CN103320480A - Method for preparing beta-alanine by coupled enzymatic reaction - Google Patents
Method for preparing beta-alanine by coupled enzymatic reaction Download PDFInfo
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- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 title claims abstract description 190
- 229940000635 beta-alanine Drugs 0.000 title claims abstract description 95
- 238000000034 method Methods 0.000 title claims abstract description 35
- 238000006911 enzymatic reaction Methods 0.000 title claims abstract description 20
- 238000006243 chemical reaction Methods 0.000 claims abstract description 75
- 102000004190 Enzymes Human genes 0.000 claims abstract description 36
- 108090000790 Enzymes Proteins 0.000 claims abstract description 35
- 235000019297 ammonium fumarate Nutrition 0.000 claims abstract description 31
- CKKXWJDFFQPBQL-SEPHDYHBSA-N azane;(e)-but-2-enedioic acid Chemical compound N.N.OC(=O)\C=C\C(O)=O CKKXWJDFFQPBQL-SEPHDYHBSA-N 0.000 claims abstract description 31
- 239000001729 Ammonium fumarate Substances 0.000 claims abstract description 22
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims abstract description 11
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 9
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- AGSPXMVUFBBBMO-UHFFFAOYSA-N beta-aminopropionitrile Chemical compound NCCC#N AGSPXMVUFBBBMO-UHFFFAOYSA-N 0.000 description 7
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- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 4
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the biotechnical field, and in particular relates to a method for preparing beta-alanine by a coupled enzymatic reaction. The method comprises the following steps: by using ammonium fumarate as a raw material, mixing bacterial cells respectively comprising aspartase and aspartic acid-alpah-decarboxylase or a crude enzyme liquor of the two enzymes with an ammonium fumarate aqueous liquor, wherein pH of the ammonium fumarate aqueous liquor is 7.0-7.5; performing enzymatic reaction at 25-55 DEG C; obtaining high purity beta-alanine by separating converted products by isoelectric point crystallization or a method combining isoelectric point crystallization or ion exchange resin. The beta-alanine is prepared by coupled enzymatic reaction, and the method has the advantages of wide source of raw materials, low cost, simplicity and convenience in operation, short enzymatic conversion time, low production cost and the like.
Description
One, technical field
The invention belongs to biological technical field, be specifically related to the method that a kind of double-enzyme method prepares Beta-alanine.
Two, background technology
Beta-alanine is the precursor of synthetic pantothenic acid, and pantothenic acid is a kind of of vitamin B group, consists of the part of coenzyme A, and is all very important for various in-house endogenous metabolism energy exchanges.Along with the gradually intensification to Beta-alanine understanding, Beta-alanine is used increasingly extensive in fields such as medicine, food, makeup, feeds, also in continuous growth, so Beta-alanine is with a wide range of applications and develops value to the market requirement of Beta-alanine.
Preparation method according to the bibliographical information Beta-alanine mainly contains chemical synthesis and biological enzyme at present.
1, chemical synthesis
Chemical synthesis has vinyl cyanide method, propylene acid system, succinimide (fourth two phthalimides) edman degradation Edman, β-aminopropionitrile method, two cyanoethyl amine methods.Wherein β-aminopropionitrile method and vinyl cyanide method cost of material are cheap, but separate relatively difficulty, particularly Beta-alanine and separation of by-products more complicated after the intermediate product hydrolysis, and hydrolytic process generates a large amount of inorganic salt.The processing condition of the method require harsh, and other side reaction easily occurs, and suitability for industrialized production and environment protection treating difficulty are large, and production cost is high.
Buc etc. generate β-aminopropionitrile with vinyl cyanide and ammoniacal liquor generation aminating reaction, are hydrolyzed under acidity or alkaline condition, through separating the preparation Beta-alanine.
2, biological enzyme
(1) nitrile degrading enzyme
River Li Yang etc. utilize the microorganism catalysis β-aminopropionitrile hydrolysis Beta-alanine that produces the organic nitrile degrading enzyme, microorganism
Alcaligenes sp.OMT-MY14,
Achromobatrer xerosisIF012668,
Pseodonocardia thermophilaATCC19285,
AminoBacter aminobranceATCC23314 can the catalysis β-aminopropionitrile be hydrolyzed, wherein
Alcaligenes sp.The OMT-MY14 catalytic effect is best, and at 30 ℃, its free cell is catalysis 1.0% β-aminopropionitrile hydrolysis reaction 1 h in damping fluid, and Beta-alanine concentration reaches 47 mmol/L.
The field coerce new youth wait utilize microorganism as
Micrococcus lysodeikticusATCC4698,
Micrococcus luicusATCC12698,
Corynebacrerium glutamscumATCC21253 etc. transform the beta-amino propyl alcohol, 30 ℃ of reaction 30 h, and Beta-alanine concentration reaches 4.0 mg/mL.
(2) aspartic acid propylhomoserin-α-decarboxylase
Asparagus fern ammonia-α-decarboxylase enzyme process production technique be take L-Aspartic acid as raw material, decarboxylase as catalyzer one the step biochemical reaction, its equipment is simple, reaction conditions is gentle, enzyme activity is high, speed of response is fast, specificity is strong.Decarboxylation Production by Enzymes Beta-alanine has become the direction of in recent years people's concern and research.
Wiliamson(J. Biol. Chem. 1979,254:8074-8082) etc. take microorganism wet thallus or crude enzyme liquid as the enzyme source, take L-Aspartic acid as substrate, 0.1 mol/L potassium phosphate buffer (pH 7.5), in 42 ℃ of Enzymatic transformation, react 20 min, obtain containing the reaction solution of Beta-alanine, make Beta-alanine through separation and Extraction, aspartic acid-α-decarboxylase
KmValue is about 160 μ mol/L.
Cronan(J. Biol. Chem. 1980,141 (3): 1291-1297) take microorganism wet thallus or crude enzyme liquid as the enzyme source, take L-Aspartic acid as substrate, 0.1mol/L potassium phosphate buffer (pH 6.8), in 37 ℃ of Enzymatic transformation, reaction 1～2 h obtains containing the reaction solution of Beta-alanine, makes Beta-alanine through separation and Extraction, and aspartic acid-α-decarboxylase Km value is about 80 μ mol/L.
(Zhejiang Polytechnical University's journal such as Hong Min, 2011,39 (3): 252-256) take L-Aspartic acid as substrate, (pET-28c (+)-panD) enzyme process has synthesized Beta-alanine, and enzyme work reaches 186 U with aspartic acid-α-decarboxylase gene engineering bacteria.
GAO Lijuan (Zhejiang Polytechnical University's master thesis, 2007) is take L-Aspartic acid as substrate, and (pET-21c (+)-panD) enzyme process has synthesized Beta-alanine, and enzyme work reaches 224.96 U with aspartic acid-α-decarboxylase gene engineering bacteria.
Three, summary of the invention
The problem that the present invention need to solve provides a kind of method that directly prepares efficiently, cheaply Beta-alanine take ammonium fumarate as the raw material double-enzyme method.The present invention prepares Beta-alanine take ammonium fumarate as raw material with L-Aspartase and the two enzyme couplings of aspartic acid-α-decarboxylase.
The present invention can reach by the following technical programs:
The double-enzyme method conversion preparation method of Beta-alanine the steps include:
(1) will have respectively the bacterial strain of L-Aspartase and aspartic acid-α-decarboxylase, and in substratum, cultivate and produce highly active L-Aspartase and aspartic acid-α-decarboxylase;
(2) will have the wet thallus of L-Aspartase and aspartic acid-α-decarboxylase or crude enzyme liquid by the certain hour order respectively with the ammonium fumarate aqueous solution of pH 7.0～7.5, add again proper amount of surfactant, under 25 ℃～55 ℃ conditions, carry out enzymatic reaction; The ammonium fumarate aqueous solution that perhaps will have wet thallus or crude enzyme liquid elder generation and the pH 7.0～7.5 of aspartase activity, add tensio-active agent, concentration obtains the aspartic acid ammonium salt solid after 25 ℃～55 ℃ enzymatic reactions, wet thallus or the crude enzyme liquid that will have aspartic acid-α-decarboxylase mix with the aspartic acid ammonium salt aqueous solution again, add tensio-active agent, carry out enzymatic reaction under 25 ℃～55 ℃ conditions; Obtain Beta-alanine.
The bacterial strain with aspartase activity in the above-mentioned steps (1) is
Escherichia coli str.K-12 substr. MG1655 or
Escherichia fergusoniiATCC 35469; Bacterial strain with aspartic acid-α-decarboxylase is
Bacillus cereusATCC 10987 or
Corynebacterium glutamicumATCC 13032.
Culture medium carbon source in the above-mentioned steps (1) adopts glucose, maltose, sucrose and/or fructose, and total carbon source quality concentration is 1～30 g/L in the substratum; Nitrogenous source adopts extractum carnis, yeast extract paste, corn steep liquor, peptone and/or soya-bean cake hydrolyzed solution, and total nitrogen source quality concentration is 1～30 g/L in the substratum.
The concentration of the ammonium fumarate aqueous solution or aspartic acid ammonium salt aqueous solution is 30 g/L～300 g/L in the above-mentioned steps (2), the tensio-active agent that adds is tween-80 or cetyl trimethylammonium bromide (CTAB) or Triton X-100 (OP), and its concentration is 0.05 g/L～5.0 g/L.
At present, the production of China's L-Aspartic acid mainly is to be obtained through the L-Aspartase ammonification by ammonium fumarate, the method operational path is very ripe, and the production of Beta-alanine mainly is by chemosynthesis or adopts aspartic acid-α-decarboxylase, obtains Beta-alanine take L-Aspartic acid as the raw material decarboxylation.One step enzyme method production cost is with the chemical synthesis process production cost is more approaching at present, and be that production cost that the double-enzyme catalysis of raw material prepares Beta-alanine is compared and exceeded much with ammonium fumarate, also be unfavorable for simultaneously solving the environmental issue that produces in the raw material L-Aspartic acid production process.
The present invention is take ammonium fumarate as substrate, with L-Aspartase converted product L-Aspartic acid as aspartic acid-α-decarboxylase reaction substrate, prepared Beta-alanine by the cascade reaction that once feeds intake, double-enzyme method catalysis, this technique has been saved the separation and Extraction process of L-Aspartic acid, acid and alkali consumption and facility investment have been reduced, meet the requirement of environmental protection and cleaner production, implementation result is outstanding, has good using value.Further work can by genetic engineering means with the gene recombination of L-Aspartase and aspartic acid-α-two kinds of enzymes of decarboxylase on an expression vector, realize the low-cost scale operation Beta-alanine of bacterium double-enzyme method product.
The present invention compared with prior art has following advantage:
(1) the present invention has adopted highly active L-Aspartase bacterial strain
Escherichia coli str.K-12 substr. MG1655 or
Escherichia fergusoniiATCC 35469 and highly active aspartic acid-α-decarboxylase bacterial strain
Bacillus cereusATCC 10987 or
Corynebacterium glutamicumATCC 13032.Can high efficient expression L-Aspartase and aspartic acid-α-decarboxylase in the preferred substratum, make the synthetic Beta-alanine of two enzyme couplings that higher catalytic rate and transformation efficiency be arranged, wherein the ammonium fumarate molar yield reaches more than 99%, and L-Aspartic acid decarboxylation efficient reaches more than 98%.
(2) the present invention utilizes ammonium fumarate to be raw material, has reduced production cost, and two enzyme coupling production have been accelerated throughput rate, have good economic benefit, environmental benefit and social benefit.
(3) the present invention with L-Aspartase aminating reaction product directly as the substrate of aspartic acid-α-decarboxylase, saved the separation and Extraction process of L-Aspartic acid, when significantly reducing acid and alkali consumption, also improved product yield, simplified production process, meet the requirement of environmental protection and cleaner production, implementation result is outstanding, has higher using value.
The advantages such as (4) the synthetic Beta-alanine of enzyme process has the reaction conditions gentleness, and the enzyme stereoselectivity is strong, and catalytic efficiency is high, and technical process is simple are fit to suitability for industrialized production.
Four, embodiment
Embodiment one
1, with 100 ml
Escherichia coli str.Centrifugal L-Aspartase wet thallus 1.5 g and 1000 ml that obtain of K-12 substr. MG1655 fermented liquid
Corynebacterium glutamicumCentrifugal aspartic acid-the α that obtains of ATCC 13032 fermented liquids-decarboxylase wet thallus 17 g, join in the 1000 ml conversion fluids, contain 150 g/L ammonium fumarates, 0.5 g/L OP in the conversion fluid, pH 7.0,37 ℃ of enzymatic reaction 16 h, paper chromatography detect and determine reaction end.Beta-alanine concentration is 85.4 g/L in the rear conversion fluid of reaction end, is 96% to the ammonium fumarate molar yield.
2, centrifugal 15 min of conversion fluid 4000 r/min are removed thalline, supernatant liquor is warmed up to 70 ℃～80 ℃, adds activated carbon decolorizing, and destainer is concentrated into 120 ml, the subcooling crystallization, and suction filtration, washing, oven dry get solid Beta-alanine 49.6 g.
3, will separate by 732 type cation exchange resin columns after the crystalline mother solution dilution, with 2.5% ammoniacal liquor wash-out Beta-alanine saturated adsorption post, collection contains elutriant 630 ml of Beta-alanine, the activated carbon decolorizing final vacuum is concentrated into 80 ml, add while hot long-pending 95% ethanol of diploid, subcooling crystallization 8 h, suction filtration, washing, oven dry, get solid Beta-alanine 26.1 g, recycled behind the crystalline mother solution Recycled ethanol.Twice crystallization is total to get Beta-alanine 75.7 g, yield 88.6%.
Embodiment two
1, with 200 ml
Escherichia coli str.Centrifugal L-Aspartase wet thallus 3.5 g and 1000 ml that obtain of K-12 substr. MG1655 fermented liquid
Corynebacterium glutamicumCentrifugal aspartic acid-the α that obtains of ATCC 13032 fermented liquids-decarboxylase wet thallus 19 g, join in the 1000 ml conversion fluids, contain 100 g/L ammonium fumarates, 0.05 g/L tween-80 in the conversion fluid, pH 7.5,37 ℃ of enzymatic reaction 12 h, paper chromatography detect and determine reaction end.Beta-alanine concentration is 57.5 g/L in the rear conversion fluid of reaction end, is 97% to the ammonium fumarate molar yield.
2, centrifugal 15 min of conversion fluid 4000 r/min are removed thalline, supernatant liquor is warmed up to 70 ℃～80 ℃, adds activated carbon decolorizing, and destainer is concentrated into 80 ml, the subcooling crystallization, and suction filtration, washing, oven dry get solid Beta-alanine 32.3 g.
3, will separate by 732 type cation exchange resin columns after the crystalline mother solution dilution, with 2.0% ammoniacal liquor wash-out Beta-alanine saturated adsorption post, collection contains elutriant 500 ml of Beta-alanine, the activated carbon decolorizing final vacuum is concentrated into 65 ml, add while hot long-pending 95% ethanol of diploid, subcooling crystallization 8 h, suction filtration, washing, oven dry, get solid Beta-alanine 18.5 g, recycled behind the crystalline mother solution Recycled ethanol.Twice crystallization is total to get Beta-alanine 50.8 g, yield 88.3%.
Embodiment three
1, with 100 ml
Escherichia coli str.Centrifugal L-Aspartase wet thallus 1.4 g that obtain of K-12 substr. MG1655 fermented liquid join in the 1000 ml conversion fluids, contain 200 g/L ammonium fumarates, 5 g/L tween-80s in the conversion fluid, then 7.0,30 ℃ of enzymatic reaction 8 h of pH add 1000 ml in conversion fluid
Corynebacterium glutamicumCentrifugal aspartic acid-the α that obtains of ATCC 13032 fermented liquids-decarboxylase wet thallus 16 g continue reaction 20 h, and paper chromatography detects determines reaction end.Beta-alanine concentration is 113.9 g/L in the rear conversion fluid of reaction end, is 96% to the ammonium fumarate molar yield.
2, centrifugal 15 min of conversion fluid 4000 r/min are removed thalline, supernatant liquor is warmed up to 70 ℃～80 ℃, adds activated carbon decolorizing, and destainer is concentrated into 155 ml, the subcooling crystallization, and suction filtration, washing, oven dry get solid Beta-alanine 66.2 g.
3, will separate by 732 type cation exchange resin columns after the crystalline mother solution dilution, with 2.5% ammoniacal liquor wash-out Beta-alanine saturated adsorption post, collection contains elutriant 860 ml of Beta-alanine, the activated carbon decolorizing final vacuum is concentrated into 105 ml, add while hot long-pending 95% ethanol of diploid, subcooling crystallization 8 h, suction filtration, washing, oven dry, get solid Beta-alanine 32.6 g, recycled behind the crystalline mother solution Recycled ethanol.Twice crystallization is total to get Beta-alanine 98.8 g, yield 86.7%.
Embodiment four
1, with 100 ml
Escherichia fergusoniiCentrifugal L-Aspartase wet thallus 1.6 g that obtain of ATCC 35469 fermented liquids join in the 1000 ml conversion fluids, contain 300 g/L ammonium fumarates, 0.5 g/L CTAB in the conversion fluid, and then 7.2,40 ℃ of enzymatic reaction 4 h of pH add 1000 ml
Bacillus cereusCentrifugal aspartic acid-the α that obtains of ATCC 10987 fermented liquids-decarboxylase wet thallus 18 g continues reaction 26 h, and paper chromatography detects determines reaction end.Beta-alanine concentration is 172.6 g/L in the rear conversion fluid of reaction end, is 97% to the fumaric acid molar yield.
2, centrifugal 15 min of conversion fluid 4000 r/min are removed thalline, supernatant liquor is warmed up to 70～80 ℃, adds activated carbon decolorizing, and destainer is concentrated into 240 ml, the subcooling crystallization, and suction filtration, washing and drying get solid Beta-alanine 102 g.
3, will separate by 732 type cation exchange resin columns after the crystalline mother solution dilution, with 2% ammoniacal liquor wash-out Beta-alanine and adsorption column, collection contains elutriant 1200 ml of Beta-alanine, the activated carbon decolorizing final vacuum is concentrated into 130 ml, add while hot long-pending 95% ethanol of diploid, subcooling crystallization 8 h, suction filtration, washing and drying get solid Beta-alanine 48.9 g, recycled behind the crystalline mother solution Recycled ethanol.Twice crystallization is total to get Beta-alanine 150.9 g, yield 87.4%.
Embodiment five
1, with 200 ml
Escherichia fergusoniiCentrifugal L-Aspartase wet thallus 2.9 g that obtain of ATCC 35469 fermented liquids join in the 1000 ml conversion fluids, contain 250 g/L ammonium fumarates, 0.01 g/L OP in the conversion fluid, and then 7.0,25 ℃ of enzymatic reaction 10 h of pH add 1000 ml
Corynebacterium glutamicumCentrifugal aspartic acid-the α that obtains of ATCC 13032 fermented liquids-decarboxylase wet thallus 15 g continues reaction 20 h, and paper chromatography detects determines reaction end.Beta-alanine concentration is 142.4 g/L in the rear conversion fluid of reaction end, is 96% to the ammonium fumarate molar yield.
2, centrifugal 15 min of conversion fluid 4000 r/min are removed thalline, supernatant liquor is warmed up to 70～80 ℃, adds activated carbon decolorizing, and destainer is concentrated into 190 ml, the subcooling crystallization, and suction filtration, washing and drying get solid Beta-alanine 74.2 g.
3, will separate by 732 type cation exchange resin columns after the crystalline mother solution dilution, with 3% ammoniacal liquor wash-out Beta-alanine and adsorption column, collection contains elutriant 1100 ml of Beta-alanine, the activated carbon decolorizing final vacuum is concentrated into 130 ml, add while hot long-pending 95% ethanol of diploid, subcooling crystallization 8 h, suction filtration, washing and drying get solid Beta-alanine 43.0 g, recycled behind the crystalline mother solution Recycled ethanol.Twice crystallization is total to get Beta-alanine 117.2 g, yield 82.3%.
Embodiment six
1, with 100 ml
Escherichia coli str.Centrifugal L-Aspartase wet thallus 1.4 g and 1000 ml that obtain of K-12 substr. MG1655 fermented liquid
Bacillus cereusCentrifugal aspartic acid-the α that obtains of ATCC 10987 fermented liquids-decarboxylase wet thallus 14 g, join in the 1000 ml conversion fluids, contain 150 g/L ammonium fumarates, 3 g/L OP in the conversion fluid, pH 7.0,45 ℃ of enzymatic reaction 15 h, paper chromatography detect and determine reaction end.Beta-alanine concentration is 86.3 g/L in the rear conversion fluid of reaction end, is 97% to the ammonium fumarate molar yield.
2, centrifugal 15 min of conversion fluid 4000 r/min are removed thalline, supernatant liquor is warmed up to 70～80 ℃, adds activated carbon decolorizing, and destainer is concentrated into 120 ml, the subcooling crystallization, and suction filtration, washing and drying get solid Beta-alanine 47.2 g.
3, will separate by 732 type cation exchange resin columns after the crystalline mother solution dilution, with 2.5% ammoniacal liquor wash-out Beta-alanine and adsorption column, collection contains elutriant 760 ml of Beta-alanine, the activated carbon decolorizing final vacuum is concentrated into 85 ml, add while hot long-pending 95% ethanol of diploid, subcooling crystallization 8 h, suction filtration, washing and drying get solid Beta-alanine 25.9 g, recycled behind the crystalline mother solution Recycled ethanol.Twice crystallization is total to get Beta-alanine 73.1 g, yield 84.7%.
Embodiment seven
1, with 100 ml
Escherichia fergusoniiCentrifugal L-Aspartase wet thallus 1.5 g and 1000 ml that obtain of ATCC 35469 fermented liquids
Corynebacterium glutamicumCentrifugal aspartic acid-the α that obtains of ATCC 13032 fermented liquids-decarboxylase wet thallus 15 g, join in the 1000 ml conversion fluids, contain 200 g/L ammonium fumarates, 0.5 g/L CTAB in the conversion fluid, pH 7.0,40 ℃ of enzymatic reaction 16 h, paper chromatography detect and determine reaction end.Beta-alanine concentration is 113.9 g/L in the rear conversion fluid of reaction end, is 96% to the ammonium fumarate molar yield.
2, centrifugal 15 min of conversion fluid 4000 r/min are removed thalline, supernatant liquor is warmed up to 70～80 ℃, adds activated carbon decolorizing, and destainer is concentrated into 145 ml, the subcooling crystallization, and suction filtration, washing and drying get solid Beta-alanine 59.6 g.
3, will separate by 732 type cation exchange resin columns after the crystalline mother solution dilution, with 3% ammoniacal liquor wash-out Beta-alanine and adsorption column, collection contains elutriant 800 ml of Beta-alanine, the activated carbon decolorizing final vacuum is concentrated into 110 ml, add while hot long-pending 95% ethanol of diploid, subcooling crystallization 8 h, suction filtration, washing and drying get solid Beta-alanine 35.5 g, recycled behind the crystalline mother solution Recycled ethanol.Twice crystallization is total to get Beta-alanine 95.1 g, yield 83.5%.
Embodiment eight
1, with 100 ml
Escherichia coli str.Centrifugal L-Aspartase wet thallus 1.5 g that obtain of K-12 substr. MG1655 fermented liquid join in the 1000 ml conversion fluids, contain 50 g/L ammonium fumarates, 0.1 g/L CTAB in the conversion fluid, and then 7.1,55 ℃ of enzymatic reaction 2 h of pH add 1000 ml
Corynebacterium glutamicumCentrifugal aspartic acid-the α that obtains of ATCC 13032 fermented liquids-decarboxylase wet thallus 15 g continues reaction 5 h, and paper chromatography detects determines reaction end.Beta-alanine concentration is 28.8 g/L in the rear conversion fluid of reaction end, is 97% to the fumaric acid molar yield.
2, centrifugal 15 min of conversion fluid 4000 r/min are removed thalline, supernatant liquor is warmed up to 70 ℃～80 ℃, adds activated carbon decolorizing, and destainer is concentrated into 40 ml, the subcooling crystallization, and suction filtration, washing, oven dry get solid Beta-alanine 16.6 g.
3, will separate by 732 type cation exchange resin columns after the crystalline mother solution dilution, with 2.0% ammoniacal liquor wash-out Beta-alanine and adsorption column, collection contains elutriant 240 ml of Beta-alanine, the activated carbon decolorizing final vacuum is concentrated into 30 ml, add while hot long-pending 95% ethanol of diploid, subcooling crystallization 8 h, suction filtration, washing, oven dry, get solid Beta-alanine 7.5 g, recycled behind the crystalline mother solution Recycled ethanol.Twice crystallization is total to get Beta-alanine 24.1 g, yield 83.7%.
Embodiment nine
1, with 100 ml
Escherichia coli str.Centrifugal L-Aspartase wet thallus 1.4 g that obtain of K-12 substr. MG1655 fermented liquid, join in the 1000 ml conversion fluids, contain 150 g/L ammonium fumarates, 0.1 g/L OP in the conversion fluid, pH 7.3,45 ℃ of enzymatic reaction 10 h, paper chromatography detects determines reaction end, after reaction is finished 70 ℃～80 ℃ of enzymatic conversion liquid intensifications, activated carbon decolorizing, vacuum filtration, filtrate concentrating is got aspartic acid ammonium salt solid 143 g.
2, with 1000 ml
Corynebacterium glutamicumCentrifugal aspartic acid-the α that obtains of ATCC 13032 fermented liquids-decarboxylase wet thallus 17 g, join in the 1000 ml conversion fluids, contain 143 g/L aspartic acid ammonium salts, 0.1 g/L CTAB in the conversion fluid, 40 ℃ of enzymatic reaction 15 h, paper chromatography detects determines reaction end, after reaction finishes in the conversion fluid Beta-alanine concentration be 73.2 g/L, be 96% to the molar yield of aspartic acid ammonium salt.
3, centrifugal 15 min of conversion fluid 4000 r/min are removed thalline, supernatant liquor is warmed up to 70 ℃～80 ℃, adds activated carbon decolorizing, and destainer is concentrated into 93 ml, the subcooling crystallization, and suction filtration, washing, oven dry get solid Beta-alanine 40.8 g.
4, will separate by 732 type cation exchange resin columns after the crystalline mother solution dilution, with 2.0% ammoniacal liquor wash-out Beta-alanine and adsorption column, collection contains elutriant 650 ml of Beta-alanine, the activated carbon decolorizing final vacuum is concentrated into 80 ml, add while hot long-pending 95% ethanol of diploid, subcooling crystallization 8 h, suction filtration, washing, oven dry, get solid Beta-alanine 19.5 g, recycled behind the crystalline mother solution Recycled ethanol.Twice crystallization is total to get Beta-alanine 60.3 g, yield 82.4%.
Claims (5)
1. two enzyme couplings prepare the method for Beta-alanine, it is characterized in that being made of following steps:
The bacterial strain that (1) will have respectively aspartase activity and aspartic acid-α-decarboxylase is cultivated in substratum, produces highly active L-Aspartase and aspartic acid-α-decarboxylase;
(2) will have the wet thallus of aspartase activity and aspartic acid-α-decarboxylase or crude enzyme liquid by the certain hour order respectively with the ammonium fumarate aqueous solution of pH 7.0～7.5, add again tensio-active agent, carry out enzymatic reaction under 25 ℃～55 ℃ conditions; The ammonium fumarate aqueous solution that perhaps will have wet thallus or crude enzyme liquid elder generation and the pH 7.0～7.5 of aspartase activity, add tensio-active agent, concentration obtains the aspartic acid ammonium salt solid after 25 ℃～55 ℃ enzymatic reactions, wet thallus or the crude enzyme liquid that will have aspartic acid-α-decarboxylase mix with the aspartic acid ammonium salt aqueous solution again, add tensio-active agent, carry out enzymatic reaction under 25 ℃～55 ℃ conditions; The Beta-alanine that the method separating reaction that utilizes at last isoelectric point crystallizing or isoelectric point crystallizing to be combined with Ion Exchange Resin Phase generates.
2. a kind of pair of enzyme coupling according to claim 1 prepares the method for Beta-alanine, it is characterized in that the described bacterial strain with aspartase activity of step (1) is
Escherichia coli str.K-12 substr. MG1655 or
Escherichia fergusoniiATCC 35469; Bacterial strain with aspartic acid-α-decarboxylase is
Bacillus cereusATCC 10987 or
Corynebacterium glutamicumATCC 13032.
3. a kind of pair of enzyme coupling according to claim 1 prepares the method for Beta-alanine, it is characterized in that the culture medium carbon source in the above-mentioned steps (1) adopts glucose, maltose, sucrose and/or fructose, and total carbon source quality concentration is 1～30 g/L in the substratum; Nitrogenous source adopts extractum carnis, yeast extract paste, corn steep liquor, peptone and/or soya-bean cake hydrolyzed solution, and total nitrogen source quality concentration is 1～30 g/L in the substratum.
4. a kind of pair of enzyme coupling according to claim 1 prepares the method for Beta-alanine, it is characterized in that the described ammonium fumarate aqueous solution of step (2) or aspartic acid ammonium salt aqueous solution concentration are 30 g/L～300 g/L.
5. a kind of pair of enzyme coupling according to claim 1 prepares the method for Beta-alanine, it is characterized in that the used tensio-active agent of step (2) is tween-80 or cetyl trimethylammonium bromide or Triton X-100, concentration is 0.05 g/L～5.0 g/L.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104195193A (en) * | 2014-09-10 | 2014-12-10 | 中国科学院天津工业生物技术研究所 | Method for preparing beta-alanine through enzymatically catalyzing hydrolysis of high-concentration beta-aminopropionitrile |
| CN106755155A (en) * | 2016-12-07 | 2017-05-31 | 江南大学 | A kind of method for preparing beta Alanine with double enzyme series connection |
| CN107012180A (en) * | 2017-06-01 | 2017-08-04 | 南京大学 | A kind of method that beta Alanine is prepared as raw material multienzyme coupling using maleic acid |
| CN107760735A (en) * | 2017-12-13 | 2018-03-06 | 湖北新生源生物工程有限公司 | A kind of method that L glutamic acid is separated from electrodialysis fresh water |
| WO2020187256A1 (en) * | 2019-03-20 | 2020-09-24 | 广安摩珈生物科技有限公司 | Methods for preparing β-alanine, β-alanine salt and pantothenate |
| CN111718969A (en) * | 2019-03-20 | 2020-09-29 | 广安摩珈生物科技有限公司 | Process for producing beta-alanine |
| CN113355370A (en) * | 2021-05-25 | 2021-09-07 | 华南理工大学 | Preparation method of fumaric acid and preparation method of amino acid |
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| CN104195193A (en) * | 2014-09-10 | 2014-12-10 | 中国科学院天津工业生物技术研究所 | Method for preparing beta-alanine through enzymatically catalyzing hydrolysis of high-concentration beta-aminopropionitrile |
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| CN107012180A (en) * | 2017-06-01 | 2017-08-04 | 南京大学 | A kind of method that beta Alanine is prepared as raw material multienzyme coupling using maleic acid |
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| WO2020187256A1 (en) * | 2019-03-20 | 2020-09-24 | 广安摩珈生物科技有限公司 | Methods for preparing β-alanine, β-alanine salt and pantothenate |
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| CN113355370A (en) * | 2021-05-25 | 2021-09-07 | 华南理工大学 | Preparation method of fumaric acid and preparation method of amino acid |
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