CN101914584A - Method for producing L-tryptophan - Google Patents
Method for producing L-tryptophan Download PDFInfo
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Abstract
The invention discloses a method for producing L-tryptophan, which comprises the following steps of: performing primary culture and shaking culture on corynebacterium glutamicum strains to obtain shaking liquid strains; preparing glucose liquid from corn serving as a raw material by steps of soaking, pulping, liquefying, saccharifying and the like, wherein the glucose liquid is used as a carbon source of a culture medium and adds sugar for fermentation flow; performing secondary culture on the shaking liquid strains to obtain secondary strains, and performing fermentation culture on the secondary strains to obtain L-tryptophan fermentation liquid; and extracting and refining the L-tryptophan fermentation liquid to obtain the L-tryptophan. The acid yield of the strains can reach 40g/L at most by controlling the composition of the culture medium and the fermentation conditions; the glucose liquid is produced by using the corn, so the production cost is low; the fluctuation of the glucose content of the culture medium is low and the saccharic acid conversion rate of a fermentation acid production level box is improved by continuously adding the glucose liquid; and the extracting and refining process flows are reasonable, the purity of the L-tryptophan product can reach over 99 percent, and the yield reaches over 75 percent.
Description
Technical field
The present invention relates to a kind of amino acid whose production method, be specifically related to a kind of fermentation method for producing of L-tryptophane, belong to the microbial fermentation technology field.
Background technology
The L-tryptophane is the neutral die aromatischen Aminosaeuren that contains indyl, formal name used at school: tryptophane; English name: Tryptophan; Molecular formula is: C
11H
12N
2O
2, relative molecular mass 204.21,289 ℃ of fusing points.Its structural formula is as follows:
The L-tryptophane is white or little yellow crystal or crystalline powder; The odorless mildly bitter flavor.Slightly soluble in the water, soluble,very slightly in ethanol, insoluble in chloroform, easily molten in formic acid, in sodium hydroxide solution or dilute hydrochloric acid, dissolve.It is present in the natural protein widely.The L-tryptophane also is widely used in aspects such as medicine, food, feed except that playing an important role in Nutrition and Metabolism.In recent years, along with the developing that tryptophane is used at aspects such as medicine, food and feeds, market demand constantly increases.
The L-tryptophane is one of eight kinds of essential amino acids in human body and the animal life activity, and the growing of humans and animals, metabolism are played an important role, and is called as second indispensable amino acid.Be widely used in aspects such as medicine, food and feed.In vivo, can synthesize hormone and physiologically active substances such as serotonine, indolylacetic acid, nicotinic acid, pigment, alkaloid and coenzyme from the L-tryptophane.These hormones and physiologically active substance participate in the specific vital movement of organism.Wherein indolylacetic acid is a kind of plant growth hormones, and serotonine is vertebrate a kind of neurotransmitter, and its content in neural system has substantial connection with the excitement and the holddown of nerve, and it also is a kind of angiotonin simultaneously.Therefore, tryptophane has the stress of elimination, improves effects such as sleeper effect, prevention and treatment pellagra, medically is being used as amino acid injection and mixed amino acid.In addition, because tryptophane is the amino acid of easy famine in some vegetable-proteins, available it take nutrient fortified food and make fodder additives, this has important effect to the utilization ratio that improves plant protein, it is the third-largest feed interpolation amino acid continue methionine(Met) and Methionin after.In addition, the effect that the L-tryptophane also has mildew-resistant, sterilization and stops oxidation can be used as the fish preservation agent.
The production method of L-tryptophane has chemical synthesis, proteolysis method, Enzymatic transformation method and fermentation method.Because chemical synthesis and proteolysis method need a large amount of organic solvents, have problems such as raw material sources and environmental pollution, seldom use aborning; Enzymatic transformation method cost is higher, and sewage quantity is big, but because production concentration height, yield height, purity height, advantage such as by product is few, purification operations is easy also have producer using now.Fermentation production of L-tryptophan has become the important method of industrialized production of L-tryptophan at present owing to advantages such as easy are lacked, controlled to its productive rate height, cycle.With the Corynebacterium glutamicum be fermented bacterium prepare the L-tryptophane have in output height, the product by product few, be convenient to advantages such as product separation and Extraction, thereby Corynebacterium glutamicum becomes the most important production of L-tryptophane fermentation industry bacterium.The thirties in 20th century, Japan just carried out fermentation production of L-tryptophan research on a large scale, mainly laid particular emphasis on traditional selection by mutation technology and changed strain properties, improved L-tryptophane output, was up to 7.2g/L, and is less to L-tryptophane fermentation research in recent years.And China starts from the seventies to the research of the compound of amino acid series, and along with the L-tryptophane market requirement is increased sharply, increasing scientific research institution is to the comprehensive deep research of L-tryptophane fermentation having carried out.Show that according to investigations there is a big difference for present domestic L-tryptophane fermentation level and advanced international standard.Major cause is that breeding technique, production technique fall behind, and the bacterial classification acid yield is not high; Fermentation period is long, transformation efficiency and to extract comprehensive yield low.Therefore, exploitation high yield L-tryptophane engineering bacteria and high-efficient production technology are very necessary.
Summary of the invention
The present invention is directed to the deficiency of above-mentioned production L-tryptophane technology, the production method of a kind of acid yield height, L-tryptophane that production cost is low is provided.
The present invention is achieved by the following measures:
A kind of production method of L-tryptophane is characterized in that may further comprise the steps at least:
(1) is bacterial classification with Corynebacterium glutamicum (Corynebacterium glutamicum), after elementary cultivation, carries out shake-flask culture and must shake a bottle liquid spawn;
(2) be raw material with the corn, make Glucose Liquid through steps such as immersion, defibrination, liquefaction, saccharification, the gained Glucose Liquid is as the carbon source and the sugaring of fermentation stream of substratum;
(3) will shake bottle liquid spawn carry out secondary cultivate second class inoculum, then second class inoculum is carried out fermentation culture and gets L-tryptophane fermented liquid;
(4) L-tryptophane fermented liquid is extracted, refining L-tryptophane.
The above-mentioned bottle strain cultivation of shaking may further comprise the steps at least:
A. serve as female the kind with Corynebacterium glutamicum (Corynebacterium glutamicum), press 0.1%-0.2%(v/v) inoculum size insert in the primary culture medium, under 30 ℃-33 ℃, leave standstill cultivation 20-24h and get elementary bacterial classification; The primary culture medium component is counted by weight: glucose 1%, and dipotassium hydrogen phosphate 0.2%, sal epsom 0.1%, yeast soak powder 2%, agar 2%, water 94.7%, pH7.0-7.2;
The elementary spawn culture base fluid that b. will obtain is inoculated in the shake-flask culture base, cultivates on reciprocating type shaking table, gets OD
600Bottle liquid spawn that shakes for 0.6-0.8; Inoculum size is 0.1%-0.2% (v/v), and shaking speed is 120-140 rev/min, and culture temperature is 30 ℃-33 ℃, and incubation time is 16-18h, the shake-flask culture base is formed: glucose 2%, and dipotassium hydrogen phosphate 0.2%, sal epsom 0.1%, yeast soak powder 1%, beans are dense 2%, water 94.7%, pH 7.0-7.2.
The preparation of above-mentioned Glucose Liquid may further comprise the steps at least:
A. corn is put into steeping tank, soak 3-5h down at 50-55 ℃;
B. after soaking corn is worn into serum;
C. corn milk is liquefied with liquefaction injector high temperature under 105 ℃ of-108 ℃ of conditions, insulation is kept 90-120min and is got liquefier in keeping jar then;
D. the liquefier behind the step c is pumped into saccharifying tank, adding liquid saccharifying enzyme carries out saccharification and gets saccharification liquid, and liquid saccharifying enzyme add-on is the pure starch of 80-120 unit/g, and saccharification temperature is 55-65 ℃, and pH is 3.8-4.5, and saccharification time is 33-36h; Used saccharifying enzyme claims glucoamylase again, and saccharifying enzyme is a kind of customary title, and formal name used at school is α-1,4-glucose hydrolysis enzyme;
E. use pressure filter press filtration saccharification liquid after the saccharification, get Glucose Liquid; The press filtration temperature is 60 ℃-70 ℃, and pressure filter pressure is 0.15MPa-0.3MPa;
In the above-mentioned Glucose Liquid preparation process, the fineness of step b gained serum is the 30-35 order, and concentration is 18 ° of B é-20 ° B é; Step c Jet liquefier pressure 0.25-0.45MPa; The used pressure filter of step e is a plate-and-frame filter press.
Above-mentioned preparation L-tryptophane fermented liquid may further comprise the steps at least:
A. will shake bottle liquid-spawn inoculation to the second class inoculum substratum by the inoculum size of 0.1-0.2% (v/v), and under 30 ℃-33 ℃, the condition of pH6-8, pressure 0.03-0.1MPa, ventilate and cultivate 14h-16h, OD
600Be the second class inoculum of 0.7-1.0, ventilating ratio is 1:0.2-0.4v/vmin; The second class inoculum medium component: glucose 5%, dipotassium hydrogen phosphate 0.2%, sal epsom 0.1, beans are dense 1.5%, water 93.2%, pH 7.0-7.2,121 ℃-125 ℃, 15min steam sterilizing;
B. the inoculum size by 10-15% (v/v) is inoculated into the enterprising wind fermentation culture that works of fermention medium with second class inoculum, add Glucose Liquid with the Continuous Flow add mode simultaneously, make the concentration of Glucose Liquid remain at 4-5wt%, get L-tryptophane fermented liquid after fermentation stops; Fermentation culture conditions: 30 ℃-33 ℃ of temperature, pH 6-8, pressure 0.03 MPa-0.1MPa, ventilating ratio 1:0.2-0.8 v/vmin, fermentation time 38h-40h; Fermentation culture based component: glucose 15%, dipotassium hydrogen phosphate 0.2%, sal epsom 0.1%, corn steep liquor 1.0%, water 83.7%, pH 7.0-7.2,121 ℃-125 ℃, 15min steam sterilizing.
In the above-mentioned L-tryptophane fermented liquid preparation process, the air that feeds among step a and the step b is the sterile air after 0.25-0.3Mpa compression, filtration, and sterile air compression, the used equipment of filtration are general air compressor and air-purification system; Among the step b, the concentration of Glucose Liquid is 500-700g/L, and Glucose Liquid adds behind 118 ℃, 10min steam sterilizing.
Above-mentioned L-tryptophane broth extraction, refining may further comprise the steps at least:
A. L-tryptophane fermented liquid being heated to 60 ℃-70 ℃, is 2-4 with sour adjust pH, through microfiltration membrane degerming, ultra-filtration membrane removal of impurities decolouring, gets cleaner liquid;
B. above-mentioned cleaner liquid is flowed into ion exchange column and adsorb, carry out wash-out with eluent then, get the high dope of L-tryptophane;
The high dope of c.L-tryptophane concentrates, after reduction vaporization concentrates, obtains L-tryptophane feed liquid through reverse osmosis membrane dehydration;
D.L-tryptophane feed liquid is crystallization 10h-12h under 0 ℃-4 ℃, pH 5.8-6.0, gets elementary L-tryptophane crystal through centrifugation then;
E. elementary L-tryptophane crystal is dissolved again, is made into the L-tryptophane solution of 30g/L-50g/L, then with L-tryptophane solution with ultra-filtration membrane decolour removal of impurities, stir decrease temperature crystalline, centrifugation, dry L-tryptophane product.
In above-mentioned L-tryptophane broth extraction, the treating process, among the step a, used acid is industrial sulphuric acid or technical hydrochloric acid; The microfiltration membrane aperture is zirconium oxide film, pellumina or ceramic membrane, aperture 0.15-0.35um, and preferred aperture is the ceramic membrane of 0.22um, micro-filtration condition: 60 ℃-70 ℃ of temperature, pressure 0.2MPa-0.35Mpa, fermented liquid flow velocity 60 L/m
2H-150L/m
2H; Ultra-filtration membrane is rolled film or hollow-fibre membrane, and molecular weight cut-off is 8-10 ten thousand D, and preferred molecular weight cut-off is the rolled film of 100,000 D, ultrafiltration condition: 40 ℃-60 ℃ of temperature, pressure 0.2MPa-0.35Mpa, fermented liquid flow velocity 50 L/m
2H-100L/m
2H.Micro-filtration condition optimization: 60 ℃ of temperature, pressure 0.25Mpa, fermented liquid flow velocity 100 L/m
2H-120L/m
2H; Ultrafiltration condition optimization: 50 ℃ of temperature, pressure 0.25Mpa, fermented liquid flow velocity 60-80L/m
2H.
In above-mentioned L-tryptophane broth extraction, the treating process, among the step b, weighting material is a storng-acid cation exchange resin in the ion exchange column, the strong-acid ion exchange resin or 001 * 7 storng-acid cation exchange resin of preferred sulfonic acid type, adsorption temp is 25-35 ℃, and the cleaner liquid flow velocity is 1.0-2.5v/vmin; The used eluent of wash-out is ammoniacal liquor or the 0.5-1.5mol/L sodium hydroxide solution of 2-3mol/L, and elution flow rate is 0.5-1.5 v/vmin.Optimum condition: adsorption temp is 30 ℃, and the cleaner liquid flow velocity is 1.5-2.0v/vmin; The used eluent of wash-out is the ammoniacal liquor of 2-3mol/L, and elution flow rate is 1.0-1.2v/vmin.
In above-mentioned L-tryptophane broth extraction, the treating process, among the step c, reverse osmosis membrane cellulose acetate membrane or polyamide composite film (aperture is greater than 5 dusts), molecular weight cut-off are smaller or equal to 100, and dehydration concentrates condition: pressure 0.5-0.6MPa, temperature 50-60 ℃; Reduction vaporization concentrates condition: temperature 70-90 ℃, vacuum tightness-0.06---0.1MPa.Dehydration concentrates condition optimization: pressure 0.55MPa, 55 ℃ of temperature; Reduction vaporization concentrates condition optimization: 80 ℃ of temperature, vacuum tightness-0.08MPa.
In above-mentioned L-tryptophane broth extraction, the treating process, among the step e, ultra-filtration membrane is rolled film or hollow-fibre membrane, molecular weight cut-off is 4000-6000D, preferred molecular weight cut-off is the rolled film of 5000D, decolouring removal of impurities condition: 40 ℃-60 ℃ of temperature, pressure 0.2MPa-0.35Mpa, flow velocity 50 L/m
2.h-80L/m
2.h; After the solution decolouring removal of impurities, be cooled to 0 ℃ of-4 ℃ of crystallization 10h-12h, centrifugation gets smart level L-tryptophane crystal then, and smart level L-tryptophane crystal is dry 2-4h under 70 ℃-80 ℃, the condition of vacuum tightness-0.06 Mpa～-0.1 Mpa, L-tryptophane product.Preferably, ultra-filtration membrane decolouring removal of impurities condition: 60 ℃ of temperature, pressure 0.3Mpa, flow velocity 60-70L/m
2.h; After the solution decolouring removal of impurities, be cooled to 0 ℃ of crystallization 10h-12h, centrifugation gets smart level L-tryptophane crystal then, and smart level L-tryptophane crystal is dry 2-4h under 75 ℃, the condition of vacuum tightness-0.08 Mpa, L-tryptophane product.
Advantage of the present invention is: be fermented bacterium with common Corynebacterium glutamicum 1., form and fermentation condition by the control substratum, the bacterial classification acid yield is increased substantially, reach as high as 40g/L; 2. utilize Maize Production Glucose Liquid technology reasonable, constant product quality, production cost is low; 3. Continuous Flow adds Glucose Liquid, makes the content fluctuation of glucose in the substratum little, can be controlled at best content, is beneficial to the generation of microbial growth and tunning, and the horizontal box glucose acid invert ratio of fermentation and acid is improved; 4. the extraction and purification process flow process is reasonable, and L-tryptophane product purity can reach more than 99%, and yield reaches more than 75%.
Embodiment
Below by specific embodiment the present invention is further set forth, should be understood that, following explanation only is in order to explain the present invention, its content not to be limited.If no special instructions, described inoculum size is the volume ratio inoculum size.
The used substratum of the embodiment of the invention is composed as follows:
The primary culture medium component is counted by weight: glucose 1%, and dipotassium hydrogen phosphate 0.2%, sal epsom 0.1%, yeast soak powder 2%, agar 2%, water 94.7%, pH7.0-7.2.
The shake-flask culture base is formed by weight: glucose 2%, and dipotassium hydrogen phosphate 0.2%, sal epsom 0.1%, yeast soak powder 1%, and beans are dense 2%, water 94.7%, pH 7.0-7.2.
The second class inoculum medium component is by weight: glucose 5%, and dipotassium hydrogen phosphate 0.2%, sal epsom 0.1, beans are dense 1.5%, water 93.2%, pH 7.0-7.2,121 ℃-125 ℃, 15min steam sterilizing.
The fermentation culture based component is by weight: glucose 15%, dipotassium hydrogen phosphate 0.2%, sal epsom 0.1%, corn steep liquor 1.0%, water 83.7%, pH 7.0-7.2,121 ℃-125 ℃, 15min steam sterilizing.
Embodiment 1
1, shake a bottle strain cultivation:
1. the eggplant bottle of elementary bacterium culture medium being packed into inserts the Corynebacterium glutamicum bacterial classification by 0.1% inoculum size then, obtains elementary bacterial classification after cultivating 24h leaving standstill under 32 ℃ of conditions;
2. the shake-flask culture base is put into and shaken bottle, the elementary spawn culture base fluid that the step obtains in the access is cultivated on reciprocating type shaking table, inoculum size is 0.1%, 120 rev/mins of rotating speeds, 32 ℃ of temperature, time cycle 18h, obtain and shake a bottle liquid spawn, gained bacterial classification microscopy thalline is even, sturdy, pollution-free, (range: 10mm, wavelength: 600nm) getting OD is 0.68 with 752 spectrophotometer measurements.
2, Glucose Liquid production:
1. with corn soak time 4h in steeping tank, 50 ℃ of holding temperatures;
2. with sand mill corn is worn into corn milk, its fineness is 30 orders, and concentration reaches 18 ° of B é;
3. with corn milk with the high temperature liquefaction under 105 ℃ of conditions of liquefaction injector, enter then and keep a jar insulation and keep 120min and get liquefier, Jet liquefier pressure is 0.25MPa;
4. liquefier is pumped into saccharifying tank, adding liquid saccharifying enzyme carries out saccharification and gets saccharification liquid, and liquid saccharifying enzyme add-on is the pure starch of 80-100 unit/g, and saccharification temperature remains 60 ℃, and pH is 3.8, saccharification period 36h;
5. use plate-and-frame filter press press filtration saccharification liquid after the saccharification, 70 ℃ of press filtration temperature, pressure 0.25MPa, getting concentration is the Glucose Liquid of 250-350g/L, Glucose Liquid concentrates the stream sugaring that is used as in next step fermented liquid production through dewatering.
3, L-tryptophane fermented liquid is produced:
1. the second class inoculum substratum is pumped into the secondary seed jar behind steam sterilizing, insert after being cooled to 33 ℃ and shake a bottle liquid spawn, inoculum size is 0.1%, simultaneously will be through the 0.25Mpa compression, the sterile air after filtering imports the secondary seed jar; This moment ventilating ratio 1:0.3v/V.min, pH6.8, pressure 0.05MPa cultivates 16h, obtains second class inoculum, second class inoculum microscopy thalline is even, and is sturdy, pollution-free, with 752 spectrophotometer measurements (range: 10mm, wavelength: must OD be 0.8 600nm);
2. fermention medium is pumped into fermentor tank behind steam sterilizing, insert second class inoculum after being cooled to 33 ℃ and carry out ventilating fermentation, bubbling air is above-mentioned sterile air in 1., simultaneously will be through 118 ℃, the mode that the Glucose Liquid of 600g/L behind the 10min steam sterilizing adds with Continuous Flow adds in the fermentor tank, make that Glucose Liquid concentration remains between the 4-5% in the fermentor tank, after stream adds end, glucose concn drops to 0-0.5g/L steadily, fermentation stops, after stopping, fermentation gets L-tryptophane fermented liquid, inoculum size is 10%, ventilating ratio is 1:0.5v/V.min, and pH 6.8(feeds liquefied ammonia control pH value), pressure 0.05MPa, fermentation 38h produces acid and reaches 38.6g/L, transformation efficiency 18.2%.
4, the L-tryptophane extracts refining:
1. L-tryptophane fermented liquid is sent into the extraction storage tank, be heated to 70 ℃, transferring pH value with industrial sulphuric acid is 4, through microfiltration membrane degerming, ultra-filtration membrane removal of impurities decolour cleaner liquid, used microfiltration membrane is the ceramic membrane of aperture 0.22um, 70 ℃ of micro-filtration controlled temperature, pressure 0.25Mpa, flow velocity 90 L/m
2.h; Used ultra-filtration membrane is that molecular weight cut-off is the rolled film of 100,000 D, 50 ℃ of ultrafiltration filter controlled temperature, pressure 0.30Mpa, flow velocity 80L/m
2.h;
2. cleaner liquid enters ion exchange column, filling 001 * 7 storng-acid cation exchange resin in the post, is that 1.8v/Vmin adsorbs at 30 ℃ of flow velocitys, and wash-out is carried out with the ammoniacal liquor of 2.5mol/L in the absorption back, elution flow rate is controlled at 1.0v/Vmin, obtains the high dope of L-tryptophane.
3. the high dope of L-tryptophane concentrates through reverse osmosis membrane dehydration that (reverse osmosis membrane is the cellulose acetate membrane of molecular weight cut-off≤100D, pressure 0.5MPa, 50 ℃ of temperature), reduction vaporization concentrates back (80 ℃ of temperature, vacuum tightness-0.08MPa), obtain L-tryptophane feed liquid.
4. L-tryptophane feed liquid is sent into crystallizer and is stirred and to be cooled to 0 ℃, to transfer that PH is 6.0, obtain crystal behind the time 10h, with the centrifugation of crystallization feed liquid, elementary L-tryptophane crystal;
5. elementary L-tryptophane crystal is dissolved again, and the L-tryptophane solution that configuration content reaches 40g/L fully dissolves it; Elementary L-tryptophane crystal after the abundant dissolving is decoloured, sends into after the removal of impurities elaboration L-tryptophane crystallizer through ultra-filtration membrane stir the companion and be cooled to 0 ℃, crystallization time 12h obtains smart level L-tryptophane crystal; Ultra-filtration membrane is the rolled film of 5000D for the ultra-filtration membrane molecular weight cut-off, 60 ℃ of temperature, pressure 0.30Mpa, flow velocity 80L/m
2.h;
6. an essence level L-tryptophane crystal is separated with whizzer, carry out Vacuumdrier oven dry 3 hours then under the condition of 80 ℃ of temperature, vacuum tightness-0.08Mpa, pulverize at last, pack, testing product purity is 99.2% to obtain qualified L-tryptophane product, yield 77.2%.
Embodiment 2
1, shake a bottle strain cultivation:
1. the eggplant bottle of elementary bacterium culture medium being packed into inserts the Corynebacterium glutamicum bacterial classification by 0.2% inoculum size then, obtains elementary bacterial classification after cultivating 20-24h leaving standstill under the 30-33 ℃ of condition;
2. the shake-flask culture base is put into and shaken bottle, the elementary spawn culture base fluid that the step obtains in the access is cultivated on reciprocating type shaking table, inoculum size is 0.15%, 140 rev/mins of rotating speeds, temperature 30-33 ℃, time cycle 16h, obtain and shake a bottle liquid spawn, gained bacterial classification microscopy thalline is even, sturdy, pollution-free, (range: 10mm, wavelength: 600nm) getting OD is 0.6 with 752 spectrophotometer measurements.
2, Glucose Liquid production:
1. with corn soak time 3-5h in steeping tank, 55 ℃ of soaking temperatures;
2. with sand mill corn is worn into corn milk, its fineness is the 32-35 order, and concentration reaches 20 ° of B é;
3. with corn milk with the high temperature liquefaction under 108 ℃ of conditions of liquefaction injector, enter then and keep a jar insulation and keep 90min and get liquefier, Jet liquefier pressure is 0.45MPa;
4. liquefier is pumped into saccharifying tank, adding liquid saccharifying enzyme carries out saccharification and gets saccharification liquid, and liquid saccharifying enzyme add-on is the pure starch of 100-120 unit/g, and saccharification temperature is 55 ℃, and pH is 4.5, saccharification period 33h;
5. use plate-and-frame filter press press filtration saccharification liquid after the saccharification, 60 ℃ of press filtration temperature, pressure 0.3MPa, getting concentration is the Glucose Liquid of 300-350g/L, Glucose Liquid concentrates the stream sugaring that is used as in next step fermented liquid production through dewatering.
3, L-tryptophane fermented liquid is produced:
1. the second class inoculum substratum is pumped into the secondary seed jar behind steam sterilizing, insert after being cooled to 30-33 ℃ and shake a bottle liquid spawn, inoculum size is 0.2%, simultaneously will be through the 0.3Mpa compression, the sterile air after filtering imports the secondary seed jar; This moment ventilating ratio 1:0.4v/V.min, pH8, pressure 0.03MPa cultivates 14h, obtains second class inoculum, second class inoculum microscopy thalline is even, and is sturdy, pollution-free, with 752 spectrophotometer measurements (range: 10mm, wavelength: must OD be 0.7 600nm);
2. fermention medium is pumped into fermentor tank behind steam sterilizing, insert second class inoculum after being cooled to 30-33 ℃ and carry out ventilating fermentation, bubbling air is above-mentioned sterile air in 1., simultaneously will be through 118 ℃, the mode that the Glucose Liquid of 500g/L behind the 10min steam sterilizing adds with Continuous Flow adds in the fermentor tank, make that Glucose Liquid concentration remains between the 4-5% in the fermentor tank, after stream adds end, glucose concn drops to 0-0.5g/L steadily, fermentation stops, after stopping, fermentation gets L-tryptophane fermented liquid, inoculum size is 15%, ventilating ratio is 1:0.8v/V.min, and pH 6(feeds liquefied ammonia control pH value), pressure 0.1MPa, fermentation 40h produces acid and reaches 39.2g/L, transformation efficiency 18.9%.
4, the L-tryptophane extracts refining:
1. L-tryptophane fermented liquid is sent into the extraction storage tank, be heated to 60 ℃, transferring pH value with technical hydrochloric acid is 2, through microfiltration membrane degerming, ultra-filtration membrane removal of impurities decolour cleaner liquid, used microfiltration membrane is the zirconium oxide film of aperture 0.15-0.2um, 60 ℃ of micro-filtration controlled temperature, pressure 0.35Mpa, flow velocity 150 L/m
2.h; Used ultra-filtration membrane is that molecular weight cut-off is the hollow-fibre membrane of 80,000 D, 60 ℃ of ultrafiltration filter controlled temperature, pressure 0.20Mpa, flow velocity 50L/m
2.h;
2. cleaner liquid enters ion exchange column, filling the strong-acid ion exchange resin of sulfonic acid type in the post, is that 1.0v/Vmin adsorbs at 25 ℃ of flow velocitys, and wash-out is carried out with the ammoniacal liquor of 2.0mol/L in the absorption back, elution flow rate is controlled at 1.5v/Vmin, obtains the high dope of L-tryptophane.
3. the high dope of L-tryptophane concentrates through reverse osmosis membrane dehydration that (reverse osmosis membrane is the polyamide composite film of molecular weight cut-off≤100D, the aperture is greater than 5 dusts, pressure 0.6MPa, 60 ℃ of temperature), reduction vaporization concentrates back (90 ℃ of temperature, vacuum tightness-0.06MPa), obtain L-tryptophane feed liquid.
4. L-tryptophane feed liquid is sent into crystallizer and is stirred and to be cooled to 4 ℃, to transfer that PH is 5.8, obtain crystal behind the time 12h, with the centrifugation of crystallization feed liquid, elementary L-tryptophane crystal;
5. elementary L-tryptophane crystal is dissolved again, and the L-tryptophane solution that configuration content reaches 30g/L fully dissolves it; Elementary L-tryptophane crystal after the abundant dissolving is decoloured, sends into after the removal of impurities elaboration L-tryptophane crystallizer through ultra-filtration membrane stir the companion and be cooled to 4 ℃, crystallization time 10h obtains smart level L-tryptophane crystal; Ultra-filtration membrane is the rolled film of 4000D for the ultra-filtration membrane molecular weight cut-off, 40 ℃ of temperature, pressure 0.2Mpa, flow velocity 50L/m
2.h;
6. an essence level L-tryptophane crystal is separated with whizzer, carry out Vacuumdrier oven dry 4 hours then under the condition of 70 ℃ of temperature, vacuum tightness-0.06Mpa, pulverize at last, pack, testing product purity is 99.3% to obtain qualified L-tryptophane product, yield 77.8%.
Embodiment 3
1, shake a bottle strain cultivation:
1. the eggplant bottle of elementary bacterium culture medium being packed into inserts the Corynebacterium glutamicum bacterial classification by 0.15% inoculum size then, obtains elementary bacterial classification after cultivating 20-24h leaving standstill under the 30-33 ℃ of condition;
2. the shake-flask culture base is put into and shaken bottle, the elementary spawn culture base fluid that the step obtains in the access is cultivated on reciprocating type shaking table, inoculum size is 0.2%, 130 rev/mins of rotating speeds, temperature 30-33 ℃, time cycle 16h, obtain and shake a bottle liquid spawn, gained bacterial classification microscopy thalline is even, sturdy, pollution-free, (range: 10mm, wavelength: 600nm) getting OD is 0.8 with 752 spectrophotometer measurements.
2, Glucose Liquid production:
1. with corn soak time 3-5h in steeping tank, 53 ℃ of soaking temperatures;
2. with sand mill corn is worn into corn milk, its fineness is the 30-32 order, and concentration reaches 19 ° of B é;
3. with corn milk with the high temperature liquefaction under 105 ℃ of conditions of liquefaction injector, enter then and keep a jar insulation and keep 110min and get liquefier, Jet liquefier pressure is 0.3MPa;
4. liquefier is pumped into saccharifying tank, adding liquid saccharifying enzyme carries out saccharification and gets saccharification liquid, and liquid saccharifying enzyme add-on is the pure starch of 120 units/g, and saccharification temperature is 65 ℃, and pH is 4.0, saccharification period 36h;
5. use plate-and-frame filter press press filtration saccharification liquid after the saccharification, 65 ℃ of press filtration temperature, pressure 0.15MPa, getting concentration is the Glucose Liquid of 330g/L, Glucose Liquid concentrates the stream sugaring that is used as in next step fermented liquid production through dewatering.
3, L-tryptophane fermented liquid is produced:
1. the second class inoculum substratum is pumped into the secondary seed jar behind steam sterilizing, insert after being cooled to 30-33 ℃ and shake a bottle liquid spawn, inoculum size is 0.15%, simultaneously will be through the 0.3Mpa compression, the sterile air after filtering imports the secondary seed jar; This moment ventilating ratio 1:0.2v/V.min, pH6, pressure 0.1MPa cultivates 16h, obtains second class inoculum, second class inoculum microscopy thalline is even, and is sturdy, pollution-free, with 752 spectrophotometer measurements (range: 10mm, wavelength: must OD be 1.0 600nm);
2. fermention medium is pumped into fermentor tank behind steam sterilizing, insert second class inoculum after being cooled to 30-33 ℃ and carry out ventilating fermentation, bubbling air is above-mentioned sterile air in 1., simultaneously will be through 118 ℃, the mode that the Glucose Liquid of 700g/L behind the 10min steam sterilizing adds with Continuous Flow adds in the fermentor tank, make that Glucose Liquid concentration remains between the 4-5% in the fermentor tank, after stream adds end, glucose concn drops to 0-0.5g/L steadily, fermentation stops, after stopping, fermentation gets L-tryptophane fermented liquid, inoculum size is 12%, ventilating ratio is 1:0.2v/V.min, and pH 8(feeds liquefied ammonia control pH value), pressure 0.03MPa, fermentation 40h produces acid and reaches 39.5g/L, transformation efficiency 19.0%.
4, the L-tryptophane extracts refining:
1. L-tryptophane fermented liquid is sent into the extraction storage tank, be heated to 65 ℃, transferring pH value with technical hydrochloric acid is 3, through microfiltration membrane degerming, ultra-filtration membrane removal of impurities decolour cleaner liquid, used microfiltration membrane is the pellumina of aperture 0.3-0.35um, 60 ℃ of micro-filtration controlled temperature, pressure 0.2Mpa, flow velocity 60 L/m
2.h; Used ultra-filtration membrane is that molecular weight cut-off is the hollow-fibre membrane of 100,000 D, 40 ℃ of ultrafiltration filter controlled temperature, pressure 0.35Mpa, flow velocity 100L/m
2.h;
2. cleaner liquid enters ion exchange column, filling the strong-acid ion exchange resin of sulfonic acid type in the post, is that 2.5v/Vmin adsorbs at 35 ℃ of flow velocitys, and wash-out is carried out with the sodium hydroxide solution of 0.5mol/L in the absorption back, elution flow rate is controlled at 0.5v/Vmin, obtains the high dope of L-tryptophane.
3. the high dope of L-tryptophane concentrates through reverse osmosis membrane dehydration that (reverse osmosis membrane is cellulose acetate membrane or the polyamide composite film of molecular weight cut-off≤100D, pressure 0.55MPa, 55 ℃ of temperature), reduction vaporization concentrates back (70 ℃ of temperature, vacuum tightness-0.1MPa), obtain L-tryptophane feed liquid.
4. L-tryptophane feed liquid is sent into crystallizer and is stirred and to be cooled to 2 ℃, to transfer that PH is 5.8, obtain crystal behind the time 12h, with the centrifugation of crystallization feed liquid, elementary L-tryptophane crystal;
5. elementary L-tryptophane crystal is dissolved again, and the L-tryptophane solution that configuration content reaches 50g/L fully dissolves it; Elementary L-tryptophane crystal after the abundant dissolving is decoloured, sends into after the removal of impurities elaboration L-tryptophane crystallizer through ultra-filtration membrane stir the companion and be cooled to 0 ℃, crystallization time 12h obtains smart level L-tryptophane crystal; Ultra-filtration membrane is the rolled film of 6000D for the ultra-filtration membrane molecular weight cut-off, 50 ℃ of temperature, pressure 0.35Mpa, flow velocity 60L/m
2.h;
6. an essence level L-tryptophane crystal is separated with whizzer, carry out Vacuumdrier oven dry 2 hours then under the condition of 75 ℃ of temperature, vacuum tightness-0.1Mpa, pulverize at last, pack, testing product purity is 99.1% to obtain qualified L-tryptophane product, yield 76.9%.
Embodiment 4
1, shake a bottle strain cultivation:
1. the eggplant bottle of elementary bacterium culture medium being packed into inserts the Corynebacterium glutamicum bacterial classification by 0.2% inoculum size then, obtains elementary bacterial classification after cultivating 24h leaving standstill under 32 ℃ of conditions;
2. the shake-flask culture base is put into and shaken bottle, the elementary spawn culture base fluid that the step obtains in the access is cultivated on reciprocating type shaking table, inoculum size is 0.15%, 140 rev/mins of rotating speeds, 32 ℃ of temperature, time cycle 18h, obtain and shake a bottle liquid spawn, gained bacterial classification microscopy thalline is even, sturdy, pollution-free, (range: 10mm, wavelength: 600nm) getting OD is 0.68 with 752 spectrophotometer measurements.
2, Glucose Liquid production: with embodiment 1.
3, L-tryptophane fermented liquid is produced: with embodiment 3.
4, the L-tryptophane extracts refining:
1. L-tryptophane fermented liquid is sent into the extraction storage tank, be heated to 70 ℃, transferring pH value with industrial sulphuric acid is 4, through microfiltration membrane degerming, ultra-filtration membrane removal of impurities decolour cleaner liquid, used microfiltration membrane is the ceramic membrane of aperture 0.22um, 60 ℃ of micro-filtration controlled temperature, pressure 0.25Mpa, flow velocity 120 L/m
2.h; Used ultra-filtration membrane is that molecular weight cut-off is the rolled film of 100,000 D, 50 ℃ of ultrafiltration filter controlled temperature, pressure 0.25Mpa, flow velocity 60L/m
2.h;
2. cleaner liquid enters ion exchange column, filling 001 * 7 storng-acid cation exchange resin in the post, is that 1.5v/Vmin adsorbs at 30 ℃ of flow velocitys, and wash-out is carried out with the ammoniacal liquor of 3.0mol/L in the absorption back, elution flow rate is controlled at 1.2v/Vmin, obtains the high dope of L-tryptophane.
3. the high dope of L-tryptophane concentrates through reverse osmosis membrane dehydration that (reverse osmosis membrane is cellulose acetate membrane or the polyamide composite film of molecular weight cut-off≤100D, pressure 0.55MPa, 55 ℃ of temperature), reduction vaporization concentrates back (80 ℃ of temperature, vacuum tightness-0.08MPa), obtain L-tryptophane feed liquid.
4. L-tryptophane feed liquid is sent into crystallizer and is stirred and to be cooled to 0 ℃, to transfer that PH is 6.0, obtain crystal behind the time 10h, with the centrifugation of crystallization feed liquid, elementary L-tryptophane crystal;
5. elementary L-tryptophane crystal is dissolved again, and the L-tryptophane solution that configuration content reaches 40g/L fully dissolves it; Elementary L-tryptophane crystal after the abundant dissolving is decoloured, sends into after the removal of impurities elaboration L-tryptophane crystallizer through ultra-filtration membrane stir the companion and be cooled to 0 ℃, crystallization time 12h obtains smart level L-tryptophane crystal; Ultra-filtration membrane is the rolled film of 5000D for the ultra-filtration membrane molecular weight cut-off, 60 ℃ of temperature, pressure 0.30Mpa, flow velocity 70L/m
2.h;
6. an essence level L-tryptophane crystal is separated with whizzer, carry out Vacuumdrier oven dry 3 hours then under the condition of 75 ℃ of temperature, vacuum tightness-0.08Mpa, pulverize at last, pack, testing product purity is 99.7% to obtain qualified L-tryptophane product, yield 78.9%.
Embodiment 5
1, shakes a bottle strain cultivation: with embodiment 2.
2, Glucose Liquid production: with embodiment 2.
3, L-tryptophane fermented liquid is produced: with embodiment 2.
4, the L-tryptophane extracts refining:
1. L-tryptophane fermented liquid is sent into the extraction storage tank, be heated to 70 ℃, transferring pH value with industrial sulphuric acid is 4, through microfiltration membrane degerming, ultra-filtration membrane removal of impurities decolour cleaner liquid, used microfiltration membrane is the ceramic membrane of aperture 0.22um, 60 ℃ of micro-filtration controlled temperature, pressure 0.25Mpa, flow velocity 100 L/m
2.h; Used ultra-filtration membrane is that molecular weight cut-off is the rolled film of 100,000 D, 50 ℃ of ultrafiltration filter controlled temperature, pressure 0.25Mpa, flow velocity 60L/m
2.h;
2. cleaner liquid enters ion exchange column, filling 001 * 7 storng-acid cation exchange resin in the post, is that 2.0v/vmin adsorbs at 30 ℃ of flow velocitys, and wash-out is carried out with the aqueous sodium hydroxide solution of 1.5mol/L in the absorption back, elution flow rate is controlled at 1.2v/Vmin, obtains the high dope of L-tryptophane.
Other are with embodiment 2.Gained L-tryptophane product purity is 99.5%, yield 79.5%.
Embodiment 6
1, shakes a bottle strain cultivation: with embodiment 3.
2, Glucose Liquid production: with embodiment 3.
3, L-tryptophane fermented liquid is produced:
1. the second class inoculum substratum is pumped into the secondary seed jar behind steam sterilizing, insert after being cooled to 33 ℃ and shake a bottle liquid spawn, inoculum size is 0.15%, simultaneously will be through the 0.3Mpa compression, the sterile air after filtering imports the secondary seed jar; This moment ventilating ratio 1:0.5v/V.min, pH6.8, pressure 0.05MPa cultivates 16h, obtains second class inoculum, second class inoculum microscopy thalline is even, and is sturdy, pollution-free, with 752 spectrophotometer measurements (range: 10mm, wavelength: must OD be 0.9 600nm);
2. fermention medium is pumped into fermentor tank behind steam sterilizing, insert second class inoculum after being cooled to 32 ℃ and carry out ventilating fermentation, bubbling air is above-mentioned sterile air in 1., simultaneously will be through 118 ℃, the mode that the Glucose Liquid of 700g/L behind the 10min steam sterilizing adds with Continuous Flow adds in the fermentor tank, make that Glucose Liquid concentration remains between the 4-5% in the fermentor tank, after stopping, fermentation gets L-tryptophane fermented liquid, inoculum size is 15%, ventilating ratio is 1:0.6v/V.min, pH 6.8(feeds liquefied ammonia control pH value), pressure 0.05MPa, fermentation 40h produces acid and reaches 40.0g/L, transformation efficiency 19.8%.
4, the L-tryptophane extracts refining: with embodiment 3, gained L-tryptophane product purity is 99.5%, yield 78.3%.
Claims (10)
1. the production method of a L-tryptophane is characterized in that may further comprise the steps at least:
(1) is bacterial classification with Corynebacterium glutamicum (Corynebacterium glutamicum), after elementary cultivation, carries out shake-flask culture and must shake a bottle liquid spawn;
(2) be raw material with the corn, make Glucose Liquid through steps such as immersion, defibrination, liquefaction, saccharification, the gained Glucose Liquid is as the carbon source and the sugaring of fermentation stream of substratum;
(3) will shake bottle liquid spawn carry out secondary cultivate second class inoculum, then second class inoculum is carried out fermentation culture and gets L-tryptophane fermented liquid;
(4) L-tryptophane fermented liquid is extracted, refining L-tryptophane.
2. production method according to claim 1 is characterized in that shaking a bottle strain cultivation and may further comprise the steps at least:
A. serve as female the kind with Corynebacterium glutamicum (Corynebacterium glutamicum), press 0.1%-0.2%(v/v) inoculum size insert in the primary culture medium, under 30 ℃-33 ℃, leave standstill cultivation 20-24h and get elementary bacterial classification; The primary culture medium component is counted by weight: glucose 1%, and dipotassium hydrogen phosphate 0.2%, sal epsom 0.1%, yeast soak powder 2%, agar 2%, water 94.7%, pH7.0-7.2;
The elementary spawn culture base fluid that b. will obtain is inoculated in the shake-flask culture base, cultivates on reciprocating type shaking table, gets OD
600Bottle liquid spawn that shakes for 0.6-0.8; Inoculum size is 0.1%-0.2% (v/v), and shaking speed is 120-140 rev/min, and culture temperature is 30 ℃-33 ℃, and incubation time is 16-18h, the shake-flask culture base is formed: glucose 2%, and dipotassium hydrogen phosphate 0.2%, sal epsom 0.1%, yeast soak powder 1%, beans are dense 2%, water 94.7%, pH 7.0-7.2.
3. production method according to claim 1 is characterized in that the preparation of Glucose Liquid may further comprise the steps at least:
A. corn is put into steeping tank, soak 3-5h down at 50-55 ℃;
B. after soaking corn is worn into serum;
C. corn milk is liquefied with liquefaction injector high temperature under 105 ℃ of-108 ℃ of conditions, insulation is kept 90-120min and is got liquefier in keeping jar then;
D. the liquefier behind the step c is pumped into saccharifying tank, adding liquid saccharifying enzyme carries out saccharification and gets saccharification liquid, and liquid saccharifying enzyme add-on is the pure starch of 80-120 unit/g, and saccharification temperature is 55-65 ℃, and pH is 3.8-4.5, and saccharification time is 33-36h;
E. use pressure filter press filtration saccharification liquid after the saccharification, get Glucose Liquid; The press filtration temperature is 60 ℃-70 ℃, and pressure filter pressure is 0.15MPa-0.3MPa.
4. production method according to claim 3 is characterized in that: in the Glucose Liquid preparation process, the fineness of step b gained serum is the 30-35 order, and concentration is 18 ° of B é-20 ° B é; Step c Jet liquefier pressure 0.25-0.45MPa; The used pressure filter of step e is a plate-and-frame filter press.
5. production method according to claim 1 is characterized in that preparing L-tryptophane fermented liquid and may further comprise the steps at least:
A. will shake bottle liquid-spawn inoculation to the second class inoculum substratum by the inoculum size of 0.1-0.2% (v/v), and under 30 ℃-33 ℃, the condition of pH6-8, pressure 0.03-0.1MPa, ventilate and cultivate 14h-16h, OD
600Be the second class inoculum of 0.7-1.0, ventilating ratio is 1:0.2-0.4v/vmin; The second class inoculum medium component: glucose 5%, dipotassium hydrogen phosphate 0.2%, sal epsom 0.1, beans are dense 1.5%, water 93.2%, pH 7.0-7.2,121 ℃-125 ℃, 15min steam sterilizing;
B. the inoculum size by 10-15% (v/v) is inoculated into the enterprising wind fermentation culture that works of fermention medium with second class inoculum, add Glucose Liquid with the Continuous Flow add mode simultaneously, make the concentration of Glucose Liquid remain at 4-5wt%, get L-tryptophane fermented liquid after fermentation stops; Fermentation culture conditions: 30 ℃-33 ℃ of temperature, pH 6-8, pressure 0.03 MPa-0.1MPa, ventilating ratio 1:0.2-0.8 v/vmin, fermentation time 38h-40h; Fermentation culture based component: glucose 15%, dipotassium hydrogen phosphate 0.2%, sal epsom 0.1%, corn steep liquor 1.0%, water 83.7%, pH 7.0-7.2,121 ℃-125 ℃, 15min steam sterilizing.
6. production method according to claim 5 is characterized in that: in the L-tryptophane fermented liquid preparation process, the air that feeds among step a and the step b is the sterile air after 0.25-0.3Mpa compression, filtration; Among the step b, the concentration of Glucose Liquid is 500-700g/L, and Glucose Liquid adds behind 118 ℃, 10min steam sterilizing.
7. production method according to claim 1 is characterized in that L-tryptophane broth extraction, refining may further comprise the steps at least:
A. L-tryptophane fermented liquid being heated to 60 ℃-70 ℃, is 2-4 with sour adjust pH, through microfiltration membrane degerming, ultra-filtration membrane removal of impurities decolouring, gets cleaner liquid;
B. above-mentioned cleaner liquid is flowed into ion exchange column and adsorb, carry out wash-out with eluent then, get the high dope of L-tryptophane;
The high dope of c.L-tryptophane concentrates, after reduction vaporization concentrates, obtains L-tryptophane feed liquid through reverse osmosis membrane dehydration;
D.L-tryptophane feed liquid is crystallization 10h-12h under 0 ℃-4 ℃, pH 5.8-6.0, gets elementary L-tryptophane crystal through centrifugation then;
E. elementary L-tryptophane crystal is dissolved again, is made into the L-tryptophane solution of 30g/L-50g/L, then with L-tryptophane solution with ultra-filtration membrane decolour removal of impurities, stir decrease temperature crystalline, centrifugation, dry L-tryptophane product.
8. production method according to claim 7 is characterized in that: in L-tryptophane broth extraction, the treating process, among the step a, used acid is industrial sulphuric acid or technical hydrochloric acid; The microfiltration membrane aperture is 0.15-0.35um, the micro-filtration condition: 60 ℃-70 ℃ of temperature, pressure 0.2MPa-0.35Mpa, fermented liquid flow velocity 60 L/m
2H-150L/m
2H; The ultra-filtration membrane molecular weight cut-off is 8-10 ten thousand D, the ultrafiltration condition: 40 ℃-60 ℃ of temperature, pressure 0.2MPa-0.35Mpa, fermented liquid flow velocity 50 L/m
2H-100L/m
2H;
Among the step b, weighting material is a storng-acid cation exchange resin in the ion exchange column, and adsorption temp is 25-35 ℃, and the cleaner liquid flow velocity is 1.0-2.5v/vmin; The used eluent of wash-out is ammoniacal liquor or the 0.5-1.5mol/L sodium hydroxide solution of 2-3mol/L, and elution flow rate is 0.5-1.5 v/vmin;
Among the step c, the reverse osmosis membrane molecular weight cut-off is smaller or equal to 100, and dehydration concentrates condition: pressure 0.5-0.6MPa, temperature 50-60 ℃; Reduction vaporization concentrates condition: temperature 70-90 ℃, vacuum tightness-0.06～-0.1MPa;
Among the step e, the ultra-filtration membrane molecular weight cut-off is 4000-6000D, decolouring removal of impurities condition: 40 ℃-60 ℃ of temperature, pressure 0.2MPa-0.35Mpa, flow velocity 50-80L/m
2H; After the solution decolouring removal of impurities, be cooled to 0 ℃ of-4 ℃ of crystallization 10h-12h, centrifugation gets smart level L-tryptophane crystal then, and smart level L-tryptophane crystal is dry 2-4h under 70 ℃-80 ℃, the condition of vacuum tightness-0.06 Mpa～-0.1 Mpa, L-tryptophane product.
9. preparation method according to claim 8 is characterized in that: among the step a, microfiltration membrane is zirconium oxide film, pellumina or ceramic membrane, the micro-filtration condition: 60 ℃ of temperature, pressure 0.25Mpa, fermented liquid flow velocity 100 L/m
2H-120L/m
2H; Ultra-filtration membrane is rolled film or hollow-fibre membrane, the ultrafiltration condition: 50 ℃ of temperature, pressure 0.25Mpa, fermented liquid flow velocity 60-80L/m
2H;
Among the step b, weighting material is the strong-acid ion exchange resin of sulfonic acid type in the ion exchange column, and adsorption temp is 30 ℃, and the cleaner liquid flow velocity is 1.5-2.0v/vmin; The used eluent of wash-out is the ammoniacal liquor of 2-3mol/L, and elution flow rate is 1.0-1.2v/vmin;
Among the step c, reverse osmosis membrane is cellulose acetate membrane or polyamide composite film, and dehydration concentrates condition: pressure 0.55MPa, 55 ℃ of temperature; Reduction vaporization concentrates condition: 80 ℃ of temperature, vacuum tightness-0.08MPa;
Among the step e, ultra-filtration membrane rolled film or hollow-fibre membrane, decolouring removal of impurities condition: 60 ℃ of temperature, pressure 0.3Mpa, flow velocity 60-70L/m
2.h; After the solution decolouring removal of impurities, be cooled to 0 ℃ of crystallization 10h-12h, centrifugation gets smart level L-tryptophane crystal then, and smart level L-tryptophane crystal is dry 2-4h under 75 ℃, the condition of vacuum tightness-0.08 Mpa, L-tryptophane product.
10. according to claim 7,8 or 9 described production methods, it is characterized in that: among the step a, microfiltration membrane is that the aperture is the ceramic membrane of 0.22um, and ultra-filtration membrane is that molecular weight cut-off is the rolled film of 100,000 D; Among the step b, weighting material is 001 * 7 storng-acid cation exchange resin in the ion exchange column; Among the step e, ultra-filtration membrane is that molecular weight cut-off is the rolled film of 5000D.
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| CN103409477A (en) * | 2013-07-18 | 2013-11-27 | 天津科技大学 | Method for improving saccharic acid conversion rate in L-tryptophan fermentation process |
| CN105274161A (en) * | 2014-07-14 | 2016-01-27 | 武汉中粮食品科技有限公司 | Method for preparing sugar |
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| CN106174596A (en) * | 2016-08-11 | 2016-12-07 | 塔城市星河生物工程有限责任公司 | A kind of production method of food plant amino acid dry powder |
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| CN114231572A (en) * | 2021-12-10 | 2022-03-25 | 南通紫琅生物医药科技有限公司 | N-acetyl-DL-tryptophan biosynthetic enzyme conversion process |
| CN114958936A (en) * | 2022-06-30 | 2022-08-30 | 厦门大学 | Method for producing L-tryptophan by using cellulose as carbon source through fed-batch fermentation |
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