CN1314798C - L-leucine high-yield bacterium and fermentation method using the same for L-leucine production - Google Patents
L-leucine high-yield bacterium and fermentation method using the same for L-leucine production Download PDFInfo
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- CN1314798C CN1314798C CNB2005100404311A CN200510040431A CN1314798C CN 1314798 C CN1314798 C CN 1314798C CN B2005100404311 A CNB2005100404311 A CN B2005100404311A CN 200510040431 A CN200510040431 A CN 200510040431A CN 1314798 C CN1314798 C CN 1314798C
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Abstract
The present invention relates to an L-leucine high-yield bacterium and a fermentation method thereof for producing the L-leucine, which belongs to the technical field of biological engineering. The present invention has a Corynebacterium glutamicum JH-27 strain; the Corynebacterium glutamicum JH-27 strain is obtained in a mode that Corynebacterium glutamicum ATCC13032 is used as an original strain and is respectively mutagenized by diethyl sulfate (DES), nitrosoguanidine (NTG) and ultraviolet (UV); then structural analogs of branched chain amino acids are respectively added to a basic culture medium, and sulfaguanidine is added to a complete culture medium; the mutagenized strain is obtained by selection via the basic culture medium and the complete culture medium. The L-leucine high-yield bacterium resists sulfaguanidine and structural analogs of branched chain amino acids. Compared with an original strain, the bacterial strain has the advantage that when the fermentation method of the bacterial strain is used for producing L-leucine, the bacterial strain has the ability of accumulating L-leucine at a high level. The yield of L-leucine is from 30 to 32 g/L after the bacterial strain is treated by JH-27 shaking culture for 60h, and the yield of L-leucine is 40 g/L after the bacterial strain is fermented in a 5L fermentation tank for 55 hours.
Description
Technical field
A kind of L-leucine high yield bacterium and use its producing L-leucine by fermentation, relate to the L-leucine high yield bacterium of a kind of anti-Sulphaguanidine and branched-chain amino acid analog and selection thereof and produce the L-leucine, belong to technical field of bioengineering with this strain fermentation method.
Background technology
The L-leucine is a kind of in common 18 seed amino acids, also is one of 8 kinds of indispensable amino acids of human body, has important purposes in industries such as medicine, food, feed, makeup.Present domestic L-leucine production method is based on the proteolysis extraction method, and as the disclosed technology of CN1056830C, it adopts hair-hydrolyzation to prepare Gelucystine, again preparation L-leucine from waste liquid.Because natural protein raw material amino acid is formed complicated, the extraction process complexity, seriously polluted, yield is lower, and Product Safety is poor, has stimulated biological fermentation process to produce the research enthusiasm of L-leucine Technology greatly.(little wild BUGY CRAXONE CN1072721C) is cultivated Escherichia and Corynebacterium to little wild BUGY CRAXONE in substratum, make the cell sugared together and acetic acid or the generation of its reactant salt and the accumulation L-leucine 27g/L in reaction solution that are produced.In wild wise youth (the wise youth in middle open country CN1100146C) utilizes 5,5, and 5-trifluoro leucine has the Escherichia fermentative production L-leucine of resistance, and acid production rate is 3.9g/L.(Marcovich CN1222576A) selects the mutant strain (Val of Escherichia to Marcovich
r, Al
r, Hleu
r, Leu
r) the leucic output of L-be 1.5～1.7g/L.Zhang Suzhen (microbiology circular, 1989,16 (5): 264～267) the corynebacterium crenatum mutant strain of seed selection such as, produce sour 20g/L.(JOURNAL OF MICROBIOLOGY, 1988,8 (1): 22～25) utilize nitrosoguanidine mutagenesis to handle brevibacterium flavum, shake flask fermentation produces acid and reaches 13.5g/L Gong Jinxiang.Qi Xiulan etc. (Shenyang Pharmacy College's journal, 1993,10 (4), 279～285) utilize the protoplastis of ultraviolet ray, Rifampin, lithium chloride mutagenesis brevibacterium flavum, and screening obtains the L-leucine and produces bacterial strain, shake the acid of bottle product and can reach 24.5g/L.Liu Dangsheng (Shenyang Pharmacy College's journal, 1993,10 (1): 41～44) screen a strain alpha-amino group beta-hydroxy valeric acid and a rifampicin resistance bacterial strain, produce acid in 16 liters of fermentor tanks and can reach 21.8g/L.(food and fermentation industries, 2004,30 (3): 61-65) bottle that shakes of the mutant strain TK0303 of the brevibacterium flavum of seed selection produces acid and can reach 28.3g/L Song Chaoxian etc.More than the acid yield of the leucic production of Bao Dao L-bacterium also has certain distance apart from suitability for industrialized production, the amino acid manufacturer of present China is the L-leucine product of useless fermentative Production still, and the L-leucine that sell the domestic market is all produced with extraction method.Therefore the good L-leucine of seed selection is produced the research emphasis that bacterium remains the fermentation of L-leucine.
Summary of the invention
The L-leucine high yield bacterium that the objective of the invention is to propose a kind of Sulphaguanidine and branched-chain amino acid analog resistance is selection and with this bacterium producing L-leucine by fermentation extremely.
Technical solution of the present invention: the present invention proposes to produce the leucic bacterial strain of L-, its name is called Corynebacterium glutamicum (Corynebacterium glutamicum) JH-27, this bacterial strain is to be starting strain with Corynebacterium glutamicum (Corynebacteriumglutamicum) ATCC13032, respectively through ethyl sulfate (DES), nitrosoguanidine (NTG) and ultraviolet (UV) mutagenic treatment, in minimum medium, add the analog of branched-chain amino acid, in perfect medium, add Sulphaguanidine, get through cultivating screening and multiple sieve.It is carried out the resistance checking, prove conclusively this bacterial strain and have following hereditary property: leucine hydroxamate resistance (LeuHx
r), resistance concentration is 5～10g/L; 2-thiazole L-Ala resistance (2-TA
r), resistance concentration is 5～10g/L; Butyrine resistance (α-AB
r), resistance concentration is 20～40g/L; , beta-hydroxy leucine resistance (β-HL
r), resistance concentration is 10～15g/L; Sulphaguanidine resistance (SG
r), resistance concentration is 15～25g/L.
Starting strain: starting strain is Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032.
Various substratum used in mutagenic treatment and the screening process consist of:
Inclined-plane and seed culture medium: glucose 1g, peptone 10g, extractum carnis 10g, yeast extract paste 5g, NaCl2.5g, agar powder 20g is settled to 1L with distilled water, pH7.0～7.2.
Perfect medium: glucose 5g, peptone 10g, extractum carnis 10g, yeast extract paste 5g, NaCl2.5g, agar powder 20g is settled to 1L with distilled water, pH7.0～7.2.
Minimum medium (MM): glucose 20g, ammonium sulfate 10g, KH
2PO
43H
2O1g, MgSO
47H
2O0.4g, FeSO
47H
2O0.01g, MnSO
4H
2O0.01g, V
H200 μ g, V
B1200 μ g, agar powder 20g is settled to 1L with distilled water, pH7.0～7.2.
Screening culture medium (CM): the analog that adds branched-chain amino acid at the MM substratum; On the basis of perfect medium, add Sulphaguanidine.
The used substratum of producing L-leucine by fermentation consists of:
Seed culture medium: glucose 20g, corn steep liquor 25ml, ammonium sulfate 2g, KH
2PO
43H
2O1.5g, V
H200 μ g, V
B1300 μ g are settled to 1L with tap water.pH7.0～7.2。
Fermention medium: glucose 130～170g, corn steep liquor 20～4ml, ammonium sulfate 20～40g, KH
2PO
43H
2O0.5～1.5g, MgSO
47H
2O0.2～0.6g, MnSO
4H
2O0.05～0.15g, V
H25～75 μ g, V
B1250～350 μ g, CaCO
320～40g is settled to 1L with tap water, pH7.0～7.2.
Bacterial strain is transferred in the fermention medium with 16～15% (W/W) inoculum size, under aeration condition, cultivates pH6～8 with shake-flask culture or fermentor tank ventilation; 30～35 ℃ of culture temperature, incubation time are 50～65 hours, and the shake-flask culture rotating speed is 200rpm, or the fermentor cultivation air flow is 3L/min, and mixing speed is 600rpm, and the L-leucine accumulates in fermentor tank.
Beneficial effect of the present invention: from the Sulphaguanidine of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 seed selection and resistant mutant strain Corynebacterium glutamicum (Corynebacterium glutamicum) JH-27 of branched-chain amino acid analog, show the leucic ability of the high-level L-of accumulation, this is owing to removed the feedback repression of three branched-chain amino acids with respect to starting strain.The test of the producing L-leucine by fermentation by shaking bottle level and 5L fermentor tank level with this bacterium, the leucic output of L-reaches 30～32g/L, 40g/L respectively, has industrial applicibility and is worth.
Description of drawings
Fig. 1 seed selection pedigree chart.
The biological material specimens preservation
A kind of bacterial strain of producing L-Leu, this bacterial strain are Corynebacterium glutamicum (Corynebacterium Glutamicum) JH-27 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, Its deposit number is CGMCC NO.1378, and preservation date is on May 24th, 2005.
Embodiment
Embodiment 1 preparation has the mutant strain of resistance to the leucine hydroxamate.
With starting strain Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 ethyl sulfate (1%, 30 ℃, 30 minutes) carry out conventional mutagenic treatment, be coated on then on the minimum medium of the leucine hydroxamate that contains 5-10g/L, cultivated 2～4 days for 30 ℃, the bacterium colony that picking is grown on this substratum, as the mutant strain that the leucine hydroxamate is had resistance, carry out the leucic fermentation test of L-then, in these mutant strains, the highest mutant strain of L-leucine output is called Corynebacterium glutamicum (Corynebacteriumglutamicum) L1-68, and the leucic output of its L-is 8.9g/L.
Embodiment 2 preparations have the mutant strain of resistance to 2-thiazole L-Ala.
Corynebacterium glutamicum (Corynebacterium glutamicum) L1-68 ethyl sulfate (1% with embodiment 1 acquisition, 30 ℃, 25 minutes) carry out conventional mutagenic treatment, be coated on then on the minimum medium of the 2-thiazole L-Ala that contains 5-10g/L, cultivated 2～4 days for 30 ℃, the bacterium colony that picking is grown on this substratum, as the mutant strain that 2-thiazole L-Ala is had resistance, carry out the leucic fermentation test of L-then, in these mutant strains, the highest mutant strain of L-leucine output is called Corynebacterium glutamicum (Corynebacteriumglutamicum) L2-132, and the leucic output of its L-is 13g/L.
Embodiment 3 preparations have the mutant strain of resistance to butyrine.
Corynebacterium glutamicum (Corynebacterium glutamicum) L2-132 nitrosoguanidine (0.3% with embodiment 2 acquisitions, 30 ℃, 45 minutes) carry out conventional mutagenic treatment, be coated on then on the minimum medium of the butyrine that contains 20-40g/L, cultivated 2～4 days for 30 ℃, the bacterium colony that picking is grown on this substratum, as the mutant strain that butyrine is had resistance, carry out the leucic fermentation test of L-then, in these mutant strains, the highest mutant strain of L-leucine output is called Corynebacterium glutamicum (Corynebacteriumglutamicum) L3-206, and the leucic output of its L-is 18g/L.
Embodiment 4 preparations have the mutant strain of resistance to the beta-hydroxy leucine.
Corynebacterium glutamicum (Corynebacterium glutamicum) L3-206 nitrosoguanidine (0.3% with embodiment 3 acquisitions, 30 ℃, 30 minutes) carry out conventional mutagenic treatment, be coated on then on the leucic minimum medium of the beta-hydroxy that contains 10-15g/L, cultivated 2～4 days for 30 ℃, the bacterium colony that picking is grown on this substratum, as the mutant strain that the beta-hydroxy leucine is had resistance, carry out the leucic fermentation test of L-then, in these mutant strains, the highest mutant strain of L-leucine output is called Corynebacterium glutamicum (Corynebacteriumglutamicum) L4-89, and the leucic output of its L-is 25g/L.
Embodiment 5 preparations have the mutant strain of resistance to Sulphaguanidine.
Corynebacterium glutamicum (Corynebacterium glutamicum) L4-89 that embodiment 4 is obtained uses ultraviolet (15W earlier, 30cm, 6 minutes) carry out conventional mutagenic treatment, use nitrosoguanidine (0.2% again, 30 ℃, 20 minutes) carry out conventional mutagenic treatment, be coated on then on the perfect medium of the Sulphaguanidine that contains 15-25g/L, cultivated 2～4 days for 30 ℃, the bacterium colony that picking is grown on this substratum, as the mutant strain that Sulphaguanidine is had resistance, carry out the leucic fermentation test of L-then, in these mutant strains, the highest mutant strain of L-leucine output is called Corynebacterium glutamicum (Corynebacterium glutamicum) JH-27, and its L-is leucic to be 30～32g/L for measuring.
Embodiment 6 is with the JH-27 producing L-leucine by fermentation.
Go into seed culture medium (25ml/500ml triangular flask) from fresh inclined-plane inoculation one ring JH-27 bacterial classification, at 30 ℃, 200rpm cultivates after 14 hours down, inserts in the fermention medium with 10-15% inoculum size (V/V).The composition of seed culture medium and fermention medium as described in this manual.
Shake-flask culture: the liquid amount of fermention medium is 20ml in the 500ml triangular flask, and rotating speed 200rpm, fermentation time are 60 hours, and the leucic output of L-is 30～32g/L.
Embodiment 7 synthesizes the L-leucine with JH-27 at the 5L fermentor tank.
Inclined-plane seed culture and fermentation inoculation condition are all with embodiment 6, and 5L fermentation cylinder for fermentation culture volume is 3L.Air flow is 3L/min, and mixing speed is 600rpm, and leavening temperature is 30-35 ℃, and fermentation time is 50-55 hour.L-leucine output is 40g/L.
Claims (2)
1. produce the leucic bacterial strain of L-for one kind, its classification called after Corynebacterium glutamicum (Corynebacteriumglutamicum) JH-27, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, its deposit number is CGMCC NO.1378.
2. produce the leucic method of L-with the leucic strain fermentation method of the described production of claim 1 L-for one kind, it is characterized in that fermention medium is: glucose 130～170g, corn steep liquor 20～40ml, ammonium sulfate 20～40g, KH
2PO
43H
2O 0.5～1.5g, MgSO
47H
2O 0.2～0.6g, MnSO
4H
2O 0.05～0.15g, V
H25～75 μ g, V
B1250～350 μ g, CaCO
320～40g is settled to 1L with tap water, pH7.0～7.2; The CGMCCNO.1378 bacterial strain is transferred in the fermention medium with 10%～15% (weight) inoculum size, under aeration condition, cultivates pH6～8 with shake-flask culture or fermentation ventilation; 30～35 ℃ of culture temperature, incubation time are 50～65 hours, and the shake-flask culture rotating speed is 200 rev/mins, or the fermentor cultivation air flow is 3L/ minute, and mixing speed is 600 to change part clocks, and the L-leucine accumulates in fermented liquid.
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102093962B (en) * | 2009-08-28 | 2012-08-08 | 吉林大学 | Lactococcus lactis induced strain and breeding method thereof |
| CN103031265B (en) * | 2013-01-11 | 2014-04-16 | 新泰市佳禾生物科技有限公司 | Corynebacterium glutamicum mutant strain and application thereof in production of L-leucine by fermentation method |
| KR101796830B1 (en) * | 2015-08-25 | 2017-11-13 | 씨제이제일제당 (주) | A microorganism having enhanced L-leucine production and process for preparing L-leucine using the same |
| CN106754807B (en) * | 2016-12-29 | 2020-10-30 | 廊坊梅花生物技术开发有限公司 | L-leucine producing strain and method for producing L-leucine |
| CN108841758B (en) * | 2018-07-13 | 2021-11-26 | 大连医诺生物股份有限公司 | Corynebacterium glutamicum mutant strain and application thereof in L-leucine production |
| CN109294966A (en) * | 2018-10-26 | 2019-02-01 | 江南大学 | A kind of the Corynebacterium glutamicum recombinant bacterium and its construction method of high yield L-Leu |
| CN114908079A (en) * | 2019-03-22 | 2022-08-16 | 三峡大学 | Breeding method of corynebacterium glutamicum capable of producing isoleucine at high yield |
| CN116731950A (en) * | 2023-08-10 | 2023-09-12 | 中国科学院天津工业生物技术研究所 | Corynebacterium glutamicum stress-resistant engineering bacterium and application thereof in production of acidic bio-based chemicals |
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Non-Patent Citations (3)
| Title |
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| L-亮氨酸产生菌的选育及其发酵条件优化 宋超先，陈宁，谢玉锋，柴凌云，张克旭，天津科技大学学报，第19卷第1期 2004 * |
| L-亮氨酸产生菌的选育及其发酵条件优化 宋超先，陈宁，谢玉锋，柴凌云，张克旭，天津科技大学学报，第19卷第1期 2004;一株L-亮氨酸产生菌的研究 刘党生 来彩霞 李福德，沈阳药学院学报，第11卷第2期 1994 * |
| 一株L-亮氨酸产生菌的研究 刘党生 来彩霞 李福德，沈阳药学院学报，第11卷第2期 1994 * |
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Address after: 214000 Donggang Town, Xishan District, Wuxi City, Jiangsu Province Patentee after: WUXI JINGHAI AMINO ACID CO., LTD. Address before: 214000 Donggang Town, Xishan District, Wuxi City, Jiangsu Province Patentee before: Wuxi Jinghai Amino Acid Co., Ltd. |
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