CN105603017A - Method for producing trans-4-hydroxyl-L-proline by means of fermentation by aid of recombinant corynebacterium acetoacidophilum - Google Patents
Method for producing trans-4-hydroxyl-L-proline by means of fermentation by aid of recombinant corynebacterium acetoacidophilum Download PDFInfo
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- CN105603017A CN105603017A CN201610035787.4A CN201610035787A CN105603017A CN 105603017 A CN105603017 A CN 105603017A CN 201610035787 A CN201610035787 A CN 201610035787A CN 105603017 A CN105603017 A CN 105603017A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/24—Proline; Hydroxyproline; Histidine
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- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
- C12Y114/11002—Procollagen-proline dioxygenase (1.14.11.2), i.e. proline-hydroxylase
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Abstract
The invention discloses a method for producing trans-4-hydroxyl-L-proline by means of fermentation by the aid of recombinant corynebacterium acetoacidophilum. Recombinant plasmids carried by the recombinant corynebacterium acetoacidophilum have proline hydroxylase genes (P4H) capable of catalyzing proline to generate trans-4-hydroxyproline. The method has the advantage that the free L-proline can be converted into the trans-4-hydroxyl-L-proline by the aid of proline-4-hydroxylase in cells of the recombinant corynebacterium acetoacidophilum under the condition of existence of alpha-ketoglutaric acid and ferrous ions. The invention further discloses application of corynebacterium acetoacidophilum to producing the trans-4-hydroxyl-L-proline.
Description
Technical field
Utilize restructuring to have a liking for the method that acetic acid rod bacillus fermentation is produced trans-4-hydroxy-l-proline, belong to microbiological genetic engineering field.
Background technology
Trans-4-Hydroxyproline is widely used in the aspects such as medicine, chemical industry, animal feed and beauty culture. Aspect medical, trans-4-Hydroxyproline is doneFor raw material can be used for synthetic antiphlogistic, Carbapenems. On chemical industry, trans-4-Hydroxyproline can be used as the synthetic multiple polymers of chiral raw material. ?Animal feed application is upper, adds trans-4-Hydroxyproline and can prevent because of the synthetic not enough malnutrition causing of glycine. Trans-4-HydroxyprolineFor superior cosmetics, can eliminate the redox state of oxidant and adjustment cell.
At present, domestic production is trans-and the main method of 4-Hydroxyproline is biological extraction method, utilizes animal protein source, if gelatin, pigskin are raw material,After acid, basic hydrolysis, extract trans-4-Hydroxyproline. The method purification step is long, and cost is high, and waste pollution is serious; And chemical synthesis processSynthetic cost is high, is also not suitable for industrial production. Along with the development of DNA recombinant technique and the discovery of microbial resources, biosynthesis enzyme process is producedHydroxyproline becomes industrial very promising production method.
Biological synthesis process can, by free L-PROLINE by the catalysis of proline hydroxylase, be converted into hydroxyl-L-PROLINE, but in biological cell,Free proline is also a kind of carbon source that can be utilized, and its accumulation is more limited, selects the bacterial strain that can produce in a large number L-PROLINE as hydroxylIt is very important that base-L-PROLINE is produced bacterial strain.
Kinoshita in 1956 etc. are separated to the microorganism MicrococcusglutamicusNo.534 that can accumulate glutamic acid at first from soil, fromAfter this, produce glutamic acid bacterial classification and found successively, according to features such as Physiology and biochemistries, this class is produced to glutamic acid bacterium at present and be referred to as C.glutamicum. ThisThe Corynebacterium acctoacidophlum C.acetoacidophilum that research institute uses is also one wherein. Corynebacterium glutamicum is gram-positive bacteria, is oneAs generally recognized as safe material (GRAS), be the amino acid whose important bacterial classification of industrial production. And this bacterium no pathogenicity, do not produce spore, self extracellular proteaseSecretion disappearance, foodsafety is good.
It is the anti-type of a strain two [sulphaguanidine (SG) that laboratory preservation of bacteria strain is had a liking for acetic acid rod bacillus (Corynebacteriumacetoacidophilum) NO.45And Thioproline (L-S-Pro) has resistance] bacterial strain, this bacterial strain is via dithyl sulfate (DES), nitrosoguanidine (NTG) mutagenic treatment step by stepObtain. This bacterium L-PROLINE output is higher, can be used as hydroxyl-L-PROLINE and produces bacterium.
The method that acetic acid rod bacillus fermentation is produced trans-4-hydroxy-l-proline is had a liking in utilization restructuring provided by the present invention, has important commercial ApplicationBe worth.
Summary of the invention
While producing hydroxyl-L-PROLINE for biological synthesis process, need external source to add L-PROLINE and cause the shortcoming that production cost is higher, order of the present inventionBe to select acetic acid rod bacillus (Corynebacteriumacetoacidophilum) NO.45 that has a liking for of high yield L-PROLINE to carry out bacterium as producing bacterial strainStrain structure, the acetic acid rod bacillus fermentation of having a liking for that obtains restructuring is produced trans-4-hydroxy-l-proline.
The present invention be a kind of by select high yield L-PROLINE have a liking for acetic acid rod bacillus (Corynebacteriumacetoacidophilum) NO.45 withReach the biological synthesis method of high yield trans-4-hydroxy-l-proline. It is characterized in that: that selects high yield L-PROLINE has a liking for acetic acid rod bacillus(Corynebacteriumacetoacidophilum) NO.45, as producing bacterial strain, has a MCS, can be present in multiple Host StrainsPk19mobsacB carries out bacterial strain structure as expression vector, makes organism can obtain a large amount of L-PROLINEs, reaches high yield hydroxyl-L-PROLINEObject.
Proline hydroxylase gene (P4H) in the present invention contains independently efficient promoter-tryptophan Gene expression (Ptrp2), from existingOn recombinant vector, enzyme is cut acquisition.
As expression vector, can be independently duplicated in host cell, for example, have pk19mobsacB, pk18mobsacB etc.
As host cell, to express genes of interest, except using corynebacterium, Colibacter, pseudomonad, bacillus, fermentThe bacterial strains such as female bacterium, also can use zooblast as host.
The present invention can be applied in the production of trans-4-Hydroxyproline, and the culture medium of cultivating recombinant bacterium will contain the utilizable carbon source of microorganism, nitrogenSource, inorganic salts etc., can be natural medium, can be also synthetic media.
As the utilizable nitrogenous source of microorganism, can be ammonium salt class or other nitrogen-containing compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, can also haveThe organic nitrogen sources such as corn steep liquor, yeast extract, peptone, dregs of beans.
As the utilizable inorganic salts of microorganism, there are potassium dihydrogen phosphate, magnesium chloride, ferrous sulfate, calcium chloride etc.
The cultivation of recombinant bacterium need to shake or stir to maintain the required amount of oxygen of thalli growth. Cultivation temperature is at 20-37 DEG C, incubation time 12-72Hour.
Method of the present invention be by restructuring have a liking for acetic acid rod bacillus fermentation trans-4-Hydroxyproline, there is important industrial application value.
Brief description of the drawings
Fig. 1 is the structure of single expression vector pk19mobsacB-Ptrp2-HYP.
Fig. 2 is the acquisition containing single expression vector bacterial strain.
Detailed description of the invention
General explanation: in detailed description of the invention, enzyme used is all bought from TaKaRa company, sanprep pillar DNA extraction agentBox and DNA gel reclaim kit all purchased from the raw work in Shanghai, and each operation is completely according to the explanation of kit.
Seed culture medium (%): glucose 3, beef extract 1.2, peptone 1, urea 0.1, yeast extract 0.5, NaCl0.5, biotin 200ug/L, pH7.0-7.2。
Kan (Amp) resistant panel: 1% (W/V) tryptone, 0.5% (W/V) yeast extract, 1% (W/V) NaCl, 1.5% (W/V)Agarose, kanamycin sulfate (ampicillin) 50 μ g/mL.
5 × KCM (electric shock buffer solution): 0.5MKCl, 0.15MCaCl2，0.25MMgCl2。
The quantitative assay of trans-4-Hydroxyproline: after zymotic fluid is centrifugal, get supernatant, be added in 10mL test tube, afterwards after being diluted to 2.5mLAdd 1mL toluene-sodium-sulfonchloramide (toluene-sodium-sulfonchloramide solution: 1.41g toluene-sodium-sulfonchloramide is dissolved in 10mL water, adds successively 10mL normal propyl alcohol and 80mL bufferingSolution, wherein cushioning liquid formula is: by 50g citric acid, 26.3gNaOH and 146.1g crystallization sodium acetate are water-soluble to 1L, then with 200ML water and 300mL normal propyl alcohol mix), after shaking up, place at room temperature 20min, then add 1mL developer (developer: take 10gParadime thylaminobenzaldehyde, uses 35mL high chloro acid dissolution, slowly adds 65mL isopropyl alcohol), shake up the rear 60 DEG C of water-baths that rapidly test tube moved toIn pot, insulation 20min, then use cold water cooling, finally measure light absorption value at 560nm wavelength place with ultraviolet specrophotometer.
Embodiment 1: the acquisition of the recombinant plasmid (PUC19-Ptrp2-HYP) that contains H4P
Get the bacterial strain that the contains object plasmid activation of preserving in laboratory, in 37 DEG C, 220rpm cultivates 12-16 hour. The bacterium liquid of getting after cultivation is carriedGet plasmid, all operations all carries out in strict accordance with description.
Embodiment 2: the acquisition of the expression vector pk19mobsacB that contains H4P gene
With EcoRI, two kinds of enzymes of BamHI double digestion processing PUC19-Ptrp2-HYP, pk19mobsacB respectively, enzyme is cut the recovery of product glue and is obtained linePtrp2-HYP and the pk19mobsacB fragment of property, be placed in both after 16 DEG C of connections spend the night with T4DNA ligase, by the company of 10 μ LConnect liquid and proceed to e. coli jm109, the single bacterium colony cultivation extraction plasmid after picking transforms carries out enzyme and cuts checking, verifies that correct recombinant plasmid ispk19mobsacB-Ptrp2-HYP。
Embodiment 3: the acquisition of the recombinant bacterial strain (pk19mobsacB-Ptrp2-HYP) that contains H4P gene
Pk19mobsacB-Ptrp2-HYP recombinant plasmid transformed is had a liking for to acetic acid rod bacillus C.acetoacidophilumNO.45, obtain recombinant bacterial strainC.acetoacidophilumNO.45(pk19mobsacB-Ptrp2-HYP)。
Embodiment 4: the fermenting experiment of recombinant bacterial strain
Shaking flask is cultivated: picking recombination bacillus coli list bacterium colony, is inoculated in seed culture medium (sulfur acid kanamycins 50 μ g/mL) 30 DEG C, 220rpmCultivate after 12h, by 5% (V/V) inoculum concentration access contain 30mL fermentation medium [14% (W/V) glucose, 1.5% (W/V) ammonium chloride,2% (W/V) corn steep liquor, 3% (W/V) sodium glutamate, 0.15% (W/V) dipotassium hydrogen phosphate, 100 μ g/mL biotins, 100 μ g/mL thiamines,PH7.0～7.2] 250mL shaking flask in, in rotary shaking table, 30 DEG C, 220rpm are cultivated 36h. Get zymotic fluid and detect trans-4-HydroxyprolineConcentration. The assay method of trans-4-Hydroxyproline refers to the general explanation of embodiment. Fermentation results shows, C.acetoacidophilumNO.45(pk19mobsacB-Ptrp2-HYP) trans-4-Hydroxyproline initial fermentation output reaches 106mg/L.
Claims (5)
1. the method utilizing restructuring to have a liking for acetic acid rod bacillus fermentation to produce trans-4-hydroxy-l-proline, wherein restructuring has a liking for that acetic acid rod bacillus carriesRecombinant plasmid is to plasmid pk19mobsacB by proline hydroxylase gene (P4H) restructuring with the trans-4-Hydroxyproline of catalysis proline generationUpper, obtain having the bioactive recombinant microorganism of synthesis of trans-4-Hydroxyproline.
2. as claimed in claim 1, there is synthesis of trans-4 hydroxyproline biologically active and refer to, biological cell have can utilize external source add orThe proline of oneself accumulation, under proline hydroxylase, KG and ferrous ion acting in conjunction, is converted into trans by free L-PROLINEThe ability of-CHP.
3. carrier claimed in claim 1, multicopy plasmid be including, but not limited to pk19mobsacB, pk18mobsacB, pET-28a, pUC19Deng.
4. the production method of trans-4-Hydroxyproline, is characterized in that the recombinant microorganism described in claim 1,2 to cultivate on culture medium, willThe culture, thalline or their handled thing that arrive, as enzyme source, under the condition existing, are L-by conversion of glucose at glucose in aqueous mediumProline, then be converted into trans-4-Hydroxyproline from L-PROLINE, last the generate trans-4-Hydroxyproline that extracts from this aqueous medium.
5. as described in claim 1 and 2, biological cell comprises anyly can express proline hydroxylase, independently produces KG, by dried meat ammoniaAcid is converted into the organism of hydroxyl-L-PROLINE, comprises and has a liking for acetic acid rod bacillus, Escherichia coli, yeast, animal and plant cells etc.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610035787.4A CN105603017A (en) | 2016-01-19 | 2016-01-19 | Method for producing trans-4-hydroxyl-L-proline by means of fermentation by aid of recombinant corynebacterium acetoacidophilum |
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| CN201610035787.4A CN105603017A (en) | 2016-01-19 | 2016-01-19 | Method for producing trans-4-hydroxyl-L-proline by means of fermentation by aid of recombinant corynebacterium acetoacidophilum |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108220259A (en) * | 2016-12-22 | 2018-06-29 | 清华大学 | L-PROLINE 4- hydroxylases |
| CN109504665A (en) * | 2018-11-22 | 2019-03-22 | 江西师范大学 | A method of improving fish scale gelatin moral character |
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| CN103275998A (en) * | 2013-06-14 | 2013-09-04 | 河北博伦特药业有限公司 | Gene segment with coded and highly-active trans-4-hydroxyl-L-prolyl hydroxylase and application thereof |
| CN104278047A (en) * | 2013-07-04 | 2015-01-14 | 江南大学 | Method for enhancing activity of trans-4-hydroxyproline biosynthesis system containing recombinant DNA |
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2016
- 2016-01-19 CN CN201610035787.4A patent/CN105603017A/en active Pending
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| CN103275998A (en) * | 2013-06-14 | 2013-09-04 | 河北博伦特药业有限公司 | Gene segment with coded and highly-active trans-4-hydroxyl-L-prolyl hydroxylase and application thereof |
| CN104278047A (en) * | 2013-07-04 | 2015-01-14 | 江南大学 | Method for enhancing activity of trans-4-hydroxyproline biosynthesis system containing recombinant DNA |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108220259A (en) * | 2016-12-22 | 2018-06-29 | 清华大学 | L-PROLINE 4- hydroxylases |
| CN108220259B (en) * | 2016-12-22 | 2021-05-07 | 清华大学 | L-proline-4-hydroxylase |
| CN109504665A (en) * | 2018-11-22 | 2019-03-22 | 江西师范大学 | A method of improving fish scale gelatin moral character |
| CN109504665B (en) * | 2018-11-22 | 2022-08-23 | 江西师范大学 | Method for improving quality of fish scale gelatin |
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Application publication date: 20160525 |