CN101603063A - The fixation of microbial cell enzyme process prepares the method for the amino lipid acid of L-2- - Google Patents
The fixation of microbial cell enzyme process prepares the method for the amino lipid acid of L-2- Download PDFInfo
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- CN101603063A CN101603063A CNA2009101817625A CN200910181762A CN101603063A CN 101603063 A CN101603063 A CN 101603063A CN A2009101817625 A CNA2009101817625 A CN A2009101817625A CN 200910181762 A CN200910181762 A CN 200910181762A CN 101603063 A CN101603063 A CN 101603063A
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- lipid acid
- acetyl
- amino lipid
- acid
- fixation
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention is the method for preparing the amino lipid acid of L-2-with the immobilized cell enzyme process that the amino lipid acid of N-acetyl-DL-2-is raw material.The microorganism cells that contains L-Aminoacylase that fermentation obtains is adorned post with the embedding of different fixing carrier; make the amino fatty aqueous acid of certain density N-acetyl-DL-2-with the certain speed immobilized cell packed column of flowing through; collect effluent liquid; condensing crystal or get amino lipid acid of L-2-and the amino lipid acid of N-acetyl-D-2-with the cationic exchange column separating purification, wherein the amino lipid acid of N-acetyl-D-2-is used for the enzyme post once more and splits after racemization.The molar yield of the amino lipid acid of N-acetyl-L-2-reaches more than 96%, and the amino lipid acid yield of L-2-is higher than 85%.Production technique of the present invention is simple, microorganism cells utilization ratio height, be fit to suitability for industrialized production.
Description
One, technical field
The present invention relates to utilize the fixation of microbial cell enzyme process to prepare the method for the amino lipid acid of L-2-; the present invention relates in particular and will contain the microorganism cells immobilization of L-Aminoacylase; effect by the fixation cell intracellular enzyme is split as amino lipid acid of L-2-and the amino lipid acid of N-acetyl-D-2-with the amino lipid acid of substrate N-acetyl-DL-2-, the amino lipid acid of N-acetyl-D-2-after racemization once more enzyme process split and can obtain the amino lipid acid of L-2-again.Belong to technical field of enzyme engineering.
Two, technical background
The 2-aminobutyric acid is to suppress the aliphatic amino acid that human nerve information is transmitted, the clinical apoplexy sequela that can be used for, and have hypotensive effect; The 2-aminobutyric acid still is a kind of important chemical material and medicine intermediate, and it is synthetic to be widely used in medicine, example hydrochloric acid Tibutol, Levetiracetam etc.2-aminovaleric acid (norvaline) is the key intermediate of synthetic drugs perindopril, has also found norvaline in a kind of anti-fungus polypeptide that this external producing bacillus subtilis is given birth to.2-aminohexanoic acid (nor-leucine) also is a kind of widely used pharmaceutical intermediate.According to present reported in literature, more than the preparation method of the amino lipid acid of three kinds of 2-mainly contain following three kinds:
1, chemical method
2007, Xi Qiang etc. (Wuhan Engineering Univ's journal, Vol.29, No.4,2007) were that raw material has synthesized the 2-bromo-butyric acid with butanic acid and bromine, again with 2-bromo-butyric acid and ammoniacal liquor prepared in reaction the DL-2-aminobutyric acid, its yield is 62.5%.
2007, Chen Xinzhi etc. (CN 101007772A, 2007) were that raw material has at first prepared amino valeronitrile with butyraldehyde-n, sodium cyanide and ammonium chloride, obtain the L-norvaline through steps such as itrile group amidation, the fractionation of L-tartrate, hydrolysis.
2006, Chen Xinzhi etc. (CN 100427460C, 2006) were raw material with positive valeric acid, obtain racemize 2-amino valeramide through chloride, bromination, ammonification, utilized L-tartrate to split posthydrolysis and obtained the L-norvaline.
2004, Wang Xu (CN 100352801C, 2004) was main starting raw material with butyraldehyde-n and acetone cyanohydrin, obtained racemize 2-amino valeramide through cyano groupization, ammonification, cyano group amidation successively, utilized L-tartrate to split posthydrolysis and obtained the L-norvaline.
1991, (Bull.Chen.Soc.Jpn. such as Tadashi, 64,1991) be to be total to molten thing with N-acetyl-L-L-Ala ammonium salt, with substitute crystallization process in ethanol optical resolution N-acetyl-DL-aminobutyric acid ammonium salt, N-acetyl-DL-norvaline ammonium salt and N-acetyl-Amicar ammonium salt, wherein N-acetyl-L-norvaline ammonium salt yield reaches 80%.
1986, Compagnone etc. (J.Org.Chem., 51 (10), 1986) sloughed methylthio group with the L-methionine(Met) and generate the L-2-aminobutyric acid under the effect of T-1 Raney's nickel.
1978, (Aust.J.Chem., 31 (1) such as Jeffery, 1978) utilize the method for mercury electrode its corresponding keto-acid amino acid (or amino acid sodium) of electrical catalyze reduction in ammoniacal liquor or ammonium chloride solution, prepared the DL-2-aminobutyric acid, productive rate only is 48%, and has by product L-glutamic acid to generate.
1970, Mahendra etc. (Canadian Journal of Chemistry, 48,1970) with the method asymmetric synthesis of prussic acid addition schiff bases norvaline, nor-leucine and leucine, yield 40%-60%.Its concrete steps are: utilize methylbenzylamine and alkanoic to prepare schiff bases, the prussic acid addition obtains amino-nitrile then, and acid hydrolysis obtains the amino acid that N-replaces, and hydrogenation catalyst obtains phenylethane and a-amino acid.
Nineteen sixty-five, Babievskii etc. (Seriya Khimicheskaya, No.1,1965) get DL-2-aminobutyric acid methyl esters with the reaction product of nitroacetic acid methyl esters, acetaldehyde and Acetyl Chloride 98Min. behind shortening, prepared the DL-2-aminobutyric acid after the hydrolysis, productive rate is low to also have the by product Threonine to generate.
1954, Losse etc. (Chemische Berichte, 87 (9), 1954) utilized chiral separation agent D-tartrate or dibenzoyl-D-tartrate to split the DL-2-aminobutyric acid and obtain its corresponding optical isomer respectively.
2, fermentation method
2000, Tomoihiro etc. (JP 2000279163 (A), 1983) utilized high red colouring agent for food, also used as a Chinese medicine to produce the L-2-aminobutyric acid.
1997, (Applied and Environmental Microbiology such as Andrey, Dec.1997) utilized recombination bacillus coli biosynthesizing L-leucine, L-Xie Ansuan, L-norvaline, L-methionine(Met), L-phenylalanine and L-tyrosine, yield is more than 80%.
Nineteen eighty-three, the microbe fermentation method that the pretty grade in Chinese and Western (JP 63287493 (A), 1983) adopts Colibacter or Corynebacterium glutamicum to belong to has prepared the L-2-aminobutyric acid.
1977, (Applied and Environmental Microbiology was Aug.1977) with Serratia marcescens nor-leucine tolerance mutant strain fermentation method accumulation nor-leucine for Masahiko etc.
3, enzyme process
2009, burnt celebrating just waited (CN 200910030982.8,2009) to utilize L-Aminoacylase microbe-derived or the genetically engineered source, is that substrate has prepared the L-2-aminobutyric acid with N-acetyl-DL-2-aminobutyric acid, and yield reaches more than 78%.
2008, and Daniel etc. (Eur.J.Org.Chem., 2008,3506-3512) utilize Sumizyme MP A dynamic resolution DL-valine ester under the condition that aldehyde exists to obtain the L-norvaline.
2008, Miao Weijuan etc. (process engineering journal, 8 (1), 2008) were substrate with glycerol dehydrogenase and leucine dehydrogenase coupling with glycerine and 2-butanone acid, synthesized 1 simultaneously, 3-otan and L-2-aminobutyric acid.
1999, (Bioorganic ﹠amp such as Fotheringham; Medicinal Chemistry, 7 (10), 1999) utilizing the e. coli k12 somatic cells is that substrate has synthesized the L-2-aminobutyric acid by transamination reaction with L-Threonine and L-aspartic acid.
1989; (J.Am.Chem.Soc. such as Chenault; 1989,6354-6364) the L-Aminoacylase I with pig kidney and aspergillus oryzae source has split N-acetyl-DL-aminobutyric acid, N-acetyl-50 several N-acetyl-a-amino acids and derivatives thereof such as DL-norvaline.
1945; (Journal of Biological Chemistry such as Greenstein; 182 (2); 1945) utilize the L-Aminoacylase asymmetric hydrolysis N-chloracetyl racemize nor-leucine, norvaline and the butyrine that extract in the pig kidney to obtain its corresponding L-type and D-type isomer, wherein the yield of L-type and D-type is respectively 70% and 60%.
Utilize free cell or enzymatic conversion to prepare the amino lipid acid of L-2-, can contain a small amount of tropina or other impurity in the conversion fluid, be unfavorable for the separation and purification of product, the repeating utilization factor of somatic cells or enzyme is not high yet.
Up to the present, the microorganism cells enzyme process that utilizes immobilization the to contain L-Aminoacylase method for preparing the amino lipid acid of L-2-yet there are no report.
Three, summary of the invention
1, goal of the invention
The object of the invention is to provide a kind of fixation of microbial cell enzyme process to prepare the method for the amino lipid acid of L-2-.
2, technical scheme
A kind of method of utilizing the fixation of microbial cell enzyme process to prepare the amino lipid acid of L-2-is characterized in that preparation process is as follows:
(1) cell fixation
Somatic cells that contains L-Aminoacylase and embedding medium that fermentation is obtained mix, and add curing molding in the forming agent, or add reinforcer behind the curing molding again and carry out intensive treatment, promptly get immobilized cell;
(2) the amino lipid acid bio-transformation of N-acetyl-DL-2-
Immobilized cell is adorned post, make the amino fatty aqueous acid of the N-acetyl-DL-2-enzyme post of flowing through carry out bio-transformation, collect effusive conversion fluid, conversion fluid repeats upper prop, and the amino fatty acid response of N-acetyl in conversion fluid-L-2-is complete;
(3) the amino lipid acid separation and purification of L-2-
Above-mentioned conversion fluid is removed impurity with activated carbon decolorizing, concentrate, crystallisation by cooling or with separating with cationic exchange coloum after the decolorization and impurity removal by active carbon matter, obtain amino lipid acid of L-2-and the amino lipid acid of N-acetyl-D-2-, wherein the amino lipid acid of N-acetyl-D-2-gets the amino lipid acid of N-acetyl-DL-2-and is used for the fractionation of enzyme post once more through racemization.
The thalline that contains L-Aminoacylase described in the above-mentioned steps (1) is cunninghamella echinulata 9980, aspergillus oryzae, genetic engineering bacterium 1016 or genetic engineering bacterium DM202.
Somatic cells described in the above-mentioned steps (1) and the mixed concentration of embedding medium are 10～200g/L, and the immobilization time is 1～48h in forming agent, and the reinforcer treatment time is 10～180min.
Embedding medium described in the above-mentioned steps (1) is sodium alginate, carrageenin, gelatin or chitosan, and described forming agent is calcium chloride, Repone K or sodium radio-phosphate,P-32 solution, and described reinforcer is a glutaraldehyde.Wherein embedding medium sodium alginate or carrageenan concentrations are 10～50g/L, and forming agent calcium chloride or potassium chloride solution concentration are 10～60g/L; The embedding medium gelatin concentration is 50～200g/L, and the concentration of colloid reinforcer glutaraldehyde is 5～30g/L; The embedding medium chitosan concentration is 10～100g/L, and forming agent is the sodium radio-phosphate,P-32 solution of pH6～8.
The amino lipid acid of N-acetyl described in the above-mentioned steps (2)-DL-2-is N-acetyl-DL-2-aminobutyric acid, N-acetyl-DL-2-aminovaleric acid or N-acetyl-DL-2-hexosamine.
The amino fatty aqueous acid concentration of N-acetyl described in the above-mentioned steps (2)-DL-2-is 50～400g/L, pH6.0～9.0, and the speed of the enzyme post of flowing through is 100～1000mL/h, invert point is 25～60 ℃.
3, beneficial effect
The present invention utilizes the fixation of microbial cell enzyme process to split the amino lipid acid of N-acetyl-DL-2-and obtains the amino lipid acid of L-2-, the reaction conditions gentleness, enzymatic conversion method efficient height, product is easy to separation and purification and immobilized cell is reusable, has advantages such as production cost is low, technical process simple, suitable suitability for industrialized production.
Four, embodiment
Embodiment 1 fixation of microbial cell enzyme process prepares the method for L-2-aminobutyric acid
Get 1000mL cunninghamella echinulata 9980 fermented liquids, the centrifugal 20min of 2000r/min collects thalline, thalline weight in wet base 40g.Wash in the 700mL 30g/L sodium alginate aqueous solution, mixing splashes in the 1000mL 20g/L calcium chloride water with the sodium alginate aqueous solution that syringe will contain thalline then, removes calcium chloride solution after solidifying 2h, and with physiological saline washing three times.
With the gained immobilized cell diameter 4cm that packs into, in the glass column of high 50cm.1000mL10%N-acetyl-DL-2-aminobutyric acid sodium water solution is with the 300mL/h enzyme post of flowing through, 45 ℃ of controlled temperature, and pH6.5, it is complete until substrate conversion that effluent liquid repeats upper prop.
To transform completely effluent liquid and be warming up to 70 ℃ and add activated carbon decolorizings, vacuum concentration is to 150mL, crystallisation by cooling, vacuum filtration gets L-2-aminobutyric acid crude product, dry 26g; The 26g crude product is joined in 150mL 75% ethanol, and agitator treating, vacuum filtration get L-2-aminobutyric acid 18.3g; With ethanol filtrate evaporation concentration, concentrated solution and crude product mother liquor are merged, transfer pH2.0 with 6mol/L hydrochloric acid, crystallisation by cooling, vacuum filtration gets N-acetyl-D-2-aminobutyric acid crude product, dry 47g, the crude product Recycling Mother Solution is applied mechanically.N-acetyl-D-2-aminobutyric acid is used for the enzyme post once more and splits after racemization.
Embodiment 2 fixation of microbial cell enzyme process prepare the method for L-norvaline
Get 1000mL aspergillus oryzae fermented liquid, the centrifugal 20min of 2000r/min collects thalline, thalline weight in wet base 34g.Wash in the 600mL 30g/L sodium alginate aqueous solution, mixing splashes in the 1000mL 20g/L calcium chloride water with the sodium alginate aqueous solution that syringe will contain thalline then, removes calcium chloride solution after solidifying 2h, and with physiological saline washing three times.
With the gained immobilized cell diameter 4cm that packs into, in the glass column of high 50cm.1000mL10%N-acetyl-DL-norvaline sodium water solution is with the 300mL/h enzyme post of flowing through, 45 ℃ of controlled temperature, and pH7.5, it is complete until substrate conversion that effluent liquid repeats upper prop.
To transform completely effluent liquid and be warming up to 70 ℃ and add activated carbon decolorizings, vacuum concentration is to 180mL, crystallisation by cooling, vacuum filtration gets L-norvaline crude product, dry 31g; The 31g crude product is joined in 180mL 75% ethanol, and agitator treating, vacuum filtration get L-norvaline 24.7g; With ethanol filtrate evaporation concentration, concentrated solution and crude product mother liquor are merged, transfer pH2.0 with 6mol/L hydrochloric acid, crystallisation by cooling, vacuum filtration gets N-acetyl-D-norvaline crude product, dry 51.5g, the crude product Recycling Mother Solution is applied mechanically.N-acetyl-D-norvaline is used for the enzyme post once more and splits after racemization.
Embodiment 3 fixation of microbial cell enzyme process prepare the method for L-nor-leucine
Get 1000mL cunninghamella echinulata 9980 fermented liquids, the centrifugal 20min of 2000r/min collects thalline, thalline weight in wet base 41g.Wash in the 700mL 30g/L sodium alginate aqueous solution, mixing splashes in the 1000mL 20g/L calcium chloride water with the sodium alginate aqueous solution that syringe will contain thalline then, removes calcium chloride solution after solidifying 2h, and with physiological saline washing three times.
With the gained immobilized cell diameter 4cm that packs into, in the glass column of high 50cm.1000mL10%N-acetyl-Amicar sodium water solution is with the 300mL/h enzyme post of flowing through, 45 ℃ of controlled temperature, and pH7.0, it is complete until substrate conversion that effluent liquid repeats upper prop.
To transform completely effluent liquid and be warming up to 70 ℃ and add activated carbon decolorizings, vacuum concentration is to 200mL, crystallisation by cooling, vacuum filtration gets L-nor-leucine crude product, dry 32.3g; The 32.3g crude product is joined in 200mL 75% ethanol, and agitator treating, vacuum filtration get L-nor-leucine 25g; With ethanol filtrate evaporation concentration, concentrated solution and crude product mother liquor are merged, transfer pH2.0 with 6mol/L hydrochloric acid, crystallisation by cooling, vacuum filtration gets N-acetyl-D-nor-leucine crude product, dry 55g, the crude product Recycling Mother Solution is applied mechanically.N-acetyl-D-nor-leucine is used for the enzyme post once more and splits after racemization.
Embodiment 4 fixation of microbial cell enzyme process prepare the method for L-2-aminobutyric acid
Get 1000mL aspergillus oryzae fermented liquid, the centrifugal 20min of 2000r/min collects thalline, thalline weight in wet base 36g.Wash in the 600mL 30g/L carrageenin aqueous solution, mixing splashes in the 1000mL 40g/L potassium chloride solution with the carrageenin aqueous solution that syringe will contain thalline then, removes Klorvess Liquid after solidifying 4h, and with physiological saline washing three times.
With the gained immobilized cell diameter 4cm that packs into, in the glass column of high 50cm.1000mL20%N-acetyl-DL-2-aminobutyric acid sodium water solution is with the 200mL/h enzyme post of flowing through, 45 ℃ of controlled temperature, and pH7.0, it is complete until substrate conversion that effluent liquid repeats upper prop.
To transform completely effluent liquid and be warming up to 70 ℃ and add activated carbon decolorizings, vacuum concentration is to 250mL, crystallisation by cooling, vacuum filtration gets L-2-aminobutyric acid crude product, dry 53.2g; The 53.2g crude product is joined in 250mL 75% ethanol, and agitator treating, vacuum filtration get L-2-aminobutyric acid 36g; With ethanol filtrate evaporation concentration, concentrated solution and crude product mother liquor are merged, transfer pH2.0 with 6mol/L hydrochloric acid, crystallisation by cooling, vacuum filtration gets N-acetyl-D-2-aminobutyric acid crude product, dry 98g, the crude product Recycling Mother Solution is applied mechanically.N-acetyl-D-2-aminobutyric acid is used for the enzyme post once more and splits after racemization.
Embodiment 5 fixation of microbial cell enzyme process prepare the method for L-norvaline
Get 1000mL genetic engineering bacterium 1016 fermented liquids, the centrifugal 20min of 4000r/min collects thalline, thalline weight in wet base 15g.Wash in the 300mL 30g/L carrageenin aqueous solution, mixing splashes in the 600mL 40g/L potassium chloride solution with the carrageenin aqueous solution that syringe will contain thalline then, removes Klorvess Liquid after solidifying 4h, and with physiological saline washing three times.
With the gained immobilized cell diameter 4cm that packs into, in the glass column of high 50cm.1000mL20%N-acetyl-DL-norvaline sodium water solution is with the 200mL/h enzyme post of flowing through, 45 ℃ of controlled temperature, and pH7.0, it is complete until substrate conversion that effluent liquid repeats upper prop.
To transform completely effluent liquid and be warming up to 70 ℃ and add activated carbon decolorizings, vacuum concentration is to 300mL, crystallisation by cooling, vacuum filtration gets L-norvaline crude product, dry 59g; The 59g crude product is joined in 300mL 75% ethanol, and agitator treating, vacuum filtration get L-norvaline 44g; With ethanol filtrate evaporation concentration, concentrated solution and crude product mother liquor are merged, transfer pH2.0 with 6mol/L hydrochloric acid, crystallisation by cooling, vacuum filtration gets N-acetyl-D-norvaline crude product, dry 105g, the crude product Recycling Mother Solution is applied mechanically.N-acetyl-D-norvaline is used for the enzyme post once more and splits after racemization.
Embodiment 6 fixation of microbial cell enzyme process prepare the method for L-nor-leucine
Get 1000mL genetic engineering bacterium DM202 fermented liquid, the centrifugal 20min of 4000r/min collects thalline, thalline weight in wet base 15g.Wash in the 300mL 30g/L carrageenin aqueous solution, mixing splashes in the 600mL 40g/L potassium chloride solution with the carrageenin aqueous solution that syringe will contain thalline then, removes Klorvess Liquid after solidifying 4h, and with physiological saline washing three times.
With the gained immobilized cell diameter 4cm that packs into, in the glass column of high 50cm.1000mL20%N-acetyl-Amicar sodium water solution is with the 200mL/h enzyme post of flowing through, 45 ℃ of controlled temperature, and pH7.0, it is complete until substrate conversion that effluent liquid repeats upper prop.
To transform completely effluent liquid and be warming up to 70 ℃ and add activated carbon decolorizings, vacuum concentration is to 350mL, crystallisation by cooling, vacuum filtration gets L-nor-leucine crude product, dry 63g; The 63g crude product is joined in 350mL 75% ethanol, and agitator treating, vacuum filtration get L-nor-leucine 47.6g; With ethanol filtrate evaporation concentration, concentrated solution and crude product mother liquor are merged, transfer pH2.0 with 6mol/L hydrochloric acid, crystallisation by cooling, vacuum filtration gets N-acetyl-D-nor-leucine crude product, dry 110g, the crude product Recycling Mother Solution is applied mechanically.N-acetyl-D-nor-leucine follows after racemization and is used for the fractionation of enzyme post once more.
Embodiment 7 fixation of microbial cell enzyme process prepare the method for L-2-aminobutyric acid
Get 1000mL genetic engineering bacterium 1016 fermented liquids, the centrifugal 20min of 4000r/min collects thalline, thalline weight in wet base 15g.Wash in the 300mL 60g/L aqueous gelatin solution, mixing places 4 ℃ to solidify then, solidifies the micelle that blob of viscose is cut into behind the 10h 5mm * 5mm, and with 600mL1% glutaraldehyde cross-linking 1h.Remove glutaraldehyde water solution, and wash three times with physiological saline.
With the gained immobilized cell diameter 4cm that packs into, in the glass column of high 50cm.1000mL30%N-acetyl-DL-2-aminobutyric acid sodium water solution is with the 150mL/h enzyme post of flowing through, 45 ℃ of controlled temperature, and pH7.0, it is complete until substrate conversion that effluent liquid repeats upper prop.
To transform completely effluent liquid and be warming up to 70 ℃ and add activated carbon decolorizings, vacuum concentration is to 350mL, crystallisation by cooling, vacuum filtration gets L-2-aminobutyric acid crude product, dry 79.4g; The 79.4g crude product is joined in 350mL 75% ethanol, and agitator treating, vacuum filtration get L-2-aminobutyric acid 54.8g; With ethanol filtrate evaporation concentration, concentrated solution and crude product mother liquor are merged, transfer pH2.0 with 6mol/L hydrochloric acid, crystallisation by cooling, vacuum filtration gets N-acetyl-D-2-aminobutyric acid crude product, dry 153g, the crude product Recycling Mother Solution is applied mechanically.N-acetyl-D-2-aminobutyric acid is used for the enzyme post once more and splits after racemization.
Embodiment 8 fixation of microbial cell enzyme process prepare the method for L-2-aminobutyric acid
Get 1000mL genetic engineering bacterium DM202 fermented liquid, the centrifugal 20min of 4000r/min collects thalline, thalline weight in wet base 15g.Wash in the 500mL 20g/L chitosan aqueous solution, mixing, the chitosan solution that will contain thalline then splashes into pH7.5, solidifies 24h in the 40g/L sodium radio-phosphate,P-32 solution.Remove sodium radio-phosphate,P-32 solution, and wash three times with physiological saline.
With the gained immobilized cell diameter 4cm that packs into, in the glass column of high 50cm.1000mL30%N-acetyl-DL-2-aminobutyric acid sodium water solution is with the 150mL/h enzyme post of flowing through, 45 ℃ of controlled temperature, and pH7.5, it is complete until substrate conversion that effluent liquid repeats upper prop.
To transform completely effluent liquid and be warming up to 70 ℃ and add activated carbon decolorizings, vacuum concentration is to 350mL, crystallisation by cooling, vacuum filtration gets L-2-aminobutyric acid crude product, dry 77.8g; The 77.8g crude product is joined in 350mL 75% ethanol, and agitator treating, vacuum filtration get L-2-aminobutyric acid 52g; With ethanol filtrate evaporation concentration, concentrated solution and crude product mother liquor are merged, transfer pH2.0 with 6mol/L hydrochloric acid, crystallisation by cooling, vacuum filtration gets N-acetyl-D-2-aminobutyric acid crude product, dry 158g, the crude product Recycling Mother Solution is applied mechanically.N-acetyl-D-2-aminobutyric acid is used for the enzyme post once more and splits after racemization.
Claims (9)
1, a kind of fixation of microbial cell enzyme process prepares the method for the amino lipid acid of L-2-, and its preparation process is as follows:
(1) cell fixation
Somatic cells that contains L-Aminoacylase and embedding medium that fermentation is obtained mix, and add curing molding in the forming agent, or add reinforcer behind the curing molding again and carry out intensive treatment, promptly get immobilized cell;
(2) the amino lipid acid bio-transformation of N-acetyl-DL-2-
Immobilized cell is adorned post, make the amino fatty aqueous acid of the N-acetyl-DL-2-enzyme post of flowing through carry out bio-transformation, collect effusive conversion fluid, conversion fluid repeats upper prop, and the amino fatty acid response of N-acetyl in conversion fluid-L-2-is complete;
(3) the amino lipid acid separation and purification of L-2-
Above-mentioned conversion fluid is removed impurity with activated carbon decolorizing, concentrate, crystallisation by cooling or with separating with cationic exchange coloum after the decolorization and impurity removal by active carbon matter, obtain amino lipid acid of L-2-and the amino lipid acid of N-acetyl-D-2-, wherein the amino lipid acid of N-acetyl-D-2-gets the amino lipid acid of N-acetyl-DL-2-and is used for the fractionation of enzyme post once more through racemization.
2, fixation of microbial cell enzyme process according to claim 1 prepares the method for the amino lipid acid of L-2-, and the thalline that it is characterized in that containing described in the step (1) L-Aminoacylase is cunninghamella echinulata 9980, aspergillus oryzae, genetic engineering bacterium 1016 or genetic engineering bacterium DM202.
3, fixation of microbial cell enzyme process according to claim 1 prepares the method for the amino lipid acid of L-2-, it is characterized in that somatic cells described in the step (1) and the mixed concentration of embedding medium are 10～200g/L, the immobilization time is 1～48h in forming agent, and the reinforcer treatment time is 10～180min.
4, fixation of microbial cell enzyme process according to claim 1 prepares the method for the amino lipid acid of L-2-, it is characterized in that the embedding medium described in the step (1) is sodium alginate, carrageenin, gelatin or chitosan, described forming agent is calcium chloride, Repone K or sodium radio-phosphate,P-32 solution, and described reinforcer is a glutaraldehyde.
5, fixation of microbial cell enzyme process according to claim 1 prepares the method for the amino lipid acid of L-2-, it is characterized in that the amino lipid acid of the N-acetyl described in the step (2)-DL-2-is N-acetyl-DL-2-aminobutyric acid, N-acetyl-DL-2-aminovaleric acid or N-acetyl-DL-2-hexosamine.
6, fixation of microbial cell enzyme process according to claim 1 prepares the method for the amino lipid acid of L-2-, it is characterized in that the amino fatty aqueous acid concentration of the N-acetyl described in the step (2)-DL-2-is 50～400g/L, pH6.0～9.0, the speed of enzyme post of flowing through is 100～1000mL/h, and invert point is 25～60 ℃.
7, fixation of microbial cell enzyme process according to claim 4 prepares the method for the amino lipid acid of L-2-, the concentration that it is characterized in that described embedding medium sodium alginate or carrageenin is 10～50g/L, and the concentration of forming agent calcium chloride or potassium chloride solution is 10～60g/L.
8, fixation of microbial cell enzyme process according to claim 4 prepares the method for the amino lipid acid of L-2-, and the concentration that it is characterized in that described embedding medium gelatin is 50～200g/L, and the concentration of colloid reinforcer glutaraldehyde is 5～30g/L.
9, fixation of microbial cell enzyme process according to claim 4 prepares the method for the amino lipid acid of L-2-, and the concentration that it is characterized in that described embedding medium chitosan is 10～100g/L, and forming agent is the sodium radio-phosphate,P-32 solution of pH6.0～8.0.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636294A (en) * | 2017-02-28 | 2017-05-10 | 滨海瀚鸿生化有限公司 | Process for producing unnatural amino acid products through coupling reaction of immobilized bi-enzyme |
| CN106769364A (en) * | 2016-12-06 | 2017-05-31 | 国家海洋局南海环境监测中心 | A kind of phytoplankton sample embedding medium and its application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106769364A (en) * | 2016-12-06 | 2017-05-31 | 国家海洋局南海环境监测中心 | A kind of phytoplankton sample embedding medium and its application |
| CN106769364B (en) * | 2016-12-06 | 2019-07-26 | 国家海洋局南海环境监测中心 | A kind of phytoplankton sample embedding medium and its application |
| CN106636294A (en) * | 2017-02-28 | 2017-05-10 | 滨海瀚鸿生化有限公司 | Process for producing unnatural amino acid products through coupling reaction of immobilized bi-enzyme |
| CN106636294B (en) * | 2017-02-28 | 2020-08-14 | 滨海瀚鸿生化有限公司 | Process for producing unnatural amino acid product by immobilized double-enzyme coupling reaction |
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Application publication date: 20091216 |