CN101270377A - Method for preparing <14>NL-phenylalanine with enzyme - Google Patents

Method for preparing <14>NL-phenylalanine with enzyme Download PDF

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CN101270377A
CN101270377A CNA2007100645340A CN200710064534A CN101270377A CN 101270377 A CN101270377 A CN 101270377A CN A2007100645340 A CNA2007100645340 A CN A2007100645340A CN 200710064534 A CN200710064534 A CN 200710064534A CN 101270377 A CN101270377 A CN 101270377A
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phenylalanine
thalline
ammonium sulfate
ammonia lyase
obtains
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CN101270377B (en
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袁其朋
岳海燕
汪文川
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Beijing University of Chemical Technology
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Abstract

The invention aims to provide an enzymatic preparation method of <15>NL-Phenylalanine. <15>N-ammonium sulfate and trans-cinnamic acid react to produce <15>NL-Phenylalanine under the catalyst of phenylalanine ammonia-lyase. The method of the invention has the advantages of the simple synthesis process, the higher output, compared with organic synthesis method and enzymatic method, achieving the easy substitution of N isotope, resulting in the product purity at 99 percent, the yield efficiency at 71 percent, the isotopic abundance at higher than 98 percent, the recovery rate of <15>N at 88 percent and being favorable for the large-scale production.

Description

A kind of enzyme process preparation 15The method of NL-phenylalanine
Technical field:
The present invention relates to a kind of enzyme process preparation 15The method of NL-phenylalanine.Belong to food, medicine, chemical field.
Background technology:
15The NL-phenylalanine is in the L-phenylalanine 14The N element is by isotropic substance 15The stable tagged compound that N replaced, "dead", use and be not subjected to time limitation, to compare with radioactivity amino acid, safety, convenient is particularly suited for the infant, is a kind of good tracer agent.Because the L-phenylalanine is one of necessary for human body amino acid,, can synthesizes and have the active enzyme of important biomolecule, hormone etc. as the primitive of synthetic protein.Therefore, adopt 15The NL-phenylalanine utilizes amino acid whose metabolism as tracer agent, to opening in the organism and intracellular physical and chemical processes, understands the metabolism of organism and has played key effect.
Mosoni etc. [20]Report [U- 14C] phenylalanine is marked on the wheat cell wall xylogen, the metabolism of xylogen in the degraded of research cell wall.Phenols transforms lessly in the ruminating animal forage digestive process, only may transform at certain functional group usefulness [U- 14C] phenylalanine mark xylogen, traceable this trace transforms, and estimates and ruminates the degree that phenols transforms in the fermentation and determine transformation mechanism.Curtius [21]Deuterium-labeled phenylalanine is a tracer agent on first Application ring in 1972, adopt the GC-MS analytical technology to set up the method for measuring human body phenylalanine-4-hydroxylase vigor, and the pku patients carried out load test, measured this enzyme activity, this enzyme activity can react the severity of hepatopathy, and the diagnoses and treatment of hepatopathy is had the certain significance.
The preparation foreign study of mark phenylalanine is more.LeMaster D.M.et al. is by Escherichia coli. fermentation, with the DL-[1-of mark 13C] lactate biosynthesizing L-[3,4- 13C 2] phenyl-[1- 13C] alanine; The Halldin C.et al. phenyl-[3-of phenylalanine transaminase method with mark 11C] phenyl-pyruvic acid and 11CO 2Change ammonia and generate L-phenyl-[3- 11C] alanine; Tanimura K.et al. passes through the Pd/C catalytic reduction reaction with cyclo[(Z)-Δ phe-D-Ala] and 2H 2Synthetic D-phenyl-[2,3- 2H 2] alanine; Fujihara H.et al. with the N-acetyl-DL-phenylalanine and 2H 2O synthesizes L-phenyl-[2- 2H] alanine, yield is up to 97%.Phenylalanine ammonia lyase method label L-phenylalanine also has report abroad.Usefulness such as Dai Tengchang 2Spike of H-phenylalanine and GC-MS analytical technology are measured phenylalanine-4-hydroxylase vigor in the human body, are used for the diagnosis and the treatment of hepatopathy.
Up to now, 15The preparation of NL-phenylalanine, unique relevant report is with the synthetic [2-of phenylalanine ammonia lyase method both at home and abroad 13C, 15N] the L-phenylalanine.Hadener A. and Tamm Ch. [2- 13C] styracin and 15NH 4Cl synthesizes L-phenyl-[2- 13C, 15N] alanine, purity 62.9%, yield 29%, 87.8%, 15NH 4Cl obtains reclaiming.The phenylalanine purity and the yield of this research mark are all low, the mark nitrogenous source 15NH 4Cl consumption height, nitrogenous source retrieving arrangement (Parnas-Wagner) complexity is operated wayward.Therefore, improve 15The preparation technology of NL-phenylalanine improves purity, yield and the abundance of marked product, and efficient recovery mark nitrogenous source has very important meaning to the expansion production of labeled amino acid.
Among the preparation method of other referential common phenylalanine, extraction method can not be introduced isotropic substance; The DL-phenylalanine that chemical synthesis obtains need split, complex steps, and by product is many, separate complex, yield is low.The fermentation method nitrogenous source is many and complicated, is difficult to obtain, and preparation cycle is long, yields poorly.And complicated because nitrogenous source is many, influence the product abundance.
Summary of the invention:
The purpose of this invention is to provide a kind of enzyme process preparation 15The method of NL-phenylalanine.By the phenylalanine ammonia lyase Enzymatic transformation 15N-ammonium sulfate and trans-cinnamic acid generate 15The NL-phenylalanine simplifying technology, improves 15Efficient recovery mark nitrogenous source in the time of the output of NL-phenylalanine, purity and isotopic abundance.
The present invention obtains to contain the thalline of phenylalanine ammonia lyase by fermenting, with 15N-ammonium sulfate is the mark nitrogenous source, and with trans-cinnamic acid generation enzymatic reaction, separation and purification obtains 15The NL-phenylalanine, and by ammonium salt retrieving arrangement recovery mark nitrogenous source.
Concrete steps are as follows:
The fermentation culture of A thalline
The yeast strain that will contain phenylalanine ammonia lyase, cultivated 20-30 hour in the 25-30 ℃ of following solid medium, place seed culture medium then, cultivated 20-24 hour down for 25-30 ℃, move to and produce in the enzyme substratum, cultivated 21-24 hour down for 25-30 ℃, centrifugal and with the physiological saline washing, obtain to contain the thalline of phenylalanine ammonia lyase;
The component of used solid medium and content are: the agar of quality percentage composition 1.5-2.5%, and 8-12 ° of Be (° Be is the ripple woods, and pol unit measures with B) Fructus Hordei Germinatus soaks powder, and all the other are water.
The component of seed culture medium and quality percentage composition are: glucose 0.5-2%, peptone 0.5-2.0%, yeast extract paste 0.5-2.0%, K 2HPO 40.05-0.2%, NaCl 0.4-0.6%, all the other are water.
The component and the quality percentage composition that produce the enzyme substratum are: glucose 0.5-2%, corn steep liquor 2.0-5.0%, soybean cake powder 1.0-3.0%, NaCl 0.4-0.6%, K 2HPO 40.05-0.2%, L-phenylalanine 0.02-0.1% transfers pH5.5 with dilute hydrochloric acid (concentration of used dilute hydrochloric acid need not strict restriction, and concentration is that 3-4% gets final product), and all the other are water.
The B conversion reaction
The thalline that contains phenylalanine ammonia lyase that steps A is obtained places conversion reaction liquid, and it is 1 that the amount of adding makes styracin and the mass ratio that contains the phenylalanine ammonia lyase thalline: 2.5-4.0, said conversion reaction liquid is for containing 15N-ammonium sulfate 0.5-0.6mol/l, sodium hydroxide 1.0-1.2mol/l, styracin quality percentage composition are the solution of 1.0-2.0%, 25-30 ℃ following conversion reaction 20-24 hour;
The C separation and purification
Converted product mixture bactofugation with step B acquisition, transfer pH to 3.0-4.0 with dilute sulphuric acid (concentration of used dilute sulphuric acid need not strict restriction, and concentration 3-4% gets final product), the centrifugal precipitation of removing, transfer pH to 5.5-6.5 again, separate through macroporous adsorbent resin, the deionized water wash-out, elutriant is transferred pH to 5.5 rotary evaporation with dilute sulphuric acid, add dehydrated alcohol, crystallisation by cooling more after filtration, obtains the crystal lyophilize 15The NL-phenylalanine;
The D nitrogenous source reclaims
With containing after the separation of step C macroporous adsorbent resin 15(unconverted mark nitrogenous source becomes in step B N-ammonium sulfate in the enzyme reaction 15N-ammonium sulfate) mixing solutions, with sodium hydroxide solution (the mass percentage concentration scope of sodium hydroxide is 20-40%, and the concentration height helps rapid reaction) reaction, the ammonia of emitting is collected with sulphuric acid soln, obtains highly purified through evaporation and oven dry 15N-ammonium sulfate, used vitriolic mass percentage concentration scope is 5-10%, too rare increase evaporation energy consumption, the too dense ammonia that is unfavorable for is collected.
Method essence of the present invention is the thalline that cultivate to obtain to contain the phenylalanine ammonia lyase high enzymatic activity by yeast fermentation, by enzymatic reaction, with the mark nitrogenous source- 15N-ammonium sulfate and trans-cinnamic acid are converted into 15The NL-phenylalanine, and make 15N source efficient recovery.Simplifying technology, improve 15Efficient recovery mark nitrogenous source in the time of the output of NL-phenylalanine, purity and isotopic abundance.
Method synthesis technique of the present invention is simple, and the enzymatic specificity is strong, and product need not split, marked product productive rate height, abundance are big, reacting final product is few, is easy to separation and purification, compares with organic synthesis method and enzyme process, the N isotropic substance substitutes easily, the purity of product can reach 99%, isotopic abundance is greater than 98%, and yield is greater than 70%, and it is simple that the mark nitrogenous source reclaims technology, the rate of recovery is 88%, is fit to scale operation.
Embodiment:
Embodiment 1:
The fermentation culture of A thalline
To contain 30 ℃ of solid culture of rhodotorula glutinis Rhodotorula glutinis 2.102 bacterial strains 20 hours of phenylalanine ammonia lyase, the component of solid medium and content are: the agar of quality percentage composition 1.5%, and 8 ° of Be Fructus Hordei Germinatus soak powder, and all the other are water.Get one and encircle in seed culture medium, the component of seed culture medium and quality percentage composition are: glucose 1.0%, peptone 1.5%, yeast extract paste 1.0%, K 2HPO 40.15%, NaCl 0.5%, and all the other are water.At 30 ℃, shaking table was cultivated 20 hours under the 150rpm, moved to 10% inoculum size and produced in the enzyme substratum, and the component and the quality percentage composition that produce the enzyme substratum are: glucose 1.0%, and corn steep liquor 3.0%, soybean cake powder 2.0%, NaCl 0.4%, K 2HPO 40.05%, L-Phe (L-phenylalanine) 0.02% is the dilute hydrochloric acid accent pH to 5.5 of 3-4% with concentration, and all the other are water.At 30 ℃, shaking table was cultivated 21 hours under the 150rpm, and is centrifugal and use the physiological saline washed twice, and the thalline that contains phenylalanine ammonia lyase of acquisition is used for conversion reaction.The thalline weight in wet base that obtains is 20g/l, and the phenylalanine ammonia lyase enzyme is 18.2 * 10 than work -3U/mg dry weight cell.
The B conversion reaction
The thalline that contains phenylalanine ammonia lyase that fermentation is obtained places conversion reaction liquid, 15The N-ammonium sulfate concentrations is 0.5mol/l, and naoh concentration is 1mol/l, and the styracin mass percentage concentration is 1.0%, and the mass ratio of styracin and wet thallus is (g/g): 1: 3.0, at 30 ℃, 150rpm was down with shaking table conversion reaction 24 hours.
The C separation and purification
The converted product mixture is transferred pH to 3.0 through bactofugation with the dilute sulphuric acid of mass percentage concentration 4%, and the mark nitrogenous source is converted into 15N-ammonium sulfate, and precipitation styracin and albumen, centrifugal disgorging is transferred pH to 5.5, separates through HP20 (resin trade names) macroporous adsorbent resin 15The NL-phenylalanine, 15N-ammonium sulfate, other inorganic salt and impurity, remove the nonpolar pigment of part, the deionized water wash-out, the elutriant mass percentage concentration is 4% dilute sulphuric acid accent pH to 5.5, concentrate through rotary evaporation, 70 ℃ add dehydrated alcohol and remove polarity pigment, crystallisation by cooling, filter, lyophilize obtains 2.10g (1L fermented liquid) 15NL-phenylalanine finished product, purity are greater than 99%, and abundance is greater than 98%, and yield is greater than 70%.
The D nitrogenous source reclaims
The ammonium salt mixing solutions is placed round-bottomed flask, with excessive mass percentage concentration be NaOH solution rapid reaction under boiling state of 30%, it is in 8% the dilution heat of sulfuric acid, to use N that the ammonia that discharges is collected in mass percentage concentration 2Drive ammonia residual in the flask to dilution heat of sulfuric acid.In the process of collecting ammonia, drip dilute sulphuric acid and regulate the pH value, make ammonia and sulfuric acid complete reaction.Collection obtains 15The N-ammoniumsulphate soln obtains highly purified solid through evaporation, oven dry 15N-ammonium sulfate. 15The N-ammonium sulfate rate of recovery is 88.2%.
Embodiment 2:
The fermentation culture of A thalline
To contain 25 ℃ of solid culture of rhodotorula glutinis Rhodotorula glutinis2.102 bacterial strain 30 hours of phenylalanine ammonia lyase, the component of solid medium and content are: the agar of quality percentage composition 2.0%, and 10 ° of Be Fructus Hordei Germinatus soak powder, and all the other are water.Get one and encircle in seed culture medium, the component of seed culture medium and quality percentage composition are: glucose 0.5%, peptone 1.0%, yeast extract paste 1.0%, K 2HPO 40.10%, NaCl 0.5%, and all the other are water.At 28 ℃, shaking table was cultivated 24 hours under the 150rpm, moved to 10% inoculum size and produced in the enzyme substratum, and the component and the quality percentage composition that produce the enzyme substratum are: glucose 0.5%, and corn steep liquor 2.0%, soybean cake powder 1.0%, NaCl 0.5%, K 2HPO 40.10%, L-Phe 0.05%, pH5.5, and all the other are water.At 28 ℃, shaking table was cultivated 22 hours under the 150rpm, and is centrifugal and use the physiological saline washed twice, and the thalline that contains PAL of acquisition is used for conversion reaction.The thalline weight in wet base that obtains is 22g/l, and the PAL enzyme is 20.4 * 10 than work -3U/mg dry weight cell.
The B conversion reaction
The thalline that contains phenylalanine ammonia lyase that fermentation is obtained places conversion reaction liquid, 15The N-ammonium sulfate concentrations is 0.6mol/l, and naoh concentration is 1.2mol/l, and styracin concentration is 2.0%, and styracin is (g/g) to the mass ratio of wet thallus: 1: 3.6,28 ℃, the shaking table conversion reaction was 20 hours under the 160rpm.
The C separation and purification
The converted product mixture is through bactofugation, and mass percentage concentration is 4% dilute sulphuric acid accent pH to 4.0, and the mark nitrogenous source is converted into 15N-ammonium sulfate, and precipitation styracin and albumen, centrifugal disgorging is transferred pH to 6.0, separates through the HP20 macroporous adsorbent resin 15The NL-phenylalanine, 15N-ammonium sulfate, other inorganic salt and impurity, remove the nonpolar pigment of part, the deionized water wash-out, the elutriant mass percentage concentration is 4% dilute sulphuric acid accent pH to 5.5, concentrate through rotary evaporation, 70 ℃ add dehydrated alcohol and remove polarity pigment, crystallisation by cooling, filter, lyophilize obtains 2.30g (1L fermented liquid) 15NL-phenylalanine finished product, purity are greater than 99%, and abundance is greater than 98%, and yield is greater than 70%.
The D nitrogenous source reclaims
The ammonium salt mixing solutions is placed round-bottomed flask, and with excessive 35%NaOH solution rapid reaction under boiling state, it is in 10% the dilution heat of sulfuric acid, to use N that the ammonia that discharges is collected in mass percentage concentration 2Drive ammonia residual in the flask to dilution heat of sulfuric acid.In the process of collecting ammonia, drip dilute sulphuric acid and regulate the pH value, make ammonia and sulfuric acid complete reaction.Collection obtains 15The N-ammoniumsulphate soln obtains highly purified solid through evaporation, oven dry 15N-ammonium sulfate. 15The N-ammonium sulfate rate of recovery is 88.4%.
Embodiment 3:
The fermentation culture of A thalline
To contain 28 ℃ of solid culture of rhodotorula glutinis Rhodotorula glutinis2.102 bacterial strain 24 hours of phenylalanine ammonia lyase, the component of solid medium and content are: the agar of quality percentage composition 2.5%, and 12 ° of Be Fructus Hordei Germinatus soak powder, and all the other are water.Get one and encircle in seed culture medium, the component of seed culture medium and quality percentage composition are: glucose 2.0%, peptone 2.0%, yeast extract paste 0.5%, K 2HPO 40.2%, NaCl0.6%, all the other are water.25 ℃, shaking table was cultivated 24 hours under the 150rpm, moved to 10% inoculum size and produced in the enzyme substratum, and the component and the quality percentage composition that produce the enzyme substratum are: glucose 0.5%, and corn steep liquor 5.0%, soybean cake powder 3.0%, NaCl 0.6%, K 2HPO 40.2%, L-Phe 0.1%, pH5.5, and all the other are water.25 ℃, shaking table was cultivated 24 hours under the 150rpm, and is centrifugal and use the physiological saline washed twice, and the thalline that contains PAL of acquisition is used for conversion reaction.The thalline weight in wet base that obtains is 24g/l, and the PAL enzyme is 17.8 * 10 than work -3U/mg dry weight cell.
The B conversion reaction
The thalline that contains phenylalanine ammonia lyase that fermentation is obtained places conversion reaction liquid, 15The N-ammonium sulfate concentrations is 0.5mol/l, and naoh concentration is 1mol/l, and styracin concentration is 1.5%, and styracin is (g/g) to the mass ratio of wet thallus: 1: 4.0,30 ℃, the shaking table conversion reaction was 22 hours under the 150rpm.
The C separation and purification
The converted product mixture is through bactofugation, is that 4% dilute sulphuric acid is transferred pH to 4.0 with mass percentage concentration, and the mark nitrogenous source is converted into 15N-ammonium sulfate, and precipitation styracin and albumen, centrifugal disgorging is transferred pH to 6.5, separates through the HP20 macroporous adsorbent resin 15The NL-phenylalanine, 15N-ammonium sulfate, other inorganic salt and impurity, remove the nonpolar pigment of part, the deionized water wash-out, the elutriant mass percentage concentration is 4% dilute sulphuric acid accent pH to 5.5, concentrate through rotary evaporation, 70 ℃ add dehydrated alcohol and remove polarity pigment, crystallisation by cooling, filter, lyophilize obtains 2.05g (1L fermented liquid) 15NL-phenylalanine finished product, purity are greater than 99%, and abundance is greater than 98%, and yield is greater than 70%.
The D nitrogenous source reclaims
The ammonium salt mixing solutions is placed round-bottomed flask, with excessive mass percentage concentration be NaOH solution rapid reaction under boiling state of 25%, it is in 5% the dilution heat of sulfuric acid, to use N that the ammonia that discharges is collected in mass percentage concentration 2Drive ammonia residual in the flask to dilution heat of sulfuric acid.In the process of collecting ammonia, drip dilute sulphuric acid and regulate the pH value, make ammonia and sulfuric acid complete reaction.Collection obtains 15The N-ammoniumsulphate soln obtains highly purified solid through evaporation, oven dry 15N-ammonium sulfate. 15The N-ammonium sulfate rate of recovery is 88.1%.

Claims (5)

1. enzyme process preparation 15The method of NL-phenylalanine obtains to contain the thalline of phenylalanine ammonia lyase by fermenting, with 15N-ammonium sulfate is the mark nitrogenous source, with trans-cinnamic acid generation enzymatic reaction, obtains through separation and purification 15The NL-phenylalanine, unconverted mark nitrogenous source reclaims; Concrete steps and method are:
The fermentation culture of A thalline
The yeast strain that will contain phenylalanine ammonia lyase, cultivated 20-30 hour in the 25-30 ℃ of following solid medium, place seed culture medium then, cultivated 20-24 hour down for 25-30 ℃, move to and produce in the enzyme substratum, cultivated 21-24 hour down for 25-30 ℃, centrifugal and with the physiological saline washing, obtain to contain the thalline of phenylalanine ammonia lyase;
The B conversion reaction
The thalline that contains phenylalanine ammonia lyase that steps A is obtained places conversion reaction liquid, and it is 1 that the amount of adding makes trans-cinnamic acid and the mass ratio that contains the phenylalanine ammonia lyase thalline: 2.5-4.0, said conversion reaction liquid is for containing 15N-ammonium sulfate 0.5-0.6mol/l, sodium hydroxide 1.0-1.2mol/l, trans-cinnamic acid quality percentage composition are the solution of 1.0-2.0%, 25-30 ℃ following conversion reaction 20-24 hour;
The C separation and purification
Behind the converted product mixture bactofugation with step B acquisition, transfer pH to 3.0-4.0 with dilute sulphuric acid, the centrifugal precipitation of removing is transferred pH to 5.5-6.5 with sodium hydroxide solution again, separate through macroporous adsorbent resin, the deionized water wash-out, elutriant is transferred pH to 5.5 with dilute sulphuric acid, behind rotary evaporation, add dehydrated alcohol, crystallisation by cooling more after filtration, obtains the crystal lyophilize 15The NL-phenylalanine;
The D nitrogenous source reclaims
With containing after the separation of step C macroporous adsorbent resin 15The mixing solutions of N-ammonium sulfate, with the sodium hydroxide solution reaction, the ammonia of emitting is collected with sulphuric acid soln, obtains highly purified through evaporation and oven dry 15N-ammonium sulfate.
2. according to the method for claim 1, it is characterized in that: the said yeast strain that contains phenylalanine ammonia lyase is rhodotorula glutinis Rhodotorula glutinis 2.102 bacterial strains.
3. according to the method for claim 1 or 2, it is characterized in that: the component of solid medium and content are: the agar of quality percentage composition 1.5-2.5%, and 8-12 ° of Be Fructus Hordei Germinatus soaks powder, and all the other are water.
4. according to the method for claim 1 or 2, it is characterized in that: the component of seed culture medium and quality percentage composition are: glucose 0.5-2.0%, peptone 0.5-2.0%, yeast extract paste 0.5-2.0%, K 2HPO 40.05-0.2%, NaCl 0.4-0.6%, all the other are water.
5. according to the method for claim 1 or 2, it is characterized in that: the component and the quality percentage composition that produce the enzyme substratum are: glucose 0.5-2.0%, corn steep liquor 2.0-5.0%, soybean cake powder 1.0-3.0%, NaCl0.4-0.6%, K 2HPO 40.05-0.2%, L-phenylalanine 0.02-0.1% transfers pH to 5.5 with dilute hydrochloric acid, and all the other are water.
CN2007100645340A 2007-03-19 2007-03-19 Method for preparing <14>NL-phenylalanine with enzyme Expired - Fee Related CN101270377B (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105647835A (en) * 2014-08-08 2016-06-08 厦门大学 Method for preparing chiral phenylalanine by marine strain catalysis asymmetric reduction
CN106417194A (en) * 2016-12-09 2017-02-22 广州微因生物科技有限公司 N15 stable isotope labeling method for insect protein quantification and tracing

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1229332C (en) * 2003-04-30 2005-11-30 上海化工研究院 Extraction process of 15N-L-phenylalanine

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105647835A (en) * 2014-08-08 2016-06-08 厦门大学 Method for preparing chiral phenylalanine by marine strain catalysis asymmetric reduction
CN105647835B (en) * 2014-08-08 2019-01-08 厦门大学 A kind of method that marine bacteria strain catalytic asymmetric reduction prepares chiral phenylalanine
CN106417194A (en) * 2016-12-09 2017-02-22 广州微因生物科技有限公司 N15 stable isotope labeling method for insect protein quantification and tracing

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