CN102352390A - Fermentation process for producing stable isotope carbon 13 completely labeled L-shaped leucine - Google Patents

Fermentation process for producing stable isotope carbon 13 completely labeled L-shaped leucine Download PDF

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CN102352390A
CN102352390A CN2011102985593A CN201110298559A CN102352390A CN 102352390 A CN102352390 A CN 102352390A CN 2011102985593 A CN2011102985593 A CN 2011102985593A CN 201110298559 A CN201110298559 A CN 201110298559A CN 102352390 A CN102352390 A CN 102352390A
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fermentation
thalline
zymotechnique
glucose
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张亮
杜晓宁
李良君
宋明鸣
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Shanghai Research Institute of Chemical Industry SRICI
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Shanghai Research Institute of Chemical Industry SRICI
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Abstract

The invention relates to a fermentation process for producing stable isotope carbon 13 completely labeled L-shaped leucine, which is characterized in that after the bottle shaking fermentation or fermentation tank fermentation is adopted for carrying out fermentation culture on thalli, the isotope C13 completely labeled L-shaped leucine is obtained through separation and extraction, wherein the fermentation tank fermentation is adopted when the product demands are great, and conversely, the bottle shaking fermentation is adopted. Compared with the prior art, the process has the advantages that the acid yield of the obtained <13>C stable isotope labeled L-shaped leucine is correspondingly improved by more than 50 percent than the acid yield adopting a fully synthesized culture medium for fermentation, in addition, the abundance decrease of <13>C can be reduced to a value lower than 0.5 percent, the production cost is reduced, and the fermentation and extraction process is simplified. Raw materials are sufficiently utilized, and the low-abundance and high-abundance <13>C dextrose can be used for meeting the requirements of products with different abundances.

Description

The leucic zymotechnique of production stability isotropic substance carbon 13 all mark L types
Technical field
The invention belongs to stable isotope tagged compound production field; Relate to the microbial fermentation production technique, especially relate to the leucic zymotechnique of a kind of production stability isotropic substance carbon 13 all mark L types.
Background technology
L-leucine-U- 13C 6Production can adopt organic synthesis method, precursor fermentation method, enzyme process and extraction method, direct fermentation etc.Adopt synthesis method technology loaded down with trivial details, and synthetic that obtain is the DL type leucine-U-of racemization 13C 6, need optical resolution just can obtain target compound L-leucine-U- 13C 6, make 13The C raw material availability reduces greatly, and cost rises.Hydrolysis method is usually used in preparing aminoacids complex, separate very difficulty of all amino acid.Production by Enzymes L-leucine-U- 13C 6Need alpha-oxo-caproic acid, ammonium formiate- 13C is as raw material, and ultimate capacity is not very high, and the raw material ammonium formiate- 13C costs an arm and a leg, and is not easy to obtain.And adopt common direct fermentation production, a large amount of enrichments of target amino acid separate simply relatively, but owing to add multiple organic carbon source in the seed, fermentating formula, and these organic carbon sources generally mostly are mixture, very difficult mark, usually make the L-leucine that obtains- 13The abundance of C product descends a lot, does not reach product requirement, and if do not add these materials, the acid yield of bacterial classification can reduce again greatly, and production cost is sharply risen,
If adopt conventional fermentation process, 13Though C stable isotope label L-leucine output is higher relatively, 13The C 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 13C glucose, cost are very high.Though adopt the full-synthetic culture medium fermentation right 13The influence of C abundance is little, and the acid amount is too low to make but produce 13The C raw material availability is not high.Therefore, screening is applicable to L-leucine-U- 13C 6The processing condition of producing are the keys of this technology.At present, in carbon 13 marker fields, also do not see the working method patent and the bibliographical information of related prods.
Summary of the invention
The object of the invention is exactly to provide a kind of for the defective that overcomes above-mentioned prior art existence to make 13The C raw material is utilized effectively, and abundance is descended seldom, so that under the situation that does not influence abundance, realize the leucic zymotechnique of production stability isotropic substance carbon 13 all mark L types of high yield.
The object of the invention can be realized through following technical scheme:
The leucic zymotechnique of production stability isotropic substance carbon 13 all mark L types after employing shake flask fermentation or ferment tank carry out fermentation culture to thalline, obtains isotropic substance carbon 13 all mark L type leucines through separation and Extraction again.
Described thalline is applicable to excellent bacillus, the brevibacterium sp that L type leucine is produced, and can adopt and force its permeability of cell membrane of CONTROL PROCESS control, comprises brevibacterium flavum, Corynebacterium crenatum, intestinal bacteria, serratia marcescens or Corynebacterium glutamicum.
Described shake flask fermentation may further comprise the steps:
Thalline is inoculated on the activation medium flat board, cultivated 16-30 hour in the biochemical incubator down for 30 ℃, after waiting to grow up to bacterium colony; In the intact fermention medium of prior sterilization, every bottle of fermention medium connects a ring thalline with colony inoculation, and the liquid amount of fermention medium is: the bottled 20-60ml fermention medium of 500ml triangle; The fermention medium of having inoculated is placed on the swing shaking table; 25-35 ℃ of control leavening temperature, continuously fermented 72-96 hour by shaking speed 180-240 rev/min.
Described ferment tank may further comprise the steps:
(1) thalline is inoculated on the activation medium flat board; Cultivated 16-30 hour in the biochemical incubator down for 30 ℃; Wait to grow up to behind the bacterium colony colony inoculation is equipped with the shaking in the bottle of seed culture medium in sterilized, cultivated 10-24 hour, obtain seed liquor at 28-30 ℃ of following shaking table;
(2) the fermentation initial temperature is 25-35 ℃, and initial pH6.8-7.2 adopts big inoculum size (2-10wt%), low-glucose addition, pressure control zymotechnique; Seed liquor is inoculated in the sterilized fermentor tank that fermention medium is housed by 2-10% (volume ratio) begins fermentation, controlled temperature 25-37 ℃, air flow 0.5-1.5VVM; Tank pressure 0.01-0.05MPa; Dissolved oxygen 5-30% (volume ratio), pH are 6.5-7.5, and with the skimmer froth breaking; The fermentation whole process is controlled pH at 7.0-7.5 with adding liquefied ammonia, and stream adds 50wt%'s 13C glucose, the control fermented liquid 13C glucose concn 3-6wt%, fermentation time 32-70 hour.
The dull and stereotyped slant activation substratum that adopts of described activation medium; The prescription of this slant activation substratum comprises: glucose: 1-5g/l, peptone: 5-15g/l, Carnis Bovis seu Bubali cream: 3-15g/l, sodium-chlor: 1-10g/l, agar: 15-30g/l; Regulate pH=6.5-8.0 with 2mol/l sodium hydroxide, sterilization is 10-20 minute in 110-130 ℃ of high-pressure sterilizing pot.
Described fermention medium with 13C glucose is carbon source, in the initial formulation 13The C glucose concn is 6-14wt%; With in ammonium chloride, ammonium sulfate, an ammonium nitrate, ammonium acetate or the urea one or more is initial nitrogenous source; Concentration is 2-5wt%; The fermentation middle and later periods adds urea, ammoniacal liquor or liquefied ammonia adjusting pH and replenishes nitrogenous source; Add high abundance thalline hydrolyzed solution as organic carbon source and nitrogenous source, addition is 0.2-2wt%.Do not add or add denier steeping water, yeast extract paste, peptone organic carbon source, add a kind of getting final product separately, addition is below 0.2wt%; Add phosphoric acid salt, magnesium salts, manganese salt and ferrous salt according to different strain; Add pure vitamin H 10-50ug/L, VB1 200-2000ug/L, VB6 100-1000ug/L; Pantothenic acid 100-500ug/L, nicotinic acid 100-600ug/L.
The prescription of described fermention medium comprises: 13C glucose: 80-120g/l, (NH 4) 2SO 4: 20-40g/l, MgSO 47H 2O:4-8mg/L, MnSO 4H 2O:2-6mg/L, FeSO 47H 2O:2-6mg/L, KH 2PO 4: 1g/l, pure vitamin H: 30ug/L, VB1:400ug/L, VB6:10ug/L, pantothenic acid: 50-300ug/l, nicotinic acid: 50-300ug/l, steeping water: 0.1-3.0g/l, 13C high abundance thalline hydrolyzed solution: 0.5-5g/l regulates pH=6.5-8.0 with 2mol/l sodium hydroxide, and sterilization is 10-20 minute in 110-130 ℃ of high-pressure sterilizing pot.
The prescription of described seed culture medium comprises: glucose: 20-50g/l, urea: 0.5-3g/l, ammonium sulfate: 2-8g/l, KH 2PO 4: 0.1-1g/l, steeping water: 5-50g/l, MgSO 47H 2O:1-10mg/L regulates pH=6.5-8.0 with 2mol/l sodium hydroxide, and sterilization is 10-20 minute in 110-130 ℃ of high-pressure sterilizing pot.
Described high abundance thalline hydrolyzed solution adopts following technology to obtain: centrifugal collection high abundance 13The bacterium mud that C stable isotope fermented liquid is produced is collected bacterium mud and is put into there-necked flask, and the sulfuric acid that adds 5mol/l disappears and boils, disappear boil 15 hours after; After the cooling, add 2mol/l sodium hydroxide, regulate the pH value to neutral; Centrifugal, collect clear liquid, be high abundance thalline hydrolyzed solution.
Described separation and Extraction adopts isoelectric precipitation or ion exchange method etc. to extract step by step and obtains 13C stable isotope label L-leucine solution is caught up with ammonia through vacuum concentration then and is adopted ethanol low temperature crystallization, vacuum-drying to obtain product.
Compared with prior art, the present invention is through changing fermentating formula and CONTROL PROCESS, and the present invention obtains 13C 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 13The C abundance descends and can reduce to below 0.5%, have in addition descend hardly, improved greatly 13C glucose utilization rate has reduced production cost, simultaneously, is guaranteeing product 13Under the C abundance condition, adopt the once method of inoculation, simplified fermentation and extraction process,, saved raw material greatly, reduced cost, the present invention's low abundance capable of using and high abundance because raw material is fully used 13C glucose satisfies different abundance product requirements.
Embodiment
Below in conjunction with specific embodiment the present invention is elaborated.
Embodiment 1
Use bacterial classification for 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, Carnis Bovis seu Bubali cream: 3g/l, sodium-chlor: 5g/l, agar: 20g/l;
Slant activation substratum: glucose: 3g/l, peptone: 10g/l, Carnis Bovis seu Bubali cream: 10g/l, sodium-chlor: 5g/l, agar: 20g/l;
Fermention medium: 13C glucose: 100g/l, (NH 4) 2SO 4: 20g/l, MgSO 4.7H 2O:6mg/L, MnSO 4H 2O:4mg/L, FeSO 47H 2O:4mg/L, KH 2PO 4: 1g/l, VH:10ug/L, VB1:400ug/L, pantothenic acid: 100ug/l, nicotinic acid: 100ug/l, corn oar: 0.5g/l, 13C high abundance thalline hydrolyzed solution 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 for use.
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 300ml 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 220rmp and fermented again 42 hours at 30 ℃, 160rmp bottom fermentation.Produce L-leucine-U- 13C 6Reach 18g/l.
With above-mentioned fermented liquid spinning (4000rpm, 10 minutes), resulting supernatant is regulated pH=2 with hydrochloric acid, last Zeo-karb (H +Type) absorption, the washing back is with 0.1N ammoniacal liquor wash-out, with the L-leucine-U-that obtains 13C 6Single spot elutriant concentrates, activated carbon decolorizing, and the crystal vacuum-drying that recrystallize and filtration obtain obtains L-leucine-U- 13C 6Solid 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 high abundance requirement of products greater than 99%.
Embodiment 2
Use bacterial classification for 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, Carnis Bovis seu Bubali cream: 3g/l, sodium-chlor: 5g/l, agar: 20g/l;
Slant activation substratum: glucose: 3g/l, peptone: 10g/l, Carnis Bovis seu Bubali cream: 10g/l, sodium-chlor: 5g/l, agar: 20g/l;
Fermention medium: 13C glucose: 100g/l, (NH 4) 2SO 4: 35g/l, MgSO 4.7H 2O:6mg/L, MnSO 4.H 2O:2mg/L, FeSO 4.7H 2O:2mg/L, KH 2PO 4: 1g/l, VH:25ug/L, VB6:100ug/L, pantothenic acid: 300ug/l, nicotinic acid: 400ug/l, peptone: 1g/l, 13C high abundance thalline hydrolyzed solution 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 for use.
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 400ml 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 ℃, 220rmp bottom fermentation 80 hours.Produce L-leucine-U- 13C 6Reach 20g/l.
With above-mentioned fermented liquid spinning (4000rpm, 10 minutes), resulting supernatant is regulated pH=2 with hydrochloric acid, last Zeo-karb (H +Type) absorption, the washing back is with 0.1N ammoniacal liquor wash-out, with the L-leucine-U-that obtains 13C 6Single spot elutriant concentrates, activated carbon decolorizing, and the crystal vacuum-drying that recrystallize and filtration obtain obtains L-leucine-U- 13C 6Solid 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 high abundance requirement of products 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, Carnis Bovis seu Bubali cream: 3g/l, sodium-chlor: 5g/l, agar: 20g/l;
Slant activation substratum: glucose: 3g/l, peptone: 10g/l, Carnis Bovis seu Bubali cream: 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 4.7H 2O:5mg/L
Fermention medium: 13C glucose: 110g/l, (NH 4) 2SO:40g/l, MgSO 4.7H 2O:6mg/L, MnSO 4.H 2O:2mg/L, FeSO 4.7H 2O:2mg/L, KH 2PO 4: 1g/l, VH:50ug/L, VB1:2000ug/L, pantothenic acid: 200ug/l, nicotinic acid: 500ug/l, steeping water: 1g/l, 13C high abundance thalline hydrolyzed solution 15g/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 for use.
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 60% is with 30% (volume ratio) NH 3H 2O regulates PH=7.2, and with the skimmer 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% 13C glucose control fermented liquid 13C glucose concn 5%, fermentation time 70 hours.Produce L-leucine-U- 13C 6Reach 19g/l.
With above-mentioned fermented liquid spinning (4000rpm, 10 minutes), resulting supernatant is regulated pH=2 with hydrochloric acid, last Zeo-karb (H +Type) absorption, the washing back is with 0.1N ammoniacal liquor wash-out, with the L-leucine-U-that obtains 13C 6Single spot elutriant concentrates, activated carbon decolorizing, and the crystal vacuum-drying that recrystallize and filtration obtain obtains L-leucine-U- 13C 6Solid 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 high abundance requirement of products greater than 99%.
Embodiment 4
Use bacterial classification for intestinal bacteria 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, Carnis Bovis seu Bubali cream: 3g/l, sodium-chlor: 5g/l, agar: 20g/l;
Slant activation substratum: glucose: 3g/l, peptone: 10g/l, Carnis Bovis seu Bubali cream: 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 4.7H 2O:5mg/L;
Fermention medium: 13C glucose: 90g/l, (NH 4) 2SO 4: 50g/l, MgSO 4.7H 2O:5mg/L, MnSO 4.H 2O:2mg/L, FeSO 4.7H 2O:2mg/LKH 2PO 4: 1g/l, H:20ug/L, VB1:200ug/L, pantothenic acid: 200ug/l, nicotinic acid: 300ug/l steeping water: 1g/l, 13C high abundance thalline hydrolyzed solution 20g/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 for use.
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) NH 3H 2O regulates PH=7.2, and with the skimmer 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% 13C glucose control fermented liquid 13C glucose concn 3%, fermentation time 55 hours.Produce L-leucine-U- 13C 6Reach 19g/l.
With above-mentioned fermented liquid spinning (4000rpm, 10 minutes), resulting supernatant is regulated pH=2 with hydrochloric acid, last Zeo-karb (H +Type) absorption, the washing back is with 0.1N ammoniacal liquor wash-out, with the L-leucine-U-that obtains 13C 6Single spot elutriant concentrates, activated carbon decolorizing, and the crystal vacuum-drying that recrystallize and filtration obtain obtains L-leucine-U- 13C 6Solid 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 high abundance requirement of products greater than 99%.
Embodiment 5
The leucic zymotechnique of production stability isotropic substance carbon 13 all mark L types after the employing shake flask fermentation carries out fermentation culture to thalline, obtains isotropic substance carbon 13 all mark L type leucines through separation and Extraction again.The thalline that adopts is applicable to excellent bacillus, the brevibacterium sp that L type leucine is produced; And can adopt and force its permeability of cell membrane of CONTROL PROCESS control; Comprise brevibacterium flavum, Corynebacterium crenatum, intestinal bacteria, serratia marcescens or Corynebacterium glutamicum, present embodiment adopts brevibacterium flavum.
Shake flask fermentation specifically may further comprise the steps:
Brevibacterium flavum is inoculated on the activation medium flat board, cultivated 16 hours in the biochemical incubator down for 30 ℃, after waiting to grow up to bacterium colony; In the intact fermention medium of prior sterilization, every bottle of fermention medium connects a ring thalline with colony inoculation, and the liquid amount of fermention medium is: the bottled 20ml fermention medium of 500ml triangle; The fermention medium of having inoculated is placed on the swing shaking table; 25 ℃ of control leavening temperatures, 180 rev/mins of shaking speed were continuously fermented 72 hours.
Wherein, The dull and stereotyped slant activation substratum that adopts of activation medium; The prescription of this slant activation substratum comprises: glucose: 1g/l, peptone: 5g/l, Carnis Bovis seu Bubali cream: 3g/l, sodium-chlor: 1g/l, agar: 15g/l; Regulate PH=6.5 with 2mol/l sodium hydroxide, sterilization is 10 minutes in 110 ℃ of high-pressure sterilizing pots.
Fermention medium with 13C glucose is carbon source, in the initial formulation 13The C glucose concn is 6wt%; With ammonium chloride is initial nitrogenous source, and concentration is 2wt%, and the fermentation middle and later periods adds urea, ammoniacal liquor or liquefied ammonia adjusting pH and replenishes nitrogenous source, adds high abundance thalline hydrolyzed solution as organic carbon source and nitrogenous source, and addition is 0.2wt%.Do not add or add organic carbon sources such as denier steeping water, yeast extract paste, peptone, add a kind of getting final product separately, addition is below 0.2wt%; Add phosphoric acid salt, magnesium salts, manganese salt and ferrous salt according to different strain; Add pure vitamin H 10ug/L, VB1 200ug/L, VB6 100ug/L; Pantothenic acid 100ug/L, nicotinic acid 100ug/L.The prescription of fermention medium comprises: 13C glucose: 80g/l, (NH 4) 2SO 4: 20g/l, MgSO 47H 2O:4mg/L, MnSO 4H 2O:2mg/L, FeSO 47H 2O:2mg/L, KH 2PO 4: 1g/l, pure vitamin H: 30ug/L, VB1:400ug/L, VB6:10ug/L, pantothenic acid: 50ug/l, nicotinic acid: 50ug/l, steeping water: 0.1g/l, 13C high abundance thalline hydrolyzed solution: 0.5g/l regulates PH=6.5 with 2mol/l sodium hydroxide, and sterilization is 10 minutes in 110 ℃ of high-pressure sterilizing pots.
High abundance thalline hydrolyzed solution adopts following technology to obtain: centrifugal collection high abundance 13The bacterium mud that C stable isotope fermented liquid is produced is collected bacterium mud and is put into there-necked flask, and the sulfuric acid that adds 5mol/l disappears and boils, disappear boil 15 hours after, after the cooling, add 2mol/l sodium hydroxide, regulate pH value to neutrality.Centrifugal, collect clear liquid, be high abundance thalline hydrolyzed solution.Separation and Extraction adopts isoelectric precipitation or ion exchange method etc. to extract step by step and obtains 13C stable isotope label L-leucine solution is caught up with ammonia through vacuum concentration then and is adopted ethanol low temperature crystallization, vacuum-drying to obtain product.
Embodiment 6
The leucic zymotechnique of production stability isotropic substance carbon 13 all mark L types after the employing shake flask fermentation carries out fermentation culture to thalline, obtains isotropic substance carbon 13 all mark L type leucines through separation and Extraction again.The thalline that adopts is applicable to excellent bacillus, the brevibacterium sp that L type leucine is produced; And can adopt and force its permeability of cell membrane of CONTROL PROCESS control; Comprise brevibacterium flavum, Corynebacterium crenatum, intestinal bacteria, serratia marcescens or Corynebacterium glutamicum, present embodiment adopts Corynebacterium crenatum.
Shake flask fermentation may further comprise the steps:
Corynebacterium crenatum is inoculated on the activation medium flat board, cultivated 30 hours in the biochemical incubator down for 30 ℃, after waiting to grow up to bacterium colony; In the intact fermention medium of prior sterilization, every bottle of fermention medium connects a ring thalline with colony inoculation, and the liquid amount of fermention medium is: the bottled 60ml fermention medium of 500ml triangle; The fermention medium of having inoculated is placed on the swing shaking table; 35 ℃ of control leavening temperatures, 240 rev/mins of shaking speed were continuously fermented 96 hours.
The dull and stereotyped slant activation substratum that adopts of activation medium; The prescription of this slant activation substratum comprises: glucose: 5g/l, peptone: 15g/l, Carnis Bovis seu Bubali cream: 15g/l, sodium-chlor: 10g/l, agar: 30g/l; Regulate pH=8.0 with 2mol/l sodium hydroxide, sterilization is 20 minutes in 130 ℃ of high-pressure sterilizing pots.
Fermention medium with 13C glucose is carbon source, in the initial formulation 13The C glucose concn is 14wt%; With ammonium sulfate and ammonium acetate is initial nitrogenous source, and concentration is 5wt%, and the fermentation middle and later periods adds urea, ammoniacal liquor or liquefied ammonia adjusting pH and replenishes nitrogenous source, adds high abundance thalline hydrolyzed solution as organic carbon source and nitrogenous source, and addition is 2wt%.Do not add or add organic carbon sources such as denier steeping water, yeast extract paste, peptone, add a kind of getting final product separately, addition is below 0.2wt%; Add phosphoric acid salt, magnesium salts, manganese salt and ferrous salt according to different strain; Add pure vitamin H 50ug/L, VB1 2000ug/L, VB6 1000ug/L; Pantothenic acid 500ug/L, nicotinic acid 600ug/L.
The prescription of fermention medium comprises: 13C glucose: 120g/l, (NH 4) 2SO 4: 40g/l, MgSO 47H 2O:8mg/L, MnSO 4H 2O:6mg/L, FeSO 47H 2O:6mg/L, KH 2PO 4: 1g/l, pure vitamin H: 30ug/L, VB1:400ug/L, VB6:10ug/L, pantothenic acid: 50-300ug/l, nicotinic acid: 300ug/l, steeping water: 3.0g/l, 13C high abundance thalline hydrolyzed solution: 5g/l regulates pH=8.0 with 2mol/l sodium hydroxide, and sterilization is 20 minutes in 130 ℃ of high-pressure sterilizing pots.
High abundance thalline hydrolyzed solution adopts following technology to obtain: centrifugal collection high abundance 13The bacterium mud that C stable isotope fermented liquid is produced is collected bacterium mud and is put into there-necked flask, and the sulfuric acid that adds 5mol/l disappears and boils, disappear boil 15 hours after, after the cooling, add 2mol/l sodium hydroxide, regulate pH value to neutral.Centrifugal, collect clear liquid, be high abundance thalline hydrolyzed solution.
Described separation and Extraction adopts isoelectric precipitation or ion exchange method etc. to extract step by step and obtains 13C stable isotope label L-leucine solution is caught up with ammonia through vacuum concentration then and is adopted ethanol low temperature crystallization, vacuum-drying to obtain product.
Embodiment 7
The leucic zymotechnique of production stability isotropic substance carbon 13 all mark L types after the employing ferment tank carries out fermentation culture to thalline, obtains isotropic substance carbon 13 all mark L type leucines through separation and Extraction again.The thalline that adopts is applicable to excellent bacillus, the brevibacterium sp that L type leucine is produced; And can adopt and force its permeability of cell membrane of CONTROL PROCESS control; Comprise brevibacterium flavum, Corynebacterium crenatum, intestinal bacteria, serratia marcescens or Corynebacterium glutamicum, present embodiment adopts intestinal bacteria.
Ferment tank may further comprise the steps:
(1) intestinal bacteria is inoculated on the activation medium flat board, cultivated 16 hours in the biochemical incubators down for 30 ℃, wait to grow up to behind the bacterium colony colony inoculation is equipped with the shaking in the bottle of seed culture medium in sterilized, cultivated 10 hours, obtain seed liquor at 28 ℃ of following shaking tables;
(2) the fermentation initial temperature is 25 ℃, and initial pH is 6.8, adopts big inoculum size (2wt%), low-glucose addition, pressure control zymotechnique; Seed liquor is inoculated in the sterilized fermentor tank that fermention medium is housed by 2% (volume ratio) begins fermentation, 25 ℃ of controlled temperature, air flow 0.5VVM; Tank pressure 0.01MPa, dissolved oxygen 5% (volume ratio), pH are 6.5; And with the skimmer froth breaking, the fermentation whole process is controlled pH 7.0 with adding liquefied ammonia, and stream adds 50wt%'s 13C glucose, the control fermented liquid 13C glucose concn 3wt%, fermentation time 32 hours.
Wherein, The dull and stereotyped slant activation substratum that adopts of activation medium; The prescription of this slant activation substratum comprises: glucose: 1g/l, peptone: 5g/l, Carnis Bovis seu Bubali cream: 3g/l, sodium-chlor: 1g/l, agar: 15g/l; Regulate pH=6.5 with 2mol/l sodium hydroxide, sterilization is 10 minutes in 110 ℃ of high-pressure sterilizing pots.
Fermention medium with 13C glucose is carbon source, in the initial formulation 13The C glucose concn is 6wt%; With an ammonium nitrate and urea is initial nitrogenous source, and concentration is 2wt%, and the fermentation middle and later periods adds urea, ammoniacal liquor or liquefied ammonia adjusting pH and replenishes nitrogenous source, adds high abundance thalline hydrolyzed solution as organic carbon source and nitrogenous source, and addition is 0.2wt%.Do not add or add organic carbon sources such as denier steeping water, yeast extract paste, peptone, add a kind of getting final product separately, addition is below 0.2wt%; Add phosphoric acid salt, magnesium salts, manganese salt and ferrous salt according to different strain; Add pure vitamin H 10ug/L, VB1 200ug/L, VB6 100ug/L; Pantothenic acid 100ug/L, nicotinic acid 100ug/L.The prescription of fermention medium comprises: 13C glucose: 80g/l, (NH 4) 2SO 4: 20g/l, MgSO 47H 2O:4mg/L, MnSO 4H 2O:2mg/L, FeSO 47H 2O:2mg/L, KH 2PO 4: 1g/l, pure vitamin H: 30ug/L, VB1:400ug/L, VB6:10ug/L, pantothenic acid: 50ug/l, nicotinic acid: 50ug/l, steeping water: 0.1g/l, 13C high abundance thalline hydrolyzed solution: 0.5g/l regulates pH=6.5 with 2mol/l sodium hydroxide, and sterilization is 10 minutes in 110 ℃ of high-pressure sterilizing pots.
The prescription of seed culture medium comprises: glucose: 20g/l, urea: 0.5g/l, ammonium sulfate: 2g/l, KH 2PO 4: 0.1g/l, steeping water: 5g/l, MgSO 47H 2O:1mg/L regulates pH=6.5 with 2mol/l sodium hydroxide, and sterilization is 10 minutes in 110 ℃ of high-pressure sterilizing pots.
High abundance thalline hydrolyzed solution adopts following technology to obtain: centrifugal collection high abundance 13The bacterium mud that C stable isotope fermented liquid is produced is collected bacterium mud and is put into there-necked flask, and the sulfuric acid that adds 5mol/l disappears and boils, disappear boil 15 hours after, after the cooling, add 2mol/l sodium hydroxide, regulate pH value to neutral.Centrifugal, collect clear liquid, be high abundance thalline hydrolyzed solution.Separation and Extraction adopts isoelectric precipitation or ion exchange method etc. to extract step by step and obtains 13C stable isotope label L-leucine solution is caught up with ammonia through vacuum concentration then and is adopted ethanol low temperature crystallization, vacuum-drying to obtain product.
Embodiment 8
The leucic zymotechnique of production stability isotropic substance carbon 13 all mark L types after the employing ferment tank carries out fermentation culture to thalline, obtains isotropic substance carbon 13 all mark L type leucines through separation and Extraction again.The thalline that adopts is applicable to excellent bacillus, the brevibacterium sp that L type leucine is produced; And can adopt and force its permeability of cell membrane of CONTROL PROCESS control; Comprise brevibacterium flavum, Corynebacterium crenatum, intestinal bacteria, serratia marcescens or Corynebacterium glutamicum, present embodiment adopts Corynebacterium glutamicum.
Ferment tank may further comprise the steps:
Described ferment tank may further comprise the steps:
(1) Corynebacterium glutamicum is inoculated on the activation medium flat board, cultivated 30 hours in the biochemical incubators down for 30 ℃, wait to grow up to behind the bacterium colony colony inoculation is equipped with the shaking in the bottle of seed culture medium in sterilized, cultivated 24 hours, obtain seed liquor at 30 ℃ of following shaking tables;
(2) the fermentation initial temperature is 35 ℃, and initial pH7.2 adopts big inoculum size (10wt%), low-glucose addition, pressure control zymotechnique; Seed liquor is inoculated in the sterilized fermentor tank that fermention medium is housed by 10% (volume ratio) begins fermentation, 37 ℃ of controlled temperature, air flow 1.5VVM; Tank pressure 0.05MPa, dissolved oxygen 30% (volume ratio), pH are 7.5; And with the skimmer froth breaking, the fermentation whole process is controlled pH 7.5 with adding liquefied ammonia, and stream adds 50wt%'s 13C glucose, the control fermented liquid 13C glucose concn 6wt%, fermentation time 70 hours.
The dull and stereotyped slant activation substratum that adopts of activation medium; The prescription of this slant activation substratum comprises: glucose: 5g/l, peptone: 15g/l, Carnis Bovis seu Bubali cream: 15g/l, sodium-chlor: 10g/l, agar: 30g/l; Regulate pH=8.0 with 2mol/l sodium hydroxide, sterilization is 20 minutes in 130 ℃ of high-pressure sterilizing pots.
Fermention medium with 13C glucose is carbon source, in the initial formulation 13The C glucose concn is 14wt%; With ammonium acetate, ammonium sulfate and urea is initial nitrogenous source, and concentration is 5wt%, and the fermentation middle and later periods adds urea, ammoniacal liquor or liquefied ammonia adjusting pH and replenishes nitrogenous source, adds high abundance thalline hydrolyzed solution as organic carbon source and nitrogenous source, and addition is 2wt%.Do not add or add organic carbon sources such as denier steeping water, yeast extract paste, peptone, add a kind of getting final product separately, addition is below 0.2wt%; Add phosphoric acid salt, magnesium salts, manganese salt and ferrous salt according to different strain; Add pure vitamin H 50ug/L, VB1 2000ug/L, VB6 1000ug/L; Pantothenic acid 500ug/L, nicotinic acid 600ug/L.
The prescription of fermention medium comprises: 13C glucose: 120g/l, (NH 4) 2SO 4: 40g/l, MgSO 47H 2O:8mg/L, MnSO 4H 2O:6mg/L, FeSO 47H 2O:6mg/L, KH 2PO 4: 1g/l, pure vitamin H: 30ug/L, VB1:400ug/L, VB6:10ug/L, pantothenic acid: 50-300ug/l, nicotinic acid: 300ug/l, steeping water: 3.0g/l, 13C high abundance thalline hydrolyzed solution: 5g/l regulates PH=8.0 with 2mol/l sodium hydroxide, and sterilization is 20 minutes in 130 ℃ of high-pressure sterilizing pots.
The prescription of seed culture medium comprises: glucose: 50g/l, urea: 3g/l, ammonium sulfate: 8g/l, KH 2PO 4: 1g/l, steeping water: 50g/l, MgSO 47H 2O:10mg/L regulates pH=8.0 with 2mol/l sodium hydroxide, and sterilization is 20 minutes in 130 ℃ of high-pressure sterilizing pots.
High abundance thalline hydrolyzed solution adopts following technology to obtain: centrifugal collection high abundance 13The bacterium mud that C stable isotope fermented liquid is produced is collected bacterium mud and is put into there-necked flask, and the sulfuric acid that adds 5mol/l disappears and boils, disappear boil 15 hours after, after the cooling, add 2mol/l sodium hydroxide, regulate pH value to neutral.Centrifugal, collect clear liquid, be high abundance thalline hydrolyzed solution.Separation and Extraction adopts isoelectric precipitation or ion exchange method etc. to extract step by step and obtains 13C stable isotope label L-leucine solution is caught up with ammonia through vacuum concentration then and is adopted ethanol low temperature crystallization, vacuum-drying to obtain product.

Claims (10)

1. the leucic zymotechnique of production stability isotropic substance carbon 13 all mark L types is characterized in that, this zymotechnique obtains isotropic substance carbon 13 all mark L type leucines through separation and Extraction after adopting shake flask fermentation or ferment tank that thalline is carried out fermentation culture again.
2. the leucic zymotechnique of production stability isotropic substance carbon 13 all mark L types according to claim 1; It is characterized in that; Described thalline is applicable to excellent bacillus, the brevibacterium sp that L type leucine is produced; And can adopt and force its permeability of cell membrane of CONTROL PROCESS control, comprise brevibacterium flavum, Corynebacterium crenatum, intestinal bacteria, serratia marcescens or Corynebacterium glutamicum.
3. the leucic zymotechnique of production stability isotropic substance carbon 13 all mark L types according to claim 1 is characterized in that described shake flask fermentation may further comprise the steps:
Thalline is inoculated on the activation medium flat board, cultivated 16-30 hour in the biochemical incubator down for 30 ℃, after waiting to grow up to bacterium colony; In the intact fermention medium of prior sterilization, every bottle of fermention medium connects a ring thalline with colony inoculation, and the liquid amount of fermention medium is: the bottled 20-60ml fermention medium of 500ml triangle; The fermention medium of having inoculated is placed on the swing shaking table; 25-35 ℃ of control leavening temperature, continuously fermented 72-96 hour by shaking speed 180-240 rev/min.
4. the leucic zymotechnique of production stability isotropic substance carbon 13 all mark L types according to claim 1 is characterized in that described ferment tank may further comprise the steps:
(1) thalline is inoculated on the activation medium flat board; Cultivated 16-30 hour in the biochemical incubator down for 30 ℃; Wait to grow up to behind the bacterium colony colony inoculation is equipped with the shaking in the bottle of seed culture medium in sterilized, cultivated 10-24 hour, obtain seed liquor at 28-30 ℃ of following shaking table;
(2) the fermentation initial temperature is 25-35 ℃, and initial pH6.8-7.2 adopts big inoculum size (2-10wt%), low-glucose addition, pressure control zymotechnique; Seed liquor is inoculated in the sterilized fermentor tank that fermention medium is housed by 2-10% (volume ratio) begins fermentation, controlled temperature 25-37 ℃, air flow 0.5-1.5VVM; Tank pressure 0.01-0.05MPa; Dissolved oxygen 5-30% (volume ratio), pH are 6.5-7.5, and with the skimmer froth breaking; The fermentation whole process is controlled pH at 7.0-7.5 with adding liquefied ammonia, and stream adds 50wt%'s 13C glucose, the control fermented liquid 13C glucose concn 3-6wt%, fermentation time 32-70 hour.
5. according to claim 3 or the leucic zymotechnique of 4 described production stability isotropic substance carbon 13 all mark L types; It is characterized in that; The dull and stereotyped slant activation substratum that adopts of described activation medium; The prescription of this slant activation substratum comprises: glucose: 1-5g/l, peptone: 5-15g/l, Carnis Bovis seu Bubali cream: 3-15g/l, sodium-chlor: 1-10g/l, agar: 15-30g/l; Regulate pH=6.5-8.0 with 2mol/l sodium hydroxide, sterilization is 10-20 minute in 110-130 ℃ of high-pressure sterilizing pot.
6. according to claim 3 or the leucic zymotechnique of 4 described production stability isotropic substance carbon 13 all mark L types, it is characterized in that, described fermention medium with 13C glucose is carbon source, in the initial formulation 13The C glucose concn is 6-14wt%; With in ammonium chloride, ammonium sulfate, an ammonium nitrate, ammonium acetate or the urea one or more is initial nitrogenous source; Concentration is 2-5wt%; The fermentation middle and later periods adds urea, ammoniacal liquor or liquefied ammonia adjusting pH and replenishes nitrogenous source; Add high abundance thalline hydrolyzed solution as organic carbon source and nitrogenous source, addition is 0.2-2wt%.Do not add or add denier steeping water, yeast extract paste, peptone organic carbon source, add a kind of getting final product separately, addition is below 0.2wt%; Add phosphoric acid salt, magnesium salts, manganese salt and ferrous salt according to different strain; Add pure vitamin H 10-50ug/L, VB1 200-2000ug/L, VB6 100-1000ug/L; Pantothenic acid 100-500ug/L, nicotinic acid 100-600ug/L.
7. according to claim 3 or the leucic zymotechnique of 4 described production stability isotropic substance carbon 13 all mark L types, it is characterized in that the prescription of described fermention medium comprises: 13C glucose: 80-120g/l, (NH 4) 2SO 4: 20-40g/l, MgSO 47H 2O:4-8mg/L, MnSO 4H 2O:2-6mg/L, FeSO 47H 2O:2-6mg/L, KH 2PO 4: 1g/l, pure vitamin H: 30ug/L, VB1:400ug/L, VB6:10ug/L, pantothenic acid: 50-300ug/l, nicotinic acid: 50-300ug/l, steeping water: 0.1-3.0g/l, 13C high abundance thalline hydrolyzed solution: 0.5-5g/l regulates pH=6.5-8.0 with 2mol/l sodium hydroxide, and sterilization is 10-20 minute in 110-130 ℃ of high-pressure sterilizing pot.
8. the leucic zymotechnique of production stability isotropic substance carbon 13 all mark L types according to claim 4 is characterized in that the prescription of described seed culture medium comprises: glucose: 20-50g/l, urea: 0.5-3g/l, ammonium sulfate: 2-8g/l, KH 2PO 4: 0.1-1g/l, steeping water: 5-50g/l, MgSO 47H 2O:1-10mg/L regulates pH=6.5-8.0 with 2mol/l sodium hydroxide, and sterilization is 10-20 minute in 110-130 ℃ of high-pressure sterilizing pot.
9. the leucic zymotechnique of production stability isotropic substance carbon 13 all mark L types according to claim 6 is characterized in that, described high abundance thalline hydrolyzed solution adopts following technology to obtain: centrifugal collection high abundance 13The bacterium mud that C stable isotope fermented liquid is produced is collected bacterium mud and is put into there-necked flask, and the sulfuric acid that adds 5mol/l disappears and boils, disappear boil 15 hours after; After the cooling, add 2mol/l sodium hydroxide, regulate the pH value to neutral; Centrifugal, collect clear liquid, be high abundance thalline hydrolyzed solution.
10. the leucic zymotechnique of production stability isotropic substance carbon 13 all mark L types according to claim 1 is characterized in that, described separation and Extraction adopts isoelectric precipitation or ion exchange method etc. to extract step by step and obtains 13C stable isotope label L-leucine solution is caught up with ammonia through vacuum concentration then and is adopted ethanol low temperature crystallization, vacuum-drying to obtain product.
CN2011102985593A 2011-09-28 2011-09-28 Fermentation process for producing stable isotope carbon 13 completely labeled L-shaped leucine Pending CN102352390A (en)

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CN101235402A (en) * 2007-02-01 2008-08-06 上海化工研究院 Fermentation technique for producing stability isotope 15N marking L-leucine
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