CN108239664A - A kind of technique for preparing 4- hydroxy-L-threonines - Google Patents

A kind of technique for preparing 4- hydroxy-L-threonines Download PDF

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CN108239664A
CN108239664A CN201810060525.2A CN201810060525A CN108239664A CN 108239664 A CN108239664 A CN 108239664A CN 201810060525 A CN201810060525 A CN 201810060525A CN 108239664 A CN108239664 A CN 108239664A
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ala
leu
threonine
asp
val
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CN108239664B (en
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黄华
王俊梅
张新帅
何遂平
刘建
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Gansu ruidilin biological Co.,Ltd.
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Shenzhen Reed Forest Biotechnology Co Ltd
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Abstract

The present invention relates to technical field of biochemistry, disclose the technique that a kind of immobilised enzymes series process prepares 4 hydroxyl L threonines.The present invention couples L threonine aldolases to complete conversion using L threonine kinases, using Adk and PaP or its fusion protease and the in due course circular regeneration ATP of cheap polyphosphoric acids, reaction process is simple and convenient to operate, product quality is stablized, it is preferable to realize the continuous conversion of raw material and the regeneration of coacetylase TP, coordinate stable immobilised enzymes, it can recycle repeatedly, so as to which production cost greatly reduce.Present invention process effectively avoids the complicated processes such as multistep radical protection in chemical synthesising technology, chiral resolution; the influence that the impurity such as metabolin caused by microbial fermentation, albumen purify later product is also avoided simultaneously, is very suitable for industrialization large-scale production.

Description

A kind of technique for preparing 4- hydroxy-L-threonines
Technical field
The present invention relates to technical field of biochemistry, and in particular to a kind of technique for preparing 4- hydroxy-L-threonines.
Background technology
4- hydroxy-L-threonines are a kind of hydroxylated amino acid, molecular formula C4H9NO4, molecular weight 135.L- Soviet Unions ammonia Acid is a kind of essential amino acid, is mainly used for medicine, chemical reagent, food additive, feed addictive etc..And hydroxyl The threonine of change is few in nature, is primarily present in pseudomonad (pseudomonas) organism.Many document report hydroxyls The amino acid of base can also be widely used as food, feed addictive, treat the active ingredient and some chemistry of diabetes The starting material of industrial goods；Therefore cheap, the such compound of batch production is laid a solid foundation for its popularization and application.
The synthesis of 4- hydroxy-L-threonines mainly has chemical synthesis, oxidation Enzyme optrode, biological fermentation process etc., industry Upper common chemical synthesis is using glyceraldehyde or D-mannital by multistep protection, functionalization and chiral resolution system Standby, synthesis technology is cumbersome, ultimate yield is low, environmental pollution is big (chemical route I, II)；A small amount of document report utilizes specific oxidation Enzyme direct hydroxylating L-threonine generation 4- hydroxy-L-threonines, but this method due to oxidizing ferment self structure it is unstable, Activated centre iron (FeII) circular regeneration complexity and high product concentration to the inhibiting effect of enzymatic activity the defects of and it is serious Restrict its amplification application；Indivedual documents are also reported is metabolized Tianmen using Pseudomonas aeruginosa (pseudomonas andropogonis) Winter propylhomoserin produces 4- hydroxy-L-threonines, but this method is also limited only to theoretical research since yield is too low, and strain is System improvement optimization will be taken a substantial amount of time and resource (chemical route III) with reaching industrial needs.
Invention content
In view of this, the purpose of the present invention is to provide a kind of techniques for preparing 4- hydroxy-L-threonines so that the work Skill using multienzyme is continuous, concerted catalysis with cheap raw material in extremely short route in high yield be converted into specific product 4- hydroxyls Base-L-threonine, while realize the continuous conversion of raw material and the regeneration of coacetylase TP, avoid the multistep in chemical synthesising technology The complicated processes such as radical protection, chiral resolution.
To achieve the above object, the present invention provides following technical solution：
A kind of technique for preparing 4- hydroxy-L-threonines, including：
Step 1, glycollic aldehyde and glycine generate 4- hydroxy-L-threonines under the catalysis of L-threonine aldolase；
4- hydroxy-L-threonines in step 2, step 1 ATP, threonine kinase catalysis under generate 4- hydroxyl phosphorus Acid-L-threonine and ADP；
ADP in step 3, step 2 is in the catalysis of polyphosphoric acids, adenosine acid kinase and polyphosphoric acids-AMP phosphotransferases Lower generation ATP, re-enters into step 2；4- dis-L-threonine in step 2 and sodium salt generation 4- dis- Then L-threonine sodium generates 4- hydroxy-L-threonines under the catalysis of pyrophosphohydrolase.
For asking for the universal very complicated of existing 4- hydroxy-L-threonines preparation process and industrial applications inefficiency Topic, the present invention are total to using L-threonine aldolase, threonine kinase, adenosine acid kinase and polyphosphoric acids-AMP phosphotransferases With preparation 4- hydroxy-L-threonines, and realize the continuous conversion of raw material and the regeneration of coacetylase TP.
The reaction route figure of technique of the present invention is shown in Fig. 1, the present invention using L-threonine aldolase (EC4.1.2.5, TA glycollic aldehyde and glycine condensation generation 4- hydroxy-L-threonines and its non-corresponding isomers can) be catalyzed；It is caused using threonine Enzyme (EC 2.7.1.-, TK) living can selective phosphorylation 4- hydroxy-L-threonines to 4- dis-L-threonine, effectively Back condensation reaction is dragged to complete.At the same time, using adenosine acid kinase (EC 2.7.4.3, Adk) and polyphosphoric acids- AMP phosphotransferases (EC 2.7.4.-, Pap) can efficiently utilize cheap polyphosphoric acids circular regeneration in the reaction system Adenosine triphosphate atp, this both can effectively control the dosage of ATP in reaction system and reduce high concentration adenosine diphosphate (ADP) ADP is to the inhibiting effect of enzyme catalyst, so as to improve yield, reduce cost；Finally utilize pyrophosphohydrolase (EC 3.6.1.1, IPH) processing 4- dis-L-threonine, final purified product 4- dis-L-threonine.
In order to be conducive to the progress of reaction, in step 1, L- is better achieved by adding in pyridoxal phosphate in the present invention The catalysis reaction of threonine aldolase, i.e. glycollic aldehyde and glycine are urged L-threonine aldolase and pyridoxal phosphate Change lower generation 4- hydroxy-L-threonines；Pyridoxal phosphate is preferably pyridoxal phosphate sodium.
Similary preferably to be carried out based on the considerations of promoting to react, step 2 is：In step 1 4- hydroxy-L-threonines ATP, Threonine kinase, Mg2+And K+Catalysis under generation 4- dis-L-threonine and ADP.Wherein, the Mg2+And K+By energy The free Mg of enough release2+And K+Inorganic salts provide, such as magnesium chloride, potassium chloride, magnesium nitrate, potassium nitrate, magnesium sulfate, potassium sulfate Etc..
Preferably, in step 3 of the present invention, 4- dis-L-threonine is changed into 4- dis-L-threonine Sodium, then the process through enzymolysis generation 4- hydroxy-L-threonines, can carry out as follows：
4- dis-L-threonine in step 2 with barium salt is precipitated, Tris- hydrochloric acid is then redissolved in and delays Fliud flushing adds in inorganic sodium reaction, is filtered to remove barium salt precipitation, obtains 4- dis-L-threonine sodium；4- dis- L-threonine sodium generates 4- hydroxy-L-threonines under the catalysis of pyrophosphohydrolase.In this step, it can not only realize anti- Process is answered, but also the purification process to 4- dis-L-threonine can be increased while reaction, makes entirely to react more Add high-efficient simple, also allow for subsequent purification process.
Wherein, the 4- dis-L-threonine in a manner that barium salt precipitates for can by adding in barium oxalate etc. Dissolubility barium salt forms 4- dis-L-threonine precipitated barium, and then addition can react to form the inorganic of precipitation with barium again Sodium salt, generation 4- dis-L-threonine sodium.The inorganic sodium to form precipitation can be reacted with barium by, which adding, to be led according to chemistry Conventional barium salt precipitation, such as BaSO in domain4、BaCO3、BaSO3、BaSiO3、Ba3(PO4)2、BaF2, corresponding selection Na2SO4、 Na3PO4、Na2CO3、Na2SO3、Na2SiO3、NaF。
In the specific embodiment of the invention, the present invention is for adenosine acid kinase (EC 2.7.4.3, Adk) and poly phosphorus Acid-AMP phosphotransferases (EC 2.7.4.-, Pap) are formed ATP regeneration fusion enzyme Adk-PaP, that is, are passed through using fusion means Then the gene assembling of two enzymes gives expression to one in cell and includes the fusion enzyme of two enzymes, while have the work(of Adk and PaP Energy.
In present invention process, all reactions preferably use Tris- hydrochloride buffers, and for reaction dissolvent, pH value in reaction is 6.0-8.5.Wherein, Tris- hydrochloride buffers preferably use the Tris- hydrochloride buffers of 25mM, pH7.0-7.5.
In present invention process, the polyphosphoric acids is preferably using 25 poly-, 0.1M phosphoric acid polyphosphoric acids.
Technology based on immobilised enzymes, the invention also provides by the L-threonine aldolase, threonine kinase, gland Thuja acid kinases be fixed on polyphosphoric acids-AMP phosphotransferases (including merging enzyme Adk-PaP) on carrier formed immobilised enzymes into Row reaction.After immobilised enzymes, technique of the present invention can carry out recycling re-using to the immobilised enzymes for participating in reaction, again The immobilised enzymes enzyme activity used still keeps higher level.
In addition, carrying out the preparation of 4- hydroxy-L-threonines according to technique of the present invention, it can finally reach nearly 80% In high yield, it is and also relatively simple in purifying.
By above technical scheme it is found that the present invention couples L-threonine aldolase to turning completely using L-threonine kinase Change, using Adk and PaP or its fusion protease and the in due course circular regeneration ATP of cheap polyphosphoric acids, reaction process is simple, behaviour Make convenient, product quality to stablize, preferably realize the continuous conversion of raw material and the regeneration of coacetylase TP, coordinate stable consolidate Surely change enzyme, can recycle repeatedly, so as to which production cost greatly reduce.Present invention process effectively avoids chemical synthesis The complicated processes such as multistep radical protection, chiral resolution in technology, at the same also avoid metabolin caused by microbial fermentation, The influence that the impurity such as albumen purify later product is very suitable for industrialization large-scale production.
Description of the drawings
Fig. 1 show the reaction route figure of technique of the present invention；
Fig. 2 show the nucleus magnetic hydrogen spectrum of identification 4- dis-L-threonine1H-NMR；
Fig. 3 show the nuclear-magnetism phosphorus spectrum of identification 4- dis-L-threonine31P-NMR；
Fig. 4 show the nucleus magnetic hydrogen spectrum of identification 4- hydroxy-L-threonines1H-NMR；
Fig. 5 show the nuclear-magnetism carbon spectrum of identification 4- hydroxy-L-threonines31C-NMR。
Specific embodiment
The invention discloses a kind of technique for preparing 4- hydroxy-L-threonines, those skilled in the art can use for reference herein Content is suitably modified technological parameter realization.In particular, it should be pointed out that all similar substitutions and modifications are to people in the art It is it will be apparent that they are considered as being included in the present invention for member.The present invention technique by preferred embodiment into Description is gone, related personnel can significantly not depart from the content of present invention, process as described herein changed in spirit and scope It is dynamic or suitably change with combining, to realize and using the technology of the present invention.
The step of technique of the present invention, is intended to clearly describe the reaction route of core, is not intended to limit entire reaction and uses One-step method or multistep processes carry out, such as the generation ATP parts of the step 1 of technique of the present invention and step 2 and step 3, Disposably all reaction raw materials can be put into and carried out.
In a specific embodiment, the present invention is reacted using fusion enzyme Adk-PaP.Used L-threonine aldehyde Contracting enzyme, threonine kinase, adenosine acid kinase, polyphosphoric acids-AMP phosphotransferases, pyrophosphohydrolase have that its is corresponding EC is numbered, and specific amino acid sequence is successively such as SEQ ID NO:Shown in 1-5, the sequence of fusion enzyme Adk-PaP is passes through connection The SEQ ID NO of small peptide connection:3-4 sequences, i.e. SEQ ID NO:3 sequences+connection small peptide+SEQ ID NO:4 sequences,；At this In invention specific embodiment, the connection short peptide sequence such as SEQ ID NO:Shown in 6, while it can also be used in fusion enzyme field Other common connection small peptides.
Each enzyme of the present invention can be artificial synthesized according to sequence progress, can also pass through respective encoding gene structure It builds recombinant plasmid and carries out cell transformation, such as：
Copper is coveted with Escherichia coli (Escherichia coli) BL21 bacterial strains (the being purchased from general biology) DNA of extraction and hookworm Bacterium (Cupriavidus necator ATCC-17699D) chromosome (being purchased from ATCC companies) is template, is gone out by PCR amplification Then TA, TK and IPH genetic fragment are connected to pET28a matter by corresponding digestion (such as BamhI and XhoI, NdeI and XhoI) (biological wind is purchased from grain), and wherein ATP regeneration enzymes Adk-PaP encoding genes are directly bought and are subcloned from general biotech firm On pET28a plasmids.Four sections of gene orders and then E.coli BL21 (DE3) bacterial strain (general biology) is transferred to, confirms correct bacterium It falls in culture to the LB culture solutions of the kalamycin containing 50uM；0.2mM isopropyl-β-D- sulphur is added in after cell rises to logarithmic phase It is expressed 4 hours for galactopyranoside (IPTG) inducible protein, last and then collection, smudge cells, high speed centrifugation clear liquid is ten Dialkyl sulfonates-polyacrylamide gel electrophoresis (SDS-PAGE) confirm protein expression.Confirm the seed culture of protein expression Base, which can also be linked into 10L cultivation and fermentation tanks, to be grown to after logarithmic phase with 0.5mM IPTG induced expressions 6 hours, is collected wet thin Born of the same parents 200g.LB culture mediums are configured to：1% tryptone, 0.5% dusty yeast, 1%NaCl, 1% dipotassium hydrogen phosphate, 1% phosphoric acid hydrogen Dipotassium and 5% glycerine.
Aldolase (TA), kinase (TK), pyrophosphohydrolase (IPH) clasmatosis clear liquid are first gradually added 60% saturation Ammonium sulfate precipitation be precipitated, then solid is slowly dissolve into the Tris buffer solutions of 25mM pH8.0, through G25 size exclusion columns Desalination (being purchased from Sigma) recycles DEAE Seplite FF (Xi'an Lan Xiao companies) anion-exchange column isolated just pure afterwards Change TA, TK, IPH enzyme.
The sodium radio-phosphate,P-32 solution that 100ml0.5M pH 7.0 are added in enzyme Adk-PaP clasmatosis clear liquids is merged in 500ml, so After be gradually added dimethoxy-ethane until albumen precipitation is complete, obtain fusion enzyme Adk-PaP after purification.
Each enzyme of the present invention can refer to the immobilised enzymes preparation method of this field routine, in the present invention when immobilizing In specific embodiment, the present invention carries out as follows：
1st, aldolase (TA), kinase (TK), pyrophosphohydrolase (IPH)
1000U purifying enzyme be dissolved in the potassium phosphate solution of 1L 100mM pH 8.0, then add in 60mM phenoxy acetic acids with And 200 grams of LX-1000EP epoxy resin filter out immobilised enzymes to buffer solution after being stirred at room temperature 24 hours, finally with clear water with And respectively to wash twice rear low temperature drying for use for 8.0 phosphate buffers of 100mM pH.Immobilization aldolase (TA), kinase (TK), Pyrophosphohydrolase (IPH) has the activity of liquid enzymes 20-50%.
2nd, fusion enzyme Adk-PaP
Fusion enzyme Adk-PaP after purification adds in 80ml25% glutaraldehyde water solutions, and mixed solution is in 4 DEG C of gentle agitations 16 Adk-PaP enzymes precipitation linked solid CLEAs is made in hour filtering；To improve cross-linking enzyme mechanical strength, tetra- methoxy silane first of 500ml Alcoholic solution instills the CLEAs phosphoric acid solutions of 500ml stirrings until clarification, room temperature filter immobilization Adk-PaP after static 6 hours Enzyme.Immobilization Adk-PaP enzymes have the activity of liquid enzymes 65%.
According to the reaction route of present invention process, the dosage of each reactive material can be adjusted according to actual conditions, in order to most Big efficient activity, the present invention provides the molar ratios of following each reactive material：
Glycollic aldehyde:Glycine:Pyridoxal phosphate:Atriphos:Polyphosphoric acids:Aldolase (TA, can immobilization): Threonine kinase (TK, can immobilization):ATP circular regenerations enzyme (Adk-PaP, can immobilization)=(100000-1000000): (100000-1000000):(5,000-20,000):(10,000-100,000):(100000-1000000):(6-60):(8- 80):(10-100)；
4- dis-L-threonine sodium:Pyrophosphohydrolase (can immobilization)=(100000-1000000):(5- 200)；
Wherein, the enzyme activity of each enzyme preferably uses the enzyme of 1000-50000U, in order to balance efficiency and and cost, can be into One step preferably using 1000-20000U, further uses 1000-10000U or 1000-5000U；In a specific embodiment, Immobilization aldolase (TA) of the present invention is 2000U, and immobilization threonine kinase (TK) is 2500U, immobilization fusion enzyme Adk- PaP is 3000U, and immobilization pyrophosphohydrolase is 1000U.
With reference to embodiment, the present invention is further explained.
Embodiment 1：Technique of the present invention
30 grams of hydroxyls are successively added in trishydroxymethylaminomethane hydrochloric acid (Tris.HCl) solution of 5L 25mM pH7.5 Acetaldehyde (0.1M), 37.5 grams of glycine (0.1M), 670 milligrams of pyridoxal phosphate sodium (0.5mM), 2.5 grams of adenosine triphosphate atps (1mM), 51 grams of polyphosphoric acids (Sigma, 25 is poly-, 0.1M phosphoric acid), 23.5 grams of magnesium chlorides (50mM), 9.3 grams of potassium chloride (25mM)； After pH value is adjusted to 7.5,2000U immobilizations aldolase (TA), 2500U immobilization 4- hydroxy-L-threonines kinases (TK) And 3000U immobilization ATP circular regenerations enzymes (Adk-PaP) are sequentially added reaction system and start to react, it is small that reaction is stirred at room temperature 6 When after solution is taken quantitatively to detect residual hydroxy groups acetaldehyde with naphthoresorcinol method, the results showed that the reaction was complete, and (active unit U represents every 1 μM of required enzyme amount of substrate of minute conversion).Product is identified using nuclear magnetic resonance, as a result sees Fig. 2 and Fig. 3, nuclear-magnetism knot Fruit shows that target product is 4- dis-L-threonine.
Filtering reacting liquid recycles immobilised enzymes, and 127.5 grams of barium oxalate (0.5mol) precipitation 4- hydroxyls are added in filtrate Base phosphoric acid-L-threonine and other phosphoric acid impurity.The trishydroxymethylaminomethane that precipitation solid is then dissolved in pH1.0 delays It rushes in solution, adds in 71 grams of anhydrous sodium sulfates (0.5mol) and insoluble barium sulfate is precipitated；Solid is filtered to remove, by filtered fluid pH tune Loading D201 anion resins exchange column (brilliant auspicious chemical industry) separation, purifying is eluted to obtain with gradient ammonium hydrogencarbonate aqueous solution after to 7.0 4- dis-L-threonine sodium.Last G25 size exclusion column desalinations obtain 99 grams of 4- dis-L-threonine sodium white Powder, yield is up to 84%.Immobilised enzymes retains 60% original activity after recycling 12 times.
50 grams of 4- dis-L-threonine sodium are dissolved in 1L 25mM pH7.0 trishydroxymethylaminomethane hydrochloric acid to delay Fliud flushing then adds in 1.9 grams of magnesium chlorides, the stirring of 1000U immobilization pyrophosphohydrolases mixed at room temperature three hours, is recovered by filtration solid Surely change enzyme, filtrate loading D201 anion resin exchange columns, product is that 4- hydroxy-L-threonines are loading efflux.Product is dense 26 grams of white foam solids, yield 78% are obtained after contracting desalination, nuclear-magnetism qualification result is shown in Fig. 4 and Fig. 5.Immobilization pyrophosphoric acid water It solves enzyme and recycles 10 activity reservations 80%.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should It is considered as protection scope of the present invention.
Sequence table
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Ile Ala Leu Val Lys Glu Arg Ile Ala Gln Glu Asp Cys Arg Asn Gly
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Glu Glu Thr Val Arg Lys Arg Leu Val Glu Tyr His Gln Met Thr Ala
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Arg Phe Leu Tyr Val Lys Ala Asp Pro Pro His Leu Phe Asn Leu Lys
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Met His Ala Ser Lys Pro Leu Asp Asp Thr Leu Phe Asp Glu Tyr Val
115 120 125
Ser Asn Met Arg Ala Phe Glu Gln Asp Leu Lys Asn Asn Asn Val Asp
130 135 140
Val Leu Lys Val Trp Phe Asp Leu Ser Trp Lys Ser Leu Gln Lys Arg
145 150 155 160
Leu Asp Asp Met Asp Pro Ser Glu Val His Trp His Lys Leu His Gly
165 170 175
Leu Asp Trp Arg Asn Lys Lys Gln Tyr Asp Thr Leu Gln Lys Leu Arg
180 185 190
Thr Arg Phe Thr Asp Asp Trp Gln Ile Ile Asp Gly Glu Asp Glu Asp
195 200 205
Leu Arg Asn His Asn Phe Ala Gln Ala Ile Leu Thr Ala Leu Arg His
210 215 220
Cys Pro Glu His Glu Lys Lys Ala Ala Leu Lys Trp Gln Gln Ala Pro
225 230 235 240
Ile Pro Asp Ile Leu Thr Gln Phe Glu Val Pro Gln Ala Glu Asp Ala
245 250 255
Asn Tyr Lys Ser Glu Leu Lys Lys Leu Thr Lys Gln Val Ala Asp Ala
260 265 270
Met Arg Cys Asp Asp Arg Lys Val Val Ile Ala Phe Glu Gly Met Asp
275 280 285
Ala Ala Gly Lys Gly Gly Ala Ile Lys Arg Ile Val Lys Lys Leu Asp
290 295 300
Pro Arg Glu Tyr Glu Ile His Thr Ile Ala Ala Pro Glu Lys Tyr Glu
305 310 315 320
Leu Arg Arg Pro Tyr Leu Trp Arg Phe Trp Ser Lys Leu Gln Ser Asp
325 330 335
Asp Ile Thr Ile Phe Asp Arg Thr Trp Tyr Gly Arg Val Leu Val Glu
340 345 350
Arg Val Glu Gly Phe Ala Thr Glu Val Glu Trp Gln Arg Ala Tyr Ala
355 360 365
Glu Ile Asn Arg Phe Glu Lys Asn Leu Ser Ser Ser Gln Thr Val Leu
370 375 380
Ile Lys Phe Trp Leu Ala Ile Asp Lys Asp Glu Gln Ala Ala Arg Phe
385 390 395 400
Lys Ala Arg Glu Ser Thr Pro His Lys Arg Phe Lys Ile Thr Glu Glu
405 410 415
Asp Trp Arg Asn Arg Asp Lys Trp Asp Asp Tyr Leu Lys Ala Ala Ala
420 425 430
Asp Met Phe Ala His Thr Asp Thr Ser Tyr Ala Pro Trp Tyr Ile Ile
435 440 445
Ser Thr Asn Asp Lys Gln Gln Ala Arg Ile Glu Val Leu Arg Ala Ile
450 455 460
Leu Lys Gln Leu Lys Ala Asp Arg Asp Thr Asp
465 470 475
<210> 5
<211> 176
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 5
Met Ser Leu Leu Asn Val Pro Ala Gly Lys Glu Leu Pro Glu Asp Ile
1 5 10 15
Tyr Val Val Ile Glu Ile Pro Ala Asn Ala Asp Pro Ile Lys Tyr Glu
20 25 30
Ile Asp Lys Asp Thr Gly Ala Leu Phe Val Asp Arg Phe Met Ser Thr
35 40 45
Ala Met Phe Tyr Pro Cys Asn Tyr Gly Tyr Ile Asn His Thr Leu Ser
50 55 60
Leu Asp Gly Asp Pro Val Asp Val Leu Val Pro Thr Pro Tyr Pro Leu
65 70 75 80
Gln Pro Gly Ser Val Ile Arg Cys Arg Pro Val Gly Val Leu Lys Met
85 90 95
Thr Asp Glu Ala Gly Glu Asp Ala Lys Leu Val Ala Val Pro His Thr
100 105 110
Lys Leu Ser Lys Glu Tyr Asp His Ile Lys Asp Val Asn Asp Leu Pro
115 120 125
Glu Leu Leu Lys Ala Gln Ile Ala His Phe Phe Glu His Tyr Lys Asp
130 135 140
Leu Glu Lys Gly Lys Trp Val Lys Val Glu Gly Trp Asp Asn Ala Glu
145 150 155 160
Ala Ala Lys Ala Glu Ile Ile Ala Ser Phe Glu Arg Ala Ala Lys Lys
165 170 175
<210> 6
<211> 9
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 6
Ser Ser Gly Leu Val Pro Arg Gly Ser
1 5

Claims (8)

1. a kind of technique for preparing 4- hydroxy-L-threonines, which is characterized in that including：
Step 1, glycollic aldehyde and glycine generate 4- hydroxy-L-threonines under the catalysis of L-threonine aldolase；
4- hydroxy-L-threonines in step 2, step 1 ATP, threonine kinase catalysis under generation 4- dis-L- Threonine and ADP；
ADP in step 3, step 2 is raw under the catalysis of polyphosphoric acids, adenosine acid kinase with polyphosphoric acids-AMP phosphotransferases Into ATP, re-enter into step 2；4- dis-L-threonine in step 2 is revived with sodium salt generation 4- dis-L- Then propylhomoserin sodium generates 4- hydroxy-L-threonines under the catalysis of pyrophosphohydrolase.
2. technique according to claim 1, which is characterized in that step 1 is：
Glycollic aldehyde generates 4- hydroxyls-L- Soviet Unions ammonia with glycine under the catalysis of L-threonine aldolase and pyridoxal phosphate Acid.
3. technique according to claim 1, the step 1-3 is using Tris- hydrochloride buffers as reaction dissolvent.
4. technique according to claim 1, the step 1-3 reacts under pH value 6.0-8.5.
5. technique according to claim 1, which is characterized in that step 2 is：
4- hydroxy-L-threonines are in ATP, threonine kinase, Mg in step 12+And K+Catalysis under generation 4- dis-L- Threonine and ADP.
6. technique according to claim 5, which is characterized in that the Mg2+And K+It is provided by inorganic salts.
7. technique according to claim 1, which is characterized in that step 3 is：
ADP generates ATP under the catalysis of polyphosphoric acids, adenosine acid kinase and polyphosphoric acids-AMP phosphotransferases in step 2, again It enters in step 2；
4- dis-L-threonine in step 2 with barium salt is precipitated, is then redissolved in Tris- hydrochloride buffers, Inorganic sodium reaction is added in, barium salt precipitation is filtered to remove, obtains 4- dis-L-threonine sodium；4- dis-L- Soviet Unions ammonia Sour sodium generates 4- hydroxy-L-threonines under the catalysis of pyrophosphohydrolase.
8. technique according to claim 1, which is characterized in that the L-threonine aldolase, threonine kinase, adenylate Kinases is immobilised enzymes with polyphosphoric acids-AMP phosphotransferases.
CN201810060525.2A 2018-01-22 2018-01-22 Process for preparing 4-hydroxy-L-threonine Active CN108239664B (en)

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