CN103865940B - A kind of ILE '-hydroxylase gene and genetic engineering bacterium and application - Google Patents
A kind of ILE '-hydroxylase gene and genetic engineering bacterium and application Download PDFInfo
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- CN103865940B CN103865940B CN201410132444.0A CN201410132444A CN103865940B CN 103865940 B CN103865940 B CN 103865940B CN 201410132444 A CN201410132444 A CN 201410132444A CN 103865940 B CN103865940 B CN 103865940B
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- ile
- hydroxyisoleucine
- ido
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- hydroxylase
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- OSCCDBFHNMXNME-UHFFFAOYSA-N gamma-hydroxyisoleucine Natural products CC(O)C(C)C(N)C(O)=O OSCCDBFHNMXNME-UHFFFAOYSA-N 0.000 claims abstract description 29
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention belongs to biological technical field, be specifically related to the ILE '-hydroxylase gene of one catalysis ILE generation specifically (2S, 3R, 4S)-4-hydroxyisoleucine, and the genetic engineering bacterium that contains this gene. Utilize this bacterial strain to produce (2S, 3R, 4S)-4-hydroxyisoleucine, can overcome that the recovery rate that extraction method exists is low, separation and purification is difficult, Raw Material Demand amount is large, high in cost of production is not enough; Chemical synthesis severe reaction conditions, the problem that step is many, separation difficulty yield is low; And enzymatic conversion method rate is low and the problem that contains the accessory substances such as a large amount of butyrines, make only to exist in zymotic fluid (2S, 3R, the 4-hydroxyisoleucine of 4S)-a kind of configuration of 4-hydroxyisoleucine, and further improve transformation efficiency by the method that ILE hydroxylase encoding gene is crossed expression, thereby reach the object of simplifying technique, reducing production costs.
Description
Technical field:
The present invention relates to one catalysis ILE (L-Ile) generation specifically (2S, 3R, 4S)-4-hydroxyl differentLeucic ILE '-hydroxylase gene and the genetic engineering bacterium that contains this gene, utilize this bacterial strain produce (2S, 3R,4S)-4-hydroxyisoleucine product is single, has higher conversion ratio, belongs to biological technical field.
Background technology:
4-hydroxyisoleucine (4-Hydroxyisoleucine, 4-HIL) is that one is mainly present in Trigonella plantILE hydroxylate in seed. Research shows that 4-hydroxyisoleucine has the promotion insulin that concentration of glucose relies onActive and the promotion myocyte of secretion absorbs, accelerates the effect of fat metabolism, reducing blood lipid and liver function protecting to blood sugar. CauseThis, as the ideal medicament of effective prevention and treatment diabetes and obesity, before 4-hydroxyisoleucine has a wide range of applicationsScape and the market demand.
The production method of the 4-hydroxyisoleucine of having reported at present comprises extraction method, chemical synthesis and enzyme process, butOnly extraction method is for suitability for industrialized production. Research shows that the 4-hydroxyisoleucine configuration of the method acquisition is more, but only (2S, 3R,4S)-4-hydroxyisoleucine has above-mentioned BA, (is only 0.091-thereby make the method exist recovery rate low0.6%), separation and purification difficulty, Raw Material Demand amount is large, high in cost of production is not enough. The report of the synthetic 4-hydroxyisoleucine of chemical methodMore, but be mainly to generate through multistep chemical reaction taking glucose, menthones or 2-methyl-acetoacetic ester as raw material, the partyMethod severe reaction conditions, step is many, separation difficulty yield is low, and easily causes environmental pollution. Enzyme process is synthetic be with hexanal, α-Ketoglutaric acid and Pidolidone are that substrate is through 4-hydroxy-3-methyl-2-ketone group-valeric acid aldolase and branched chain amino acid aminoTransferase two-step catalysis is synthetic, in the method low conversion rate and product, contains the accessory substances such as a large amount of butyrines.
Therefore, adopting synthetic (2S, 3R, the 4S)-4-hydroxyisoleucine of specificity ILE hydroxylase catalysis isFor desirable method. Can be by ILE (L-although Haefel é etc. find in the broken liquid of fenugreek seedIsoleucine, L-Ile) be converted into the active component of (2S, 3R, 4S)-4-hydroxyisoleucine, but so far there are no follow-up studyReport. Kodera etc. (2009) reported first in bacillus thuringiensis (Bacillusthuringiensis) find energyCatalysis L-Ile generates the ILE hydroxylase of (2S, 3R, 4S)-4-hydroxyisoleucine specifically. But announce at presentGenerate the base of the ILE hydroxylase of (2S, 3R, 4S)-4-hydroxyisoleucine about catalysis L-Ile specificallyLimited because of sequence, and have no and adopt microbe transformation method to produce the report of (2S, 3R, 4S)-4-hydroxyisoleucine.
Summary of the invention:
An object of the present invention is to provide one catalysis L-Ile generation specifically (2S, 3R, 4S)-4-hydroxyl differentLeucic ILE '-hydroxylase gene (representing with ido), the nucleotide sequence of this gene is as sequence table SEQ IDNO:1Shown in. The amino acid sequence of its coded ILE hydroxylase is as shown in sequence table SEQ IDNO:2.
Another object of the present invention is to provide a kind of genetic engineering bacterium that comprises described ILE '-hydroxylase gene,Utilize gene clone technology that described ILE '-hydroxylase gene is connected on suitable carrier, and be converted into host cellExpress, acquisition can production specificity catalysis L-Ile generates the ILE of (2S, 3R, 4S)-4-hydroxyisoleucineThe genetic engineering bacterium of hydroxylase.
Another object of the present invention be for the production of current (2S, 3R, 4S)-4-hydroxyisoleucine because extracting (orReaction) isomorphism type 4-hydroxyisoleucine does not coexist in liquid, but have bioactive (2S, 3R, 4S)-4-hydroxyisoleucine denseSpend lowly, cause and have the problems such as Raw Material Demand amount is large, cost is high, seriously polluted, provide a kind of with ILE and α-oneGlutaric acid is substrate, adopts the production method of synthetic (2S, 3R, the 4S)-4-hydroxyisoleucine of microbe transformation method, makes fermentationIn liquid, only there is the 4-hydroxyisoleucine of a kind of configuration of (2S, 3R, 4S)-4-hydroxyisoleucine, and pass through ILEThe method that hydroxylase encoding gene is crossed expression further improves transformation efficiency, thus reach simplify technique, reduce production costs.
One of technical scheme that the present invention solves the problems of the technologies described above is: a kind of side that builds said gene engineering bacteria is providedMethod, comprises the steps:
(1) structure of recombinant plasmid
Taking ILE hydroxylase encoding gene of the present invention as template, with IDO-3(SEQIDNO:3) andIDO-4(SEQIDNO:4) be primer, carry out pcr amplification;
IDO-3：5’-CCGGTCGACAAGGAAGCTAGATATGAAAATGAGTGGCTTTAGCATAG-3’(SalⅠ)
IDO-4：5’-CCGGAATTCTTATTTTGTCTCCTTATAAGAAAATGTTACTA-3’(EcoRⅠ)
By pcr amplification product through reclaim after carry out double digestion through Sal I and EcoR I respectively, through electrophoresis, cut glue reclaim afterBe connected to expression vector pXMJ19, obtain recombinant plasmid.
(2) structure of recombinant bacterial strain
The recombinant plasmid electricity that (1) is obtained is converted in Corynebacterium glutamicum ATCC13032 cell, obtains recombinant bacteriumStrain.
The present invention addresses the above problem two of adopted technical scheme: provide a kind of microbe transformation method produce (2S,3R, 4S) method of-4-hydroxyisoleucine, comprise the following steps:
(1) seed culture: 1-2 is propped up to the said gene engineering bacteria through fresh slant activation with oese, be seeded to and be equipped withThe 1L flask with indentation of 100mL seed culture medium, in 32-37 DEG C, 200rpm shaken cultivation 6-8h.
(2) fermented and cultured of thalline: the inoculum of step (1) is seeded to 3L fermentation is housed with 5%-10% inoculum concentrationThe 5L fermentation tank of culture medium is sent out tank and is cultivated, fermentation temperature 32-37 DEG C, and dissolved oxygen maintains 20-40%, and it is 60-that stream adds concentrationThe glucose solution of 80% (W/V), the ammoniacal liquor that stream adds 25% (W/V) regulates zymotic fluid pH to 6.8-7.2, fermented and cultured 8-12h. PressAccording to 10% inoculum concentration, primary seed solution is transferred into 5L Fermentation, liquid amount is 3L, and dissolved oxygen is controlled at 20%-30%, cultivates temperatureSpend 33 DEG C, pH is controlled at 7.0 left and right.
(3) wait ILE and the KG sterile solution of amount of substance concentration to interpolation in fermentation tank, make itFinal concentration is 0.1-0.3mol/L, continues to cultivate 18-24h by step (2). Cultivate through 24-36h, can be by 91.7% L-IleBe converted into (2S, 3R, 4S)-4-hydroxyisoleucine.
(4) after adopting DNF to (2S, 3R, 4S)-4-isoleucine derivatization, with high performance liquid chromatographyMeasure its content.
Described slant medium composition is: glucose 1.0-5.0g/L, and peptone 10-15g/L, beef extract 10-15g/L,NaCl2.0-3.0g/L, agar 25g/L, pH7.0-7.2,0.075MPa high pressure steam sterilization 30min.
Described seed culture based component is: glucose 25-30g/L, corn steep liquor 15-30mL/L, soybean meal hydrolysate 15-30mL/L，KH2PO40.5-1.0g/L，MgSO4·7H2O0.1-0.5g/L，MnSO415-30mg/L，FeSO410-30mg/L, urineElement 2-5/L, pH7.0-7.2,0.075MPa high pressure steam sterilization 30min.
Described fermented and cultured based component is: glucose 60-80g/L, K2HPO43.0-5.0g/L，MgSO41.0-2.0g/L，MnSO420-50mg/L，FeSO420-50mg/L, corn steep liquor 20-50mL/L, soybean meal hydrolysate 15-30mL/L, 0.075MPa high pressureSteam sterilizing 30min.
Beneficial effect:
1, the ILE hydroxylase of ido gene code of the present invention has following features:
The Km of this enzyme and Vmax are respectively 0.15mmol/L and 3.13 μ mol/min/mg; Enzyme work rises with the rising of temperatureHeight, in the time that temperature reaches 35 DEG C, its activity is the highest, and along with the continuation of temperature raises, its activity is rapid to be reduced, when temperature is higher than 65 DEG CTime activity completely lose (see figure 7); When pH7.0, activity is the highest, when pH is higher or lower than 7.0 time, and along with departing from of pH, its activity(see figure 8) sharply declines; Process 5h under 35 DEG C of conditions after, still there is 87.5% activity.
2, production method of the present invention has overcome the recovery rate low (being only 0.091-0.6%) that extraction method exists, has separatedPurification difficult, Raw Material Demand amount is large, high in cost of production is not enough; Chemical synthesis severe reaction conditions, step are many, separation difficulty is receivedRate is low, and easily causes the problem of environmental pollution; And enzymatic conversion method rate is low and product in contain a large amount of butyrines etc.The problem of accessory substance, makes only to exist in zymotic fluid the 4-hydroxyl of a kind of configuration of (2S, 3R, 4S)-4-hydroxyisoleucine different brightPropylhomoserin, and further improve transformation efficiency by the method that ILE hydroxylase encoding gene is crossed expression, thus reach letterMetallization processes, reduce production costs.
3, taking genetic engineering bacterium of the present invention as catalyst, ILE and KG are substrate, utilizeMicrobe transformation method synthesizes (2S, 3R, 4S)-4-hydroxyisoleucine, and selectivity is strong, and transformation efficiency is higher than 75%, and technique is simple and easy toFeasible, with low cost, be applicable to the green manufacturing of (2S, 3R, 4S)-4-hydroxyisoleucine.
Brief description of the drawings:
Fig. 1 recombinant plasmid pXMJ-ido design of graphics;
Fig. 2 ido gene PCR product electrophoresis pattern
Wherein M be Marker swimming lane 1 taking IDO-1 and IDO-2 as primer, grand genome as template swimming lane 2 with IDO-3 andIDO-4 is that primer, pET-ido are template;
The restriction enzyme digestion and electrophoresis collection of illustrative plates of Fig. 3 recombinant plasmid pET-ido and pXMJ-ido
Wherein M be Marker swimming lane 1 for pET-ido through Nde I and Xho I double digestion swimming lane 2 for pXMJ-ido through Sal I andEcoR I double digestion;
The recombinate SDS-PAGE electrophoresis pattern of ILE hydroxylase of Fig. 4
Wherein M is that Marker swimming lane 1 is the broken liquid of E.coliBL21 (DE3) containing empty plasmid pET-42a
Swimming lane 2 is to be the broken liquid swimming lane 4 of the BL-IDO through IPTG induction without the broken liquid swimming lane 3 of BL-IDO of IPTG inductionFor purified restructuring ILE hydroxylase;
Fig. 5 (2S, 3R, 4S)-4-isoleucine standard items and the present invention ILE hydroxylase product of recombinatingHPLC and MS collection of illustrative plates
Wherein A is (2S, 3R, 4S)-4-isoleucine standard items HPLC collection of illustrative plates
B is the product HPLC collection of illustrative plates of restructuring ILE hydroxylase
C is contrast HLPC collection of illustrative plates after DNF derivatization
D is (2S, 3R, 4S)-4-isoleucine standard items MS collection of illustrative plates
E is the product MS collection of illustrative plates of restructuring ILE hydroxylase
Retention time is that the chromatographic peak of 21.6min is the product of restructuring ILE hydroxylase
Retention time is that the chromatographic peak of 13.5min is derivating agent DNF
Retention time is that the chromatographic peak of 23.8min is ILE;
Fig. 6 double reciprocal curve;
The impact of Fig. 7 temperature on ILE hydroxylase activity of the present invention;
The impact of Fig. 8 pH on ILE hydroxylase activity of the present invention;
The recombinate heat endurance of ILE hydroxylase of Fig. 9 the present invention;
Detailed description of the invention
Embodiment 1:L-isoleucine hydroxylase encoding gene ido obtains
Adopt and extract with the following method soil metagenome:
Get the following 10cm of the University Of Science and Technology Of Tianjin's garden topsoil 10g of place soil sample and be placed in shaking flask, add 20mLDNA to extractLiquid (100mol/LTris-HCl, 100mmol/LEDTA2Na; 100mmol/LNa3PO4,1.5mol/LNaCl, 2%CTAB,PH8.0) mix. Add 500 μ L lysozymes (50mg/mL, pH8.0), mix rear 37 DEG C of water-bath 30min. Be placed in ultrasonic cleaning instrumentAfter middle processing 10min, add 1.5g sterile glass beads (diameter 1mm), vibration 5min. Add 40 μ L Proteinase Ks (20mg/mL), mixedAfter even, add 2mL20%SDS, 65 DEG C of water-bath 1h, jiggle every 15-20min again. In the centrifugal 10min of room temperature 10000g, receiveCollection supernatant is transferred in another centrifuge tube. With isopyknic phenol and the each extracting of chloroform/isoamyl alcohol (24:1) once, 10000gCentrifugal 15min. Draw supernatant and be transferred in another centrifuge tube, add 0.6 times of volume isopropyl alcohol to mix, more than precipitation 2h,The centrifugal 20min of 10000g. Collection nucleic acid precipitation, with 75% cold ethanol washing 2 times, is dissolved in appropriate amount of deionized water after being dried.
Taking above-mentioned soil metagenome DNA as template, utilize primer I DO-1(SEQIDNO:5) and IDO-2(SEQIDNO:6), by pcr amplification ido gene (ido gene PCR product electrophoresis pattern is shown in Fig. 2);
IDO-1：5’-_CCGCATATGAAAATGAGTGGCTTTAGCATAG-3’(NdeⅠ)
IDO-2：5’-CCGCTCGAGTTATTTTGTCTCCTTATAAGAAAATGTTACTA-3’(XhoⅠ)
PCR condition is: 94 DEG C 5min1 circulation, 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C 1min30 circulation, 72 DEG C of 10min1Individual circulation, reaction system is 100 μ L. Getting 10 μ LPCR products detects through 1.5% agarose gel electrophoresis.
After being reclaimed, the object fragment of pcr amplification from macro genome DNA is connected to pMDTM18-TVector is also converted intoIn Escherichia coli E.coliDH5 α competent cell, then coat the LB solid culture containing ampicillin (100 μ g/mL)Upper, be inverted and cultivate 24h in 37 DEG C. 3 monoclonals of picking, extract recombinant plasmid (by the ido fragment on 3 recombinant plasmids respectivelyCalled after ido-1, ido-2, ido-3) and measure ido sequence.
Sequencing result shows, above-mentioned 3 ido genes are all containing 723 nucleotides, 240 amino acid of encoding. Its nucleotides sequenceRow are respectively 100%, 97.47% and 96.13% with the ido gene order similarity of the B.thuringiensis2-e-2 reporting,Its amino acid sequence similarity is respectively 100%, 97.91% and 93.33%.
Embodiment 2: the structure of recombinant plasmid pET-ido and ILE hydroxylase encoding gene ido functional analysis
Extraction is carried out agarose electrophoresis after also cutting with restriction enzyme Nde I and Xho I enzyme containing the recombinant plasmid of ido-3,Cut after glue reclaims and be connected to the expression vector pET-42a cutting through same enzyme, obtain recombinant plasmid pET-ido, be converted intoE.coliBL21(DE3) in competent cell, obtain recombinant bacterial strain BL-IDO.
BL-IDO inoculum is seeded in LB fluid nutrient medium, adopts 0.1mmol/LIPTG abduction delivering 4h.Get 1mL culture and collect thalline in the centrifugal 1min of 4 DEG C of 10000g, and with 1mL buffer A (Na2HPO4·12H2O2.9g/L,NaCl8.5g/L,NaH2PO4·2H2O0.30g/L, pH7.4) use again 1mL buffer A resuspended after washing bacterial sediment 3 times. With superThe above-mentioned bacteria suspension of the broken instrument ultrasonication of sound, condition of work is: power 350W, working time 5sec, interval time 10sec, 5Circulation, in operation on ice. Get the above-mentioned broken liquid of part and carry out SDS-PAGE. After coomassie brilliant blue staining result as shown in Figure 4,There is the band of an about 29.0kDa of molecular weight in the broken liquid of BL-IDO, with ILE hydroxylase theoretical molecular28.92kDa(is containing 6 × His label) consistent.
To after centrifugal residue bacterial cell disruption liquid, get supernatant and adopt Ni-NTA affinity chromatography separation and purification restructuring L-differentLeucine hydroxylase occurs that molecular weight is about single of 29kDa after dialysis, concentrated, SDS-PAGE, coomassie brilliant blue stainingBand (Fig. 4), adopting its concentration of BCA protein quantification kit measurement is 3.5mg/mL.
Get appropriate recombinase liquid and measure its enzymatic activity. Reaction condition is: ILE 10mmol/L, KG10mmol/L，FeSO40.5mmol/L, ascorbic acid 10mmol/L, restructuring ILE hydroxylase enzyme liquid 50 μ L(are to addIsopyknic buffer A is contrast), supplement volume to 1mL by buffer A, after 35 DEG C of reaction 30min, add 10 μ L acetic acid eventuallyOnly reaction.
Adopt DNF to (2S, 3R, 4S)-4-isoleucine standard items and get above-mentioned reactant liquor derivatizationAfter, adopt high performance liquid chromatography GC-MS to identify it. Actual conditions is as follows: chromatographic column is AgilentZORBAXEclipsAAA (4.6 × 150mm, 5-Micron), 33 DEG C of column temperatures, detect wavelength 360nm. Mobile phase A and B are respectively concentration50% acetonitrile and acetate buffer (50mmol/L, pH6.4), flow velocity is 1.0mL/min. Mass spectrum condition is: electron spray ionSource, positive ion mode detects, and capillary voltage is 2000V, and sampling taper hole voltage is 15V, and extraction taper hole voltage is 3V, ion gunTemperature is 120 DEG C, and desolventizing temperature degree is 400 DEG C, and desolventizing gas velocity is 400L/h, adopts full scan mode, sweep limitsM/z=0～400. Testing result shows, produces through standard items and the restructuring ILE hydroxylase enzyme liquid catalytic reaction of derivatizationThing all occurs that retention time is the chromatographic peak of 21.6min, contrast without. Respectively this chromatographic peak is carried out to mass spectral analysis, resultShow that its karyoplasmic ratio is 311.9(Fig. 5), through being accredited as after (2S, 3R, 4S)-4-isoleucine reacts with DNF2 of generation, 4-dinitrophenyl-4-hydroxyisoleucine (its molecular weight is 313.3), therefore prove ido coded by said gene albumenThere is the activity that catalysis ILE generates (2S, 3R, 4S)-4-isoleucine.
Embodiment 3: the structure (building process is shown in Fig. 1) of recombinant plasmid pXMJ-ido and recombinant bacterial strain CG-ido
Taking recombinant plasmid pET-ido as template, utilize primer I DO-3 and IDO-4, its ido gene (ido gene PCR increasesProduct electrophoresis pattern is shown in Fig. 2);
IDO-3：5’-CCGGTCGACAAGGAAGCTAGATATGAAAATGAGTGGCTTTAGCATAG-3’(SalⅠ)
IDO-4：5’-CCGGAATTCTTATTTTGTCTCCTTATAAGAAAATGTTACTA-3’(EcoRⅠ)
PCR condition is: 94 DEG C 5min1 circulation, 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C 1min30 circulation, 72 DEG C of 10min1Individual circulation, reaction system is 100 μ L. Getting 10 μ LPCR products detects through 1.5% agarose gel electrophoresis.
Remaining 90 μ LPCR amplified productions are carried out to double digestion through Sal I and EcoR I respectively after reclaiming, through electrophoresis, cutGlue is connected to expression vector pXMJ19 after reclaiming, and obtains recombinant plasmid pXMJ-ido(restriction enzyme digestion and electrophoresis collection of illustrative plates and sees Fig. 3). Then will weighIts electricity of group plasmid pXMJ-ido is converted in Corynebacterium glutamicum ATCC13032 cell, obtains recombinant bacterial strain CG-ido.
Embodiment 4:(2S, 3R, 4S) microbial conversion of-4-hydroxyisoleucine
(1) seed culture: 1-2 is propped up to the recombinant bacterial strain CG-through the embodiment of fresh slant activation 3 gained with oeseIdo is seeded to the 1L flask with indentation that 100mL seed culture medium is housed, in 32-37 DEG C, and 200rpm shaken cultivation 6-8h.
(2) fermented and cultured of thalline: the inoculum of step (1) is seeded to 3L fermentation is housed with 5%-10% inoculum concentrationThe 5L fermentation tank of culture medium is sent out tank and is cultivated, fermentation temperature 32-37 DEG C, and dissolved oxygen maintains 20-40%, and it is 60-that stream adds concentrationThe glucose solution of 80% (W/V), the ammoniacal liquor that stream adds 25% (W/V) regulates zymotic fluid pH to 6.8-7.2, fermented and cultured 6-12h. PressAccording to 10% inoculum concentration, primary seed solution is transferred into 5L Fermentation, liquid amount is 3L, and dissolved oxygen is controlled at 20%-30%, cultivates temperatureSpend 33 DEG C, pH is controlled at 7.0 left and right.
(3) wait ILE and the KG sterile solution of amount of substance concentration to interpolation in fermentation tank, make itFinal concentration is 0.1-0.3mol/L, continues to cultivate 18-24h by step (2).
(4) after adopting DNF to (2S, 3R, 4S)-4-isoleucine derivatization, with high performance liquid chromatographyThe content of measuring it, actual conditions is as follows: chromatographic column is AgilentZORBAXEclipsAAA (4.6 × 150mm, 5-Micron), 33 DEG C of column temperatures, detect wavelength 360nm. It is 50% acetonitrile and acetate buffer that mobile phase A and B are respectively concentration(50mmol/L, pH6.4), flow velocity is 1.0mL/min. Testing result shows, the appearance time of (2S, 3R, 4S)-4-isoleucineBe about 21.6-25.2min, output is 0.099-0.112mol/L, and conversion ratio is 79.2-91.7%.
Composition is cultivated on inclined-plane: glucose 1.0-5.0g/L, and peptone 10-15g/L, beef extract 10-15g/L,NaCl2.0-3.0g/L, agar 25g/L, pH7.0-7.2,0.075MPa high pressure steam sterilization 30min.
Seed culture based component is: glucose 25-30g/L, and corn steep liquor 15-30mL/L, soybean meal hydrolysate 15-30mL/L,KH2PO40.5-1.0g/L，MgSO4·7H2O0.1-0.5g/L，MnSO415-30mg/L，FeSO410-30mg/L, urea 2-5g/L. PH7.0-7.2,0.075MPa high pressure steam sterilization 30min.
Fermented and cultured based component is: glucose 60-80g/L, K2HPO43.0-5.0g/L，MgSO41.0-2.0g/L，MnSO420-50mg/L，FeSO420-50mg/L, corn steep liquor 20-50mL/L, soybean meal hydrolysate 15-30mL/L, 0.075MPa high pressureSteam sterilizing 30min.
Embodiment 5:(2S, 3R, 4S) microbial conversion of-4-hydroxyisoleucine
(1) seed culture: the bacterium that with oese, 2 conversions through fresh slant activation is had to ILE hydroxylaseBe seeded to the 1L flask with indentation that 100mL seed culture medium is housed, in 35 DEG C, 200rpm shaken cultivation 8h.
(2) fermented and cultured of thalline: the inoculum of step (1) is seeded to 3L fermented and cultured is housed with 8% inoculum concentrationThe 5L fermentation tank of base is sent out tank and is cultivated, 35 DEG C of fermentation temperatures, and dissolved oxygen maintains 30%, and stream adds the grape that concentration is 80% (W/V)Sugar juice, the ammoniacal liquor that stream adds 25% (W/V) regulates zymotic fluid pH to 7.0, fermented and cultured 6h. According to 10% inoculum concentration by first order seedLiquid is transferred into 5L Fermentation, and liquid amount is 3L, and dissolved oxygen is controlled at 30%, 35 DEG C of cultivation temperature, and pH is controlled at 7.0.
(3) wait ILE and the KG sterile solution of amount of substance concentration to interpolation in fermentation tank, make itFinal concentration is 0.125mol/L, continues to cultivate 20h by step (2). Cultivate through 26h, CG-ido can be by 89.2% L-IleizationFor 4-hydroxyisoleucine.
(4) after adopting DNF to (2S, 3R, 4S)-4-isoleucine derivatization, with high performance liquid chromatographyThe content of measuring it, actual conditions is as follows: chromatographic column is AgilentZORBAXEclipsAAA (4.6 × 150mm, 5-Micron), 33 DEG C of column temperatures, detect wavelength 360nm. It is 50% acetonitrile and acetate buffer that mobile phase A and B are respectively concentration(50mmol/L, pH6.4), flow velocity is 1.0mL/min. Testing result shows, the appearance time of (2S, 3R, 4S)-4-isoleucineBe about 21.6min, output is 0.112mol/L, and conversion ratio is 91.7%.
Composition is cultivated on inclined-plane: glucose 4.0g/L, peptone 12.0g/L, beef extract 12.0g/L, NaCl2.5g/L, fine jadeFat 25g/L, pH7.0-7.2,0.075MPa high pressure steam sterilization 30min.
Seed culture based component is: glucose 25g/L, corn steep liquor 15mL/L, soybean meal hydrolysate 15mL/L, KH2PO40.6，MgSO4·7H2O0.2g/L，MnSO415mg/L，FeSO415mg/L, urea 2.5g/L. PH7.0,0.075MPa high steam goes outBacterium 30min.
Fermented and cultured based component is: glucose 80g/L, K2HPO43.5g/L，MgSO41.5g/L，MnSO425mg/L，FeSO425mg/L, corn steep liquor 25mL/L, soybean meal hydrolysate 15mL/L, 0.075MPa high pressure steam sterilization 30min.
Embodiment 6: zymologic property is measured
(1) enzyme activity assay
Measure the enzymatic reaction speed of embodiment 2 enzyme that produces under Different L-Ile concentration, adopt Lineweaver-Burk twoGraphing method curve plotting reciprocal (Fig. 6) also obtains equation 1/V=48.25/[S]+0.32, the K of this enzyme as calculatedmAnd VmaxBe respectively0.15mmol/L and 3.13 μ mol/min/mg, with the ILE hydroxylase of the B.thuringiensis2-e-2 reportingThere is some difference (its KmAnd Vmax0.27mmol/L and 1.13 μ mol/min/mg respectively), show that the L-of this research acquisition is different brightThe affinity of propylhomoserin hydroxylation enzyme-to-substrate and catalytic efficiency are all higher than the latter.
(2) optimal reactive temperature
Measure respectively the embodiment 2 enzyme relative activity that produces under different temperatures, the rising of its activity with temperature and raising, works as temperatureThe highest (the ILE hydroxylase optimum temperature of the B.thuringiensis2-e-2 having reported of its activity when degree reaches 35 DEG CBe 25 DEG C), along with the continuation of temperature raises, it active reduces rapidly, when temperature during higher than 65 DEG C activity completely lose (Fig. 7).
(3) optimal pH
Under optimum temperature (35 DEG C) condition, measure the impact of different pH on 2 inulinase-producing activities of embodiment, activity when pH7.0The highest (the ILE hydroxylase optimal pH of the B.thuringiensis2-e-2 having reported is 6.0), when pH higher than or lowAt 7.0 o'clock, along with departing from of pH, its activity sharply decline (Fig. 8).
(4) heat endurance
Respectively embodiment 2 enzyme that produces is placed to different time to measure its heat endurance in 25 DEG C, 35 DEG C and 45 DEG C. ResultShow, in front 8h, it keeps greater activity at 35 DEG C of optimum temperatures; Along with the increase of time, the L-that recombinates at 3 temperature is differentLeucine hydroxylase residual activity all presents downward trend, but under 25 DEG C and 35 DEG C of conditions, enzymatic activity declines slowly, in 35 DEG C of barsAfter processing 5h under part, still there is 87.5% activity. And under 45 DEG C of conditions active decline the most remarkable, at this temperature, place 5h after itsActivity almost completely loses (Fig. 9).
Claims (6)
1. the encode gene of ILE hydroxylase, its nucleotide sequence is as shown in sequence table SEQ IDNO:1.
2. the ILE hydroxylase of gene code claimed in claim 1, its amino acid sequence is as sequence table SEQ IDShown in NO:2.
3. one kind includes the genetic engineering bacterium of gene claimed in claim 1.
4. a method that builds genetic engineering bacterium described in claim 3, step is as follows:
Taking ILE hydroxylase encoding gene claimed in claim 1 as template, taking IDO-3 and IDO-4 as primer, carry outPcr amplification;
IDO-3：5’-CCGGTCGACAAGGAAGCTAGATATGAAAATGAGTGGCTTTAGCATAG-3’(SalⅠ)
IDO-4：5’-CCGGAATTCTTATTTTGTCTCCTTATAAGAAAATGTTACTA-3’(EcoRⅠ)
PCR condition is: 94 DEG C 5min1 circulation, and 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C 1min30 circulation, 72 DEG C 10min1 is followedRing, reaction system is 100 μ L, gets 10 μ LPCR products and detects through 1.5% agarose gel electrophoresis;
By pcr amplification product through reclaim after carry out double digestion through Sal I and EcoR I respectively, through electrophoresis, cut glue reclaim after be connected toExpression vector pXMJ19, obtains recombinant plasmid, then its electricity is converted in Corynebacterium glutamicum ATCC13032 cell, obtainsRecombinant bacterial strain.
5. utilize bacterial strain described in claim 3 to produce (2S, 3R, 4S)-4-hydroxyisoleucine by microbe transformation methodMethod, comprise the following steps:
(1) seed culture: 1-2 is propped up to the said gene engineering bacteria through fresh slant activation with oese, be seeded to 100mL is housedThe 1L flask with indentation of seed culture medium, in 32-37 DEG C, 200rpm shaken cultivation 6-8h;
(2) fermented and cultured of thalline: the inoculum of step (1) is seeded to 3L fermented and cultured is housed with 5%-10% inoculum concentrationThe 5L fermentation tank of base is sent out tank and is cultivated, fermentation temperature 32-37 DEG C, and dissolved oxygen maintains 20-40%, and it is 60-80%W/V that stream adds concentrationGlucose solution, the ammoniacal liquor that stream adds 25%W/V regulates zymotic fluid pH to 6.8-7.2, fermented and cultured 6-12h, according to 10% inoculationAmount transfers primary seed solution into 5L Fermentation, and liquid amount is 3L, and dissolved oxygen is controlled at 20%-30%, 33 DEG C of cultivation temperature, pHBe controlled at 7.0 left and right;
(3) wait ILE and the KG sterile solution of amount of substance concentration to interpolation in fermentation tank, make it dense eventuallyDegree is 0.1-0.3mol/L, continues to cultivate 18-24h by step (2).
6. one as claimed in claim 5 is produced the side of (2S, 3R, 4S)-4-hydroxyisoleucine by microbe transformation methodMethod, is characterized in that:
Composition is cultivated on described inclined-plane: glucose 1.0-5.0g/L, and peptone 10-15g/L, beef extract 10-15g/L,NaCl2.0-3.0g/L, agar 25g/L, pH7.0-7.2,0.075MPa high pressure steam sterilization 30min;
Described seed culture based component is: glucose 25-30g/L, and corn steep liquor 15-30mL/L, soybean meal hydrolysate 15-30mL/L,KH2PO40.5-1.0g/L，MgSO4·7H2O0.1-0.5g/L，MnSO415-30mg/L，FeSO410-30mg/L, urea 2-5g/L, pH7.0-7.2,0.075MPa high pressure steam sterilization 30min;
Described fermented and cultured based component is: glucose 60-80g/L, K2HPO43.0-5.0g/L，MgSO41.0-2.0g/L，MnSO420-50mg/L，FeSO420-50mg/L, corn steep liquor 20-50mL/L, soybean meal hydrolysate 15-30mL/L, 0.075MPa high pressureSteam sterilizing 30min.
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| CN107475267B (en) * | 2017-09-29 | 2020-07-14 | 天津科技大学 | 4-hydroxyisoleucine production plasmid and strain and synthesis method of 4-hydroxyisoleucine |
| CN109576234B (en) * | 2018-12-26 | 2021-05-07 | 天津科技大学 | Leucine-5-hydroxylase mutant and application thereof |
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| JP5246639B2 (en) * | 2006-09-28 | 2013-07-24 | 味の素株式会社 | Process for producing 4-hydroxy-L-isoleucine |
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