阅读说明：本技术 固定化酶催化法合成d-木酮糖 (Method for synthesizing D-xylulose by immobilized enzyme catalysis method ) 是由 黄华 张传封 张兴锟 于 2019-08-23 设计创作，主要内容包括：本发明公开了固定化酶催化法合成D-木酮糖,该方法从廉价的D-木糖和D-木糖醇出发,克隆了代谢途径中相关的系列酶基因,发酵生产系列酶,经过纯化和固定,然后加入相关起始原料,合成D-木酮糖。本发明采用固定化酶法,以廉价的木糖和木糖醇为原料高收率转化成D-木酮糖；通过与木酮糖激酶的偶合催化不仅实现了原料的转化完全,并且大大的方便了后期产品纯化,而固定化酶的应用则进一步降低了生产成本并有利于规模化工业生产。(The invention discloses a method for synthesizing D-xylulose by an immobilized enzyme catalysis method, which starts from cheap D-xylose and D-xylitol, clones related enzyme genes in a metabolic pathway, produces the related enzymes by fermentation, and synthesizes the D-xylulose by purifying and immobilizing the enzymes and then adding related initial raw materials. The method adopts an immobilized enzyme method, uses cheap xylose and xylitol as raw materials to convert the xylose and the xylitol into D-xylulose in high yield; the coupling catalysis with xylulokinase not only realizes the complete conversion of raw materials, but also greatly facilitates the purification of products at the later stage, and the application of immobilized enzyme further reduces the production cost and is beneficial to large-scale industrial production.)

1. A method for synthesizing D-xylulose, comprising the steps of:

(1) PCR amplifying gene segments of xylose isomerase, xylitol oxidase, xylulokinase, ATP regenerative enzyme, lactate dehydrogenase and phosphohydrolase, respectively connecting the obtained gene segments to plasmids, and transferring the plasmids into cells; carrying out resistance screening on the cells, then carrying out amplification culture step by step and inducing protein expression, and respectively collecting wet cells containing the various enzymes;

(2) crushing and centrifuging the collected wet cells under high pressure, and gradually adding ammonium sulfate into the supernatant until protein solids are separated out; centrifuging to collect protein, purifying to obtain XI, XDH, XK, LDH, PPK and AP liquid enzymes respectively;

(3) dissolving XI, XK and PPK (3.0-6.0) in a buffer solution according to an activity unit ratio of 1.0 (1.5-3.0), then adding epoxy resin, stirring at room temperature for more than 8 hours, and fixing enzyme on the epoxy resin;

mixing XDH, XK, PPK and LDH according to activity unit ratio (1.5-2.5): (1.5-3.0): (3.0-6.0): 1.0-2.0), fixing on epoxy resin according to the same steps;

the AP is independently fixed according to the same steps;

(4) adding D-xylose, adenosine disodium triphosphate, polyphosphoric acid, magnesium chloride and potassium chloride into a buffer solution, adjusting the pH value to 6.5-8.5, adding immobilized mixed enzyme (XI, XK and PPK), maintaining the pH value of a system at 6.5-8.5, stirring for 3-5 hours at 30 ℃, filtering and recovering the immobilized enzyme, and purifying the obtained D-xylulose-5-phosphate crude liquid;

adding D-xylitol, sodium pyruvate, adenosine disodium triphosphate, nicotinamide adenine dinucleotide monosodium salt, polyphosphoric acid, magnesium chloride and potassium chloride into a buffer solution, adjusting the pH value to 6.0-9.0, adding immobilized mixed enzyme (XDH, XK, LDH and PPK), maintaining the pH value of a system at 6.0-9.0, stirring at 30 ℃ for 2.5-5.5 hours, filtering and recovering immobilized enzyme, and purifying the obtained D-xylulose-5-phosphate crude liquid;

(5) adding D-xylulose-5-sodium phosphate and magnesium chloride into a buffer solution, adding immobilized AP, reacting for 1.5-3.5 hours at 20-40 ℃, filtering and recovering the immobilized AP, removing phosphorus acid-containing impurities from the reaction solution by using anion exchange resin, and eluting D-ribulose at first; and finally purifying to obtain a pure D-xylulose product.

2. The method of claim 1, wherein: the buffer solution described in step (3) is potassium phosphate solution having a pH of 8.0.

3. The method of claim 1, wherein: the buffer solution in the step (4) is Tris.HCl solution with the pH value of 8.0.

4. The method of claim 1, wherein: the buffer solution in the step (5) is Tris.HCl solution with the pH value of 7.0.

5. The method of claim 1, wherein: purifying the D-xylulose-5-phosphoric acid crude liquid in the step (4), which comprises the following steps:

adding barium oxalate with 1.1 equivalent of xylose or xylitol into the filtrate, stirring thoroughly, mixing with ethanol solution with twice filtrate volume to precipitate all the phosphorus-containing acid components, centrifuging to collect precipitate, dissolving in Tris buffer solution with pH of 1.0, adding anhydrous sodium sulfate with equivalent of barium oxalate, centrifuging to remove BaSO₄Precipitating, adjusting pH of the supernatant to 7.0, removing adenosine impurity with D201 anion exchange resin, desalting with G25 size exclusion column, and lyophilizing to obtain white solid, i.e. pure D-xylulose-5-sodium phosphate.

6. The method of claim 1, wherein: the PCR amplification in the step (1) takes Escherichia coli strain gDNA and Mycobacterium smegmatis chromosome as templates.

7. The method of claim 1, wherein: and (2) the purification is to dissolve the protein into Tris buffer solution with the pH value of 8.0, then to perform dialysis treatment in the same buffer solution, and finally to separate the protein through DEAE Seplite FF anion exchange column to obtain the purified liquid enzyme.

8. The method of claim 1, wherein: in the step (1), the culture medium adopted for resistance screening and amplification culture is an LB liquid culture medium.

9. The method of claim 8, wherein: the medium used for resistance selection also contained 50. mu.M kanamycin.

10. The method of claim 8, wherein: the amplification culture comprises 0.5mM IPTG in a culture medium and 6 hours of induced expression at 37 ℃.

Technical Field

The invention relates to a method for synthesizing D-xylulose by an immobilized enzyme catalysis method, in particular to a method for converting D-xylose and D-xylitol into D-xylulose by multi-step catalysis of immobilized enzyme.

Background

D-Xylulose (D-Xylulose) is a pentose with a molecular formula C₅H₁₀O₅Molecular weight is 150; it is an important intermediate of xylose (D-xylose) metabolism that is widely present in nature, and is converted into xylose phosphate by the action of the corresponding kinase and then enters the general pentose phosphate metabolic pathway.

D-xylulose is detected in high concentration in urine, blood, cerebrospinal fluid and other parts of human body, and has unknown functions to be explored. Meanwhile, D-xylulose can be conveniently converted into furfural (furfural) through dehydration reaction, and the furfural is a renewable biological energy source and a starting material of biochemical products which are researched at present, so that the method has great market development prospect.

The high price on the market (￥ 16000/g, Carbosynth corporation) greatly limits the further development of D-xylulose in research and industrial application fields, so it is important to develop a simple and scalable preparation method using cheap raw materials (D-xylose: ￥ 380/kg; xylitol: ￥ 555/kg, aladdin reagent) to reduce the market price.

The conventional preparation method of D-xylulose includes extraction method, chemical synthesis method and fermentation method.

However, because the abundance of D-xylulose is not high in nature, the polarity is high, the water solubility is high, and many similar sugars are mixed, the cost of the separation, extraction and purification method is high, and the method only exists in the research of early D-xylulose; later, with the gradual maturity of the chemical preparation process of sugar, xylulose gradually realizes selective oxidation synthesis by using cheap xylitol, however, due to the existence of a plurality of hydroxyl groups on xylitol, the selective protection, oxidation and deprotection of a substrate-OH in the method leads the whole synthesis process to be complicated and complex, and the production cost is high.

The enzyme method or fermentation method for preparing the compound has unique advantages because a plurality of chiral centers on the saccharide compound are not required to be considered, and the Ajinomoto company in Japan utilizes a yeast of Gluconobacter (Gluconobacter) to ferment and produce D-xylulose (US 6221634B1) by taking Arabitol (D-Arabitol) as a raw material, but the final purification cost is high because xylitol, xylose and other similar monosaccharide impurities are mixed in the product.

Disclosure of Invention

In order to overcome the defect that the prior art cannot conveniently prepare high-purity D-xylulose at low cost, the invention aims to provide an immobilized enzyme catalysis method for synthesizing D-xylulose.

The purpose of the invention is realized by the following technical scheme:

a method of synthesizing D-xylulose comprising the steps of:

(1) amplifying gene fragments of Xylose Isomerase (XI), xylitol oxidase (XDH), Xylulokinase (XK), ATP regenerating enzyme (PPK), Lactate Dehydrogenase (LDH) and phosphohydrolase (AP) by PCR, connecting the obtained gene fragments to plasmids respectively, and transferring the plasmids into cells; carrying out resistance screening on the cells, then carrying out amplification culture step by step and inducing protein expression, and respectively collecting wet cells containing the various enzymes;

the PCR amplification is carried out by taking Escherichia coli (Escherichia coli DH5a) strain gDNA and Mycobacterium smegmatis (Mycobacterium smegmatis ATCC 700084) chromosome as templates;

the plasmid is preferably pET28 a;

the cell is preferably E.coli BL21(DE3) strain;

in the step (1), the culture medium adopted for resistance screening and amplification culture is an LB liquid culture medium; the culture medium for resistance selection also contains 50 mu M kanamycin;

the amplification culture is carried out, wherein the culture medium contains 0.5mM IPTG, and the induction expression is carried out for 6 hours at 37 ℃;

(2) crushing and centrifuging the collected wet cells under high pressure, and gradually adding ammonium sulfate into the supernatant until protein solids are separated out; centrifuging to collect protein, purifying to obtain XI, XDH, XK, LDH, PPK and AP liquid enzymes respectively;

the centrifugation is 10000-;

the purification comprises the steps of dissolving the protein into a Tris buffer solution with the pH value of 8.0, then carrying out dialysis treatment in the same buffer solution, and finally separating by using a DEAE Seplite FF anion exchange column to obtain purified liquid enzyme;

(3) dissolving XI, XK and PPK (3.0-6.0) in a buffer solution according to the activity unit ratio of 1.0 (1.5-3.0), then adding epoxy resin, stirring at room temperature for more than 8 hours, and fixing enzyme on the epoxy resin, wherein the immobilized enzyme has 30-70% of initial activity; in the step, the stability of three enzymes is different, and the stability of XI is high and is an equilibrium reaction; to achieve complete conversion, the enzymes in the last two steps are in excess. XI reaction is easy to carry out, enzyme stability is good, and the dosage is minimum. XK is relatively difficult, and the regeneration of ATP by PPK is critical to realize effective conversion; the combination can realize that the subsequent reaction quickly drags the equilibrium reaction of the XI in front, thereby realizing the quick and smooth conversion of the product.

Mixing XDH, XK, PPK and LDH according to activity unit ratio (1.5-2.5): (1.5-3.0): (3.0-6.0): 1.0-2.0), fixing on epoxy resin according to the same steps, wherein the fixed enzyme has 20-45% initial activity; similarly to the above, the ratio of several enzymes is adjusted to achieve rapid conversion of the system. Unlike the above, XDH stability is much worse than XI, so the amount is increased and LDH regeneration NAD is also increased⁺However, this step is very efficient and stable, and the amount of the enzyme used can be reduced appropriately. The ratio of XK to PPK is the same as above.

AP is independently fixed according to the same steps, and 90% of liquid enzyme activity is reserved after the AP is fixed;

the buffer solution is preferably potassium phosphate solution with the pH value of 8.0;

(4) adding D-xylose, adenosine disodium triphosphate, polyphosphoric acid, magnesium chloride and potassium chloride into a buffer solution, adjusting the pH value to 6.5-8.5, adding immobilized mixed enzyme (XI, XK and PPK), maintaining the pH value of a system at 6.5-8.5, stirring at 30 ℃ for 3-5 hours, filtering and recovering the immobilized enzyme (the recovery activity is 50-85%), and purifying the obtained D-xylulose-5-phosphate crude liquid;

adding D-xylitol, sodium pyruvate, adenosine disodium triphosphate, nicotinamide adenine dinucleotide monosodium salt, polyphosphoric acid, magnesium chloride and potassium chloride into a buffer solution, adjusting the pH value to 6.0-9.0, adding immobilized mixed enzyme (XDH, XK, LDH and PPK), maintaining the pH value of a system at 6.0-9.0, stirring at 30 ℃ for 2.5-5.5 hours, filtering and recovering immobilized enzyme (the recovery activity is 55-85%), and purifying the obtained D-xylulose-5-phosphate crude liquid;

the buffer solution is preferably tris (hydroxymethyl) aminomethane hydrochloric acid (Tris.HCl) solution with the pH value of 8.0;

the purification of the D-xylulose-5-phosphoric acid crude liquid comprises the following steps:

adding barium oxalate with 1.1 equivalent of xylose or xylitol into the filtrate, stirring thoroughly, mixing with ethanol solution with twice filtrate volume to precipitate all phosphorus-containing acid components (containing D-xylulose-5-phosphoric acid, AMP, ADP, and ATP), centrifuging to collect precipitate, dissolving in Tris buffer solution with pH of 1.0, adding anhydrous sodium sulfate with equivalent of barium oxalate, centrifuging to remove BaSO₄Precipitating, adjusting the pH value of the supernatant to 7.0, removing adenosine impurities by using D201 anion exchange resin (gradient elution is carried out by using 0-1N ammonium bicarbonate water solution), desalting by using a G25 size exclusion column (deionized water is used as eluent), and freeze-drying to obtain a white solid, namely a pure D-xylulose-5-sodium phosphate product;

(5) adding D-xylulose-5-sodium phosphate and magnesium chloride into a buffer solution, adding immobilized AP, reacting for 1.5-3.5 hours at 20-40 ℃, filtering and recovering the immobilized AP (with 92% of initial activity), passing the reaction solution through anion exchange resin to remove phosphorus acid-containing impurities, and eluting D-ribulose at first; finally, purifying to obtain a pure D-xylulose product;

the buffer solution in the step (5) is preferably Tris.HCl solution with the pH value of 7.0;

the purification is G25 size exclusion column desalting.

The metabolic pathways involved in the methods of the invention are shown below:

xylose isomerase (XI, EC 5.3.1.5) can convert D-xylose to D-xylulose, but due to the incomplete equilibrium reaction, the purification of xylulose is greatly hampered by the large amount of residual xylose starting material in the reaction solution. D-xylulokinase (XK, EC2.7.1.17) selectively phosphorylates D-xylulose to D-xylulose 5-phosphate, which, through coupling with an isomerase, can drive the complete conversion of xylose.

Sugar alcohol oxidase (XDH, EC 1.1.1.-) can efficiently and specifically oxidize xylitol into D-xylulose, high-yield conversion from xylitol to D-xylulose-5-phosphate can be effectively realized by coupling with the xylulokinase, and preparation of D-xylulose can be conveniently realized by hydrolysis with nonspecific phosphohydrolase (AP, EC 3.6.1.66).

In the method, adenosine triphosphate ATP and coenzyme nicotinamide adenine dinucleotide (NAD +) are expensive raw materials, and in order to reduce the cost, cyclic regeneration of the coenzyme can be effectively realized by introducing ATP regenerative enzyme (PPK, EC2.7.4.1) and polyphosphoric acid Pi (n), lactate dehydrogenase (LDH, EC 1.1.1.28) and pyruvic acid into a reaction system, so that the using amount of the coenzyme is greatly reduced.

Compared with the prior art, the invention has the following advantages and effects:

the method adopts an immobilized enzyme method, uses cheap xylose and xylitol as raw materials to convert the xylose and the xylitol into D-xylulose in high yield; the coupling catalysis with xylulokinase not only realizes the complete conversion of raw materials, but also greatly facilitates the purification of products at the later stage, and the application of immobilized enzyme further reduces the production cost and is beneficial to large-scale industrial production.

Drawings

FIG. 1 is an SDS-PAGE gel chromatogram of purified protein; wherein the leftmost lane is a three-color prestained protein standard (10-180 kDa).

FIG. 2 shows the purification of D-xylulose 5-phosphate at 600M Varian D₂In O solution¹H-NMR spectrum.

FIG. 3 shows the purification of D-xylulose 5-phosphate at 600M Varian D₂In O solution¹³C-NMR spectrum.

FIG. 4 shows the purification of D-xylulose 5-phosphate at 600M Varian D₂Mass spectrum in O solution.

FIG. 5 shows the purification of D-xylulose at 600M Varian D₂In O solution¹H-NMR spectrum.

FIG. 6 shows the purification of D-xylulose at 600M Varian D₂In O solution¹³C-NMR spectrum.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.