CN105112323B - A kind of Guangdong bar bacterium for producing several amino acids and its application - Google Patents
A kind of Guangdong bar bacterium for producing several amino acids and its application Download PDFInfo
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Abstract
The invention discloses a kind of Guangdong bar bacterium and its application.Bar bacterium (Corynebacterium guangdongensis) S01 in Guangdong was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address on July 31st, 2015：Chaoyang District Institute of Microorganism, Academia Sinica of city of BeiJing, China, deposit number are CGMCC No:11162.Guangdong bar bacterium (Corynebacterium guangdongensis) S01 of the present invention can produce 15 kinds of amino acid such as aspartic acid, threonine, glutamic acid, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine and arginine.Therefore, Guangdong bar bacterium S01 of the invention has important application value in amino acids product is developed.
Description
Technical field：
The invention belongs to microbial technology field, and in particular to a kind of bacterium new species-Guangdong that can produce several amino acids
Bar bacterium and its application.
Background technology：
Amino acid is the base unit for forming bioprotein, there is irreplaceable biological function, extensive
Applied to fields such as medicine, health care, food, feed, fertilizer, cosmetics.Sodium glutamate is first amino acid of mankind's application,
A kind of and amino acid that application is most wide in the world, volume of production and marketing is maximum.Medicine, food, feed etc. are gone in the world in recent years
Industry is quickly grown, increasing to the demand of amino acid.Current amino acids production enterprise of China reaches nearly various schools of thinkers, and total output exceedes
3000000 tons, wherein, the annual production of large amino acid products glutamic acid and its esters turns into amino acid products name pair up to 2,200,000 tons
" world's factory " in fact.Microbial fermentation is the important channel for producing amino acid.Start within 1940 using fermentation method production ammonia
Base acid, nineteen fifty-seven, Japan with bacterial fermentation carried out commercial production glutamic acid (monosodium glutamate), can be produced in the world with fermentation method at present
Amino acid have kind more than 20.With the development of modern biotechnology, increased increasingly using DNA recombination engineerings production amino acid
It is more, benefit increase.China's amino acids production technology aggregate level is larger away from international most advanced level, especially shows strain acid production rate
Low, productivity effect is poor, high quality, the amino acid of high value are few etc..Therefore, the microorganism for actively excavating production amino acid is high-quality
New resources, restructuring have the amino acids production strain of special premium properties for promoting the development of whole amino acid application field can
Play particularly important effect.
The content of the invention：
First purpose of the present invention is to provide a kind of bacterium new species that can produce several amino acids --- Guangdong bar
Bacterium (Corynebacterium guangdongensis) S01, the bacterial strain were preserved in China Microbiological bacterium on July 31st, 2015
Kind preservation administration committee's common micro-organisms center (CGMCC), address：In city of BeiJing, China Chaoyang District North Star West Road 1 institute 3
Institute of microbiology of the academy of sciences of state, deposit number are CGMCC No：11162.
Guangdong bar bacterium (Corynebacterium guangdongensis) S01 of the present invention can produce aspartic acid, Soviet Union
Propylhomoserin, glutamic acid, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylpropyl alcohol ammonia
15 kinds of amino acid such as acid, lysine, histidine and arginine.
Therefore, second object of the present invention is to provide Guangdong bar bacterium (Corynebacterium
Guangdongensis) S01 is preparing aspartic acid, threonine, glutamic acid, glycine, alanine, cystine, valine, egg
Application in propylhomoserin, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine and/or arginine.
Guangdong bar bacterium (Corynebacterium guangdongensis) S01 of the present invention can produce aspartic acid, Soviet Union
Propylhomoserin, glutamic acid, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylpropyl alcohol ammonia
15 kinds of amino acid such as acid, lysine, histidine and arginine.Therefore, Guangdong bar bacterium S01 of the invention is in exploitation amino acids
There is important application value in product.
Guangdong bar bacterium (Corynebacterium guangdongensis) S01 of the present invention was on July 31st, 2015
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address：City of BeiJing, China Chaoyang District
No. 3 Institute of Microorganism, Academia Sinica of institute of North Star West Road 1, deposit number are CGMCC No:11162.
Brief description of the drawings：
Fig. 1 is the Guangdong bar bacterium S01 of present invention transmission electron microscope photo；
The NJ phylogenetic trees for the 16S rRNA gene orders structure that Fig. 2 is the Guangdong bar bacterium S01 of the present invention.
Embodiment：
Following examples are to further explanation of the invention, rather than limitation of the present invention.
Embodiment 1：
First, Guangdong bar bacterium S01 separation.
Guangdong bar bacterium (Corynebacterium guangdongensis) S01 of the present invention is isolated from the micro- life in Guangdong Province
On the NA flat boards of the laboratory pollution of thing research institute laboratory super-clean bench, there is the pale yellow of pollution in placement process in NA flat boards
Bar bacterium S01 in Guangdong of the present invention is obtained during color bacterium colony, picking culture and the light yellow bacterium colony of identification.
Described NA culture medium prescriptions are：Peptone 10.0g, beef extract 3.0g, sodium chloride 5.0g, agar 15.0g, double steamings
Water 1000mL, pH 7.3, after being well mixed, sterilizing is standby.
2nd, Guangdong bar bacterium S01 identification.
1. phenotypic characteristic：Guangdong bar bacterium S01 is inoculated on NA culture mediums using three rides, is placed in 30 DEG C of incubators
Culture 2 days, take out observation colony characteristicses.Cellular morphology observation (Fig. 1), Gram's staining, motility experiment, catalase, oxidation
Enzyme, urase, nitrate reduction, hydrolysis aesculin, carbohydrate the measure such as fermentation referring to《Common bacteria identification handbook》.Guangdong bar
Utilizations of the bacterium S01 to different carbon nitrogen sources then employs Biolog GN III, and operation is carried out to specifications.
Cultivate basal growth test using inoculation the ride mode of single bacterium colony three, be respectively adopted NA, LB, R2A, TSA,
2216E culture mediums.
NA culture mediums：Peptone 10.0g, beef extract 3.0g, sodium chloride 5.0g, agar 15.0g, distilled water 1000mL, pH
7.3, after being well mixed, sterilizing is standby.
LB culture medium prescriptions：Trypsase 10.0g, yeast extract 5.0g, sodium chloride 10.0g, agar 15.0g, double steamings
Water 1000mL, pH 7.0, after being well mixed, sterilizing is standby.
R2A culture medium prescriptions：Yeast extract 0.5g, peptone 0.5g, sour hydrolysed casein 0.5g, glucose 0.5g,
Soluble starch 0.5g, K2HPO4 0.3g,MgSO4·7H2O 0.05g, Sodium Pyruvate 0.3g, agar powder 15g, distilled water
1000mL, pH 7.0~7.4, after being well mixed, sterilizing is standby.
TSA culture medium prescriptions：Tryptone 15.0g, soy peptone 5.0g, sodium chloride 5.0g, agar powder 15.0g are double
Water 1000mL is steamed, pH value 7.3, after being well mixed, sterilizing is standby.
2216E culture medium prescriptions are using the rich product (article No. in Qingdao sea：HB0132), after being well mixed, sterilizing is standby.
Morphological feature：Bacterium colony is in faint yellow, circular, edge rule to Guangdong bar bacterium S01 after 30 DEG C of culture 48h on NA flat boards
Then, surface is smooth, translucent.Cell is in shaft-like, atrichia, is not moved, no gemma, facultative aerobic growth (Fig. 1).
Physiological and biochemical property：Gram-positive, oxidase negative, catalase, nitrate reduction and urase are the positive,
Glucose, ribose, maltose, the fermentation and acid of lactose and raffinose are feminine gender.It is unable to gelatin hydrolysate and aesculin, Neng Gou
Grown on NA, LB, R2A, TSA and 2216E culture medium.
The Guangdong bar bacterium S01 of the present invention has been delivered into type strain with its homology highest and has carried out Physiology and biochemistry spy
The comparison of sign, as a result as shown in table 1.
The relative type strain Microbiological Characteristics of Guangdong bar bacterium S01 of table 1 compare
Note："+" represents positive；"-" represents negative；" w " represents weakly positive.
2. genetic development is analyzed：Using bacterial universal primers 27F (5 '-AGAGTTTGATCMTGGCTCAG-3 ') and 1492R
(5 '-GGYTACCTTGTTACGACTT-3 ') carry out the PCR amplifications of Guangdong bar bacterium S01 16S rRNA genes, by Shanghai English
Fine horse Bioisystech Co., Ltd is sequenced, and obtains its sequence information (its nucleotide sequence is as shown in SEQ ID NO.1), sequence is believed
Breath carries out Blast with ncbi database and compared, and obtains having delivered type sepecies information with its similarity highest.It is soft using biology
Part MEGA 5 builds Guangdong bar bacterium S01 NJ phylogenetic trees, as shown in Figure 2.
16S rRNA gene magnifications and sequence alignment find the Guangdong bar bacterium (Corynebacterium of the present invention
Guangdongensis) S01 maximum similarities type strain is Corynebacterium humireducens MFC-5T
(95.9%), less than novel species critical value 97% generally acknowledged in the world.Further genetic development analysis finds that the bacterial strain belongs to rod
Shape Bacillus (Corynebacterium), on chadogram with Corynebacterium maris JCM 17018T
And Corynebacterium doosanense JCM 17317 (94.8%)T(95.0%) stable branch is formed, is had most
Near affiliation (Fig. 2).These evidences show that Guangdong bar bacterium S01 may be a new species of corynebacterium.
3. chemical classification feature：Aliphatic acid extract and measure using MIDI companies S.O.P. (Sasser,
1990).The assay method of DNA G+C contents is with reference to Mesbah et al. (1989).
The Guangdong bar bacterium S01 of the present invention the delivered type sepecies nearest with its affiliation aliphatic acid composition has been subjected to
The comparison of composition, as a result as shown in table 2.Guangdong bar bacterium S01 (C on main content of fatty acid18:1ω 9c, C16:0And 10-
methyl C18:0) to related type strain obvious difference be present, show that Guangdong bar bacterium S01 is different from and delivered
Associative mode strain, may be a new species of corynebacterium.
The Guangdong bar bacterium S01 of table 2 and its type strain aliphatic acid ratio of components of correlation are compared with (%)
| Fatty acid | C.guangdongensis S01 | C.maris JCM 17018T | C.doosanense JCM 17317T |
| C14:0 | 1.8 | 0.4 | 0.4 |
| C16:1ω9c | 1.7 | 1.5 | 1.9 |
| C16:0 | 27.8 | 22.5 | 17.2 |
| C17:0 | / | / | 12.4 |
| C18:1ω9c | 49.8 | 55.7 | 57.8 |
| C18:0 | 9.4 | 10.6 | 10.0 |
| 10-methyl C18:0 | 9.6 | 7.1 | / |
| Summed feature 7 | / | 2.0 | / |
| C18:1ω7c | / | 0.4 | 0.4 |
Note：" Summed feature 7 " represent that the component is unknown C18.846Or C19:1ω6c；"/" represents not detect
To the component；
Comprehensive phenotypic characteristic, genetic development analysis, chemical classification feature, Guangdong bar bacterium S01 of the invention are bar-like bar
Category a new species, be named as Guangdong bar bacterium (Corynebacterium guangdongensis) S01, in
On July 31st, 2015 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address：In
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica of state, deposit number are CGMCC No:
11162。
3rd, bar bacterium S01 in Guangdong produces the measure of aminoacid ingredient.
The Guangdong bar bacterium S01 of picking present invention single bacterium colony, is seeded in 200mL liquid nutrient broth culture medium,
30 DEG C of 180rpm shaking tables shaken cultivation 2 days.Take mistake of the 5mL zymocyte liquids using the disposable filter (0.02 μm of aperture) of sterilizing
Filter, collect filtrate.The aminoacid ingredient in filtrate and content are carried out using full-automatic amino-acid analyzer L-8900 (Hitachi)
Measure analysis.Test result finds that Guangdong bar bacterium S01 can produce 15 kinds of amino acid (table 3).
The Guangdong bar bacterium fermentation production aminoacid ingredient of table 3 and content
| Amino acid profiles | Content (mg/L) |
| Aspartic acid (Asp) | 4.1 |
| Threonine (Thr) | 5.47 |
| Serine (Ser) | <0.01 |
| Glutamic acid (Glu) | 13.27 |
| Proline (Pro) | <0.01 |
| Glycine (Gly) | 23.55 |
| Alanine (Ala) | 90.84 |
| Cystine (Cys) | 24.89 |
| Valine (Val) | 86.24 |
| Methionine (Met) | 44.58 |
| Isoleucine (Ile) | 49.24 |
| Leucine (Leu) | 166.67 |
| Tyrosine (Tyr) | 98.64 |
| Phenylalanine (Phe) | 233.43 |
| Lysine (Lys) | 218.14 |
| Histidine (His) | 36.12 |
| Arginine (Arg) | 534.23 |
Found through amino-acid analyzer analysis, Guangdong bar bacterium S01 of the invention is during the Liquid Culture caused by energy
15 kinds of different amino acid, as shown in table 3.Yield is larger for arginine (534.2mg/L), phenylalanine (233.43mg/
L), lysine (218.14mg/L), leucine (166.67mg/L).Moreover it is possible to produce tyrosine, alanine, valine, different
Leucine, methionine, histidine, cystine, glycine, glutamic acid, threonine and aspartic acid.Therefore, Guangdong of the invention
Bar bacterium S01 has important application value in amino acids product is developed.
Claims (2)
- Guangdong bar bacterium 1. (Corynebacterium guangdongensis) S01, deposit number is CGMCC No：11162.
- 2. bar bacterium (Corynebacterium guangdongensis) S01 in Guangdong is preparing aspartic acid, threonine, paddy ammonia Acid, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, Application in histidine and/or arginine.
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| CN1710066A (en) * | 2005-06-07 | 2005-12-21 | 山东大学 | Glutamic acid capable of having high-yield glutamine |
| CN101517066A (en) * | 2006-07-13 | 2009-08-26 | Cj第一制糖株式会社 | Method for producing L-arginine using Corynebacterium glutamicum |
| CN101805713A (en) * | 2010-02-05 | 2010-08-18 | 北京大学 | Corynebacterium sp strain and application thereof |
| CN102382787A (en) * | 2011-12-13 | 2012-03-21 | 梅花生物科技集团股份有限公司 | Corynebacterium pekinense and application thereof |
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| CN1710066A (en) * | 2005-06-07 | 2005-12-21 | 山东大学 | Glutamic acid capable of having high-yield glutamine |
| CN101517066A (en) * | 2006-07-13 | 2009-08-26 | Cj第一制糖株式会社 | Method for producing L-arginine using Corynebacterium glutamicum |
| CN101805713A (en) * | 2010-02-05 | 2010-08-18 | 北京大学 | Corynebacterium sp strain and application thereof |
| CN102382787A (en) * | 2011-12-13 | 2012-03-21 | 梅花生物科技集团股份有限公司 | Corynebacterium pekinense and application thereof |
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