CN103013876B - L-histidine high-yielding strain and application thereof - Google Patents
L-histidine high-yielding strain and application thereof Download PDFInfo
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- CN103013876B CN103013876B CN201210539597.8A CN201210539597A CN103013876B CN 103013876 B CN103013876 B CN 103013876B CN 201210539597 A CN201210539597 A CN 201210539597A CN 103013876 B CN103013876 B CN 103013876B
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- histidine
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- serratia marcescens
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Abstract
The invention provides an L-histidine high-yielding strain and an application thereof, and belongs to the field of a bioengineering technology. The invention provides Serratia marcescens ZJZG25 which is obtained by the following steps of: by taking Serratia marcescens ATCC (American Type Culture Collection) 31026 as a starting strain, carrying out diethyl sulfate (DES), nitrosoguanidine (NTG) and ultraviolet (UV) gradual-grade mutation; and adding analogues of L-histidine including 3-amino-1,2,4-triazole, 6-purinethol, histidine methyl ester, 2-thiouracil, D-histidine and the like into a basic culture medium and screening. The L-histidine is produced by a strain fermentation method; and compared with the starting strain, the capability of the accumulating high-level L-histidine is expressed. The ZJZ 625 is subjected to shake-flask culture for 72 hours and the yield of the L-histidine can reach to 9.7 g/L. The fermentation is a 3-L fermentation tank is carried out for 60 hours and the yield of the L-histidine can reach to 18.1 g/L.
Description
Technical field
L-Histidine Producing Strain and an application thereof, belong to technical field of bioengineering.
Background technology
L-Histidine is indispensable amino acid to infant, become in human body can with synthetic L-Histidine, to adult, be therefore semi-dispensable amino acid.L-Histidine is mainly used in amino acid transfusion and comprehensive amino-acid reagent, cardiac cycle organ medicine and digestive tract ulcer medicine, treatment anaemia, rheumatism, allergy etc.L-Histidine has different physiological roles, is widely used in medicine, feed and food service industry.At present domestic is mainly with ion exchange process, to extract L-Histidine from pig Hydrolysis of dried blood powder liquid, and because cost is higher, hydrolysising loss rate is high, causes environmental pollution, is not suitable for suitability for industrialized production, therefore accelerates fermentation method of producing L-histidine extremely urgent.
Fermentation method of producing L-histidine is by mutagenic treatment, seed selection analog resistance, auxotroph or q enzyme Auxotrophie mutant.Abroad start to walk early in this respect.The waste wood in field, clear Zhi Zhongshanhe village of Japan be take Corynebacterium glutamicum as starting strain, carry out mutagenesis, directive breeding L-Histidine analog 2-thiazole L-Ala and 1,2,4-triazole L-Ala resistant mutant strain, broken bacterial strain eubolism and regulated, produced acid and reach 6-8g/L (Agric Biol Chem.1971,35 (13): 2081-2088).After again by mutant strain mutagenesis, obtain purine, pyrimidine structure analogue resistant strain, make to produce acid and reach 15g/L (Agric Biol Chem.1974,38 (11): 2091-2096).
Domestic research is in this respect started late.Zeng Ying etc. be take brevibacterium flavum as starting strain, and the enhanced variant obtaining by complex mutation can produce L-Histidine 128.28mg/L (amino acid and Biological resources, 2005,27 (1): 46-48).Gu Zhenghua [21] etc. be take Corynebacterium glutamicum and is carried out mutagenesis as starting strain, and seed selection D-His and 6-azaguanine resistant mutant strain, produce sour 1.6g/L (the journal .2002 of Wuxi Light Industry Univ., 21 (5): 533-535).
Along with the fast development of DNA recombinant technology, people start to utilize molecular biology and microbiology means to carry out bacterial classification transformation, change microbial metabolism approach, thereby improve output.Japan Chuan island utilizes the Histidine Producing Mutant AJ12111 DAN of subtilis K to clone, and obtains the complementary plasmid pAH3 of subtilis.The gene fragment that comprises hisJ from pAH3 excision, imports AJ12111 by carrier, and L-Histidine output is brought up to 8.8g/L (J Mol Biol.1988,203 (3): 585-606) from 6.3g/L.Cang Qiao repaiies etc. subtilis mutagenesis, obtains L-Histidine and produces bacterium AJ11733, is cultured to logarithmic phase, extracts DNA.Again AJ11733 mutagenesis is obtained to deficient strain AJ11732, cultivate and become the cell with absorption DNA ability.DNA is imported to AJ11733, obtain AJ11734.From AJ11734, extract L-Histidine and synthesize and antagonist tolerance gene, import AJ11733, obtain superior strain AJ11735, produce acid and brings up to 23g/L (J.Bacteriol, 1987,169 (2): 823-829) from 11g/L.
Summary of the invention
The object of this invention is to provide a kind of L-Histidine Producing Strain and application thereof.
Technical solution of the present invention is: a kind of L-Histidine Producing Strain, on April 25th, 2012, be preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC NO:M 2012144, its taxonomy called after serratia marcescens ZJZ G25Serratia marcescens ZJZ G25, address: Wuhan, China, Wuhan University.
Described bacterial strain has 3-amino-1,2,4-triazole resistance, and resistance concentration is 2mg/mL.
Described bacterial strain has Ismipur resistance, and resistance concentration is 3mg/mL.
Described bacterial strain has Histidine methyl esters resistance, and resistance concentration is 3mg/mL.
Described bacterial strain tool D-His resistance, resistance concentration is 3mg/mL.
The application method of described bacterial strain, accesses fermention medium by bacterial strain, and described fermention medium is by glucose, (NH
4)
2sO
4, corn steep liquor, KH
2pO
4, MgSO
40.5-1.5 and CaCO
320-40 forms.
Particularly, through optimizing Medium of shaking flask fermentation, be (g/L): glucose 130-160, (NH
4)
2sO
420-40, corn steep liquor 15-25, KH
2pO
41.0-1.5, MgSO
40.5-1.5, CaCO
320-40.
The fermention medium using on fermentor tank is (g/L): glucose 110-130, corn steep liquor 7.0-10.0, ammonium sulfate 20-40, potassium primary phosphate 1.0-2.0, magnesium sulfate 0.5-1.5, calcium carbonate 10.0-20.0, vitamin H 30 μ g/L, Trisodium Citrate 2, VB
14mg/L, calglucon 5-20, pH is adjusted to 7.0.
The invention provides to such an extent that produce the bacterial strain of L-Histidine, that to take serratia marcescens (Serratia marcescens) ATCC 31026 be starting strain, through ethyl sulfate (DES), nitrosoguanidine (NTG) and ultraviolet (UV) mutagenesis step by step, in inclined-plane minimum medium, add L-Histidine analog 3-amino-1,2,4-triazole, Ismipur, Histidine methyl esters, 2-thiouracil and D-His, through cultivating screening and sieving and obtain again.Starting strain: serratia marcescens (Serratia marcescens) ATCC31026.
Fermentation method of producing L-histidine used medium component is:
Seed culture medium (g/L): glucose 25, corn steep liquor 20, urea 1.25, KH
2pO
41.0, MgSO
40.5, pH is adjusted to 7.0-7.2;
Fermention medium is (g/L): glucose 130-160, (NH
4)
2sO
420-40, corn steep liquor 15-25, KH
2pO
41.0-1.5, MgSO
40.5-1.5, CaCO
320-40 (divide and disappear), pH is adjusted to 7.0.Bacterial strain is transferred to fermention medium with 10% inoculum size, and rotary shaking speed is 200-250r/min, pH 6.8-7.0,30 ℃ of culture temperature, incubation time 72h.
3L tank upper reaches adds fermentation basic medium (g/L): glucose 110-130, corn steep liquor 7.0-10.0, ammonium sulfate 20-40, potassium primary phosphate 1.0-2.0, magnesium sulfate 0.5-1.5, calcium carbonate 10.0-20.0, vitamin H 30 μ g/L, Trisodium Citrate 2, VB
14mg/L, calglucon 5-20, pH is adjusted to 7.0.
On fermentor tank, technique is: air flow is 1vvm, and it is 700r/min that 0 ~ 15h controls mixing speed, and after 15h, controlling mixing speed is 600r/min, and carries out suitable feed supplement, and L-Histidine accumulates in fermented liquid.
Beneficial effect of the present invention: from L-Histidine Analogue resistant mutant serratia marcescens (Serratia marcescens) ZJZ G25 (AMT of serratia marcescens (Serratia marcescens) ATCC 31026 seed selections
r6-MP
rhE
r2-TU
rdH
r), remove the feedback inhibition of L-Histidine, so shown the ability of the high-level L-Histidine of accumulation.With this bacterium, passed through the experiment of the fermentation method of producing L-histidine of shaking flask level and 3L fermentor tank level, L-Histidine output reaches respectively 9.7g/L, 18.1g/L, for industrialization is laid a good foundation.
Accompanying drawing explanation
Fig. 1 L-Histidine seed selection pedigree chart
Biological material specimens preservation
The bacterial strain of L-Histidine is produced in one strain, on April 25th, 2012, be preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC NO:M 2012144, its taxonomy called after serratia marcescens ZJZ G25 Serratia marcescensZJZ G25, address: Wuhan, China, Wuhan University.
Embodiment
The mensuration of embodiment 1 starting strain initial flow
By starting strain serratia marcescens (Serratia marcescens) ATCC 31026 line activation, prepare seed culture medium, and the fermention medium of transferring, recording initial flow is 2.1g/L.
Embodiment 2 preparations are to 3-amino-1,2, and 4-triazole has the mutant strain of resistance
By ethyl sulfate mutagenic treatment 20min at 30 ℃ of starting strain serratia marcescens (Serratia marcescens) ATCC 31026 use concentration 2%, be applied to 3-amino-1,2, in 4-triazole (2mg/mL) resistant panel, cultivate 36h for 30 ℃, the bacterial strain that picking grows on this substratum, as to 3-amino-1,2,4-triazole has the mutant strain of resistance.Then carry out L-Histidine fermentation test, in these mutant strains, the mutant strain that L-Histidine output is the highest is called serratia marcescens (Serratiamarcescens) A 07, and L-Histidine output is 3.5g/L.
Embodiment 3 preparations have the mutant strain of resistance to Ismipur
Nitrosoguanidine mutagenic treatment 20min at 30 ℃ of serratia marcescens (Serratia marcescens) the A 07 use 250 μ g/mL that embodiment 2 is obtained, be applied in Ismipur (3mg/mL) resistant panel, cultivate 36h for 30 ℃, the bacterial strain that picking grows on this substratum, as Ismipur being had to the mutant strain of resistance.Then carry out L-Histidine fermentation test, in these mutant strains, the mutant strain that L-Histidine output is the highest is called serratia marcescens (Serratia marcescens) M 20, and L-Histidine output is 4.6g/L.
Embodiment 4 preparations have the mutant strain of resistance to Histidine methyl esters
Serratia marcescens (Serratia marcescens) M 20 uv irradiating mutagenic treatment 40s in 30cm place under 15W ultraviolet lamp that embodiment 3 is obtained, be applied in Histidine methyl esters (3mg/mL) resistant panel, cultivate 36h for 30 ℃, the bacterial strain that picking grows on this substratum, as Histidine methyl esters being had to the mutant strain of resistance.Then carry out L-Histidine fermentation test, in these mutant strains, the mutant strain that L-Histidine output is the highest is called serratia marcescens (Serratia marcescens) H 35, and L-Histidine output is 5.2g/L.
Embodiment 5 preparations have the mutant strain of resistance to 2-thiouracil
Serratia marcescens (Serratia marcescens) H 35 that embodiment 4 is obtained is successively through ultraviolet (15W, 30cm, 40s) and ethyl sulfate (2%, 20min, 30 ℃) mutagenic treatment, be applied in 2-thiouracil (2mg/mL) resistant panel, cultivate 36h for 30 ℃, the bacterial strain that picking grows on this substratum, as 2-thiouracil being had to the mutant strain of resistance.Then carry out L-Histidine fermentation test, in these mutant strains, the mutant strain that L-Histidine output is the highest is called serratia marcescens (Serratia marcescens) T 17, and L-Histidine output is 6.9g/L.
Embodiment 6 preparations have the mutant strain of resistance to D-His
Serratia marcescens (Serratia marcescens) T 17 that embodiment 5 is obtained is successively through ultraviolet (15W, 30cm, 40s) and nitrosoguanidine (250 μ g/mL, 20min, 30 ℃) mutagenic treatment, be applied in D-His (3mg/mL) resistant panel, cultivate 36h for 30 ℃, the bacterial strain that picking grows on this substratum, as D-His being had to the mutant strain of resistance.Then carry out L-Histidine fermentation test, in these mutant strains, the mutant strain that L-Histidine output is the highest is called serratia marcescens (Serratiamarcescens) ZJZ G25, and L-Histidine output is 7.6g/L.
Embodiment 7 is with ZJZ 625 fermentation method of producing L-histidine
From fresh inclined-plane, inoculate a ring ZJZ G25 bacterial classification to seed culture medium (50mL/500mL triangular flask), 30 ℃, 200r/min, cultivates 18h, with 10%-15% inoculum size access fermention medium.The composition of seed culture medium and fermention medium as described in this description.
Shake-flask culture: 250mL triangular flask liquid amount is 15mL, 30 ℃, 200r/min, cultivates 72h, and L-Histidine output is 7.6g/L.
Embodiment 8 is with the synthetic L-Histidine of ZJZ G25 batch fermentation on 3L fermentor tank
Inclined-plane seed culture and fermentation inoculation condition are with embodiment 7, and in 3L fermentor tank, culture volume is 1.8L, and air flow is 1vvm, inoculum size 10%, 31 ± 1 ℃ of culture temperature, adopt two stage oxygen-supply control modes, before, 10h rotating speed is 700r/min, is 600r/min after 10h.Fermentation 56h, L-Histidine output is 14.1g/L.
Embodiment 9 is with the synthetic L-Histidine of ZJZ G25 fed-batch fermentation on 3L fermentor tank
In 8 pairs of stage oxygen supplys of embodiment, control on basis, when glucose concn drops to 20g/L, start feed supplement.The feeding strategy of taking is to mend 10g/L at every turn, and after feed supplement, four hours residual sugar amounts are basic identical with before feed supplement, and glucose consumption is to 20g/L, then carries out feed supplement, common feed supplement four times.Fermentation 60h, L-Histidine output is 18.1g/L.Feed supplement liquid is the glucose of 500g/L.
Be understandable that, for those of ordinary skills, can be equal to replacement or change according to technical scheme of the present invention and inventive concept thereof, and all these changes or replacement all should belong to the protection domain of the appended claim of the present invention.
Claims (2)
1. L-Histidine is produced a bacterium, has been preserved in Chinese Typical Representative culture collection center, and deposit number is CCTCC NO:M2012144.
2. described in claim 1, bacterial strain is applied in L-Histidine fermentative production.
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| CN109082449A (en) * | 2018-09-13 | 2018-12-25 | 陆培廉 | A kind of L-Histidine production technology based on plant source |
| CN111154704B (en) * | 2020-03-30 | 2023-04-11 | 河南巨龙生物工程股份有限公司 | Serratia marcescens mutant strain and method for producing histidine by fermentation |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3902966A (en) * | 1973-06-18 | 1975-09-02 | Tanabe Seiyaku Co | Fermentative preparation of L-histidine |
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2012
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3902966A (en) * | 1973-06-18 | 1975-09-02 | Tanabe Seiyaku Co | Fermentative preparation of L-histidine |
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| L-组氨酸高产菌种选育及其发酵条件研究进展;张淼 等;《发酵科技通讯》;20090731;第38卷(第3期);40－44 * |
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