CN105238708A - Bacteria for L-hydroxyproline production and application of bacteria for L-hydroxyproline production - Google Patents
Bacteria for L-hydroxyproline production and application of bacteria for L-hydroxyproline production Download PDFInfo
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Abstract
The invention discloses a strain of bacteria for L-hydroxyproline production and application of the bacteria for L-hydroxyproline production and provides Escherichia coli.HYP035 with a preservation number being CCTCC NO:M 2015401 in China Center For Type Culture Collection. According to experiments, the Escherichia coli.HYP035 can be used for L-hydroxyproline production, yield of L-hydroxyproline in fermentation broth is 45g/L, the proline conversion rate is 85%, a fermentation period is 40h, and high production value is achieved.
Description
Technical field
The present invention relates to microorganism field, be specifically related to bacterium and application thereof that L-oxyproline is produced in a strain.
Background technology
L-oxyproline is imino-acid, does not belong to 20 kinds of common amino acids, and it is the product after proline(Pro) hydroxylation, and the main moiety of collagen protein, is mainly used in the aspects such as medicine, chemical industry, animal-feed, nutrition and beauty culture.In recent years extensive concern is caused to the research and development of L-oxyproline: L-oxyproline has anti-oxidant, radiation-resistant effect, in beauty culture, can be used as cosmetics additive; L-oxyproline has antiobesity action, and future can become more satisfactory slimming medicine; L-oxyproline has different physiological roles and unique biological activity, both can be used as the medicine of various soft tissue diseases, as impaired in reticular tissue, rheumatic arthritis etc., again can accelerating wound, and treats various dermatosis; L-oxyproline, as the important component of amino acid injection, has certain curative effect to the hypoproteinemia that acute and chronic hepatitis causes; L-oxyproline participates in the emulsification of fat and the formation of red corpuscle protoheme and sphaeroprotein, has effects such as regulating fat emulsification; L-oxyproline is multi-medicament, as the synthesis material of third generation microbiotic, antitumor, hypertension and novel stomach medicine etc.Current L-oxyproline world consumption is more than 500 tons/year, and its demand is still in increasing trend year by year.
At present, the method for synthesizing L-oxyproline has chemical synthesis, biological extraction method and microbial enzyme method.The production of China L-oxyproline mainly adopts chemical hydrolysis extraction process, take animal collagen as raw material, by process preparations such as strong acid hydrolysis, nitrite-oxidizing and ion-exchanges, " three wastes " quantity discharged is large, seriously polluted, energy consumption is high, purity is low and production cost is high, is easy to by market.And chemical synthesis process synthesis step is more, raw material sources are restricted, cost is high, contaminate environment, are difficult to realize industrial production.2011, Germany scientist ChristianKlein is optimized the condition that the colibacillus engineering microbe conversion containing companion's factor produces L-oxyproline, recording the transformation efficiency that proline(Pro) is converted into L-oxyproline is 61%, its fermentation period length, substrate input amount and transformation efficiency are all lower, be not suitable for suitability for industrialized production L-oxyproline, and for the expression of enzymes of colibacillus engineering, adopt isopropylthiogalactoside (IPTG) to make inductor more, though its inducing effect is good, but expensive, be not suitable for suitability for industrialized production.Meanwhile, being that substrate direct fermentation is produced in L-oxyproline process with L-PROLINE, because Host Strains itself can consume substrate L-PROLINE, L-PROLINE utilization ratio step-down is caused.Visible, develop more energy-conservation, pollute less, that the high microbe conversion method of transformation efficiency produces L-oxyproline is imperative.
Summary of the invention
Technical problem to be solved by this invention how to produce L-oxyproline.
For solving the problems of the technologies described above, the invention provides the bacterium that a strain can produce L-oxyproline.
Bacterium provided by the present invention is intestinal bacteria HYP035 (Escherichiacoli.HYP035) CCTCCNO:M2015401.This bacterial strain is preserved in China typical culture collection center (be called for short CCTCC, address is: Wuhan, China Wuhan University) on June 25th, 2015, and deposit number is CCTCCNO:M2015401.
The application of described intestinal bacteria HYP035 (Escherichiacoli.HYP035) CCTCCNO:M2015401 in production L-oxyproline also belongs to protection scope of the present invention.
In above-mentioned application, the method of producing L-oxyproline with described intestinal bacteria HYP035 (Escherichiacoli.HYP035) CCTCCNO:M2015401 can comprise intestinal bacteria HYP035 (Escherichiacoli.HYP035) CCTCCNO:M2015401 described in fermentation culture, obtains the step of L-oxyproline.
In aforesaid method, the solute of the fermention medium that described fermentation culture uses and concentration thereof can be: glucose 3g/100mL, glycerine 0.5g/100mL, peptone 0.8g/100mL, ammonium sulfate 0.5g/100mL, dipotassium hydrogen phosphate 0.1g/100mL, sodium-chlor 0.2g/100mL, magnesium sulfate 0.02g/100mL, ferrous sulfate 0.1g/100mL, calcium chloride 0.0015g/100mL, calcium carbonate 1.5g/100mL, proline-4 .3g/100mL; Solute can be water; PH7.0-7.2.
In aforesaid method, the inoculum size of described fermentation culture can be 10% ± 3% (volume percent).
In aforesaid method, the culture condition of described fermentation culture can be 36 DEG C ± 2 DEG C and cultivates 45 ± 5h, and oxyty can be 40%-50%.
In aforesaid method, described fermentation culture also can comprise preparation primary seed solution and/or secondary seed solution.
The step of described preparation primary seed solution is as follows: intestinal bacteria (Escherichiacoli.) HYP035 mono-clonal is inoculated in LB slant medium by (1), and 36.5 DEG C (in practical application 36 DEG C ± 2 DEG C) cultivate 12h (in practical application 12h ± 2h); (2) the intestinal bacteria HYP035 inclined-plane prepared by step (1), resuspended with 5mL sterilized water, then be inoculated into the inoculum size of 2% (volume percent) (in practical application 2% ± 1%) in the seed flask (150mL seed flask liquid amount is for 30mL) that first order seed Shake flask medium is housed, 36.5 DEG C (in practical application 36 DEG C ± 2 DEG C) cultivate 12h (in practical application 12h ± 2h), obtain primary seed solution.
Described first order seed Shake flask medium solute and concentration thereof can be: Tryptones 1g/100mL, yeast extract 0.5g/100mL, sodium-chlor 1g/100mL; Solute can be water; PH7.0-7.2,121 DEG C of autoclaving 15min are cooled to 60 DEG C, add penbritin, make the concentration of penbritin in system be 100 μ g/mL.
The step of described preparation secondary seed solution is as follows: the primary seed solution 60mL that aforesaid method is prepared by (1) is equipped with in the fermentor tank (30L fermentor tank liquid amount is for 12L) of secondary seed medium with the culture transferring amount of 0.5% (volume percent) (in practical application 0.5% ± 0.2%) access, 36 DEG C (in practical application 36 DEG C ± 2 DEG C) cultivate 8h (in practical application 8h ± 2h), oxyty can be 50% (in practical application 50%-70%), obtains OD
610be about the secondary seed solution of 25 (in practical application 25 ± 2).
Described secondary seed medium solute and concentration thereof can be: peptone 1.5g/100mL, yeast extract 2.5g/100mL, potassium primary phosphate 0.24g/100mL, dipotassium hydrogen phosphate 1.6g/100mL, glycerine 0.5g/100mL; Solute can be water; PH7.0-7.2,121 DEG C of autoclaving 15min.
Result shows, utilize intestinal bacteria provided by the invention (Escherichiacoli.) HYP035 can produce L-oxyproline, in its fermented liquid, L-oxyproline output is 45g/L, and the transformation efficiency of proline(Pro) reaches 85%, has higher productive value.
Compared with prior art, tool of the present invention has the following advantages: with the promotor of strong promoter tac for Protocollagen prolyl hydroxylase gene, compare the expression amount that other promoter genes can significantly improve Protocollagen prolyl hydroxylase gene, tac is constitutive promoter, do not need to carry out abduction delivering in the process of fermentative production L-oxyproline, reduce the use of inductor, reduce environmental pollution; Improving the expression amount of Host Strains proline biosynthesis pathways metabolism key gene by introducing proC gene, improving the amount of thalline proline biosynthesis in metabolic process, the injected volume of substrate proline can be reduced; Knocking out of putA gene also can reduce the metabolism of thalline own effectively to the consumption of L-PROLINE, improve the transformation efficiency of L-oxyproline, reduce production cost, improve product yield and quality, and reducing costs, is that applicable industrial fermentation transforms the method for producing L-oxyproline.
preservation explanation
Strain name: intestinal bacteria
Latin name: (Escherichiacoli.)
Strain number: HYP035
Preservation mechanism: China typical culture collection center
Preservation mechanism is called for short: CCTCC
Address: Wuhan, China Wuhan University
Preservation date: on June 25th, 2015
Register on the books numbering: CCTCCNO:M2015401 at preservation center
Accompanying drawing explanation
Fig. 1 is the structural representation of carrier pBR322 of setting out.
Fig. 2 is the structural representation of restructuring pBR322 plasmid-tac-Hyp-proC.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Test method in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, be routine biochemistry reagent shop and buy and obtain, e. coli bl21 (DE3) plysS is purchased from NTCC Type Tissue Collection.
The carrier pBR322 that sets out in following embodiment, restriction enzyme A at II, restriction enzyme Hind III, ExTaqDNA enzyme, 10 × ExTaqBuffer and dNTP are precious biotechnology (Dalian) company limited product.
Plasmid pKD13 in following embodiment is Quan Yang bio tech ltd, Shanghai Products, and catalog number is TV00500; Plasmid pKD46 is Quan Yang bio tech ltd, Shanghai product, and catalog number is TV00858; Plasmid pCP20 is Quan Yang bio tech ltd, Shanghai product, and catalog number is TV00859.
The preparation method of chloramine-T solution: 1.41g chloramine-T (Shanghai Aladdin biochemical technology limited-liability company Products, catalog number is 104060) be dissolved in 10mL water, then add 10mL n-propyl alcohol and 80mL damping fluid successively, mixing, obtains chloramine-T solution.The preparation method of damping fluid: 50g citric acid, 26.3gNaOH and 146.1g crystallization sodium acetate are dissolved in deionized water, are settled to 1L, then mix with 200mL water and 300mL n-propyl alcohol, obtain damping fluid.
The preparation method of developer: fully dissolve 10g paradimethy laminobenzaldehyde with 35mL perchloric acid, then slowly adds 65mL Virahol, mixes.
The substratum related in following embodiment is as follows:
LB slant medium: solute and concentration thereof are: Tryptones 1g/100mL, yeast extract 0.5g/100mL, sodium-chlor 1g/100mL, agar powder 2.0g/100mL; Solute is water; PH7.0-7.2,121 DEG C of autoclaving 20min are cooled to 60 DEG C, add penbritin, make the concentration of penbritin in system be 100 μ g/mL.
First order seed Shake flask medium: solute and concentration thereof are: Tryptones 1g/100mL, yeast extract 0.5g/100mL, sodium-chlor 1g/100mL; Solute is water; PH7.0-7.2,121 DEG C of autoclaving 20min are cooled to 60 DEG C, add penbritin, make the concentration of penbritin in system be 100 μ g/mL.
Secondary seed medium: solute and concentration thereof are: peptone 1.5g/100mL, yeast extract 2.5g/100mL, potassium primary phosphate 0.24g/100mL, dipotassium hydrogen phosphate 1.6g/100mL, glycerine 0.5g/100mL; Solute is water; PH7.0-7.2,121 DEG C of autoclaving 20min.
Fermention medium: solute and concentration thereof are: glucose 3g/100mL, glycerine 0.5g/100mL, peptone 0.8g/100mL, ammonium sulfate 0.5g/100mL, dipotassium hydrogen phosphate 0.1g/100mL, sodium-chlor 0.2g/100mL, magnesium sulfate 0.02g/100mL, ferrous sulfate 0.1g/100mL, calcium chloride 0.0015g/100mL, calcium carbonate 1.5g/100mL, proline-4 .3g/100mL; Solute is water; PH7.0-7.2,121 DEG C of autoclaving 15min.
Quantitative test in following embodiment, all arranges three revision tests, results averaged.
The structure of embodiment 1, recombinant plasmid pBR322-tac-Hyp-proC
1, the double chain DNA molecule shown in artificial synthesized sequence table sequence 1.In this double chain DNA molecule, sequence 1 be restriction enzyme Aat II from 5 ' end the 1 to 8 enzyme cuts recognition site, 9 to 62 Nucleotide is tac promotor, 63 to 878 Nucleotide is the encoding gene of Protocollagen prolyl hydroxylase, 895 to 1701 Nucleotide is the encoding gene of pyrroline-5-carboxylate reductase, 1705 to 1723 Nucleotide is T7 terminator, and the 1724 to 1731 is that the enzyme of restriction enzyme Hind III cuts recognition site.
2, the double chain DNA molecule restriction enzyme A at II of step 1 synthetic and Hind III is carried out double digestion, reclaim digestion products, obtain fragment.
3, cut out a carrier pBR322 with restriction enzyme A at II and Hind III enzyme, reclaim the carrier framework of about 4360bp.
4, the carrier framework that fragment step 2 obtained and step 3 obtain is connected, and obtains recombinant plasmid pBR322-tac-Hyp-proC.
The Protocollagen prolyl hydroxylase shown in sequence 2 of recombinant plasmid pBR322-tac-Hyp-proC expressed sequence table and the pyrroline-5-carboxylate reductase shown in sequence 3 of sequence table.
The structural representation of carrier pBR322 of setting out is shown in Fig. 1.The structural representation of recombinant plasmid pBR322-tac-Hyp-proC is shown in Fig. 2.Recombinant plasmid pBR322-tac-Hyp-proC is through order-checking, as follows to restructuring pBR322 plasmid-tac-Hyp-proC structrual description: the small segment between the Aat II of the carrier pBR322 that sets out and Hind III restriction enzyme site to be substituted by the sequence 1 of sequence table from the DNA molecular shown in 5 ' end the 9 to 1723 Nucleotide.Recombinant plasmid pBR322-tac-Hyp-proC has an expression cassette, the nucleotide sequence of this expression cassette as the sequence 1 of sequence table from shown in 5 ' end the 9 to 1723, wherein the 9 to 62 Nucleotide is tac strong promoter, 63 to 878 Nucleotide is the encoding gene of coding Protocollagen prolyl hydroxylase, 895 to 1704 position Nucleotide is the encoding gene of coding pyrroline-5-carboxylate reductase, and the 1705 to 1723 Nucleotide is T7 terminator.
Knocking out and the structure of recombination bacillus coli of embodiment 2, putA gene
The nucleotide sequence of the putA gene in the genomic dna of e. coli bl21 (DE3) plysS is as shown in the sequence 4 of sequence table.
One, the acquisition of target practice DNA homology recombinant fragment
Target practice DNA homology recombinant fragment is obtained according to following step:
1, homology arm primer pair is synthesized according to the gene order of kantlex of encoding in the nucleotide sequence of putA gene and plasmid pKD13 by precious biotechnology (Dalian) company limited, homology arm primer pair comprises two primers, respectively called after putac1 and putac2.
The nucleotide sequence of putac1 and putac2 is in table 1.Wherein single underscore is the fragment upstream of putA gene, dotted line is the segments downstream of putA gene, wavy line is the fragment upstream of nucleotide sequence of kantlex of encoding in plasmid pKD13, and square frame is the segments downstream of nucleotide sequence of kantlex of encoding in plasmid pKD13.By putac1 deionized water dissolving, be mixed with the upstream primer solution that concentration is 10 μm of ol/L.By putac2 deionized water dissolving, be mixed with the downstream primer solution that concentration is 10 μm of ol/L.
2, after completing steps 1, carry out pcr amplification according to following PCR amplification system, obtain target practice DNA homology recombinant fragment.Pcr amplification condition is: 94 DEG C of 5min, 25 circulations (95 DEG C of 45S, 55 DEG C of 40S, 72 DEG C of 1.5min), 72 DEG C of 19min.
| Reactant | Consumption (μ L) |
| 10×ExTaq Buffer | 5 |
| DNTP (often kind of dNTP concentration 2.5mM) | 4 |
| Plasmid pKD13 (8ng/ μ L) | 1.5 |
| Upstream primer solution (10 μm of ol/L) | 2.5 |
| Downstream primer solution (10 μm of ol/L) | 2.5 |
| Sterilized water | 34 |
| ExTaq archaeal dna polymerase | 0.5 |
| Cumulative volume | 50 |
Two, the knocking out of putA gene
Adopt the Standard operation procedure SOP (DatsenkoAandWannerB, Proc.Nat1.Acad.Sci.U.S.A.2000,97 (12): 6640-6645) of RED recombination system to carry out knocking out of putA gene, concrete steps are as follows:
1, plasmid pKD46 is proceeded to e. coli bl21 (DE3) plysS, obtain e. coli bl21 (DE3) plysS containing plasmid pKD46.
What 2, step 1 obtained is inoculated in LB substratum (containing ammonia benzyl mycin 100 μ g/mL) containing e. coli bl21 (DE3) the plysS mono-clonal of plasmid pKD46,30 DEG C, 220rpm cultivates 12h, obtains cultivation bacterium liquid; This cultivation bacterium liquid is inoculated into LB substratum (containing ammonia benzyl mycin 100 μ g/mL) with 1:100 (volume ratio), 30 DEG C, 220rpm shaking culture is to OD
600value is 0.15, then adds pectinose, makes the concentration of pectinose in system be 10mmol/L, 30 DEG C, induction 6-8h, and collecting cell preparation electricity turns experiences polypeptide cell.
3, competent cell prepared by 100 μ L steps 2 is mixed with the target practice DNA homology recombinant fragment that 20ng step one obtains, bottom the pole cup putting into precooling, then use electroporation conversion method (2200V, 5ms) to transform; After conversion completes, add rapidly the LB substratum re-suspended cell of 1mL in pole cup, in the centrifuge tube then transferred to, 30 DEG C, 100rpm recovery cultivation 1h, obtain converted product; Be coated on by converted product on the LB solid plate containing kantlex 50 μ g/mL, 37 DEG C of static gas wave refrigerator 16h, screening obtains knocking out the positive bacterium colony of e. coli bl21 (DE3) plysS of putA gene.
4, Positive E. coli BL21 (DE3) the plysS positive bacteria single colony clone ordinary method screened is prepared into competent cell.
5, competent cell and the 20ngpcp20 plasmid of getting 100 μ L steps 4 preparations mix, and bottom the pole cup putting into precooling, then use electroporation conversion method (2500V, 5ms) carry out electricity to transform, after conversion completes, 30 DEG C, 100rpm recovery cultivation 1h, obtain converted product; Converted product is coated on the LB solid plate containing ammonia benzyl mycin 50 μ g/mL, 37 DEG C of static gas wave refrigerator 16h, obtains the positive monoclonal of genetic engineering bacterium; By above-mentioned positive monoclonal access LB substratum, 30 DEG C, 220rpm recovery to cultivate after 12h 42 DEG C, 220rpm cultivates 5h, get above-mentioned nutrient solution 5 μ L to coat on LB solid plate 37 DEG C and cultivate 12h and be separated and screen the positive colony that kalamycin resistance gene lacks, realize the seamless of puta gene and knock out.By this genetic engineering bacterium called after e. coli bl21 (DE3) plysS (Δ puta).
Through above-mentioned steps 1-5, putA gene in e. coli bl21 (DE3) plysS genomic dna carried out seamless knocking out.
Three, the structure of recombination bacillus coli
The construction step of recombination bacillus coli is as follows:
1, the competent cell of e. coli bl21 (DE3) plysS (Δ puta) is prepared.
2, the recombinant plasmid pBR322-tac-Hyp-proC2 μ L of Example 1 structure, add in the competent cell of e. coli bl21 (DE3) plysS (Δ puta) prepared by step 1, bottom the pole cup putting into precooling, then electroporation conversion method (2200V, 5ms) is used to transform; After conversion completes, add rapidly the LB substratum re-suspended cell of 1mL in pole cup, in the centrifuge tube then transferred to, 37 DEG C, 220rpm recovery cultivation 1h, obtain converted product; Converted product is coated on the LB solid plate containing ammonia benzyl mycin 100 μ g/mL, 37 DEG C of static gas wave refrigerator 12h, obtains e. coli bl21 (DE3) plysS (Δ puta) the positive bacterium colony containing recombinant plasmid pBR322-tac-Hyp-proC.
By the recombinant bacterium called after intestinal bacteria HYP035 screened, this intestinal bacteria HYP035 is preserved in China typical culture collection center on June 25th, 2015 and (is called for short CCTCC, address is: Wuhan, China Wuhan University), deposit number is CCTCCNO:M2015401.The full name of intestinal bacteria HYP035 is intestinal bacteria HYP035 (Escherichiacoli.HYP035) CCTCCNO:M2015401, hereinafter referred to as intestinal bacteria (Escherichiacoli.) HYP035.
Embodiment 3, intestinal bacteria (Escherichiacoli.) HYP035 produce L-oxyproline
One, intestinal bacteria (Escherichiacoli.) HYP035 produces L-oxyproline
The concrete steps that intestinal bacteria (Escherichiacoli.) HYP035 produces L-oxyproline are as follows:
1, intestinal bacteria (Escherichiacoli.) HYP035 mono-clonal is inoculated in LB slant medium, 36.5 DEG C (in practical application 36 DEG C ± 2 DEG C) cultivate 12h (in practical application 12h ± 2h).
2, the intestinal bacteria HYP035 inclined-plane prepared by step 1, resuspended with 5mL sterilized water, then be inoculated into the inoculum size of 2% (volume percent) (in practical application 2% ± 1%) in the seed flask (150mL seed flask liquid amount is for 30mL) that first order seed Shake flask medium is housed, 36.5 DEG C (in practical application 36 DEG C ± 2 DEG C) cultivate 12h (in practical application 12h ± 2h), obtain primary seed solution.
3, primary seed solution 60mL step 2 prepared is equipped with in the fermentor tank (30L fermentor tank liquid amount is for 12L) of secondary seed medium with the culture transferring amount of 0.5% (volume percent) (in practical application 0.5% ± 0.2%) access, 36 DEG C (in practical application 36 DEG C ± 2 DEG C) cultivate 8h (in practical application 8h ± 2h), oxyty is 50% (in practical application 50%-70%), obtains OD
610be about the secondary seed solution of 25 (in practical application 25 ± 2).
4, secondary seed solution 1.5L step 3 prepared is equipped with in the fermentor tank (30L fermentor tank liquid amount is for 15L) of fermention medium with the culture transferring amount of 10% (volume percent) (in practical application 10% ± 3%) access, 36 DEG C (in practical application 36 DEG C ± 2 DEG C) cultivate 40h (in practical application 40h ± 2h), oxyty is 40% (in practical application 40%-50%), obtains fermented liquid.In fermenting process, when the glucose content in fermentation system is less than 1.0g/L, fermentor tank automatic makeup adds the D/W that concentration is 700g/L, and in maintenance fermenting process, in fermentation system, glucose content is 0.5g/L-2.0g/L.
The fermented liquid that step 4 obtains is namely containing L-oxyproline.
Two, L-hydroxyproline content is measured
The content of L-oxyproline in the fermented liquid that determination step 4 obtains, concrete steps are as follows:
1, the making of typical curve
A) accurately take L-hydroxyproline standard product 50.0mg in 10mL volumetric flask, use 0.01mol/L dissolving with hydrochloric acid, be then settled to 100mL with 0.01mol/L hydrochloric acid, shake up, be made into 500mg/LL-hydroxyproline standard storage liquid.
B) draw 10mLL-hydroxyproline standard storage liquid in 50mL volumetric flask, be settled to 50mL with distilled water, shake up, be made into 100 μ g/mLL-oxyproline working fluids.
C) L-oxyproline working fluid continuation distilled water diluting is obtained concentration be respectively 2,4,6,8, the standardized solution of 10mg/L.
D) get step c) in each standardized solution 5ml in 10ml test tube, add 2ml chloramine-T solution, shake up, in room temperature, place 20min; Then add 2ml developer, shake up and rapidly test tube is moved in 60 DEG C of water-baths afterwards, insulation 20min, then with cold water cooling, room temperature places 30min, then measures absorbancy with spectrophotometer at 560nm wavelength place, repeats for 3 times.
With L-Hydroxyproline concentration for X-coordinate, absorbancy is ordinate zou, draws L-hydroxyproline standard curve.
2, L-oxyproline in the fermented liquid that obtains of determination step 4
(1) fermented liquid step 4 obtained 4 DEG C, the centrifugal 15min of 5000g, collect supernatant liquor, dilute 100 times, obtain supernatant dilution.
(2) add step (1) supernatant dilution 5mL and chloramine-T solution 2mL at 10mL test tube, mixing, room temperature places 20min; Then add developer 2mL, shake up and rapidly test tube is moved in 60 DEG C of water-baths afterwards, insulation 20min; With cold water cooling, room temperature places 30min; Absorbance A 560 is measured at 560nm wavelength place with spectrophotometer.
(3) according to the typical curve of step 1, the transformation efficiency of L-oxyproline output and proline(Pro) in fermented liquid is calculated.The calculation formula of the transformation efficiency of proline(Pro) is:
Result shows, utilize intestinal bacteria provided by the invention (Escherichiacoli.) HYP035 can produce L-oxyproline, in its fermented liquid, L-oxyproline output is 45g/L, and the transformation efficiency of proline(Pro) reaches 85%.Utilize intestinal bacteria provided by the invention (Escherichiacoli.) HYP035 can produce L-oxyproline to have higher productive value as seen, fermentation period is only 40h.
Claims (2)
1. intestinal bacteria HYP035 (Escherichiacoli.HYP035), its deposit number in China typical culture collection center is CCTCCNO:M2015401.
2. intestinal bacteria HYP035 (Escherichiacoli.HYP035) described in claim 1 is producing the application in L-oxyproline.
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| CN105543265A (en) * | 2016-01-19 | 2016-05-04 | 江南大学 | Method for effectively improving proline conversion rate in system for producing Cis-3-hydroxy-L-proline according to biosynthesis method |
| CN107189990A (en) * | 2017-07-06 | 2017-09-22 | 江南大学 | A kind of proline-4 hydroxylase and its application for recombinantly expressing Host Strains |
| CN107604017A (en) * | 2017-10-13 | 2018-01-19 | 广东肇庆星湖生物科技股份有限公司 | A kind of method for improving hydroxyproline earlier fermentation bacteriolyze |
| CN107699595A (en) * | 2017-11-28 | 2018-02-16 | 绍兴厚普生物科技有限责任公司 | A kind of method of Production by Microorganism Fermentation L hydroxyprolines |
| CN108330124A (en) * | 2018-03-20 | 2018-07-27 | 安徽鼎欣医药科技有限公司 | It is a kind of improve L- hydroxyl proline acid-producing bacteria kinds gene mutation and strain breeding method |
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| CN107604017A (en) * | 2017-10-13 | 2018-01-19 | 广东肇庆星湖生物科技股份有限公司 | A kind of method for improving hydroxyproline earlier fermentation bacteriolyze |
| CN107604017B (en) * | 2017-10-13 | 2020-10-16 | 广东肇庆星湖生物科技股份有限公司 | Method for improving bacterial lysis in early fermentation stage of hydroxyproline |
| CN107699595A (en) * | 2017-11-28 | 2018-02-16 | 绍兴厚普生物科技有限责任公司 | A kind of method of Production by Microorganism Fermentation L hydroxyprolines |
| CN108330124A (en) * | 2018-03-20 | 2018-07-27 | 安徽鼎欣医药科技有限公司 | It is a kind of improve L- hydroxyl proline acid-producing bacteria kinds gene mutation and strain breeding method |
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