 | 57.8 |
| C 18:0 | 9.4 | 10.6 | 10.0 |
| 10-methyl C 18:0 | 9.6 | 7.1 | / |
| Summed feature 7 | / | 2.0 | / |
| C 18:1ω7c | / | 0.4 | 0.4 |
Note: " Summedfeature7 " represents that this component is unknown C
18.846or C
19:1ω 6c; "/" represents and this component do not detected;
Comprehensive phenotypic characteristic, genetic development analysis, chemical classification feature, Guangdong coryneform bacteria S01 of the present invention is the new species that rod-like stem belongs to, by its called after Guangdong coryneform bacteria (Corynebacteriumguangdongensis) S01, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on July 31st, 2015, address: No. 3 Institute of Microorganism, Academia Sinica of No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute, deposit number is CGMCCNo:11162.
Three, Guangdong coryneform bacteria S01 produces the mensuration of aminoacid component.
Single bacterium colony of picking Guangdong of the present invention coryneform bacteria S01, is seeded in the liquid nutrient broth substratum of 200mL, 180rpm shaking table 30 DEG C of shaking culture 2 days.Get the filtration that 5mL zymocyte liquid adopts the disposable filter (0.02 μm, aperture) of sterilizing, collect filtrate.Full-automatic amino acidanalyser L-8900 (Hitachi) is adopted to carry out determination and analysis to the aminoacid component in filtrate and content.Test result finds that Guangdong coryneform bacteria S01 can produce 15 seed amino acids (table 3).
Aminoacid component and content are produced in the coryneform bacteria fermentation of table 3 Guangdong
| Amino acid profiles | Content (mg/L) |
| Aspartic acid (Asp) | 4.1 |
| Threonine (Thr) | 5.47 |
| Serine (Ser) | <0.01 |
| L-glutamic acid (Glu) | 13.27 |
| Proline(Pro) (Pro) | <0.01 |
| Glycine (Gly) | 23.55 |
| L-Ala (Ala) | 90.84 |
| Gelucystine (Cys) | 24.89 |
| α-amino-isovaleric acid (Val) | 86.24 |
| Methionine(Met) (Met) | 44.58 |
| Isoleucine (Ile) | 49.24 |
| Leucine (Leu) | 166.67 |
| Tyrosine (Tyr) | 98.64 |
| Phenylalanine (Phe) | 233.43 |
| Methionin (Lys) | 218.14 |
| Histidine (His) | 36.12 |
| Arginine (Arg) | 534.23 |
Find through amino acidanalyser analysis, the amino acid that Guangdong coryneform bacteria S01 of the present invention 15 kinds of can produce in liquid culture process are different, as shown in table 3.What output was larger is arginine (534.2mg/L), phenylalanine (233.43mg/L), Methionin (218.14mg/L), leucine (166.67mg/L).In addition, tyrosine, L-Ala, α-amino-isovaleric acid, Isoleucine, methionine(Met), Histidine, Gelucystine, glycine, L-glutamic acid, Threonine and aspartic acid can also be produced.Therefore, Guangdong coryneform bacteria S01 of the present invention has important using value in exploitation amino acids product.
Claims (2)
1. Guangdong coryneform bacteria (Corynebacteriumguangdongensis) S01, deposit number is CGMCCNo:11162.
2. Guangdong coryneform bacteria (Corynebacteriumguangdongensis) S01 is preparing the application in aspartic acid, Threonine, L-glutamic acid, glycine, L-Ala, Gelucystine, α-amino-isovaleric acid, methionine(Met), Isoleucine, leucine, tyrosine, phenylalanine, Methionin, Histidine and/or arginine.
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