CN1834228A - Yellow graminic mutant strain and its application in prodn. of L-isoleucine by fementation process - Google Patents
Yellow graminic mutant strain and its application in prodn. of L-isoleucine by fementation process Download PDFInfo
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Abstract
This invention adopts Brevibacterium flavum HL41 preserved in China Center of Industrial Culture Collection with a serial number of CICC 10135 as the starting strain to culture mutant strain TC21 possessing genetic markers of methionine (Met) deficiency and alpha-aminobutyric acid (alpha-ABr) resistance, S-2-aminoethyl-L-cysteine resistance and ethionine resistance. This invention also discloses the application of Brevibacterium flavum in the manufacture of L-isoleucine by fermentation method, which comprises the steps of: adding mutant strain TC21 into the culture medium for fermentation at a pH value of 6.0-8.0 and 20-40 deg.C, and performing vibrational culture or fermentation tank culture for 2-5 day with an inoculum concentration of 1-15% (V/V). This invention adopts mutant strain to manufacture, metal membrane to filter and ion exchange method to extract L-isoleucine, thus can significantly raise the extraction rate (higher than 50%) and purity (98%) of L-isoleucine.
Description
[technical field]: the invention belongs to the biochemical engineering field, relate to a kind of Corynebacterium (Brevibacteriumflavum) bacterial classification and use the method that this strain fermentation is produced the L-Isoleucine.
[background technology]: the chemistry of L-Isoleucine (L-Isoleucine) is called L-a-amino-beta--methylvaleric acid, the L-Isoleucine is one of eight kinds of indispensable amino acids of human body, be again one of three kinds of branched-chain amino acids, because of its special 26S Proteasome Structure and Function, therefore, the status that in human life's metabolism, has particularly important.
L-Isoleucine production method has extraction method, chemical synthesis and fermentation method three classes, but on industrial production, implement at present have only fermentation method.Because L-Isoleucine and other isomer separation difficulty that extraction method and chemical synthesis are produced, thereby all fail to realize suitability for industrialized production.
Fermentation method is exactly a metabolism of utilizing microorganism, and biosynthesizing and excess accumulation L-Isoleucine comprise and add precursor fermentation method and direct fermentation two classes.
Add the precursor fermentation method and claim microbe transformation method again.This method uses glucose as fermenting carbon source, the energy, add the i.e. suitable mesostate of some in amino acid biosynthetic pathway of special precursor substance again, to avoid the feedback regulation effect in the amino acid biosynthetic pathway, it effectively is converted into purpose amino acid through microbial process.Because its precursor substance is rare or cost an arm and a leg, seldom adopt this method to produce the L-Isoleucine at present.
Direct fermentation is to have self amino acid needed ability of synthesizing by means of microorganism, by mutagenic treatment to specified microorganisms, select auxotroph and amino acid structure analogue resistant mutant strain, with feedback inhibition and the feedback repression in the releasing metabolism adjusting, thereby reach certain amino acid whose purpose of excess accumulation.At present, Isoleucine produces bacterium mostly by glutamate-producing strain brevibacterium flavum, Corynebacterium glutamicum, brevibacterium lactofermentum mutagenic and breeding.
It is low that this method has a raw materials cost, wide material sources, and the product purity height, advantages such as reaction conditions gentleness have the potentiality of suitability for industrialized production.
[summary of the invention]: one of the object of the invention provides a kind of brevibacterium flavum (Brevibacterium flavum) of new Corynebacterium, and this bacterium has the ability that improves L-Isoleucine productive rate.
Two of the technical issues that need to address of the present invention provide the production method that adopts above-mentioned strain fermentation to produce the L-Isoleucine.
Brevibacterium flavum provided by the invention (Brevibacterium flavum) mutant strain TC21 has L-Isoleucine throughput, and genetic marker is the methionine(Met) defective, ethionine, butyrine, S-2-amino-ethyl-L-halfcystine resistance (Met
-+ Eth
r+ α-AB
r+ AEC
r).
(annotate: brevibacterium flavum (Brevibacterium flavum) mutant strain TC21 (Met
-+ Eth
r+ α-AB
r+ AEC
r) once delivered in " University Of Science and Technology Of Tianjin's journal " the 19th the 2nd phase of volume in June, 2004, now being preserved in University Of Science and Technology Of Tianjin metabolic control fermentation research department, the public can get in touch with this research department if needed straight.)
Its mutafacient system is, brevibacterium flavum HL41 with Corynebacterium (is preserved in Chinese industrial microbial strains preservation administrative center, deposit number is CICC 10135) be the bacterial classification that sets out, handle through ethyl sulfate (DES) and ultraviolet mutagenesis, directive breeding goes out a strain L-Isoleucine and produces bacterial strain TC21, and its genetic marker is the methionine(Met) defective, ethionine, butyrine, S-2-amino-ethyl-L-halfcystine resistance (Met
-+ Eth
r+ α-AB
r+ AEC
r), make it have the biosynthesis ability of L-Isoleucine.
The application of brevibacterium flavum (Brevibacterium flavum) mutant strain TC21 in fermentative Production L-Isoleucine, this method:
A, the brevibacterium flavum mutant strain is added fermentation culture in the fermention medium, wherein, carbon source content should be 1%～30% in the fermention medium, carbon-nitrogen ratio N: C should be between 5: 100～50: 100, inorganic salt content should be 0.0001～10%, the initial p H6.0 of fermention medium～8.0,20 ℃～40 ℃ of culture temperature, shaking culture or fermentor cultivation 2～5 days, inoculum size are 1%～15% (V/V); In the fermenting process, add PH6.0～8.0 that lime carbonate and stream add urea, ammoniacal liquor adjusting fermention medium, fermentation ends L-Isoleucine maximum production can reach 24.82g/L;
The extraction step of B, L-Isoleucine: adopt metal membrane filter to remove thalline and albumen above-mentioned nutrient solution, then extract by Zeo-karb, elution flow rate is 1～6 times of bed volume/h, and elutriant is 0.1～10mol/L ammoniacal liquor, or ammonium chloride solution, or ammoniumsulphate soln, or Spirit of Mindererus, or ethanolic soln, adopt gac to the filtrate processing of decolouring then, obtain L-Isoleucine crude product, adopt 95% ethanol refining at last, obtain pure product.
Above-mentioned activated carbon dosage is 0.1%～5%, and pH scope 0～14, service temperature are 0～40 ℃, and bleaching time is 1～60min.
The pH scope 0～14 of the upper prop feed liquid in the ion exchange process, service temperature are 0～40 ℃, and flow velocity is 1～6 times of bed volume/h.Ion exchange column is several and is connected in series, and effusive fermented liquid behind the upper prop is injected another radical ion exchange column.
Ion exchange resin adopts strongly acidic cation-exchange, JK006, HD-8,001 * 7, D061, D001-CC and HZ014.
Carbon source in the fermention medium is a sugar, i.e. glucose, sucrose, fructose, maltose, seminose, starch and syrup; Or sugar alcohol, glycerine; Or organic acid, acetic acid; Or low mass molecule alcohol, ethanol; Carbon source content the best is 10%～20%.
Nitrogenous source is ammonia or ammonium salt: ammonium sulfate, ammonium acetate, ammonium nitrate, ammonium phosphate, ammonium acetate or ammonium chloride; Or organonitrogen: peptone, extractum carnis, corn steep liquor, or soya-bean cake hydrolyzed solution.
Inorganic salt are potassium phosphate salt, sal epsom, lime carbonate, ferrous sulfate, or manganous sulfate.
Can add nutrition in the fermention medium, amino acid and VITAMIN.
In the fermentation liquor pretreatment process in the fermention medium, with alkali fermented liquid pH is transferred to 9～14, stir and be heated to 80 ℃, inject stainless steel tubular type film separating system behind adding 0.1%～0.5% diatomite, service temperature is controlled at 50～60 ℃, advances film pressure and membrane pressure difference 0.3～0.8kg/cm
2, filtering velocity is controlled at 200～300ml/min.
Metallic membrane in the extraction step of L-Isoleucine adopts stainless steel tubular type film separating system, and its aperture is 50nm～100nm, molecular weight cut-off 2000～50000MW, and pH scope 0～14, service temperature are 0～40 ℃, operation pressure reduction is 0.2～1.0MPa.
Utilize the method for ion exchange extraction L-Isoleucine, adopt strong acid type cationic resin to be filled in the ion exchange column, before use this plastic resin treatment is become Hydrogen or sodium type, with acid the pretreated fermented liquid pH of aforesaid method is transferred to 2～6 upper props exchange absorption, service temperature is 0～40 ℃, and flow velocity is 1～6 times of bed volume/h.
The method of purification L-Isoleucine is, to transfer to 4.5 according to the L-Isoleucine pH value of solution that the said extracted method was handled, add 0.5%～2% powdered active carbon, 60 ℃～80 ℃ 1h that decolour down, filter the back use Rotary Evaporators at 60 ℃～70 ℃ reduction vaporizations to going out crystallization, add long-pending 95% ethanol of monoploid, refrigeration is spent the night, and gets coarse crystallization after the filtration; All dissolve coarse crystallization with distilled water, add isopyknic 95% ethanol again, refrigeration is spent the night, and filters crystal, gets the crystallization of L-Isoleucine after the oven dry.
Advantage of the present invention and positively effect: the present invention utilizes brevibacterium flavum (Brevibacterium flavum) mutant strain fermentative production L-Isoleucine, adopt this production technique fermentative production L-Isoleucine at 10L full automatic control fermentor tank, can improve the productive rate of L-Isoleucine greatly, production peak can reach 25g/L.The present invention utilizes metal membrane filter method pre-treatment fermented liquid, and extracts the L-Isoleucine with ion exchange method, and purity can reach more than 98%, extraction yield 50%
In L-Isoleucine biosynthetic process, because the succinyl homoserine synthase activity is than homoserine kinase height, metabolism is preferentially carried out to the synthetic methionine direction, utilizes methionine(Met) defective (Met
-) can to make biotransformation be that Threonine and Isoleucine are preferentially synthetic, and the methionine(Met) defective, and it is removed for homoserine dehydrogenase and these two feedback repressions of homoserine kinase, also makes more carbon skeleton turn to the synthetic of Threonine and Isoleucine.
The analog S-2-amino-ethyl L-halfcystine resistance (AEC of seed selection Methionin
r) can remove Methionin, Threonine collaborative feedback inhibition to E.C. 2.7.2.4., help the accumulation of the precursor Threonine of L-Isoleucine.
(α-AB) is the analog of Xie Ansuan to butyrine.Seed selection α-AB resistant mutant strain, the releasing branched-chain amino acid of inheritability is to the collaborative feedback repression of acetohydroxy acid synthetic enzyme and the Xie Ansuan feedback inhibition to the acetohydroxy acid synthetic enzyme, also can remove the feedback regulation of Isoleucine in heredity ground to threonine dehydra(ta)se, thereby impel Threonine to be converted into Isoleucine in a large number, the result helps the accumulation of Isoleucine.
Utilize the metal membrane filter pre-treatment can effectively remove suspended substances such as thalline in the fermented liquid, heteroproteins, help the absorption of L-Isoleucine.Use HZ014 vinylformic acid strongly acidic cation-exchange, fermented liquid is acidified to pH=2 with oxalic acid, and the absorption of twin columns polyphone is with the 0.5mol/LNH of pH=9
4The Cl wash-out, after the charcoal absorption decolouring, with 95% alcohol crystal purifying, purity can reach more than 98%, extraction yield 50%.
[embodiment]:
Embodiment 1: the preparation of mutant strain TC21
Brevibacterium flavum provided by the invention (Brevibacterium flavum) mutant strain TC21, brevibacterium flavum HL41 with Corynebacterium is a starting strain, handle through ethyl sulfate (DES) and ultraviolet mutagenesis, directive breeding goes out two strain L-Isoleucines and produces bacterial strain TC21, the genetic marker of TC21 is the methionine(Met) defective, the butyrine resistance, S-2-amino-ethyl-L-halfcystine resistance, ethionine resistance (Met
-+ AEC
r+ α-AB
r+ Eth
r), it has the biosynthesis ability of L-Isoleucine.
The L-Isoleucine is produced the seed selection of bacterial strain:
The first step, mutagenesis primary dcreening operation:
Will be after the conventional mutagenesis of DES bacterial strain, make bacteria suspension through centrifuge washing, coat minimum medium flat board (glucose 20g/L, (NH after the dilution
4)
2SO
410g/L, KH
2PO
43H
2O 1g/L, MgSO
47H
2O 0.4g/L, FeSO
47H
2O 0.01g/L, MnSO
4H
2O 0.01g/L, vitamin H 100 μ g/L, VB
1100 μ g/L, agar powder 20g/L, PH 7.0～7.2), in every flat board, place an aseptic filter paper sheet that speckles with defective material liquid again, 31 ℃ of constant temperature leave standstill to be cultivated 4～6 days, selected the single bacterium colony in the inhibition zone around the filter paper, and preserved in the inclined-plane and wait to sieve.
Second step, mutagenesis are sieved again
Shake on bottle level former bacterial strain and each mutant strain are produced L-Isoleucine performance relatively
Picking one encircles former bacterial strain and each mutant strain lawn (glucose 25g/L, (NH in seed culture medium respectively from fresh inclined-plane
4)
2SO
43g/L, soya-bean cake hydrolyzed solution 21mL/L, KH
2PO
43H
2O 1.5g/L, MgSO
47H
2O0.4g/L, FeSO
47H
2O 0.01g/L, MnSO
4H
2O 0.01g/L, vitamin H 0.5mg/L, VB
12.5mg/LpH7.0～7.2), 500mL triangular flask liquid amount 55mL, 9 layers of gauze seal.Place (200r/min) on the rotary shaking table 31 ℃ of shaking culture 36 hours.With inoculum size 10%, initial pH7.0 inserts (glucose 160g/L, (NH in the fermention medium
4)
2SO
412.5g/L, KH
2PO
43H
2O 1.5g/L, MgSO
47H
2O0.5g/L, CaCO
330g/L, K
2HPO
43H
2O 3g/L,, FeSO
47H
2O 15mg/L, MnSO
4H
2O15mg/L, vitamin H 140 μ g/L, V
B11mg/L, Met 20mg/L, soya-bean cake hydrolyzed solution 20mL/L).500mL triangular flask liquid amount 25mL.31 ℃ of shaking culture 96 hours, rotating speed is 200r/min, intermittent flow adds 30% urea control pH about 7.0.Former bacterial strain and each mutant strain product L-Isoleucine performance are relatively seen Table 1.
The screening of table 1 Isoleucine superior strain and shake flask fermentation result
| Bacterial strain number | Genetic marker | Output g/L) |
| HL41 J92 H49 ISW230 TC21 | (Met -) (Met -+AEC r) (Met -+AEC r+α-AB r) (Met -+AEC r+α-AB r+Eth r) | Trace trace 3.6 9.18 21.45 |
By table 1 as seen, the product L-Isoleucine performance of mutant strain TC21 is the strongest.
Embodiment 2:
Utilize bacterial classification of the present invention to produce the L-Isoleucine:
Accumulate the L-Isoleucine by in substratum, cultivating, can effectively produce the L-Isoleucine as the above-mentioned brevibacterium flavum that obtains with in substratum.
In order to utilize brevibacterium flavum of the present invention to produce the L-Isoleucine, can utilize and contain carbon source, nitrogenous source and inorganic salt and other nutritive substance of trace such as the substratum of amino acid and VITAMIN are cultivated.
In seed culture medium, inclined-plane seed culture and seed and fermentation culture conditions are all with embodiment 1 from the lawn of fresh inclined-plane picking one ring TC21 bacterial strain, and the seed culture based component comprises: glucose 25g/L, (NH
4)
2SO
43g/L, soya-bean cake hydrolyzed solution 21mL/L, KH
2PO
43H
2O 1.5g/L, MgSO
47H
2O 0.4g/L, FeSO
47H
2O 0.01g/L, MnSO
4H
2O 0.01g/L, vitamin H 0.5mg/L, VB
12.5mg/L, pH7.0～7.2.500mL triangular flask liquid amount 55mL, 9 layers of gauze seal.Place (200r/min) on the rotary shaking table 31 ℃ of shaking culture 36 hours.With inoculum size 10%, initial pH7.0 inserts in the fermention medium, and composition comprises: glucose 160g/L, (NH
4)
2SO
412.5g/L, KH
2PO
43H
2O 1.5g/L, MgSO
47H
2O 0.5g/L, CaCO
330g/L, K
2HPO
43H
2O 3g/L,, FeSO
47H
2O 15mg/L, MnSO
4H
2O 15mg/L, vitamin H 140 μ g/L, V
B11mg/L, Met 20mg/L, soya-bean cake hydrolyzed solution 20mL/L.500mL triangular flask liquid amount 25mL.31 ℃ of shaking culture 96 hours, rotating speed is 200r/min, intermittent flow adds 30% urea control pH about 7.0.Fermentation ends L-Isoleucine maximum production is 21.45g/L
Embodiment 3
In seed culture medium, inclined-plane seed culture and seed culture condition are all with embodiment 1 from the lawn of fresh inclined-plane picking one ring TC21 bacterial strain, and the seed culture based component comprises: glucose 35g/L, (NH
4)
2SO
43g/L, soya-bean cake hydrolyzed solution 15mL/L, corn steep liquor 15mL/L, KH
2PO
4.3H
2O 1.5gg/L, MgSO
47H
2O 0.4gg/L, FeSO
47H
2O 0.01g/L, MnSO
4H
2O 0.01g/L, vitamin H 0.5mg/L, VB
12.5mg/L, urea 2g/L, pH7.0～7.2.By 15% inoculum size seed liquor 450mL is inserted in the 5L fermentor tank, the fermentation culture based component comprises: glucose 160g/L, (NH
4)
2SO
412.5g/L, corn steep liquor 30mL/L, KH
2PO
43H
2O1.5g/L, MgSO
47H
2O 0.5g/L, K
2HPO
43H
2O 3g/L,, FeSO
47H
2O 15mg/L, MnSO
4H
2O 15mg/L, vitamin H 0.1mg/L, V
B11mg/L, Met 20mg/L, soya-bean cake hydrolyzed solution 20mL/L.Initial loading liquid measure 3L, ventilation 500L/h, mixing speed 600r/min, 31 ℃ of culture temperature are by auto-feeding ammonia soln control pH7.0 ± 0.05.L-Isoleucine maximum production is 22.8g/L after the fermentation ends
Embodiment 4
In seed culture medium, inclined-plane seed culture and seed insert seed liquor 450mL in the 5L fermentor tank by 15% inoculum size all with embodiment 3 from the lawn of fresh inclined-plane picking one ring TC21 bacterial strain, and the fermentation culture based component comprises: glucose 70g/L, (NH
4)
2SO
412.5g/L, corn steep liquor 30mL/L, KH
2PO
43H
2O1.5g/L, MgSO
47H
2O 0.5g/L, K
2HPO
43H
2O 3g/L,, FeSO
47H
2O 15mg/L, MnSO
4H
2O 15mg/L, vitamin H 0.1mg/L, V
B11mg/L, Met 20mg/L, soya-bean cake hydrolyzed solution 20mL/L.Initial loading liquid measure 3L, ventilation 500L/h, mixing speed 600r/min, 31 ℃ of culture temperature are by auto-feeding ammonia soln control pH7.0 ± 0.05.Between yeast phase, survey residual sugar, reduce to 2～3g/L when residual sugar and promptly mend into concentration 80% glucose every the 2h sampling.Determine the feed supplement amount according to sugar consumption rate, keep glucose concn 2～3g/L.Feed supplement finishes, and residual sugar is lower than 2g/L and finishes fermentation.The TC21 bacterial strain enters behind the 15h in fermentation and produces the acid phase, reaches at about 60h and produces sour peak period, and its high acid amount reaches 24.82g/L.
The extraction purifying of embodiment 5 L-Isoleucines
With NaOH 10L isoleucine fermentation liquid pH is transferred to 11, stir and 80 ℃ of heating in water bath 10min, add 0.1% diatomite, pour fermented liquid into the metal membrane filter device, 50 ℃～60 ℃ of controlled temperature advance film pressure and membrane pressure difference 0.6kg/cm
2, when turnover mould difference obviously reduces, mend appropriate amount of deionized water, filtering velocity is controlled at 200～300ml/min, and it is standby that the fermented liquid after will filtering with oxalic acid or hydrochloric acid transfers to pH=2.To be processed into H
+The HZ014 resin of type is packed in the ion exchange column of diameter 2.4cm, the high 12cm of dress post, two posts polyphone, service temperature is a room temperature, and upper prop speed is 0.5BV/h, behind the upper prop, with the sour water washing resin of twice fermentating liquid volume pH=2, flow velocity is 1BV/h, washings is collected prepared upper prop once more.With ammoniacal liquor with 0.5mol/LNH
4Cl transfers to pH=9, and the 0.6BV/h wash-out is collected the elutriant of pH=2～9 parts.
PH transfers to 4.5 with elutriant, adds 1% powdered active carbon, 60 ℃ of 1h that decolour down, and use Rotary Evaporators in filtration back, adds monoploid and amasss 95% ethanol to going out crystallization at 60 ℃～70 ℃ reduction vaporizations, and refrigeration is spent the night, and gets coarse crystallization after the filtration.With a certain amount of dissolved in distilled water coarse crystallization, add isopyknic 95% ethanol again, refrigeration is spent the night, and filters crystal, gets the crystallization of L-Isoleucine after the oven dry.
Claims (10)
1, a kind of brevibacterium flavum (Brevibacterium flavum) mutant strain TC21 has L-Isoleucine throughput; Mutant strain TC21 genetic marker is the methionine(Met) defective, ethionine, butyrine, S-2-amino-ethyl-L-halfcystine resistance (Met
-+ Eth
r+ α-AB
r+ AEC
r).
2, the mutafacient system of the described brevibacterium flavum mutant strain of a kind of claim 1 TC21, it is characterized in that: the brevibacterium flavum HL41 with Corynebacterium (is preserved in Chinese industrial microbial strains preservation administrative center, deposit number is CICC 10135) be the bacterial classification that sets out, handle through ethyl sulfate (DES) and ultraviolet mutagenesis, directive breeding goes out a strain L-Isoleucine and produces bacterial strain TC21, its genetic marker is the methionine(Met) defective, ethionine, butyrine, S-2-amino-ethyl-L-halfcystine resistance (Met
-+ Eth
r+ α-AB
r+ AEC
r), make it have the biosynthesis ability of L-Isoleucine.
3, the application of the described brevibacterium flavum mutant strain of a kind of claim 1 TC21 in fermentative Production L-Isoleucine is characterized in that:
A, the brevibacterium flavum mutant strain is added fermentation culture in the fermention medium, wherein, carbon source content should be 1%～30% in the fermention medium, carbon-nitrogen ratio N: C should be between 5: 100～50: 100, inorganic salt content should be 0.0001～10%, the initial p H6.0 of fermention medium～8.0,20 ℃～40 ℃ of culture temperature, shaking culture or fermentor cultivation 2～5 days, inoculum size are 1%～15% (V/V); In the fermenting process, add PH6.0～8.0 that lime carbonate and stream add urea, ammoniacal liquor adjusting fermention medium;
The extraction step of B, L-Isoleucine: adopt metal membrane filter to remove thalline and albumen above-mentioned nutrient solution, then extract by Zeo-karb, elution flow rate is 1～6 times of bed volume/h, and elutriant is 0.1～10mol/L ammoniacal liquor, or ammonium chloride solution, or ammoniumsulphate soln, or Spirit of Mindererus, or ethanolic soln, adopt gac to the filtrate processing of decolouring then, obtain L-Isoleucine crude product, adopt 95% ethanol refining at last, obtain pure product.
4, application according to claim 3 is characterized in that the carbon source in the fermention medium is sugar, i.e. glucose, sucrose, fructose, maltose, seminose, starch and syrup; Or sugar alcohol, glycerine; Or organic acid, acetic acid; Or low mass molecule alcohol, ethanol; Carbon source content the best is 10%～20%.
5, application according to claim 3 is characterized in that the nitrogenous source in the fermention medium is ammonia or ammonium salt: ammonium sulfate, ammonium acetate, ammonium nitrate, ammonium phosphate, ammonium acetate or ammonium chloride; Or organonitrogen: peptone, extractum carnis, corn steep liquor, or soya-bean cake hydrolyzed solution.
6, application according to claim 3 is characterized in that inorganic salt are potassium phosphate salt, sal epsom, lime carbonate, ferrous sulfate, or manganous sulfate.
7, application according to claim 3, it is characterized in that in the fermentation liquor pretreatment process in the fermention medium, with alkali fermented liquid pH is transferred to 9～14, stirring also is heated to 80 ℃, inject stainless steel tubular type film separating system after adding 0.1%～0.5% diatomite, service temperature is controlled at 50～60 ℃, advances film pressure and membrane pressure difference 0.3～0.8kg/cm
2, filtering velocity is controlled at 200～300ml/min.
8, application according to claim 3 is characterized in that adopting stainless steel tubular type film separating system, and its aperture is 50nm～100nm, molecular weight cut-off 2000～50000MW, and pH scope 0～14, service temperature are 0～40 ℃, operation pressure reduction is 0.2～1.0MPa.
9, according to claim 3,7 described application, it is characterized in that utilizing the method for ion exchange extraction L-Isoleucine, adopt strong acid type cationic resin to be filled in the ion exchange column, before use this plastic resin treatment is become Hydrogen or sodium type, the fermented liquid pH that the described method of claim 7 is handled with acid transfers to 2～6 upper props exchange absorption, effusive fermented liquid behind the upper prop is injected another radical ion exchange column of polyphone.Service temperature is 0～40 ℃, and flow velocity is 1～6 times of bed volume/h.
10, application according to claim 9, the method that it is characterized in that purification L-Isoleucine is, to transfer to 4.5 according to the L-Isoleucine pH value of solution that right 9 described methods were handled, add 0.5%～2% powdered active carbon, 60 ℃～80 ℃ 1h that decolour down, the use Rotary Evaporators, adds monoploid and amasss 95% ethanol to going out crystallization at 60 ℃～70 ℃ reduction vaporizations after filtering, refrigeration is spent the night, and gets coarse crystallization after the filtration; All dissolve coarse crystallization with distilled water, add isopyknic 95% ethanol again, refrigeration is spent the night, and filters crystal, gets the crystallization of L-Isoleucine after the oven dry.
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| CN108138192A (en) * | 2015-08-25 | 2018-06-08 | Cj第制糖株式会社 | It generates the microorganism of L-Leu and generates the method for L-Leu using it |
| CN108893502A (en) * | 2018-06-28 | 2018-11-27 | 无锡晶海氨基酸股份有限公司 | A method of improving l-Isoleucine yield |
| CN109554324A (en) * | 2018-12-14 | 2019-04-02 | 江南大学 | The brevibacterium flavum recombinant bacterium and its construction method of one plant of production l-Isoleucine |
| CN109851514A (en) * | 2019-02-24 | 2019-06-07 | 内蒙古拜克生物有限公司 | A kind of isolation and purification method of l-Isoleucine |
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| CN102250977A (en) * | 2011-06-28 | 2011-11-23 | 三峡大学 | Method for preparing L-isoleucine by using immobilized cells |
| CN104357503A (en) * | 2014-11-23 | 2015-02-18 | 吉林大学 | Method for improving yield of isoleucine |
| CN108138192A (en) * | 2015-08-25 | 2018-06-08 | Cj第制糖株式会社 | It generates the microorganism of L-Leu and generates the method for L-Leu using it |
| CN108893502A (en) * | 2018-06-28 | 2018-11-27 | 无锡晶海氨基酸股份有限公司 | A method of improving l-Isoleucine yield |
| CN109554324A (en) * | 2018-12-14 | 2019-04-02 | 江南大学 | The brevibacterium flavum recombinant bacterium and its construction method of one plant of production l-Isoleucine |
| CN109851514A (en) * | 2019-02-24 | 2019-06-07 | 内蒙古拜克生物有限公司 | A kind of isolation and purification method of l-Isoleucine |
| CN109851514B (en) * | 2019-02-24 | 2019-12-13 | 内蒙古拜克生物有限公司 | Separation and purification method of L-isoleucine |
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