CN109251948B

CN109251948B - The method that immobilized enzyme catalysis method prepares D- erythrulose

Info

Publication number: CN109251948B
Application number: CN201811054510.1A
Authority: CN
Inventors: 黄华; 高睿迪
Original assignee: South China Normal University
Current assignee: South China Normal University
Priority date: 2018-09-11
Filing date: 2018-09-11
Publication date: 2019-10-08
Anticipated expiration: 2038-09-11
Also published as: CN109251948A

Abstract

The invention discloses a kind of methods for preparing D- erythrulose using immobilised enzymes continuous catalysis erythritol, method includes the following steps: amplification EDH, EuK, LDH, PPK, AP gene, connection plasmid is simultaneously transferred to cell, clasmatosis centrifuge separation is obtained into 5 kinds of enzymes after purification after inducible protein expression, by enzyme immobilization；Corresponding enzymatic hydrolysis substrate is taken, immobilization mixed enzyme is added, reaction generates D- erythrulose -4- phosphoric acid, obtains D- erythrulose -4- sodium phosphate after purification；The substrate is reacted with immobilization phosphohydrolase AP, finally obtained D- erythrulose.The method of the present invention utilizes the successive reaction of butanol carbohydrate oxidase and hydroxypropanone- kinases, realizes the disposable complete conversion of cheap erythritol to D- erythrulose -4- phosphoric acid, and then easily can generate D- erythrulose using phosphohydrolase dephosphorylation.Reaction will not generate the product of similar structures, and purity and yield are all guaranteed.

Description

The method that immobilized enzyme catalysis method prepares D- erythrulose

Technical field

The present invention relates to the preparation methods of D- erythrulose, and in particular to a kind of to utilize the red algae sugar of immobilised enzymes continuous catalysis The method that alcohol prepares D- erythrulose.

Background technique

D- erythrulose (D-erythrulose) is a kind of four carbon ketoses, molecular formula C₄H₈O₄, molecular weight 120 is It is present in a kind of inner sugar in yellow to red of red berry (being common in rasp berry), since the sugar can be with epidermis dead cell film surface egg Casamino acid effect generates brown color (brownish tint), thus is widely used and makees safe and nontoxic skin dyestuff.

D- erythrulose, which is often mixed with dihydroxyacetone (DHA) (Dihydroxyacetone), is applied to pigmenting of skin, and product is usual It is reconciled with DHA/E and dyes mark (such as: Tanfastic, Veneto product).Erythrulose tone and dihydroxyacetone (DHA) slightly have area Not, skin color can be made redder, more enthusiastic.However its expensive price seriously restricts always the application (reference of its further genralrlization Price list), thus develop it is a kind of convenient, can largely prepare the method for erythrulose and become extremely important to reduce its cost.

Various related compound price lists:

Chemical combination name	CAS number	Company	Price ($)
				D- threose (D-threose)	95-43-2	Carbosynth	350/ gram
L- threose (L-threose)	95-44-3	Carbosynth	1000/ gram
				D- erythrose (D-erythrose)	583-50-6	Carbosynth	55/ gram
L- erythrose (L-erythrose)	533-49-3	Carbosynth	420/ gram
				Erythritol (meso-erythritol)	149-32-6	Carbosynth	95/ kilogram
D- threitol (D-threitol)	2418-52-2	Carbosynth	450/ gram
				L- threitol (L-threitol)	2319-57-5	Carbosynth	200/ gram
L- erythrulose (L-erythrulose)	533-50-6	Carbosynth	1200/ kilogram
				D- erythrulose (D-erythrulose)	496-55-9	Carbosynth	2600/ gram

D- erythrulose customary preparation methods have partition method and chemical synthesis.Due to D- erythrulose yield in nature The very low and compound polarity is big, high water solubility, isolate and purify it is very troublesome, so production can not be amplified.

Chemical preparation method mainly includes following two scheme, first is that utilizing sugared (erythritol) the non-selective oxygen of its cheap alcohol Change, then isomerization is isolated and purified；But since there are many similar compound (such as threoses, erythrose and L- erythrulose) It generates, yield and to isolate and purify efficiency all very low.Another is sugared (such as ribose, glucose) using cheap pentasaccharides/six It degraded, purified step by step, efficiency and purity are also very poor.

Summary of the invention

In order to overcome chemical method to prepare the technological deficiency of D- erythrulose, the purpose of the present invention is to provide a kind of utilizations The method that immobilised enzymes continuous catalysis erythritol prepares D- erythrulose.

The purpose of the invention is achieved by the following technical solution:

A method of D- erythrulose is prepared using immobilised enzymes continuous catalysis erythritol, comprising the following steps:

(1) PCR amplification EDH (erythritol oxidizing ferment), EuK (erythrulose kinases), LDH (lactic dehydrogenase), PPK (ATP regeneration enzyme), AP (phosphohydrolase) genetic fragment, is then attached on plasmid；Plasmid is transferred in cell again；

The PCR amplification is with Escherichia coli (Escherichia coli), smegma bacillus (Mycobacterium smegmatis) or the chromosome of rhizobium melioti (Rhizobium meliloti) are template；

The PCR amplification, primer used is as shown in SEQ.ID.NO.1-NO.10；

The preferred pET23a of the plasmid, the enzymatic activity of the plasmid expression are higher；

The preferred e. coli bl21 of the cell (DE3) bacterial strain；

(2) cell for being transferred to plasmid is placed in 37 DEG C, trained in the LB liquid medium containing 100 μM of ampicillins It supports, after cell rises to logarithmic phase (OD 0.5-0.6), isopropyl-beta D-thio galactopyranoside (IPTG) induction is added Protein expression 4 hours or more, collect cell；

The preferred 0.4-0.5mM of the final concentration of the IPTG；

(3) clasmatosis, the centrifugation collected step (2), are added ammonium sulfate to final concentration of 30- by amount in supernatant 50% (W/V), until there is protein solid precipitation；Albumen is collected by centrifugation again, is dissolved into the Tris buffer of pH value 8.0, then Through G25 size exclusion column desalination, through DEAE Seplite FF anion exchange post separation, the EDH, EuK purified, LDH, PPK, AP liquid enzymes；

The DEAE Seplite FF anion exchange post separation, is to do eluent, concentration with gradient NaCl aqueous solution It is gradually promoted from 0 to 1.0M；

(4) the purifying mixed enzyme of EDH, EuK, LDH and PPK are dissolved in buffer, epoxy resin, room temperature is then added Stirring 24 hours or more, enzyme was fixed on epoxy resin, filters out epoxy resin, is washed with clear water and buffer, finally at 4 DEG C It is dry；AP is individually fixed according to same step；

In step (4), every 1000U purifying mixed enzyme is preferably added to 200 grams of epoxy resin；

The potassium phosphate solution of the preferred 100mM pH value 6.5-9.0 of the buffer；

In the purifying mixed enzyme, active unit's ratio of EDH, EuK, LDH and PPK are 1:(2-3): 1:(2-3)；

The preferred LX-1000EP epoxy resin of the epoxy resin (production of Xi'an Lan Xiao company)；

(5) erythritol, Sodium Pyruvate, polyphosphoric acids, adenosine disodium triphosphate ATP, nicotinoyl are added in buffer Amine adenine-dinucleotide mono-sodium salt, magnesium chloride and potassium chloride adjust pH value to 8.0, consolidating for EDH, EuK, LDH and PPK are added Surely change mixed enzyme, reacted 4 hours in 25-40 DEG C of gentle agitation or more, generate D- erythrulose -4- phosphoric acid；Magnesium chloride and chlorination Potassium is that EuK is reacted with ATP, and PPK reacts needs with polyphosphoric acids, they can be in conjunction with activation ATP and polyphosphoric acids.

Filtering reacting liquid recycles epoxy resin and its immobilised enzymes, and barium oxalate and twice filtrate volume are added in filtrate Ethyl alcohol, for precipitating D- erythrulose -4- phosphoric acid and adenosine phosphate (ADP, ATP)；Precipitating is collected by centrifugation, is dissolved in pH value and is less than In 7 Tris buffer, anhydrous sodium sulfate is added, insoluble barium sulfate is precipitated；It is filtered to remove insoluble barium salt, and by filtrate pH Be transferred to 7.0 in value, loading anion-exchange resin column, and with the elution of gradient ammonium hydrogencarbonate aqueous solution, ammonium hydrogencarbonate concentration from 0 by It edging up to 1.0M, the organic impurities of not phosphorous acid groups is eluted out from exchange column at first, later, the D- of the group containing monophosphate Erythrulose -4- phosphoric acid gradually is eluted out, and the ADP/ATP containing multiple phosphate groups is finally eluted；The D- being collected into D- erythrulose -4- the sodium phosphate that erythrulose -4- phosphoric acid is most purified through G25 size exclusion column desalination afterwards；

G25 size exclusion column desalination, is to do eluent with deionized water described in step (2) and (5)；

The Tris buffer, pH value preferably 1.0；

In this step, when pH value is 7.0, the smoke suppressing effect of phosphoric acid is insoluble, D- erythrulose -4- barium phosphate Insoluble, precipitating at this time is exactly the barium salt of product and the barium salt of ATP/ADP.But the barium salt can dissolve in acid condition, D- erythrulose -4- barium phosphate is dissolved under acidity, the Na of equivalent is then added₂SO₄, can generate insoluble under acidity BaSO₄, barium salt effectively can be substituted for by sodium salt by this method.

(6) D- erythrulose -4- sodium phosphate, magnesium chloride are dissolved in the Tris.HCl solution of pH value 7.0, are then added Immobilization phosphohydrolase AP, reacted 2 hours in 25-40 DEG C of gentle agitation or more, it is then separated by filtration epoxy resin and its consolidates Surely change enzyme；Filtrate chromatographs by anion-exchange resin column, and phosphorous acid compound is adsorbed on resin and D- erythrulose is direct Outflow, the D- erythrulose finally purified with G25 size exclusion column desalination；

The preferred D201 anion exchange resin of anion exchange resin described in step (5) and (6).

In the present invention, butanol carbohydrate oxidase (EDH, EC 1.1.1.-) can utilize coenzyme nicotinamide adenine dinucleotide (NAD⁺) by erythritol selective oxidation at D- erythrulose, but the reaction is to be intended to the reversible reaction of sugar alcohol raw material, only There is the ketose product of minimal amount of (< 10%) to generate.And hydroxypropanone- kinases (EuK, EC 2.7.1.209) then can use three The complete phosphoric acid of D- erythrulose is melted into D- erythrulose -4- phosphoric acid by adenosine phosphate (ATP).Both the above enzyme is combined same The catalysis of one system can be achieved with the disposable complete conversion of cheap erythritol to D- erythrulose -4- phosphoric acid, and then can be with Easily D- erythrulose is generated using phosphohydrolase (AP, EC 3.6.1.66) dephosphorylation.

Since the continuous conversion is related to expensive coenzyme nicotinamide adenine dinucleotide (NAD⁺) and Adenosine triphosphate Glycosides (ATP), therefore lactic dehydrogenase (LDH, EC 1.1.1.28) and pyruvic acid, ATP regeneration enzyme (PPK, EC 2.7.4.1) and more Polyphosphoric acid is used in real-time circulation in the reaction system respectively and regenerates two kinds of coenzyme, substantially reduces coenzyme dosage.

The present invention has the following advantages and effects with respect to the prior art:

1, the method for the present invention utilizes the successive reaction of butanol carbohydrate oxidase and hydroxypropanone- kinases, realizes cheap red algae Sugar alcohol and then can easily utilize phosphohydrolase dephosphorylation to the disposable complete conversion of D- erythrulose -4- phosphoric acid Generate D- erythrulose.Reaction will not generate the product of similar structures, and purity and yield are all guaranteed.

2, the method for the present invention also applies lactic dehydrogenase and pyruvic acid, ATP regeneration enzyme and polyphosphoric acids in reaction system In, real-time circulation regenerates two kinds of coenzyme (NAD⁺And ATP), coenzyme dosage is substantially reduced, cost has been saved.

3, the present invention enzyme used is immobilized, significantly improve enzyme stability, large-scale industrial operability and The green color index entirely produced improves the recovery utilization rate of enzyme.

Detailed description of the invention

Fig. 1 is EDH, Euk, LDH, AP, PPK genetic fragment electrophoretogram.

Fig. 2 is EDH, Euk, LDH, AP, PPK purifying protein electrophoretogram.

Fig. 3 is D- erythrulose -4- phosphoric acid in 600M Varian D₂In O solution¹H-NMR spectrum.

Fig. 4 is D- erythrulose -4- phosphoric acid in 600M Varian D₂In O solution¹³C-NMR spectrogram.

Fig. 5 is D- erythrulose -4- phosphoric acid in 600M Varian D₂Mass spectrogram in O solution.

Fig. 6 is D- erythrulose in 600M Varian D₂In O solution¹H-NMR spectrum.

Fig. 7 is D- erythrulose in 600M Varian D₂In O solution¹³C-NMR spectrogram.

Specific embodiment

Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited In this.

Embodiment

(1) fermenting and producing erythritol oxidizing ferment (EDH), erythrulose kinases (EuK), ATP regeneration enzyme (PPK), lactic acid Dehydrogenase (LDH) and phosphohydrolase (AP):

It is purchased with Escherichia coli (Escherichia coli DH5a) bacterial strain (being purchased from general biology) gDNA, ATCC of extraction The smegma bacillus (Mycobacterium smegmatis ATCC 700084) and rhizobium melioti (Rhizobium bought Meliloti Strain 1021) chromosome is template, above-mentioned primer PCR amplifies EDH, EuK, LDH, PPK and AP gene Segment (see Fig. 1) is then connected on pET23a plasmid by corresponding digestion, enzyme and (is purchased from biological wind).

The sequence amplification primer of gene:

EDH forward primer: 5 '-gtcgaaaggtatttcatatgtccaatcaag-3 ' (SEQ.ID.NO.1)；

EDH reverse primer: 5 '-ctcggctcatcacagaagctttcgctatgc-3 ' (SEQ.ID.NO.2)；

EuK forward primer: 5 '-gaaggacggacatatgacgtacctcctgaac-3 ' (SEQ.ID.NO.3)；

EuK reverse primer: 5 '-gtcggtcgggcctcgagccgggtgaggtgc-3 ' (SEQ.ID.NO.4)；

LDH forward primer: 5 '-catcactggagaaagtcatatgaaactcg-3 ' (SEQ.ID.NO.5)；

LDH reverse primer: 5 '-gaatgcaggggagcctcgagattaaaccag-3 ' (SEQ.ID.NO.6)；

PPK forward primer: 5 '-gagcgggaggaagcatatggcactcgacg-3 ' (SEQ.ID.NO.7)；

PPK reverse primer: 5 '-ctgatcgtcagctcgagggaatcacctgag-3 ' (SEQ.ID.NO.8)；

AP forward primer: 5 '-catggagaaaatcatatgaaacaaagcac-3 ' (SEQ.ID.NO.9)；

AP reverse primer: 5 '-aattcactgccgggctcgagtttatttcagc-3 ' (SEQ.ID.NO.10)；

Be transferred to E.coli BL21 (DE3) bacterial strain (general biology) by the correct plasmid of sequence verification, 37 DEG C, 4ml is cultivated in the LB culture solution containing 100 μM of ampicillins, and (OD 0.5-0.6) is added after cell rises to logarithmic phase 0.4mM isopropyl-beta D-thio galactopyranoside (IPTG) inducible protein is expressed 4 hours, and smudge cells, high speed are finally collected Centrifugation, supernatant is using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmation protein expression (see figure 2)。

Correct seed culture medium can also be linked into step by step grow to logarithmic phase in 5L cultivation and fermentation tank after use 0.5mM IPTG inducing expression 6 hours, collect wet cell 50g.LB culture medium is constituted are as follows: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate and 5% glycerol.

(2) purifying and fixation of enzyme:

The oxidizing ferment containing erythritol (EDH) of above-mentioned collection, erythrulose kinases (EuK), ATP regeneration enzyme (PPK), lactic acid The cell of dehydrogenase (LDH) and phosphohydrolase (AP) is crushed by high pressure, high speed centrifugation supernatant is by amount addition ammonium sulfate Solid is until be precipitated (30-50%W/V ammonium sulfate).

The enzyme solid after being collected by centrifugation by being slowly dissolve into the Tris buffer of 25mM pH value 8.0, then through G25 ruler Very little exclusion column desalination (being purchased from Sigma) and utilization DEAE Seplite FF (Xi'an Lan Xiao company) anion exchange post separation It obtains just purifying EDH, EuK, LDH, PPK, AP liquid enzymes.

The G25 size exclusion column desalination, is to do eluent with deionized water；

Enzyme EDH, EuK, LDH and PPK are using LX-1000EP epoxy resin (Xi'an Lan Xiao company) according to active unit 1:2: 1:2 and following methods carry out disposable mixed immobilization: 1000U purifying mixed enzyme is dissolved in the phosphoric acid of 1L 100mM pH value 8.0 In potassium solution, 200 grams of LX-1000EP epoxy resin are then added to buffer, filter out immobilization after being stirred at room temperature 24 hours It is stand-by finally respectively to wash twice rear low temperature drying with clear water and 8.0 phosphate buffer of 100mM pH value for enzyme.Immobilization mixed enzyme (EDH, EuK, LDH, PPK) is respectively provided with the activity of the 20-60% of corresponding liquid enzymes.

Phosphohydrolase then carries out independent immobilization using above-mentioned same method, and immobilization AP enzyme has liquid enzymes 80% activity.

It (3) is that raw material prepares D- erythrulose-using mixed immobilization enzyme (EDH, EuK, LDH, PPK) and erythritol 4- phosphoric acid:

9.2 grams are successively added in trishydroxymethylaminomethane hydrochloric acid (Tris.HCl) solution of 1L 25mM pH value 8.0 Erythritol (75mM), 8.8 grams of Sodium Pyruvates (80mM), 2.2 grams of adenosine disodium triphosphate ATP (4mM), 0.69 gram of niacinamide Adenine-dinucleotide mono-sodium salt (1mM), 10.3 grams of polyphosphoric acids (Sigma, 25 is poly-, 100mM monophosphate), 2.8 grams of magnesium chlorides (30mM), 1.5 grams of potassium chloride (20mM)；After pH value is adjusted to 8.0,2000U immobilization mixed enzyme (EDH, EuK, LDH, PPK) It is added at one time mixed system, takes after gentle agitation 4 hours solution to detect remaining erythritol (its oxidizing ferment at 30 DEG C wait react With NAD⁺Detect UV 340nm Change of absorption), the results showed that fully reacting.(active unit U represents 30 DEG C of 1 μM of conversions per minute Enzyme amount required for substrate)

Filtering reacting liquid recycle immobilised enzymes, in filtrate be added 19.1 grams of barium oxalates (75mmol) and with twice of body Long-pending ethyl alcohol mixed precipitation D- erythrulose -4- phosphoric acid and adenosine phosphate (AMP, ADP and ATP).The precipitation solid being collected by centrifugation It is slowly dissolved in the Tris buffer solution of pH value 1.0,10.6 grams of anhydrous sodium sulfates (75mmol) is added, insoluble sulfuric acid is precipitated Barium；After being filtered to remove barium salt and being transferred to 7.0 in supernatant pH value, then direct loading D201 anion resin exchange column is (brilliant Auspicious chemical industry), the elution separation of gradient ammonium hydrogencarbonate aqueous solution removes adenosine phosphate impurity and obtains pure D- erythrulose -4- phosphoric acid. Last G25 size exclusion column desalination obtains 13.6 grams of D- erythrulose -4- sodium phosphate weak yellow foams, and yield is up to 82% (nuclear-magnetism And mass spectrogram is shown in Fig. 3-Fig. 5).Immobilised enzymes retains 90% original activity after recycling 10 times.

(4) it using D- erythrulose -4- phosphoric acid as raw material, is hydrolyzed using immobilization phosphohydrolase (AP) and generates the red moss ketone of D- Sugar:

10 grams of D- erythrulose -4- sodium phosphates (45mM), 77mg magnesium chloride (2mM) are dissolved in 400 milliliters of 25mM pH value In 7.0 Tris.HCl solution, 500U immobilization phosphohydrolase AP is then added and is reacted, the mixed solution is slow at 30 DEG C Slowly immobilised enzymes is directly separated by filtration after 2 hours of stirring.Reaction solution flows through D201 anion resin exchange column, phosphoric acid chemical combination Object is adsorbed on resin and D- erythrulose flows directly out, and finally obtains 3.8 grams of white foams with G25 size exclusion column desalination (ultimate yield 71%, nuclear magnetic spectrogram are shown in Fig. 6-Fig. 7).Reservation 80% is first after immobilization phosphohydrolase (AP) recycles 12 Begin activity.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

Sequence table

<110>South China Normal University

<120>method that immobilized enzyme catalysis method prepares D- erythrulose

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>EDH forward primer

<400> 1

gtcgaaaggt atttcatatg tccaatcaag 30

<210> 2

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>EDH reverse primer

<400> 2

ctcggctcat cacagaagct ttcgctatgc 30

<210> 3

<211> 31

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>EuK forward primer

<400> 3

gaaggacgga catatgacgt acctcctgaa c 31

<210> 4

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>EuK reverse primer

<400> 4

gtcggtcggg cctcgagccg ggtgaggtgc 30

<210> 5

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>LDH forward primer

<400> 5

catcactgga gaaagtcata tgaaactcg 29

<210> 6

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>LDH reverse primer

<400> 6

gaatgcaggg gagcctcgag attaaaccag 30

<210> 7

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>PPK forward primer

<400> 7

gagcgggagg aagcatatgg cactcgacg 29

<210> 8

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>PPK reverse primer

<400> 8

ctgatcgtca gctcgaggga atcacctgag 30

<210> 9

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>AP forward primer

<400> 9

catggagaaa atcatatgaa acaaagcac 29

<210> 10

<211> 31

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>AP reverse primer

<400> 10

aattcactgc cgggctcgag tttatttcag c 31

Claims

1. a kind of method for preparing D- erythrulose using immobilised enzymes continuous catalysis erythritol, it is characterised in that including following Step:

(1) PCR amplification erythritol oxidizing ferment, erythrulose kinases, lactic dehydrogenase, ATP regeneration enzyme, phosphoric acid hydrolysis enzyme gene Segment is then attached on plasmid；Plasmid is transferred in cell again；

(2) cell for being transferred to plasmid is placed in 37^oC, it is cultivated in the LB liquid medium containing 100 μM of ampicillins, when After cell rises to logarithmic phase, it is added the expression of isopropyl-beta D-thio galactopyranoside inducible protein 4 hours or more, collects Cell；

(3) clasmatosis, the centrifugation collected step (2), are added ammonium sulfate to final concentration of 30-50% by amount in supernatant W/V, until there is protein solid precipitation；Albumen is collected by centrifugation, is dissolved into the Tris buffer of pH value 8.0, then is arranged through G25 size Hinder column desalination, through DEAE Seplite FF anion exchange post separation, erythritol oxidizing ferment, the erythrulose purified Kinases, lactic dehydrogenase, ATP regeneration enzyme, phosphohydrolase liquid enzymes；

(4) the purifying mixed enzyme of erythritol oxidizing ferment, erythrulose kinases, lactic dehydrogenase and ATP regeneration enzyme is dissolved in In buffer, epoxy resin is then added, is stirred at room temperature 24 hours or more, enzyme is fixed on epoxy resin, filters out asphalt mixtures modified by epoxy resin Rouge is washed with clear water and buffer, finally 4^oC is dry；Phosphohydrolase is individually fixed according to same step；

Buffer described in step (4) is the potassium phosphate solution of 100mM pH value 6.5-9.0；

(5) erythritol, Sodium Pyruvate, polyphosphoric acids, adenosine disodium triphosphate ATP, nicotinoyl amine gland are added in buffer Purine dinucleotides mono-sodium salt, magnesium chloride and potassium chloride adjust pH value to 8.0, erythritol oxidizing ferment, erythrulose are added Kinases, lactic dehydrogenase and ATP regenerate enzyme immobilizatio mixed enzyme, in 25-40^oIt the reaction of C gentle agitation 4 hours or more, generates D- erythrulose -4- phosphoric acid；The buffer is the Tris.HCl solution of pH value 8.0；

Filtering reacting liquid recycles epoxy resin and its immobilised enzymes, and the second of barium oxalate and twice of filtrate volume is added in filtrate Alcohol, for precipitating D- erythrulose -4- phosphoric acid and adenosine phosphate；Precipitating is collected by centrifugation, is dissolved in Tris buffering of the pH value less than 7 In liquid, anhydrous sodium sulfate is added, insoluble barium sulfate is precipitated；It is filtered to remove insoluble barium salt, and 7.0 will be transferred in filtrate pH value, Loading anion-exchange resin column, and with the elution of gradient ammonium hydrogencarbonate aqueous solution, ammonium hydrogencarbonate concentration is gradually increased to 1.0M from 0, The organic impurities of not phosphorous acid groups is eluted out from exchange column at first, later, the D- erythrulose -4- of the group containing monophosphate Phosphoric acid gradually is eluted out, and the ADP/ATP containing multiple phosphate groups is finally eluted；D- erythrulose-the 4- being collected into D- erythrulose -4- the sodium phosphate that phosphoric acid is most purified through G25 size exclusion column desalination afterwards；

(6) D- erythrulose -4- sodium phosphate, magnesium chloride are dissolved in the Tris.HCl solution of pH value 7.0, are then added and fix Change phosphohydrolase, in 25-40^oThe reaction of C gentle agitation 2 hours or more, then it is separated by filtration epoxy resin and its immobilised enzymes； Filtrate chromatographs by anion-exchange resin column, and phosphorous acid compound is adsorbed on resin and D- erythrulose flows directly out, most The D- erythrulose purified afterwards with G25 size exclusion column desalination.

2. according to the method described in claim 1, it is characterized by: PCR amplification described in step (1), is with Escherichia coli (Escherichia coli), smegma bacillus (Mycobacterium smegmatis) or rhizobium melioti (Rhizobium meliloti) chromosome be template.

3. according to the method described in claim 1, it is characterized by: plasmid described in step (1) is pET23a.

4. according to the method described in claim 1, it is characterized by: cell described in step (1) is e. coli strain bl21.

5. according to the method described in claim 1, it is characterized by: step (2) the isopropyl-beta D-thio galactopyranose The final concentration of 0.4-0.5 mM of glycosides.

6. according to the method described in claim 1, it is characterized by: DEAE Seplite FF anion described in step (3) is handed over Post separation is changed, is to do eluent with gradient NaCl aqueous solution, concentration is gradually promoted from 0 to 1.0 M.

7. according to the method described in claim 1, it is characterized by: in purifying mixed enzyme described in step (4), erythritol oxygen The active unit's ratio for changing enzyme, erythrulose kinases, lactic dehydrogenase and ATP regeneration enzyme is 1:2-3:1:2-3.

8. according to the method described in claim 1, it is characterized by: G25 size exclusion column described in step (3), (5) and (6) Desalination is to do eluent with deionized water.

9. according to the method described in claim 1, it is characterized by: Tris buffer described in step (5), pH value 1.0.