CN101235402B - Fermentation technique for producing stability isotope 15N marking L-leucine - Google Patents
Fermentation technique for producing stability isotope 15N marking L-leucine Download PDFInfo
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- CN101235402B CN101235402B CN2007100370545A CN200710037054A CN101235402B CN 101235402 B CN101235402 B CN 101235402B CN 2007100370545 A CN2007100370545 A CN 2007100370545A CN 200710037054 A CN200710037054 A CN 200710037054A CN 101235402 B CN101235402 B CN 101235402B
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Abstract
The invention relates to a fermentation manufacturing technique which is suitable for producing stability isotopic element 15N mark L-leucine, the technique comprises following steps: firstly, selecting parent strains, secondly, fermenting and culturing a formula, thirdly, adopting a fermentation technique, and fourthly, separating and extracting, compared with an existing technique, the 15N stability isotopic element L-leucine which is obtained through the technique has high acid yield, the 15N abundance ratio in the 15N stability isotopic element L-leucine decreases little, some even does not decrease, which simplifies the fermenting and abstracting technique, saves raw materials greatly, and lowers the cost.
Description
Technical field
The invention belongs to stable isotope tagged compound production field, relate to the microbial fermentation production technique, relate in particular to and be applicable to the production stability isotropic substance
15N label L-leucic fermentation manufacturing technique.
Background technology
15N stable isotope label L-leucic production can be adopted organic synthesis method, precursor fermentation method, enzyme process and extraction method, direct fermentation etc.Adopt synthesis method technology loaded down with trivial details, and need optical resolution to make
15The N raw material availability is lower, and cost rises.Hydrolysis method is usually used in preparing aminoacids complex, and it is very difficult to separate all amino acid.Production by Enzymes L-leucine-
15N need alpha-oxo-caproic acid, ammonium formiate-
15N is as raw material, and ultimate capacity is not very high, the raw material ammonium formiate-
15The N utilization ratio is lower.And adopt direct fermentation production, a large amount of enrichments of target amino acid, separates fairly simple, but because the very difficult mark of organic nitrogen source in the seed, fermentating formula, usually make the L-leucine-
15The abundance of N descends and does not much reach product requirement, screening be applicable to the L-leucine-
15The processing condition that N produces are keys of this technology.And adopt conventional fermentation process,
15Though N stable isotope label L-leucine output is higher relatively,
15The N abundance descends very big, often descends more than 3%, is difficult to reach the high abundance product requirement.And owing to add a large amount of
15N is inorganic nitrogen-sourced, and cost is very high.Though adopt the full-synthetic culture medium fermentation right
15The influence of N abundance is little, and the acid amount is too low to make but produce
15The N raw material availability is not high.At present, exist
15Also lose patent and bibliographical information in N stable isotope label L-leucic production field.
Summary of the invention
Purpose of the present invention is exactly to provide a kind of for the weak point that overcomes above-mentioned prior art existence
15N-L-leucine abundance height,
15The N utilization ratio is high is applicable to the production stability isotropic substance
15N label L-leucic fermentation manufacturing technique.
Purpose of the present invention can be achieved through the following technical solutions:
Be applicable to the production stability isotropic substance
15N label L-leucic fermentation manufacturing technique is characterized in that, this technology may further comprise the steps:
(1) starting strain chooses
Choose be applicable to the L-leucine-
15Excellent bacillus, brevibacterium sp that N produces, and can adopt and force CONTROL PROCESS to control its permeability of cell membrane, comprise brevibacterium flavum (Brevibacterium flavum), Corynebacterium crenatum (Corynebacterium crenatum), intestinal bacteria (Escherichia coil), serratia marcescens (Serratia marcescens Bizio), Corynebacterium glutamicum (Corynebacterium glutamicum);
(2) fermentation culture prescription
With the full-synthetic culture medium is minimum medium, adds a small amount of organic nitrogen source, is carbon source with glucose, and glucose concn is 6~14wt% in the initial formulation; With ammonium salts such as ammonium chloride, ammonium sulfate, ammonium nitrate or urea is initial nitrogenous source, the fermentation middle and later periods adds urea, ammoniacal liquor or liquefied ammonia adjusting pH and replenishes nitrogenous source, do not add or add a kind of of organic nitrogen sources such as denier corn steep liquor, yeast extract paste, peptone, casein hydrolyzate, thalline hydrolyzed solution, amino nitrogen concentration is 5~20g/L in this organic nitrogen source; Inorganic salt such as phosphoric acid salt, magnesium salts, manganese salt, ferrous salt add a certain amount of according to different strain; Vitamin H is excessive greatly in the fermentation initial formulation, and VB1 adds 50~2000ug/L, and VB6 adds 50~1000ug/L, and pantothenic acid adds 50~500ug/L, and nicotinic acid adds 20~600ug/L;
(3) zymotechnique
Adopt shake flask fermentation or ferment tank technology to get fermented liquid respectively according to product consumption;
Described shake flask fermentation is that thalline is inoculated on the activation medium flat board, cultivated 16~30 hours in the biochemical incubator down for 30 ℃, after treating that bacterium colony grows up to, thalline is inoculated in the intact fermentation culture of prior sterilization, and every bottle of fermentation culture connects a ring thalline, and the liquid amount of fermentation culture is the 3wt%~5wt% of liquid bottle, the fermentation culture of having inoculated is placed on the swing shaking table, 28~32 ℃ of leavening temperatures, 180~240 rev/mins of shaking speed were continuously fermented 72~96 hours;
Described ferment tank is that thalline is inoculated on the activation medium flat board, cultivated 16~30 hours in the biochemical incubator down for 30 ℃, after treating that bacterium colony grows up to, long good lawn is inoculated in sterilized the shaking in the bottle of seed culture medium of being equipped with, cultivated 10~24 hours at 28~32 ℃ of following shaking tables, obtain seed liquor; 28~33 ℃ of fermentation initial temperatures, big inoculum size 2~10wt%, low-glucose addition are adopted in initial pH6.8~7.2, force the control zymotechnique, seed 2~10% is inoculated in the sterilized fermentor tank that fermention medium is housed and begins fermentation by volume, control condition: 29~37 ℃ of temperature, air flow 0.5~1.5VVM, tank pressure 0.01~0.05MP, dissolved oxygen 1~95%, pH7.0~7.5 adopt interpolation liquefied ammonia mode to control fermented liquid pH value, and with the defoamer froth breaking.
(4) separation and Extraction
In the fermented liquid
15N stable isotope label L-leucic separation is adopted ion exchange method to extract and is obtained
15N stable isotope label L-leucine solution is caught up with ammonia and is adopted ethanol low temperature crystallization, vacuum-drying to obtain product by vacuum concentration.
Substratum in the described step (2) comprises slant preservation substratum, slant activation substratum, seed culture medium, fermention medium.
The prescription of described slant preservation substratum is: peptone: 10g/l, extractum carnis: 3g/l, sodium-chlor: 5g/l, agar: 20g/l.
The prescription of described slant activation substratum is: glucose: 3g/l, peptone: 10g/l, extractum carnis: 10g/l, sodium-chlor: 5g/l, agar: 20g/l.
The prescription of described seed culture medium is: glucose: 30g/l, urea: 1g/l, ammonium sulfate: 5g/l, KH
2PO
4: 0.5g/l, corn oar: 20g/l, MgSO
47H
2O:5mg/L.
The addition of organic nitrogen source is below the 0.2wt% in the described step (2).
Vitamin H is excessive greatly in the initial formulation of fermenting in the described step (2), adds pure vitamin H 100~1000ug/L.
Ferment tank Whole Process Control pH is in slight alkalinity in the described step (3), and pH is controlled at 7.0~7.5 with interpolation liquefied ammonia, and stream adds 50wt% glucose control fermented liquid glucose concn 3~6wt%, fermentation time 32~70 hours.
Compared with prior art, the present invention is by changing fermentating formula and CONTROL PROCESS, acquisition
15N stable isotope label L-leucine produces acid molar ratio and adopts the corresponding raising of full-synthetic culture medium fermentation and acid amount more than 50%, and
15N stable isotope label L-leucine
15The N abundance descends and can reduce to below 1%, have in addition descend hardly, improved greatly
15The N raw material availability has reduced production cost.Simultaneously, guaranteeing product
15Under the N abundance condition, adopt the once method of inoculation, simplified fermentation and extraction process,, saved raw material greatly, reduced cost because raw material is fully used.The present invention can utilize low abundance and high abundance
15The N inorganic raw material satisfies different abundance product requirements.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Adopt special-purpose mass spectrograph of isotropic substance or analysis of spectral method
15The N abundance, purity check adopts Kjeldahl determination.
Embodiment 1
Use bacterial classification for brevibacterium flavum (Brevibacterium flavum) ATCC39106 for setting out bacterium, the substratum of use comprises slant preservation substratum, slant activation substratum, shake flask fermentation substratum.The slant preservation substratum adopts identical with the slant activation substratum with conventional fermentation.The slant preservation substratum, slant activation substratum, the shake flask fermentation culture medium prescription that use are as follows:
Slant preservation substratum: peptone: 10g/l, extractum carnis: 3g/l, sodium-chlor: 5g/l, agar: 20g/l;
Slant activation substratum: glucose: 3g/l, peptone: 10g/l, extractum carnis: 10g/l, sodium-chlor: 5g/l, agar: 20g/l;
Fermentative medium formula: glucose: 100g/l, (
15NH
4)
2SO
4(
15N atom abundance: 10.24%): 20g/l, MgSO
4.7H
2O:6mg/L, MnSO
4H
2O:4mg/L, FeSO
47H
2O:4mg/L, KH
2PO
4: 1g/l, VH:600ug/L, VB1:400ug/L, pantothenic acid: 100ug/l, nicotinic acid: 100ug/l, corn oar: 2g/l;
With the carrier fluid amount packing fermention medium of 500ml triangular flask with 20ml.
Above substratum is all regulated pH=7.0 with 2mol/l sodium hydroxide, and sterilization is 15 minutes in 115 ℃ of high-pressure sterilizing pots, and is stand-by.
Extracting yellow tyrothricin ATCC39106 one ring thalline inserts the slant activation substratum, put into biochemical incubator, cultivated 28 hours down for 30 ℃, on the aseptic technique platform, the thalline on the slant activation substratum is inserted in the fermention medium for preparing in advance, every bottle graft one ring thalline, inoculated with nine layers of gauze and sealed, placed on the rotary shaking table, after 42 hours shaking speed has been increased to 220rpm and fermented again 42 hours at 30 ℃, 160rpm bottom fermentation.Produce
15The N-L-leucine reaches 20g/l.
With above-mentioned fermented liquid centrifugation (4000rpm, 10 minutes), resulting supernatant liquor is regulated pH=2 with hydrochloric acid, last Zeo-karb (H
+Type) absorption, washing back is with 0.1mol/l ammoniacal liquor wash-out, with the L-leucine that obtains-
15The single spot elutriant of N concentrates, activated carbon decolorizing, recrystallize and filter the crystal vacuum-drying that obtains obtain the L-leucine-
15N solid 4.22 grams.Obtain product abundance 10.19% through mass spectroscopy, abundance descends less than 0.5%, and abundance descends hardly, and purity can satisfy the requirement to the high abundance product greater than 99%.
Embodiment 2:
Use bacterial classification for Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 for setting out bacterium, the substratum of use comprises slant preservation substratum, slant activation substratum, shake flask fermentation substratum.The slant preservation substratum adopts identical with the slant activation substratum with conventional fermentation.The slant preservation substratum, slant activation substratum, the shake flask fermentation culture medium prescription that use are as follows:
Slant preservation substratum: peptone: 10g/l, extractum carnis: 3g/l, sodium-chlor: 5g/l, agar: 20g/l;
Slant activation substratum: glucose: 3g/l, peptone: 10g/l, extractum carnis: 10g/l, sodium-chlor: 5g/l, agar: 20g/l;
Fermentative medium formula: glucose: 120g/l, (
15NH
4)
2SO
4(
15N atom abundance: 99.67%): 20g/l, MgSO
47H
2O:6mg/L, MnSO
4H
2O:2mg/L, FeSO
47H
2O:2mg/L, KH
2PO
4: 1g/l, VH:600ug/L, VB6:100ug/L, pantothenic acid: 300ug/l, nicotinic acid: 400ug/l, peptone: 3g/l.
With the carrier fluid amount packing fermention medium of 500ml triangular flask with 20ml.
Above substratum is all regulated pH=7.0 with 2mol/l sodium hydroxide, and sterilization is 15 minutes in 115 ℃ of high-pressure sterilizing pots, and is stand-by.
Get Corynebacterium glutamicum ATCC13032 one ring thalline and insert the slant activation substratum, put into biochemical incubator, cultivated 24 hours down for 30 ℃, on the aseptic technique platform, the thalline on the slant activation substratum is inserted in the fermention medium for preparing in advance, every bottle graft one ring thalline, inoculated with nine layers of gauze and sealed, placed on the rotary shaking table, 30 ℃, 220rpm bottom fermentation 80 hours.Produce
15The N-L-leucine reaches 18g/l.
With above-mentioned fermented liquid centrifugation (4000rpm, 10 minutes), resulting supernatant liquor is regulated pH=2 with hydrochloric acid, last Zeo-karb (H
+Type) absorption, washing back is with 0.1mol/l ammoniacal liquor wash-out, with the L-leucine that obtains-
15The single spot elutriant of N concentrates, activated carbon decolorizing, recrystallize and filter the crystal vacuum-drying that obtains obtain the L-leucine-
15N solid 5.52 grams.Obtain product abundance 99.16% through mass spectroscopy, abundance descends less than 0.5%, and abundance descends hardly, and purity can satisfy the requirement to the high abundance product greater than 99%.
Embodiment 3:
Use bacterial classification for brevibacterium flavum ATCC39103 for setting out bacterium, the substratum of use comprises slant preservation substratum, slant activation substratum, seed culture medium, fermention medium.The slant preservation substratum adopts identical with the slant activation substratum with conventional fermentation.The slant preservation substratum, slant activation substratum, seed culture medium, the shake flask fermentation culture medium prescription that use are as follows:
Slant preservation substratum: peptone: 10g/l, extractum carnis: 3g/l, sodium-chlor: 5g/l, agar: 20g/l;
Slant activation substratum: glucose: 3g/l, peptone: 10g/l, extractum carnis: 10g/l, sodium-chlor: 5g/l, agar: 20g/l;
Seed culture medium: glucose: 30g/l, urea: 1g/l, ammonium sulfate: 5g/l, KH
2PO
4: 0.5g/l, corn oar: 20g/l, MgSO
47H
2O:5mg/L;
Fermentative medium formula: glucose: 110g/l, (
15NH
4)
2SO
4(
15N atom abundance: 99.91%): 20g/l, MgSO
47H
2O:6mg/L, MnSO
4H
2O:2mg/L, FeSO
47H
2O:2mg/L, KH
2PO
4: 1g/l, VH:600ug/L, VB1:2000ug/L, pantothenic acid: 200ug/l, nicotinic acid: 500ug/l, corn oar: 2g/l.
Above substratum is all regulated pH=7.0 with 2mol/l sodium hydroxide, and sterilization is 15 minutes in 115 ℃ of high-pressure sterilizing pots, and is stand-by.
Thalline is inoculated on the activation medium flat board, cultivated 30 hours in the biochemical incubators down for 30 ℃, treat that bacterium colony grows up to after, long good lawn is inoculated in sterilized 2 500ml that the 50ml seed culture medium respectively is housed shakes in the bottle, cultivated 10 hours at 28 ℃ of following shaking tables, obtain seed liquor.
Seed is inoculated in by 2% (volume ratio) begins fermentation in the fermentor tank of the sterilized 5000ml of being equipped with fermention medium, control condition: 29 ℃ of temperature, air flow 1.5VVM, tank pressure 0.02MP, dissolved oxygen 90% is with 30% (volume ratio)
15NH
3H
2O regulates pH=7.2, and with the defoamer froth breaking.Fermentation Whole Process Control pH is slight alkalinity (pH is controlled at 7.0~7.5, with adding liquefied ammonia control), and stream adds 50% glucose control fermented liquid glucose concn 5%, fermentation time 70 hours.Produce
15The N-L-leucine reaches 18g/l.
With above-mentioned fermented liquid centrifugation (4000rpm, 10 minutes), resulting supernatant liquor is regulated pH=2 with hydrochloric acid, last Zeo-karb (H
+Type) absorption, washing back is with 0.1mol/l ammoniacal liquor wash-out, with the L-leucine that obtains-
15The single spot elutriant of N concentrates, activated carbon decolorizing, recrystallize and filter the crystal vacuum-drying that obtains obtain the L-leucine-
15N solid 30.18 grams.Obtain product abundance 99.66% through mass spectroscopy, abundance descends less than 0.5%, and abundance descends hardly, and purity can satisfy the requirement to the high abundance product greater than 99%.
Embodiment 4:
Use bacterial classification for intestinal bacteria (Escherichia coil) ATCC21530 for setting out bacterium, the substratum of use comprises slant preservation substratum, slant activation substratum, seed culture medium, fermention medium.The slant preservation substratum adopts identical with the slant activation substratum with conventional fermentation.The slant preservation substratum, slant activation substratum, seed culture medium, the shake flask fermentation culture medium prescription that use are as follows:
Slant preservation substratum: peptone: 10g/l, extractum carnis: 3g/l, sodium-chlor: 5g/l, agar: 20g/l;
Slant activation substratum: glucose: 3g/l, peptone: 10g/l, extractum carnis: 10g/l, sodium-chlor: 5g/l, agar: 20g/l;
Seed culture medium: glucose: 30g/l, urea: 1g/l, ammonium sulfate: 5g/l, KH
2PO
4: 0.5g/l, corn oar: 20g/l, MgSO
47H
2O:5mg/L;
Fermentative medium formula: glucose: 130g/l, (
15NH
4)
2SO
4(
15N atom abundance: 99.14%): 20g/l, MgSO
47H
2O:5mg/L, MnSO
4H
2O:2mg/L, FeSO
47H
2O:2mg/L, KH
2PO
4: 1g/l, VH:1000ug/L, VB1:200ug/L, pantothenic acid: 200ug/l, nicotinic acid: 300ug/l, corn oar: 3g/l.
Above substratum is all regulated pH=7.0 with 2mol/l sodium hydroxide, and sterilization is 15 minutes in 115 ℃ of high-pressure sterilizing pots, and is stand-by.
Thalline is inoculated on the activation medium flat board, cultivated 16 hours in the biochemical incubators down for 30 ℃, treat that bacterium colony grows up to after, long good lawn is inoculated in sterilized 3 500ml that the 50ml seed culture medium respectively is housed shakes in the bottle, cultivated 30 hours at 28 ℃ of following shaking tables, obtain seed liquor.
Seed is inoculated in by 5% (volume ratio) begins fermentation in the fermentor tank of the sterilized 3000ml of being equipped with fermention medium, control condition: 32 ℃ of temperature, air flow 0.5VVM, tank pressure 0.05MP, dissolved oxygen 80% is with 30% (volume ratio)
15NH
3H
2O regulates pH=7.2, and with the defoamer froth breaking.Fermentation Whole Process Control pH is slight alkalinity (pH is controlled at 7.0~7.5, with adding liquefied ammonia control), and stream adds 50% glucose control fermented liquid glucose concn 3%, fermentation time 55 hours.Produce
15The N-L-leucine reaches 19g/l.
With above-mentioned fermented liquid centrifugation (4000rpm, 10 minutes), resulting supernatant liquor is regulated pH=2 with hydrochloric acid, last Zeo-karb (H
+Type) absorption, washing back is with 0.1mol/l ammoniacal liquor wash-out, with the L-leucine that obtains-
15The single spot elutriant of N concentrates, activated carbon decolorizing, recrystallize and filter the crystal vacuum-drying that obtains obtain the L-leucine-
15N solid 22.32 grams.Obtain product abundance 99.06% through mass spectroscopy, abundance descends less than 0.5%, and abundance descends hardly, and purity can satisfy the requirement to the high abundance product greater than 99%.
Claims (1)
1. be applicable to the production stability isotropic substance
15N label L-leucic fermentation manufacturing technique is characterized in that, this technology may further comprise the steps:
Extracting yellow tyrothricin (Brevibacterium flavum) ATCC39106 one ring thalline inserts the slant activation substratum, put into biochemical incubator, cultivated 28 hours down for 30 ℃, on the aseptic technique platform, the thalline on the slant activation substratum is inserted in the fermention medium for preparing in advance, every bottle graft one ring thalline, inoculated with nine layers of gauze and sealed, place on the rotary shaking table, at 30 ℃, the 160rpm bottom fermentation is increased to 220rpm with shaking speed after 42 hours and fermented 42 hours again, with above-mentioned fermented liquid centrifugation 10 minutes, centrifugal rotational speed is 4000rpm, and resulting supernatant liquor is regulated pH=2 with hydrochloric acid, last H
+Type cationic exchange resin adsorption, washing back be with 0.1mol/l ammoniacal liquor wash-out, with the L-leucine that obtains-
15The single spot elutriant of N concentrates, activated carbon decolorizing, recrystallize and filter the crystal vacuum-drying that obtains obtain the L-leucine-
15The N solid;
Described slant activation culture medium prescription is as follows: glucose: 3g/l, peptone: 10g/l, extractum carnis: 10g/l, sodium-chlor: 5g/l, agar: 20g/l;
Described fermentative medium formula is as follows: glucose: 100g/l, (
15NH
4)
2SO
4: 20g/l, wherein
15N atom abundance is 10.24%, MgSO
47H
2O:6mg/L, MnSO
4H
2O:4mg/L, FeSO
47H
2O:4mg/L, KH
2PO
4: 1g/l, VH:600ug/L, VB1:400ug/L, pantothenic acid: 100ug/l, nicotinic acid: 100ug/l, corn oar: 2g/l;
Described slant activation substratum and fermention medium are all regulated pH=7.0 with 2mol/l sodium hydroxide, and sterilization is 15 minutes in 115 ℃ of high-pressure sterilizing pots, with the carrier fluid amount packing of 500ml triangular flask with 20ml.
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CN101475629B (en) * | 2009-01-15 | 2011-12-14 | 上海化工研究院 | Preparation of stable isotope 15N labeling octapeptide GFL |
CN101811992B (en) * | 2010-04-02 | 2012-11-14 | 上海化工研究院 | Preparation method for semicarbazide hydrochloride labeled by stable isotope 13C and 15N |
CN102352390A (en) * | 2011-09-28 | 2012-02-15 | 上海化工研究院 | Fermentation process for producing stable isotope carbon 13 completely labeled L-shaped leucine |
CN106701853B (en) * | 2016-12-02 | 2019-09-20 | 武汉远大弘元股份有限公司 | A kind of production l-Isoleucine Corynebacterium glutamicum fermentation medium and cultural method |
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CN110862928A (en) * | 2019-12-03 | 2020-03-06 | 华东理工大学 | A kind of15Preparation method and application of N-labeled mycoprotein |
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