CN1155716C - D-aminosuccinic acid and L-alanine preparation method - Google Patents
D-aminosuccinic acid and L-alanine preparation method Download PDFInfo
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- CN1155716C CN1155716C CNB02138553XA CN02138553A CN1155716C CN 1155716 C CN1155716 C CN 1155716C CN B02138553X A CNB02138553X A CN B02138553XA CN 02138553 A CN02138553 A CN 02138553A CN 1155716 C CN1155716 C CN 1155716C
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- aspartic acid
- ala
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Abstract
The present invention provides a method for preparing D-aspartic acid and L-alanine, which belongs to the field of enzyme engineering. The preparing method aims to cultivate pseudomonas microbes and produce L-aspartic acid-beta decarboxylase with high activity. Cells containing enzyme or culture solution is mixed with DL-aspartic acid or the salt solution of the DL-aspartic acid to carry out enzyme reaction at the temperature from 32 DEG C to 45 DEG C in a free cell method. Then, reaction resultant is separated to obtain D-aspartic acid and L-alanine which have high chemical purity and optical purity by a method with isoelectric point crystal combined with ion exchange resin. The method realizes the simultaneous production of the D-aspartic acid and the L-alanine. The present invention has the advantages of simple operation, short conversion time and low production cost.
Description
Technical field
The present invention relates to the preparation method of D-aspartic acid and L-L-Ala, belong to the enzyme engineering field.
Background technology
D-aspartic acid technology of preparing has chemical asymmetric fractionation of employing (special public clear 52-54190) and preferential crystallization method (special public clear 54-25006).But these method complex process, the product optical purity is low, does not have very big practical value.
In the Production by Enzymes D-aspartic acid technology of having reported, the L-aspartic acid in the microorganism cancellation DL-aspartic acids such as utilizing mycocandida is arranged and obtain D-aspartic acid (clear 56-20753); Utilize improvement of genes to obtain the generation bacterium of L-aspartic acid-α-decarboxylase, prepare D-aspartic acid and Beta-alanine (CN 1242054A) simultaneously from the DL-aspartic acid, utilize L-Aspartase and fumarase combined action from the DL-aspartic acid, to prepare D-aspartic acid and L MALIC ACID (flat 1-285193) simultaneously in addition.Aforesaid method or enzyme are lived very low, perhaps complex process.
But L-aspartic acid-beta-decarboxylase catalysis L-aspartic acid decarboxylic reaction is produced L-L-Ala (flat 2-79909, flat 2-242690), and the bacterial classification that can produce this enzyme has Rhodopseudomonas, clostridium perfringens genus, achromobacter, Nocardia etc.Cultivate these microorganisms, utilize L-aspartic acid-beta-decarboxylation DL-aspartic acid or its salt of its generation, can produce two kinds of compounds that are widely used---D-aspartic acid and L-L-Ala.
The D-aspartic acid is as a kind of important chipal compounds, and is main as medicine synthetic precursor, intermediate in medicine is synthetic.Comprise and be used for syncillin derivative (Aspoxicillin), treatment virus infection medicine---D-aspartic acid-beta-azanol (DAH); Other purposes of D-aspartic acid also comprises inhibition arginine base succsinic acid synthase activity, prevents or treats the systemic ypotension of Sepsis or cytokine induction and be used as immunosuppressor.In addition, the L-L-Ala is as gal4 amino acid, widespread use in amino acid transfusion, and the L-L-Ala also is used as vitamin B6 synthetic main raw material and foodstuff additive.
So far have only and mention among patent special public clear 50-100289, clear 53-1831, clear 57-39791, the clear 57-132882 with L-aspartic acid-beta-decarboxylase production D-aspartic acid and L-L-Ala.The common ground of these patents all is the production that stresses to study process for fixation and L-L-Ala, needs to add expensive coenzyme pyridoxal phosphate and metal ion Co in the production D-aspartic acid process of mentioning
2+, cause production cost very high.
Summary of the invention
The objective of the invention is to avoid above-mentioned weak point of the prior art and provide a kind of efficiently, the preparation method of D-aspartic acid and L-L-Ala cheaply.
Purpose of the present invention can reach by following measure:
The microorganism that the present invention adopts is a pseudomonas strain, at fumaric acid and salt thereof or L-glutamic acid and salt thereof is to cultivate on the substratum of carbon source, generate high vigor L-aspartic acid-beta decarboxylase, vigor can transform 1 * 10 for every gram wet cell per hour
4～1 * 10
5The L-aspartic acid of μ mol, or per hour every mL cell culture fluid transforms 100～2000 μ mol L-aspartic acids; Adopt free cell method, will contain enzyme cell or nutrient solution and DL-aspartic acid or DL-aspartic acid salts solution and mix, carry out enzyme reaction under 32 ℃-45 ℃, the separating reaction resultant obtains the D-aspartic acid and the L-L-Ala of high chemical purity and optical purity.
The used culture medium carbon source of the present invention is limited to fumaric acid and salt thereof, L-glutamic acid and salt thereof, and its concentration is 10～50g/L, is preferably 10～30g/L.Nitrogenous source adopts organic nitrogen source, and as peptone and corn steep liquor, concentration is respectively 5～20g/L and 5～50g/L.
What the present invention adopted is that free cell transforms, and will contain enzyme cell or nutrient solution direct and DL-aspartic acid and salt solution mix thereof.DL-aspartic acid and salt concn thereof are 100～500g/L, and cell concn is 0.2g～2g in the 100ml mixing solutions, 32～45 ℃ of reaction 24～72h.
The present invention adopts the D-aspartic acid and the L-L-Ala of iso-electric point and ion exchange resin combined techniques separating reaction system, the ion exchange resin that adopts is Zeo-karb, comprises 001 * 7, JK004, JK006, JK008, JK010, HD-8, HZ001, HZ016 and weak base anion-exchange resin.
The present invention has following advantage compared to existing technology:
The false unicellular genus bacterial strain that the present invention adopts, in preferred substratum, cultivate and to generate L-aspartic acid-beta decarboxylase by high productivity, and do not contain aspartic acid and alanine racemase, make reaction that high transformation efficiency and high speed of reaction be arranged, the optical purity of D-aspartic acid and L-L-Ala product is 100%.
The present invention adopts free cell method to transform the DL-aspartic acid, prepares D-aspartic acid and L-L-Ala simultaneously, and is easy and simple to handle, improves speed of reaction greatly, simplifies technical process.Owing to do not use expensive coenzyme and Co
2+Deng metal ion, reduce production costs significantly.
Embodiment
Below be embodiment, the present invention will be further described for the general, but to the present invention without limits.
Embodiment 1
Unicellular bacterial strain NX-1 cultivates in following 200ml substratum with vacation:
Sodium fumarate 0.5%, ammonium fumarate 1.0%, corn steep liquor 2.0%, peptone 0.8%, K
2HPO
40.05%, MgSO
4.7H
2O 0.01%, pH7.0, and 30 ± 1 ℃ are shaken a bottle shaking culture 24h, the centrifugal wet cell that gets, every gram cell L-aspartic acid-beta decarboxylase activity is 1.42 * 10
4U.Cell is joined 200mL 10%DL-aspartic acid ammonium solution, 37 ℃ of reaction 10h.D-aspartic acid concentration is 5% in the reaction solution, and L-L-Ala concentration is 3.345%, to L-aspartic acid molar yield 100%.
With reaction solution 0.5% activated carbon decolorizing, filtrate is transferred pH3.0, is cooled to 5 ℃, and the crystallization cold water of separating out is cleaned, and gets 7.2g D-aspartic acid.Remaining mother liquor stream is crossed 001 * 7 type resin (H
+Type), the washing back is collected D-aspartic acid and L-L-Ala filtrate respectively with 1% ammonia soln wash-out, concentrates, and gets 2.3g D-aspartic acid, 6.0g L-L-Ala.
D-aspartic acid: [α]
D 25=-25.5 ° (C=1,5N HCL)
L-L-Ala: [α]
D 25=+14.4 ° of (C=6.46,1N HCL) Mp=270 ℃ (decomposition)
Embodiment 2
Unicellular bacterial strain NX-1 cultivates in following 200ml substratum with vacation:
Sodium Glutamate 2%, corn steep liquor 2.0%, peptone 0.8%, K
2HPO
40.05%, MgSO
4.7H
2O0.01%, pH7.0,30 ℃ ± 1 ℃ is shaken a bottle shaking culture 24h.L-aspartic acid-beta-the decarboxylase activity that records every mL fermented liquid is for per hour transforming 1000 μ mol L-aspartic acids.Centrifugal cell and the mixing of 200mL20%DL-aspartic acid ammonium solution, 32 ℃ of reaction 20h of getting.D-aspartic acid concentration is 10% in the reaction solution, and L-L-Ala concentration is 6.69%, is 100% to L-aspartic acid molar yield.
With the reaction solution activated carbon decolorizing, filtrate is transferred pH3.0, is concentrated in vacuo to 100mL, is cooled to 5 ℃ of placements and spends the night.Separate out crystallization, cold water washing gets the 18.6gD-aspartic acid, [α]
D 25=-25.5 ° (C=1,5NHCL).Mother liquor stream is crossed 001 * 7 type resin (H
+) type, with 1% ammonia soln wash-out, collect L-L-Ala filtrate, concentrate, get 11.6g L-L-Ala, [α]
D 25=+14.4 ° of (C=6.46,1N HCL) Mp=270 ℃ (decomposition).
Embodiment 3
With embodiment 2 culture medium prescriptions, the 1L substratum of in the 1.5L fermentor tank, packing into, insert false unicellular bacterial strain NX-1 by 0.5% inoculum size, mixing speed 500r/min, ventilating ratio 1: 0.3, cultivate 18h for 30+1 ℃, the L-aspartic acid-beta decarboxylase activity of every mL nutrient solution is for per hour transforming 1500 μ mol L-aspartic acids.Centrifugal cell and the mixing of 1000mL10%DL-aspartic acid ammonium solution, 40 ℃ of reaction 8h of getting.D-aspartic acid concentration is 5% in the reaction solution.L-L-Ala concentration is 3.345%, is 100% to the molar yield of L-aspartic acid.
Reaction solution after having reacted is centrifugal, take out cell, react in the same adding substrate solution, repeat result such as table 15 times:
The result that the D-aspartic acid is produced in the recycling of table 1 cell
Reaction times generates production concentration (w/v)
The cell access times
The L-Asp transformation efficiency
(h)
D-aspartic acid L-L-Ala
The 1st 8h 100% 5% 3.345%
The 2nd 8h 100% 5% 3.345%
The 3rd 8h 100% 5% 3.345%
The 4th 8h 100% 5% 3.345%
The 5th 8h 99.5% 4.97% 3.33%
Merge 5 secondary response liquid, use 0.5% activated carbon decolorizing, filtrate is transferred pH3.0, is concentrated in vacuo to 2000mL, is cooled to 5 ℃ of placements and spends the night, and the crystallization cold water of separating out is cleaned, and gets 233g D-aspartic acid, [α]
D 25=-25.5 ° (C=1,5N HCL).Remaining mother liquor stream is crossed 001 * 7 type resin (H
+Type), L-L-Ala filtrate is collected with 1% ammonia soln wash-out in the washing back, concentrates, and gets 145g L-L-Ala.
L-L-Ala: [α]
D 25=+14.4 ° of (C=6.46,1N HCL) Mp=270 ℃ (decomposition).
Adopt this law and process for fixation to produce D-aspartic acid leading indicator relatively as table 2:
Table 2 free cell method and process for fixation are produced D-aspartic acid leading indicator relatively
The training reaction times of obtaining cell generates the D-asparagus fern
Other
Support (h) propylhomoserin amount (g) of liquid long-pending (mL)
This law 1,000 40 145.0
Need
Immobilization method (clear 50-
200 83.3 30.5 0.1mMPLP，1mM
100289)
Co
2+
Immobilization method (clear 53-
200 83.3 31.0 need 0.1mMPLP
1831)
Immobilization method (clear 57-
1,200 25 30.4 need 0.1mMPLP
39791)
Immobilization method (clear 57-
1,200 33 30.4 need 0.1mMPLP
132882)
Claims (5)
1. the preparation method of D-aspartic acid and L-L-Ala, it is characterized in that the microorganism that the present invention adopts is a pseudomonas strain, be to cultivate on the substratum of carbon source at fumaric acid and salt thereof or L-glutamic acid and salt thereof, generate the L-aspartic acid-beta-decarboxylase of high vigor; Adopt free cell method, the solution that will contain enzyme cell or nutrient solution and DL-aspartic acid or DL-aspartate mixes, and carries out enzyme reaction under 32 ℃-45 ℃, and the separating reaction resultant obtains the D-aspartic acid and the L-L-Ala of high chemical purity and optical purity.
2. the preparation method of D-aspartic acid according to claim 1 and L-L-Ala is characterized in that adopting fumaric acid and salt thereof or L-glutamic acid and salt thereof to make carbon source, and its concentration is 10-50g/L; Peptone and corn steep liquor are made nitrogenous source, and concentration is respectively 5～20g/L and 5～50g/L.
3. the preparation method of D-aspartic acid according to claim 1 and L-L-Ala, L-aspartic acid-beta-the decarboxylase that it is characterized in that nutrient solution per hour is approximately every mL nutrient solution conversion 100-2000 μ mol L-aspartic acid, or per hour every gram wet cell transforms 1 * 10
4～1 * 10
5μ mol L-aspartic acid.
4. the preparation method of D-aspartic acid according to claim 1 and L-L-Ala, the method that it is characterized in that separating D-aspartic acid and L-L-Ala is an isoelectric point method coupled ion exchange resin method, and the ion exchange resin of employing is Zeo-karb and weak base anion-exchange resin.
5. the preparation method of D-aspartic acid according to claim 4 and L-L-Ala is characterized in that Zeo-karb, comprises 001 * 7, JK004, JK006, JK008, JK010, HD-8, HZ001, HZ016.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB02138553XA CN1155716C (en) | 2002-11-07 | 2002-11-07 | D-aminosuccinic acid and L-alanine preparation method |
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|---|---|---|---|
| CNB02138553XA CN1155716C (en) | 2002-11-07 | 2002-11-07 | D-aminosuccinic acid and L-alanine preparation method |
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|---|---|
| CN1405317A CN1405317A (en) | 2003-03-26 |
| CN1155716C true CN1155716C (en) | 2004-06-30 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102605015A (en) * | 2011-01-20 | 2012-07-25 | 烟台恒源生物工程有限公司 | L-alanine production method |
| CN102443612A (en) * | 2011-11-24 | 2012-05-09 | 蒋光玉 | Production technology of D-aspartic acid and L-alanine |
| CN108384818A (en) * | 2018-05-18 | 2018-08-10 | 南京大学 | A kind of method that enzymatic conversion method prepares D-His |
| CN108484423B (en) * | 2018-07-05 | 2021-02-09 | 秦皇岛华恒生物工程有限公司 | Method for separating and purifying L-alanine from L-alanine fermentation liquor |
| CN109628519B (en) * | 2019-01-14 | 2021-08-24 | 江南大学 | Method for preparing N-methyl-D-aspartic acid by enzymatic resolution |
| CN110407711A (en) * | 2019-08-19 | 2019-11-05 | 精晶药业股份有限公司 | A kind of D-Asp derivative and preparation method thereof |
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2002
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