CN1279176C - Process for producing L-methionine, aminoacylase strain and aminoacylase - Google Patents
Process for producing L-methionine, aminoacylase strain and aminoacylase Download PDFInfo
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- CN1279176C CN1279176C CNB021537151A CN02153715A CN1279176C CN 1279176 C CN1279176 C CN 1279176C CN B021537151 A CNB021537151 A CN B021537151A CN 02153715 A CN02153715 A CN 02153715A CN 1279176 C CN1279176 C CN 1279176C
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Abstract
The present invention relates to a production technology for generating L-methionine by utilizing the enzyme aminoacyl of a colibacillus genetic engineering bacterium (the preservation number is CGMCC. No. 0368) to carry out enzymolysis reaction. The present invention also relates to a colibacillus enzyme aminoacyl bacterial strain and a preparation method thereof, and enzyme aminoacyl derived from the colibacillus genetic engineering bacterium. The production technology of the L-methionine of the present invention comprises the steps that N-acetyl-D, L-methionine is used as a raw material; the enzyme aminoacyl and the cultured bacterium or the treatment object thereof are used as enzyme sources; the L-methionine is generated by the step of enzymolysis reaction. The L-methionine manufactured by the method can be used as the composed raw material of medicines and cosmetics, and can be widely used as an important additive, etc. in foods and feed.
Description
Technical field:
The present invention relates to production technique, L-Aminoacylase bacterial strain of a kind of L-methionine(Met) and preparation method thereof, and L-Aminoacylase.Specifically, the present invention relates to utilize intestinal bacteria L-Aminoacylase enzyme digestion reaction, generate the production technique of L-methionine(Met), intestinal bacteria L-Aminoacylase bacterial strain and preparation method thereof, and L-Aminoacylase.
Background technology:
Amino acid is to constitute the proteinic fundamental unit of organism, it is the base substance that earns a bare living, and the L-methionine(Met) is one of sulfur-bearing " needed by human eight seed amino acids ", human health there is effect of crucial importance, fields such as medical and health, foodstuff additive, daily cosmetics have been widely used in.D, and the L-methionine(Met) (D, chemical synthesis process technology L-Met) is very ripe, and production cost is lower, and world's annual production can reach more than 200,000 tons.Be mainly used in fodder additives, the import insurance and freight (CIF) was about 4000 dollars per ton in 97 years, and this helps with D, and the L-methionine(Met) is a raw material, adopted enzyme process to split and produced the L-methionine(Met).
Adopt enzyme process to split and produce the L-methionine(Met), its principles of chemistry mainly may further comprise the steps as shown in Figure 1:
1. acylation reaction step: D, L-methionine(Met) and diacetyl oxide are under the condition that water exists, and 70 ℃ acylation reactions take place generate N-acetyl-D, L-methionine(Met).
2. enzyme digestion reaction step: N-acyl-D, L-methionine(Met) are under cobalt ion participates in, and through the special hydrolysis reaction of acylase, the ethanoyl of hydrolyzing N-acetyl-L-methionine(Met) N end generates the L-methionine(Met), also remaining N-acetyl-D-methionine(Met) in the reactant.
3. racemization step: racemization takes place at 70 ℃ under the condition that acetate exists in N-acetyl-D-methionine(Met) and diacetyl oxide, generates the N-acetyl-D of equivalent, the L-methionine(Met).
4. enzyme digestion reaction step: with 2, the N-acetyl-D after the hydrolysis racemization, L-methionine(Met).
5 and 6 repeat 3 and 4 step.
By D, the L-methionine(Met) splits the acylase of producing the L-methionine(Met) through enzyme and extensively is present in animals and plants and the biology, but the acylase that the acylase I that mainly contains the pig stomach of practical application and aspergillus oryzae produce.The former is limited because of originating, and is not suitable for large-scale industrial production, and the latter is called for short the bent acylase of rice, because of it is an extracellular enzyme, and can pass through the fermentation method mass production, so the enzyme immobilization technology of being applicable to.Japan and other national this enzyme that all adopts are produced.The bent acylase of rice is in purification and immobilization process, and enzyme is lived loss greatly, and the yield of enzyme is lower, the preparation cost height.As far back as nineteen fifty-seven; the Tchiro of Japan and Chibata etc. carried out research to the character of intestinal bacteria acylase; but because the enzyme activity that wild strain produces is very low; be not suitable for suitability for industrialized production; and; the superior strain screening that produces bacterium owing to reasons such as pathways metabolisms is very difficult, up to the present all can not produce the L-methionine(Met) with microbial fermentation technology both at home and abroad.Therefore, state such as Japan still mainly adopts the bent acylase of rice to produce so far, and also there is the research report that utilizes the bent acylase of rice to prepare L-Ala in China.
Summary of the invention
The present invention has overcome above-mentioned defective, has proposed a kind of microbial fermentation technology that utilizes, and adopts high yield intestinal bacteria acylase, is applicable to the production technique of the L-methionine(Met) of suitability for industrialized production.
The present invention provides a kind of high yield coli strain simultaneously, and it has the ability of efficient generation L-Aminoacylase.
The present invention also provides a kind of preparation method of high yield coli strain.
The present invention further provides a kind of intestinal bacteria L-Aminoacylase.
The invention provides a kind of production technique of L-methionine(Met); comprise acetyl-D with N-; the L-methionine(Met) is a raw material, is the enzyme source with the L-Aminoacylase that derives from bacillus coli gene engineering bacteria XW9707 (preserving number CGMCC.No.0368), carries out the enzyme digestion reaction step and generates the L-methionine(Met).
N-acetyl-D of the present invention, L-methionine(Met) can use method preparation well known in the art, and for example by acylation reaction step: D, L-methionine(Met) and diacetyl oxide are under the condition that water exists, and 70 ℃ acylation reaction take place generate N-acetyl-D, L-methionine(Met).In order to improve the output of L-methionine(Met) better, preferably at 60 ℃ of temperature, vacuum tightness 0.1P.M..Add N-acetyl-D, the concentration of L-methionine(Met) is 25-100g/l.
Enzyme as the enzyme source can be bacillus coli gene engineering bacteria XW9707 culture of the present invention, cultivates thalline, the enzyme that extracts in the thalline or this bacterial disposing thing.The preferred thalline of cultivating.More preferably adopt acylase engineering bacteria immobilized thallus cells produce L-methionine(Met).Wherein the enzyme that extracts in the thalline can for extract in this thalline obtain have the active enzyme of L-Aminoacylase or a cured article of the refining standard of this enzyme.Accumulated the microorganism of very high L-Aminoacylase from thalline, utilized common enzyme separation, method for refining can separate and make with extra care out L-Aminoacylase separately.For example,, collect the thalline in the nutrient solution by the nutrient solution of this microorganism of centrifugation, clean this thalline after, use broken thalline such as ultrasonic disruption machine, squeezing machine, homogenizer, ball mill or organic solvent again, obtain cell-free extract.Again cell-free extract is carried out centrifugation and obtain supernatant liquor, saltout with the sulphur ammonium again, anion-exchange columns such as DEAE, agarose, the molecular sieve gel filtration method, methods such as iso-electric point electrophoretic method can obtain refining enzyme standard substance.
As the bacterial strain handled thing, can enumerate the dry thing of Lin's body, the ultrasonication thing of the tensio-active agent handled thing of the freeze-dried thing of thalline, thalline, the enzyme handled thing of thalline, thalline, the machinery of thalline grind the cured article that protein differentiation thing, thalline and the thalline of the solvent treatment thing of handled thing, thalline, thalline handle etc.
Production technique of the present invention comprises having an active culture of very high L-Aminoacylase with what separate to obtain from culture; cultivate thalline; utilize the enzyme or this bacterial disposing thing that extract in cultivation thalline, the thalline to be used as the enzyme source; in the aqueous medium of suitable enzyme reaction; add N-acetyl-D, the L-methionine(Met), hydrolysis generates accumulates the L-methionine(Met); from this aqueous medium, extract the L-methionine(Met) again, can make the L-methionine(Met) like this.
As aqueous medium, damping fluids such as water, phosphoric acid salt, carbonate, acetate, borate, Citrate trianion can have been enumerated, alcohols such as methyl alcohol, ethanol, ester classes such as acetic ester, N-BUTYL ACETATE, ketones such as acetone, amidess such as ethanamide etc.The nutrient solution that is obtained in the L-Aminoacylase manufacturing, available its stoste or diluent also can be used as aqueous medium.
In the reaction solution of enzyme effect, its enzymic activity can wait to determine, be generally 600-60000u/l, preferably 3000-18000u/l according to amount of substrate.When utilizing the culture that obtains in the L-Aminoacylase preparation, dilute 1-10 doubly, preferably dilute 2-5 and doubly use the back.
Adjust the pH value with NaOH and be about 5.6-7.5, reaction is about 0.5-20 hour about 10-54 ℃.The final concentration of preferred control cobalt ion is 5 * 10 in the enzyme digestion reaction step
-5-1.5 * 10
-4Mole.
After reaction finishes, with acid such as sulfuric acid, hydrochloric acid the pH of aqueous medium is adjusted into 5.0, adsorbs by ion exchange column, wash-out can reclaim the L-methionine(Met) from the wash-out aqueous medium.
Preferably, the intestinal bacteria L-Aminoacylase can activate through 2% toluene before enzyme digestion reaction, and it activates more than 30 minutes.
In a preferred embodiment of the invention; adopt acylase engineering bacteria immobilized cell to produce L-methionine(Met) Technology referring to (embodiment 3); described immobilization enzymolysis is reflected in the enzyme post and carries out; the preparation method of described enzyme post is as follows: stir in the doubly isopyknic 3% sodium alginate colloidal sol of adding 4-6 in the thalline suspension liquid; slowly be added dropwise to again to the 0.2-0.7M calcium chloride solution and make it to become to solidify bead; and be positioned over 0-4 ℃ of refrigerator overnight; remove calcium chloride solution; add washing 2-3 time; then the enzyme that fixes is packed in the Glass tubing, make the enzyme post.Purification, the preparation process of acylase saved in the use of immobilized cell technology, and the vigor loss of enzyme is little, and preparation cost is low.The transformation period of enzyme is more than 20 days.This Technology bent acylase enzyme immobilization technology of rice is produced the amino acid 20-40% that saves production cost.Utilize this technology through tens of batches of tests in the laboratory, obtained with D, the L-methionine(Met) is a raw material, adopt immobilization XW9707 cell technology enzyme to split the L-methionine(Met) of producing, detect through Xinjiang medicine inspecting institute, all index meets the pharmacopeia required standard of the Japan and the U.S., splits its L-methionine(Met) productive rate through the secondary enzyme and reaches more than 70%.
Utilize crystallization process racemization of the present invention to generate the N-acetyl-D of equivalent the acetyl of the N-behind the enzyme digestion reaction-D-methionine solution, the L-methionine(Met), method is as follows: N-acetyl-D-methionine solution transfers pH to be about between the 1.5-2.5 with concentrated hydrochloric acid, preferred 2.0, vacuum concentration is to doing then, optimum condition is P=0.1p.m, t=60 ℃.A large amount of industrial chemicals has been saved in the employing of crystallization process racemization technology, and makes that a few step complex processes are reduced to a step in the racemization technology, and production cost is reduced significantly.
In order to obtain higher productive rate, can utilize method of the present invention, repeat enzyme digestion reaction and racemization processing step, through twice enzymolysis, its productive rate can reach more than 70%, and through three enzymolysis, productive rate can reach more than 75%.
Can obtain by methods known in the art by the L-methionine(Met) in the solution of the present invention's acquisition, for example by decolouring, concentrating under reduced pressure, steps such as crystallization obtain.Adopt the L-methionine(Met) preparation of method manufacturing of the present invention to can be used as the constitutive material of pharmaceuticals and makeup, and as additive in food and the feed etc.
According to measuring and calculating, according to method of the present invention with D, the L-methionine(Met) is a raw material, utilize enzyme split to produce the L-methionine(Met), about 30,000 dollars of per ton costs, ten thousand dollars/ton of and 1996-1997 Japan imported L-methionine(Met) insurance and freight (CIF) (not comprising tariff) 8.9-9.5, in 80,000 dollars, 50,000 dollars of net incomes per ton, and can save a large amount of foreign exchanges, society and economic benefit are very considerable.
On the other hand, the present invention provides a kind of L-Aminoacylase superior strain XW9707 simultaneously, is deposited in China Microbial Culture Preservation Commission common micro-organisms center (CGMCC) before the applying date.Address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100080.Preservation date is on November 20th, 1998, and preserving number is CGMCC.No.0368, and it produces acylase can reach 100-200u/ml, reaches as high as 500u/ml, is 10-20 times that the original strain acidated enzyme is lived.In a preferred embodiment of the invention, utilize acylase engineering bacteria immobilized cell to produce the L-methionine(Met), this Technology bent acylase enzyme immobilization technology production of rice amino acid saves production cost more than 30%.
This high yield L-Aminoacylase bacterial strain is to utilize gene recombination technology to obtain, and the concrete hereditary property of L-Aminoacylase is referring to accompanying drawing.
According to the invention provides the method for making the L-methionine(Met), in substratum, cultivate dust Xi Shi intestinal bacteria with generation L-Aminoacylase ability, in substratum, add succsinic acid, make to generate in the substratum and the accumulation L-Aminoacylase.
As microbiological culture media of the present invention, can contain the required carbon source of microorganism, nitrogenous source, inorganic salts, microbiotic etc., as long as this microorganism can grow well, can use any in natural medium, the synthetic medium.
As carbon source, can use glucose, fructose, sucrose, contain carbohydrate such as these sugared molasses, starch or amylolysis thing, organic acids such as acetic acid, fumaric acid, succsinic acid, lactic acid, propionic acid, alcohols such as ethanol, glycerol, propyl alcohol.
As nitrogenous source, can use various mineral acids and organic acid ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium phosphate, the extract of amine, other nitrogenous compound and peptone, meat, yeast extract, W-Gum, casein water decomposition thing, soybean meal and soybean meal water decomposition thing, various fermentation thalline and its digest etc.
As other nutritive element, can use potassium phosphate,monobasic, potassium primary phosphate, trimagnesium phosphate, sodium-chlor, ferrous sulfate etc.
Culture temperature is 20-40 ℃, preferably 30 ℃-37 ℃.The pH of substratum is 5-9, preferably PH6-8.The PH of substratum adjusts and can utilize inorganic or organic acids and base solution, ammonia, PH damping fluid etc.Usually cultivated 10-24 hour in normal condition, just can be at the very high L-Aminoacylase of thalline accumulation.
The present invention also provides the preparation method of a kind of L-Aminoacylase superior strain XW9707.When cultivating described genetic engineering bacterium, can add Thioctic Acid, succsinic acid and penbritin.For making dust Xi Shi intestinal bacteria high yield L-Aminoacylase of the present invention; use succsinic acid as nutrition source, obtain in the high yield L-Aminoacylase bacterial strain in the applying gene reorganization, for keeping the stability of recombinant bacterial strain; antibiotics is added in use in substratum, for example the method for penbritin.
The present invention further provides a kind of L-Aminoacylase; it derives from bacillus coli gene engineering bacteria XW9707 (preserving number CGMCC.No.0368); its can for from this thalline, extract obtain have the active enzyme of L-Aminoacylase or a cured article of the refining standard of this enzyme.Accumulated the microorganism of very high L-Aminoacylase from thalline, utilized common enzyme separation, method for refining can separate and make with extra care out L-Aminoacylase separately.For example,, collect the thalline in the nutrient solution by the nutrient solution of this microorganism of centrifugation, clean this thalline after, use broken thalline such as ultrasonic disruption machine, squeezing machine, homogenizer, ball mill or organic solvent again, obtain cell-free extract.Again cell-free extract is carried out centrifugation and obtain supernatant liquor, saltout with the sulphur ammonium again, anion-exchange columns such as DEAE, agarose, the molecular sieve gel filtration method, methods such as iso-electric point electrophoretic method can obtain refining enzyme standard substance.Enzyme feature: molecular weight 55000, iso-electric point 5.31, KM value 0.08,65 ℃ of optimum temperutures.
Description of drawings:
Fig. 1 shows the principles of chemistry figure that the L-methionine(Met) is produced.
Fig. 2 shows L-methionine(Met) technological process of production figure.
L-Aminoacylase high reactivity thalline process flow sheet is produced in Fig. 3 demonstration.
Fig. 4 shows the hereditary property of L-Aminoacylase bacterial strain.
Embodiment:
The preparation of embodiment 1, L-Aminoacylase
(1) preparation of the amino acylase of high reactivity
1. the hereditary property of genetically engineered bacterial classification XW9707 of the present invention:
Vitamin H, sodium succinate double defect type, amicillin resistance (Apr
+), have the pALM118 plasmid, carry Arg
-The L gene.
2. the compatibility of substratum.
No. 1 nutrient agar
No. 2 bacteria culture mediums
Composition is identical with substratum 1, but does not need agar.The final concn of penbritin is 0.05g/l.
3) sodium potassium phosphoric acid buffer
1 mole of KH
2PO
4PH=6.8-7.0
With 10 moles of NaOH solution adjust pHs.
4) borate buffer
0.1 mole H
3BO
3PH=9.7.
5) cobalt chloride solution
With 0.02 mole of CoCl
2Be dissolved in distilled water.
No. 6 enzyme buffer suspension liquid
0.1 mole of sodium potassium phosphorus damping fluid
Dithiothreitol (DTT) (DTT) 1 mmole
Pheuylmethylsulfonyl fluoride (PMSF) 1 mmole
CoCl
20.1 mmole, pH=6.8-7.0.
No. 7 reaction mixtures (RM)
N-Ac D, 0.06 mole of L-Met
0.05 mole of sodium potassium phosphoric acid buffer
CoCl
20.1 mmole, pH=6.8-7.0
No. 8 O-phthalic aldehyde reagents (OPA)
60 milliliters of borate buffers
5 microlitre thiomethyl alcohols
10 milligrams of o-phthalaldehyde(OPA) (solvent is an ethanol)
Annotate: OPA reagent needs preparation on the same day.
No. 9 trichoroacetic acid(TCA) reagent (TCA)
30% trichoroacetic acid(TCA) is water-soluble.
3. screening method is as follows:
As shown in Figure 3, genetically engineered bacterial classification XW9707 is kept at No. 1 and cultivates on the inclined-plane; Be deposited at ordinary times and cultivate for No. 1 on the inclined-plane, upward apply one deck paraffin oil or Vaseline, place agar column.Before fermentation, be ready to the great-hearted slant strains of amino acylase earlier.The preparation method chooses 20-25 bacterium colony earlier as shown in Figure 3, inoculates on another clean culture dish that substratum 1 is housed.From this 40-50 bacterium colony, select 4-6 bacterial classification the most activated with amino acylase vigor mass analysis.
The mass analysis of the vigor of amino acylase is carried out according to following method.Take a clean culture dish, put two filter paper in each ware, wherein be placed on following filter paper and soaked with toluene, top filter paper soaked with No. 7 reaction mixtures.In ware, press a graticule point 20-22 point with inoculating needle, in another ware, order the same 20-22 point, add a cover, in 37 ℃ of incubators, cultivated 30 minutes.There is the filter paper of bacterium colony trace under 50-60 ℃ of condition, to dry top one deck in the ware,,, can shows purple in periphery of bacterial colonies so if enzyme has vigor with the triketohydrindene hydrate colour developing.For doing further checking, select those the same big bacterium of two ware mid points, these bacterium are inoculated on the inclined-plane 2, further test their activity.For analyzing the unit that the amino acylase enzyme is lived, the bacterium that will filter out from two wares shakes in the bottle at two ferments.Give earlier and add No. 2 bacteria culture mediums of 100-125 milliliter and inoculation in 500 milliliters of bottles.Under 37-38 ℃ of condition, cultivated 18-20 hour, 120 rev/mins of rotating speeds, centrifugation then, will not precipitate liquid separation after, with 60 milliliters of No. 6 damping fluids 250 milliliters of enzymes are dissolved as suspension.
Enzyme work is not less than the bacterial classification of 5000 units per ml can regard great-hearted as.
Such bacterial classification can be used for producing the amino acylase of production usefulness.This inclined-plane can keep the activity of the 1-2 month under 4 ℃ of conditions, and inoculating the bacterium that obtains for the first time, for the second time with this inclined-plane has activity too.But active reduction gradually with the increase of inoculation times.The inclined-plane needs to screen activated bacterial classification again by the top method of saying after twice inoculation.
4. high active enzyme producing and preserving
High active enzyme shakes bottle production with the laboratory under 3 conditions of being told about.Enzyme work can reach more than 150000 units per liter.At the above-mentioned preservation condition 80-90% that enzyme is lived and lived for initial enzyme after next week.
5. the activation of enzyme
Activating enzyme adopts the method for O for toluene.The ratio of toluene and enzyme liquid is 2: 100.After adding toluene, vortex stirred 1-2 minute.
(2) amplification of L-Aminoacylase and determination of activity
The CGMCC.No.0368 bacterial strain is transplanted to (glucose 0.3%, sodium succinate 0.3%, KH in the mother culture media
2PO
40.3%, Na
2HPO
40.6%, NaCl 0.05, NH
4Cl 0.1, MgSO
40.025%, VB1 0.001%, penbritin 0.01%), 37 ℃ of following oxygen supply shaking culture 16 hours.The seed culture fluid that 50ml is obtained is transplanted to 51 substratum (glucose 0.6%, sodium succinate 0.6%, KH again
2PO
40.3%, Na
2HPO
40.6%, NaCl 0.05, NH
4Cl 0.1, MgSO
40.025%, VB1 0.001%, penbritin 0.01%) 7 liters of small-sized fermentation jars in, under 37 ℃, change with per minute 484 and to carry out stir culture.In culturing process, keep 5 liters/minute air flow.Finish to cultivate when glucose consumption is intact in substratum.Measure the L-Aminoacylase activity of gained thalline.Record the L-Aminoacylase activity be not less than that the 6000u/g thalline can think can practical application.The activity of above-mentioned L-Aminoacylase is tested as follows:
Detected liquid joined contain N-acetyl-D, L-methionine(Met) (10g/l), CoCl
2(0.1mM), phosphoric acid buffer (0.05M, in aqueous solution pH6.8-7.0), 37 ℃ of following enzymolysis 5 minutes.Measure the L-methionine(Met) that produces with the stupid dicarbaldehyde colorimetric determination of neighbour.To generate the activity of the L-methionine(Met) of 1 μ mol in 1 hour, with 1 (u) unit representation, method is as follows:
In two test tubes, respectively add 0.5 milliliter of No. 7 reaction mixture.First test tube adds 0.01 milliliter of enzyme liquid, puts into 37 ℃ of thermostat containers; Second test tube adds 0.1 milliliter of No. 9 TCA solution in contrast immediately.First test tube heating adds 0.1 milliliter of TCA after 5 minutes then, makes the stopping of reaction.Give and add 0.01 milliliter of bacterium liquid of crossing through O for toluene in second test tube.
After reaction stops, giving in two empty test tubes respectively to add 1.25 milliliters of H
2Solution and 1.25 milliliters of No. 8 OPA reagent in O, 0.03 milliliter of test tube 1 or the test tube 2 after the vigorous stirring, were placed 5-6 minute under room temperature.The sample of getting blank (only need add water and No. 8 reagent) after 5-6 minute and containing concentration known methionine(Met) (as 0.01 milliliter of 1 grams per liter methionine(Met) and 0.01 milliliter of 22 grams per liter methionine(Met)) is in wavelength 340nm colorimetric estimation.The enzyme of 1 milliliter of bacterium liquid after O for toluene is applied flexibly following formula and is calculated:
Wherein: D
1340OD value for treatment samples;
D
2340For in the same old way OD value;
2.53 be the volume number of tested mixed solution, unit: milliliter;
60 be minute with hour gain factor;
∑ absorbs number (being 1 mmole methionine(Met)) for the mol of the methionine(Met) that together detects with sample in above-mentioned reaction;
5 is the reaction times, unit: minute;
0.000492 be the amount of enzyme liquid in 2.53 milliliters of test solutions, unit: milliliter.
The formation reaction of embodiment 2, L-methionine(Met)
Can obtain the activated L-Aminoacylase after handling more than 30 minutes with toluene (2%) culture of the CGMCC.No.0368 strain of acquisition among the embodiment 1; in 1 liter of reaction solution, add 1000 unit bacterial strain handled thing or L-Aminoacylases; 50g N-acetyl-D; the L-methionine(Met), adding sodium hydroxide adjustment pH is 6.5.Fully stir this reaction solution, under 37 ℃ of conditions, carry out enzyme digestion reaction.Till the content that enzymolysis time is controlled at L-methionine(Met) in the enzymolysis solution no longer increases, approximately need 12-16 hour.The centrifugal water-insoluble of removing under reduced pressure concentrates 1/20 (50ml) that makes it to original volume then.Add under 4 ℃ of conditions, to place in the concentrated solution and with the gained mixed solution with the cold dehydrated alcohol of equal-volume (50ml) and spend the night.Crystallization that the filtration collection is separated out and drying, obtain 18g L-methionine(Met), its fusing point is 280 to 281 ℃, [α]
D 25=-24.3 ° (c=1,6NHCl).Determine the enzymolysis number of times according to generating the methionine(Met) needs in the concrete experiment.
The formation reaction of embodiment 3, L-methionine(Met)
The nutrient solution that obtains among the embodiment 1 is carried out centrifugal, obtain thalline.(0.1M potassiumphosphate, sodium suspend in pH6.8) at phosphoric acid buffer with this thalline.Utilize the O for toluene suspension liquid of ultrasonic wave or 2%, with the activity of activating enzyme.Then the activated suspension liquid is added in 5 times of isopyknic 3% sodium alginate colloidal sols and stir, slowly be added dropwise to again to the 0.5M calcium chloride solution and make it to become to solidify bead, and be positioned over 4 ℃ of refrigerator overnight, remove calcium chloride solution, add washing 2-3 time, then the enzyme that fixes is packed in the Glass tubing, make the enzyme post.Again under 37 ℃ with N-acetyl-D, L-methionine solution (50g/l adjusts pH6.5 with sodium hydroxide) obtains the L-methionine solution of 20g/l by the enzyme post.Be evaporated to 1/20 of original volume then, add isopyknic dehydrated alcohol and mixing again, the gained mixed solution is filtered down at 4 ℃.Filter and collect the crystallization and the oven dry of separating out, obtain the crystallization of 17.8g L-methionine(Met), the rate of recovery is 89%.
Embodiment 4, L-methionine(Met) production technique
Schema is referring to accompanying drawing 2.
1. synthetic N-acetyl-D, the L-methionine(Met):
Volume is that 1 liter three-necked bottle (is adorned prolong on one side, adorn thermometer on one side) the middle 200 gram D that add, L-methionine(Met) (D, the content of L-methionine(Met) is not less than 96%) (1.28 moles), 0.34 rise distilled water, stir down fully at 50 ℃, whipped state dripped 0.24 liter of (2.5 moles) diacetyl oxide down, 70 ℃ of following stirring reactions 2 hours.
Reaction process is observed with the thin plate chromatography.Developing agent is propyl carbinol: H
2O: acetate=3: 1: 1; The colour developing ninhydrin; Chromatoplate silicon-dioxide chromatography plate (SiO
2).Methionine(Met) concentration constantly reduces with the carrying out of reaction.Add diacetyl oxide after 2 hours, methionine(Met) concentration is answered＜10 grams per liters, totally 0.58 liter of acidylate productive rate 97%, reaction solution in the reaction solution.The productive rate of the 1st step acidylate is the 94-97% of theoretical value.
2. concentrate N-acetyl-D, L-methionine solution
0.58 liter with the 1st step concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) to thick liquid.In thick liquid, add little water and evaporate to dryness, repeat twice (removal diacetyl oxide).Add 1.5 premium on currency in the thick liquid of last evaporate to dryness.The 2nd step productive rate is 99% (the mechanicalness loss is only arranged).
3. adsorb unreacted D with resin cation (R.C.), the L-methionine(Met)
1.7 liters of solution that previous step obtains are crossed the ion exchange column that 0.5 liter of strongly-acid 732 resin cation (R.C.) is housed, and direction is for from top to bottom, flow velocity 0.5-0.7 liter/hour.After treating that solution has been crossed post, add 0.5 premium on currency again and cross post, obtain 2 liters of lower prop effluent liquid.
Observe the absorption situation with chromatography, analytical method is with the 1st step.The content of methionine(Met) is no more than 0.1% in the solution of collecting.
Annotate: ion exchange column can repeatedly use.When resin was full of amino acid, with 8% ammoniacal liquor wash-out, evaporation concentration, with the D that obtains, the L-methionine(Met) dropped into for the 1st step.
4. enzymolysis N-acetyl-D, the L-methionine(Met)
2 liters of N-acetyl-D in the 3rd step, L-methionine solution are with the 10 moles of NaOH pH=6.8 that neutralizes, and adding distilled water to volume is 5 liters; The enzyme liquid that adds 25 milliliters of 0.02 mole of cobalt chloride solutions then and cross (wherein containing 133000 units activity acyltransferases) through O for toluene.Reacted 12-14 hour, and treated stopped reaction after the methionine(Met) concentration stabilize.Add acetate pH is transferred to 4-4.5.
Reaction solution is 5.2 liters, and methionine(Met) concentration 15-16 grams per liter (meter 80.5 grams) is 87% of initial reaction solution N-acetyl-L-methionine(Met) amount.
5.L-the adsorption and desorption of methionine(Met):
5.2 liters of solution that the 4th step obtained are crossed the ion exchange column (1.8 liters of amount of resin) that 732 resins are housed from top to bottom with 0.8 liter/hour speed.Situation according to the content inspection of methionine(Met) in effluent liquid absorption.After treating that complete soln has been crossed, cross post with 1 premium on currency again, the 6.2 up-flow fluids (methionine(Met) concentration is less than 0.1 grams per liter) that obtain drop into the 5-1 step.Subsequently with distillation washing post, until effluent liquid pH=6-7; Again with the L-methionine(Met) of 1.5 liter of 8% ammoniacal liquor wash-out absorption.After ammoniacal liquor is crossed post, with distillating washing resin to effluent liquid pH=7-8.The elutriant of collecting 1.2 liters (L-methionine(Met) concentration is no more than the 6-7 grams per liter) dropped into for the 6th step.
The productive rate in the 5th step is 94-96%.
6. concentrate the L-methionine solution
With 1.2 liters of elutriant vacuum concentration (p=0.1p.m., t=50 ℃).After catching up with ammonia to finish, change 0.4 liter of L-methionine solution of concentrated solution over to the 7th step.The productive rate in the 6th step is 99% (only being the mechanicalness loss).
7. decolouring
In 0.4 liter of L-methionine solution, add water to 2 liters.Add 50 gram gacs then, stirred 0.5 hour down, mixture was dropped into for the 8th step at 50 ℃.
8. suction filtration
With mixed solution filter paper vacuum filtration.With the charcoal on the 200ml hot water wash suction funnel.Filtrate dropped into for the 9th step for 2.2 liters.
Productive rate is 97-98%.
9. ultrafiltration
With 2.2 liters of L-methionine solutions of previous step millipore filtration vacuum filtration with 0.22 micron.Exhaust back with 100 ml water filter wash films.Obtain 2.3 liters of filtrates.The 9th step productive rate is 99% (the mechanicalness loss is only arranged).
10. concentrate
With 2.3 liters of filtrate vacuum concentration (p=0.1p.m, t=50 ℃) to 0.6 liter.
The 10th step productive rate is 99% (mechanicalness loss).
11,12. crystallizations and fractional crystallization
0.6 liter of suspension is added equal-volume ethanol, placed 6-12 hour down at 5-10 ℃.Vacuum filtration (P=0.1p.m uses glass funnel) obtains the crystal of 75 gram L-methionine(Met).Two step productive rates are 80%.
Vacuum concentration (P=0.1p.m, t=50 ℃) mother liquor makes ethanol evaporation; The L-methionine content changes it over to the 7th step for 20% of the preceding solution of evaporation in the remaining solution.
13. oven dry:
The crystal that will wet places vacuum drying oven to dry, 50 ℃ of temperature, and air pressure P=0.08Mpa was dried 4-6 hour.Productive rate 99% (mechanicalness loss).
5-1. concentrate and racemization N-acetyl-D-methionine solution
6.2 liters of N-acetyl-D-methionine solutions in the 5th step are transferred pH=2.0 with concentrated hydrochloric acid, and vacuum concentration (P=0.1p.m, t=60 ℃) is to doing then.
The solid that obtains is dropped into the 5-2 step.
Productive rate is 99% (mechanicalness loss).
5-2. washing:
Solid is washed with less water, collect filtrate and drop into the 5-1 step.
Collect solid, adding distil water to 1.4 liter is with 10 moles of NaOH accent pH ≠ 6-6.5.This solution is dropped into enzymolysis once more in the 4th step.
Behind three enzymolysis, the ultimate production of L-methionine(Met) is 150 grams, and productive rate is 75%.
The preferred forms of above-mentioned steps 4 by the immobilized bacterium somatocyte, concrete grammar is as follows: enzymolysis N-acetyl-D, the L-methionine(Met) is regulated pH=6.8 with 10 moles of NaOH, and adding distilled water to 5 liter adds 25 milliliters of 0.02 mole of cobalt chloride solutions again.Under 37 ℃, cross enzyme post (0.8 liter of enzyme column volume contains 133000 units activity acyltransferases) from bottom to top with 0.5 liter/hour flow velocity.
Effluent liquid methionine(Met) concentration 15-16 grams per liter.
Annotate: the enzyme post uses 20 days transformation period.Wherein, the process for fixation of enzyme is referring to embodiment 3.
Claims (12)
1. the production technique of a L-methionine(Met), it comprises acetyl-D with N-, the L-methionine(Met) is a raw material, carries out the enzyme digestion reaction step with the L-Aminoacylase that derives from bacillus coli gene engineering bacteria CGMCC.No.0368.
2. the production technique of L-methionine(Met) as claimed in claim 1, it further comprises remaining N-acetyl-D-methionine(Met) in the enzyme digestion reaction step is carried out the crystallization racemization: N-acetyl-D-methionine solution is transferred pH=1.5-2.5, and vacuum concentration is to doing then.
3. production technique as claimed in claim 1 or 2; it is characterized in that the enzyme digestion reaction step adopts immobilized cell; comprise N-acetyl-D; the L-methionine(Met) is under cobalt ion participates in; in aqueous medium; by containing the immobilized thallus cell enzymolysis process of L-Aminoacylase, generate the L-methionine(Met), till the content that enzymolysis time is controlled at L-methionine(Met) in the enzymolysis solution no longer increases.
4. production technique as claimed in claim 3, it is characterized in that described immobilization enzymolysis is reflected in the enzyme post carries out, and described enzyme column filling is solidified through calcium chloride solution by thalline suspension liquid and 3% sodium alginate colloidal sol and forms.
5. production technique as claimed in claim 4 is characterized in that enzymic activity is 600-60000u/l in the enzyme digestion reaction step.
6. production technique as claimed in claim 5 is characterized in that the pH value is 5.6-7.5 in the enzyme digestion reaction step, reacts down at 10-54 ℃ and carries out 0.5-20 hour.
7. production technique as claimed in claim 6, the final concentration that it is characterized in that cobalt ion in the enzyme digestion reaction step is 5 * 10
-5-1.5 * 10
-4Mole.
8. production technique as claimed in claim 1 or 2 is characterized in that the enzyme digestion reaction step adopts enzyme to live and is 600-60000u/l L-Aminoacylase or bacterial strain handled thing.
9. production technique as claimed in claim 8, wherein L-Aminoacylase for extract in this thalline obtain have the active enzyme of L-Aminoacylase or a cured article of the refining standard of this enzyme; The bacterial strain handled thing comprises the dry thing of thalline, and the ultrasonication thing of the tensio-active agent handled thing of the freeze-dried thing of thalline, thalline, the enzyme handled thing of thalline, thalline, the machinery of thalline grind the cured article of protein differentiation thing, thalline and the thalline processing of the solvent treatment thing of handled thing, thalline, thalline.
10. production technique as claimed in claim 1 or 2 is characterized in that the intestinal bacteria L-Aminoacylase activates through 2% toluene before enzyme digestion reaction, and it activates more than 30 minutes.
11. one kind has the ability bacillus coli gene engineering bacteria CGMCC.No.0368 that generates L-Aminoacylase.
12. the preparation method of the described bacillus coli gene engineering bacteria of claim 11 adds Thioctic Acid, succsinic acid and penbritin when it is characterized in that cultivating described genetic engineering bacterium.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831482A (en) * | 2010-04-20 | 2010-09-15 | 天津金耀集团有限公司 | Application of nanofiltration membrane in splitting D,L-amino acid by using acyltransferase or D-amino acylase |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100392090C (en) * | 2005-06-24 | 2008-06-04 | 上海化工研究院 | Enzyme method for detaching and preparing L-methionine-15N and D-methionine-15N |
| CN102051401A (en) * | 2010-04-01 | 2011-05-11 | 山东阳成生物科技有限公司 | High-efficiency acylation production method of L-methionine nonaqueous solution |
| CN102628075B (en) * | 2012-02-24 | 2014-08-13 | 上海瀚鸿化工科技有限公司 | Method for producing chiral amino acid by penicillin acylase resolution and product thereof |
| CN102628076B (en) * | 2012-02-27 | 2013-08-07 | 滨海瀚鸿生化有限公司 | Preparation technology of chiral amino acid |
| CN102925530A (en) * | 2012-11-04 | 2013-02-13 | 宁波市远发生物工程有限公司 | L-methionine preparation method |
| CN104152524A (en) * | 2014-08-08 | 2014-11-19 | 山东阳成生物科技有限公司 | Production technology of L-methionine |
| CN106380433B (en) * | 2016-08-26 | 2018-04-20 | 宁夏紫光天化蛋氨酸有限责任公司 | A kind of isolation and purification method of N acetyl D, L methionine |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831482A (en) * | 2010-04-20 | 2010-09-15 | 天津金耀集团有限公司 | Application of nanofiltration membrane in splitting D,L-amino acid by using acyltransferase or D-amino acylase |
| CN101831482B (en) * | 2010-04-20 | 2012-11-07 | 天津金耀集团有限公司 | Application of nanofiltration membrane in splitting D,L-amino acid by using acyltransferase or D-amino acylase |
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