阅读说明：本技术 一种阿洛酮糖3-差向异构酶固定化酶、其固定化方法与应用 (Psicose 3-epimerase immobilized enzyme, and immobilization method and application thereof ) 是由 朱玥明 陈朋 孙媛霞 于 2020-09-18 设计创作，主要内容包括：本发明提供了一种阿洛酮糖3-差向异构酶固定化酶,还提供了该阿洛酮糖3-差向异构酶的固定化方法。本发明通过将阿洛酮糖3-差向异构酶或其融合标签酶与固定化树脂相结合,制备获得阿洛酮糖3-差向异构酶固定化酶。该固定化酶可应用于批次或连续反应催化果糖高效转化为D-阿洛酮糖,提高了酶的重复使用率和使用周期,显著降低了D-阿洛酮糖的生产成本,具有广泛应用前景。(The invention provides a psicose 3-epimerase immobilized enzyme and an immobilization method of the psicose 3-epimerase. The psicose 3-epimerase immobilized enzyme is prepared by combining the psicose 3-epimerase or fusion tag enzyme thereof with immobilized resin. The immobilized enzyme can be applied to batch or continuous reaction to catalyze fructose to be efficiently converted into D-psicose, so that the reuse rate and the use period of the enzyme are improved, the production cost of the D-psicose is obviously reduced, and the immobilized enzyme has wide application prospect.)

1. A psicose 3-epimerase immobilized enzyme is characterized in that the immobilized enzyme is prepared by combining psicose 3-epimerase derived from rumen bacteria and immobilized resin.

2. A psicose 3-epimerase immobilized enzyme according to claim 1, wherein the psicose 3-epimerase immobilized by the immobilized enzyme comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID No. 1.

3. A psicose 3-epimerase immobilized enzyme according to claim 1, wherein the psicose 3-epimerase immobilized by the immobilized enzyme has a lysine-rich tag, preferably AKAKAKAKAK, added to the carboxyl terminus of the protein represented by SEQ ID No. 1.

4. A psicose 3-epimerase characterized in that the psicose 3-epimerase has a lysine-rich tag, preferably AKAKAKAKAK, added to the carboxy terminus of the protein represented by SEQ ID No. 1.

5. The psicose 3-epimerase immobilized enzyme of claim 1, wherein the carrier of the immobilized enzyme is a macroporous resin.

6. The psicose 3-epimerase immobilized enzyme of claim 1, wherein the carrier of the immobilized enzyme is ES-1, ES-103, ES-108, ESR-1, ESR-2 or ESQ-1.

7. The psicose 3-epimerase immobilized enzyme of claim 1, wherein the carrier of the immobilized enzyme is an epoxy-based macroporous resin.

8. The psicose 3-epimerase immobilized enzyme of claim 1, wherein the carrier of the immobilized enzyme is ES-108.

9. The psicose 3-epimerase immobilized enzyme of claim 1, which is prepared by the following method:

(1) resin activation: activating the macroporous resin in potassium phosphate buffer solution with pH 7.5-8.0;

(2) preparation of enzyme: preparing enzyme solution by fermenting engineering bacteria for heterologous expression of the psicose 3-epimerase, wherein the engineering bacteria can be escherichia coli or bacillus subtilis;

(3) immobilization: adding the activated resin into the enzyme solution of the psicose 3-epimerase for immobilization, wherein the immobilization temperature is 20-40 ℃, and the immobilization time is 12-24 h.

(4) And (3) crosslinking: crosslinking the immobilized enzyme by using a glutaraldehyde solution with the concentration of 0.2-0.5%, and washing and draining off water to obtain the psicose 3-epimerase immobilized enzyme.

10. Use of a psicose 3-epimerase immobilized enzyme of claim 1 for producing psicose.

11. Use of a psicose 3-epimerase immobilized enzyme according to claim 6 for producing psicose, wherein the fructose concentration in the raw material is 20 to 75%.

12. Use of a psicose 3-epimerase immobilized enzyme according to claim 6 for producing psicose, wherein the conversion temperature is 40-70 ℃.

13. Use of a psicose 3-epimerase immobilized enzyme for producing psicose according to claim 6, wherein Mn is added to the reaction system²⁺Or Co²⁺Ion concentration of 0.2-2.0 mM.

14. Use of a psicose 3-epimerase immobilized enzyme according to claim 6 for producing psicose, wherein the conversion means is a batch reaction or a continuous reaction.

Technical Field

The invention belongs to the technical field of industrial biology, and particularly relates to a psicose 3-epimerase immobilized enzyme, a preparation method of the immobilized enzyme, and application of the immobilized enzyme in production of psicose.

Background

D-psicose (D-allolose) is an epimer at the carbon-3 position of D-fructose, the sweet taste of the D-psicose is 70% of that of sucrose, but the calorie is only 0.2cal/g, and the D-psicose has special physiological functions of improving lipid metabolism, reducing postprandial blood sugar and the like, and the mouthfeel and characteristics of the D-psicose are close to those of the sucrose, so the D-psicose (D-allolose) is an ideal substitute of the sucrose. The related reports indicate that allulose is a weak inhibitor of alpha-glucosidase, alpha-amylase, maltase and sucrase, can inhibit the metabolism of starch and disaccharides into monosaccharides in the gastrointestinal tract, and also inhibits the absorption of glucose by the body through transporters in the intestinal tract. Therefore, the psicose has a potential hypoglycemic effect and can reduce postprandial hyperglycemia of a human. Allulose may be used as a low calorie sweetener in beverages, yogurt, ice cream, baked goods, and other high calorie foods. After the U.S. Food and Drug Administration (FDA) certified D-psicose as gras (general recognited as safe), a draft of guidelines was released in 2019, 4/16, allowing food manufacturers to exclude psicose from total and added sugar counts on the nutritional data label, which made psicose a greater concern to the market.

Psicose is found in very small amounts in nature and is also known as a functional "rare sugar". At present, the method for producing the psicose is mainly an enzymatic conversion method, namely, the psicose is obtained by carrying out conversion reaction by using psicose 3-epimerase or tagatose 3-epimerase and taking fructose as a substrate. With the discovery and directed evolution of a large number of psicose 3-epimerases or tagatose 3-epimerases, a batch of high-quality enzymes with high catalytic activity and strong thermostability have been applied to the industrial production of psicose.

With the advance of industrial production of psicose, immobilization of psicose 3-epimerase or tagatose 3-epimerase has been attracting attention. The immobilized enzyme can increase the reuse rate and the use period of the enzyme, is more suitable for a continuous production process, and is one of the key factors for further reducing the production cost of the psicose. In addition, compared with free enzyme, the immobilized enzyme can avoid other components in the enzyme liquid from being mixed into the product, thereby simplifying the allulose purification process. Methods for immobilizing an engineered strain producing psicose 3-epimerase are disclosed in granted patents CN102869783B and CN104160023B of CJ first sugar manufacturing co. In both of the two patents, the calcium alginate embedding method is used for immobilizing enzyme-producing engineering bacteria, so that the service cycle of immobilized cells is prolonged, but the greatest defect of the embedding method is that the binding force between enzyme or cells and a carrier is weak, and the leakage of the enzyme or cells is possibly caused. In addition, the preparation process of the calcium alginate microspheres is complex, the hardness of the calcium alginate microspheres is poor, and the calcium alginate microspheres are not beneficial to long-term use. Therefore, an immobilized enzyme process of the psicose 3-epimerase more suitable for industrial large-scale production needs to be developed, and the reuse rate and the use period of the immobilized enzyme are greatly increased, so that the production cost of the psicose is reduced.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the psicose 3-epimerase immobilized enzyme and the preparation method of the immobilized enzyme, and the method can realize simple, efficient and low-cost immobilization of the psicose 3-epimerase, thereby greatly improving the reuse rate and the use cycle of the psicose 3-epimerase.

The invention adopts the following technical scheme:

the invention provides a psicose 3-epimerase immobilized enzyme, which is prepared by combining psicose 3-epimerase derived from rumen bacteria and enzyme protein obtained by molecular modification of the psicose 3-epimerase with immobilized resin.

According to an embodiment of the present invention, the enzyme immobilized with the psicose 3-epimerase immobilized enzyme is psicose 3-epimerase derived from ruminal bacteria or a mutant thereof, which comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID No. 1.

According to an embodiment of the present invention, the psicose 3-epimerase or a mutant thereof immobilized on the psicose 3-epimerase immobilized enzyme is obtained by fermentation culture of an engineered strain of Escherichia coli or an engineered strain of Bacillus subtilis carrying a gene encoding the enzyme or a mutant thereof.

According to an embodiment of the present invention, the carrier of the psicose 3-epimerase immobilized enzyme is a weakly basic macroporous resin capable of forming covalent bonding with psicose 3-epimerase without destroying the enzyme activity, and the macroporous resin may be of an epoxy type or an amino type, but is not limited to these two types.

According to the embodiment of the invention, the enzyme immobilized by the psicose 3-epimerase immobilized enzyme is a modified enzyme of the psicose 3-epimerase derived from rumen bacteria, and by introducing more basic amino acids, the covalent bonding between the enzyme and a carrier epoxy group is improved on the premise of not changing the enzyme activity, so that the immobilization effect is enhanced, and the immobilization efficiency is improved; if AKAKAKAKAK labels are added at the carboxyl terminal of the enzyme, the immobilization efficiency is greatly improved by the crosslinking of free amino groups in lysine and epoxy groups on the resin.

The invention also provides a preparation method of the psicose 3-epimerase immobilized enzyme, which comprises the following specific steps:

(1) resin activation: activating the macroporous resin in potassium phosphate buffer solution with pH 7.5-8.0;

(2) preparation of enzyme: preparing enzyme solution by fermenting engineering bacteria for heterologous expression of the psicose 3-epimerase, wherein the engineering bacteria can be escherichia coli or bacillus subtilis;

(3) immobilization: adding the activated resin into an enzyme solution of the psicose 3-epimerase for immobilization, wherein the immobilization temperature is 20-40 ℃, and the immobilization time is 12-24 h;

(4) and (3) crosslinking: crosslinking the immobilized enzyme by using a glutaraldehyde solution with the concentration of 0.2-0.5%, and washing and draining off water to obtain the psicose 3-epimerase immobilized enzyme.

The invention also provides a specific application of the psicose 3-epimerase immobilized enzyme in the production of psicose.

According to the embodiment of the invention, the psicose 3-epimerase immobilized enzyme can convert fructose into psicose, the concentration of fructose in the raw material is 20-75%, and the raw material can be pure fructose solution, or fructose-containing mixed syrup (such as high fructose syrup), or fructose-containing plant extract (such as fruit juice).

According to the embodiment of the invention, the temperature of the conversion reaction for converting fructose into psicose by the psicose 3-epimerase immobilized enzyme is 40-70 ℃, the low-temperature reaction can improve the reuse rate or the service cycle of the immobilized enzyme, and the high-temperature reaction can help to improve the reaction conversion rate.

According to the embodiment of the invention, Mn is added into a reaction system for converting fructose into psicose by using psicose 3-epimerase immobilized enzyme²⁺Or Co²⁺The ion concentration is 0.2-2.0mM, and the addition of the metal ion is helpful for improving the reuse rate or the service cycle of the immobilized enzyme.

According to the embodiment of the invention, the conversion mode of converting fructose into psicose by the psicose 3-epimerase immobilized enzyme can be batch reaction or continuous reaction.

Advantageous effects

The invention establishes an immobilization method of psicose 3-epimerase, obtains an immobilized enzyme of the psicose 3-epimerase, and compared with free enzyme, the immobilized enzyme has obviously improved thermal stability. The psicose 3-epimerase immobilized enzyme can be used for producing psicose by taking fructose or various substrates rich in fructose as raw materials, batch or continuous reaction can be adopted in the conversion process, the reuse rate or the use period of the enzyme is greatly improved, the production cost is reduced, and the psicose 3-epimerase immobilized enzyme has wide application prospect.

Drawings

FIG. 1 for improving the thermostability of psicose 3-epimerase immobilized enzyme by adding a tag

FIG. 2 reuse rate of psicose 3-epimerase-immobilized enzyme

Detailed Description

As used herein, "psicose" and "D-psicose", "fructose" and "D-fructose" all have the same meaning and are used interchangeably.

As used herein, the psicose 3-epimerase refers to a wild-type protein or a mutant protein of the psicose 3-epimerase derived from rumen bacteria.

Macroporous resins conventionally used in the art may be used in the present invention, and the macroporous resins described in the examples are epoxy-based macroporous resins or amino-based macroporous resins. The macroporous resin may be produced or commercially available, and may be, for example, at least one of ES-1, ES-103, ES-108, ESR-1, ESR-2, or ESQ-1 available from Tianjin Nankai and science and technology Co.

The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Example 1 immobilization method of psicose 3-epimerase

200g of a resin (ES-1, ES-103, ES-108, ESR-1, ESR-2 or ESQ-1) was weighed out and activated in 2L of PBS buffer (dipotassium hydrogenphosphate 22.8g, potassium dihydrogenphosphate 2.75g, volume 1000mL, pH about 7.5), and after stirring appropriately for about 2 hours, the activated resin was obtained by filtration.

The psicose 3-epimerase is derived from rumen bacteria, the amino acid sequence of the enzyme is shown in SEQ ID No.1, and the production of the enzyme is realized by fermentation of engineering strains carrying wild-type protein or mutant protein genes of the enzyme. The engineering strain can be Escherichia coli or Bacillus subtilis. Producing intracellular enzyme by using escherichia coli, and obtaining a crude enzyme solution by homogenizing and crushing thalli under high pressure; bacillus subtilis produces extracellular enzyme, and the supernatant of the fermentation liquid is the crude enzyme liquid. The crude enzyme solution was obtained by using psicose 3-epimerase and an appropriate amount of activated resin, and was stirred at room temperature overnight. Psicose 3-epimerase was immobilized using different resins, and the immobilized enzyme activity thereof is shown in table 1. The results show that the psicose 3-epimerase can be well immobilized by using different types of macroporous resin of Tianjin Nankai and science and technology Co Ltd, wherein the enzyme activity of the epoxy resin ES-108 immobilized enzyme is highest.

TABLE 1 different resin immobilized enzyme Activity

Example 2 improvement of immobilization efficiency and thermostability of psicose 3-epimerase

In order to further improve the immobilization efficiency of the psicose 3-epimerase and enhance the binding force of the enzyme and a resin carrier, an AKAKAKAKAK label is added at the carboxyl terminal of the psicose 3-epimerase. The amino group on the side chain of the basic amino acid (lysine) on the label increases the binding site of the enzyme and the epoxy group on the resin, thereby improving the immobilization efficiency. As a result of experiments, it was found that the immobilization efficiency of the unlabeled psicose 3-epimerase and the ES-108 resin was 74.7%, while the immobilization efficiency of the AKAKAKAKAK-labeled psicose 3-epimerase and the ES-108 resin reached 100%. The experimental result also shows that the specific enzyme activity of the psicose 3-epimerase is not obviously changed after the label is added.

And further determining the thermal stability of the immobilized enzyme of the psicose 3-epimerase added with or without the label at 60 ℃, specifically, preserving the heat of the immobilized enzyme at 60 ℃ for a certain time, then adding a fructose solution to determine the enzyme activity, and calculating the residual enzyme activity of the immobilized enzyme after heat preservation by taking the enzyme activity of the immobilized enzyme without heat preservation as 100%. The results are shown in fig. 1, and it can be seen that the thermal stability of the labeled psicose 3-epimerase immobilized enzyme is significantly improved compared with that of the unlabeled immobilized enzyme, and the immobilized enzyme still has more than 90% of activity after being incubated at 60 ℃ for 6 hours. The half-life of the immobilized enzyme was calculated, with the half-life of the immobilized enzyme added with the tag at 60 ℃ being 47.9h, while the half-life of the immobilized enzyme added without the tag at 60 ℃ being only 14.4 h. The addition of the label can be seen to contribute to the remarkable improvement of the heat stability of the immobilized enzyme. The application of the tagged psicose 3-epimerase immobilized enzyme was investigated to determine its effect in batch reactions and continuous reactions.

Example 3 reuse efficiency of psicose 3-epimerase-immobilized enzyme

Measuring the reuse times of the immobilized enzyme by using batch reaction, adding the immobilized enzyme by taking 50% (w/v) fructose solution as a substrate, wherein the enzyme adding amount is 10% (w/w), and the reaction temperatureSampling and determining the conversion rate after reacting for 2 hours at 55 ℃; then filtering to obtain immobilized enzyme, adding the immobilized enzyme into a fresh 50% (w/v) fructose solution for a second reaction, and sampling to determine the conversion rate after reacting for 2 hours; the above steps were repeated until more than 50 cycles of reaction. The result is shown in fig. 2, and it can be seen that the psicose 3-epimerase immobilized enzyme can catalyze the epimerization reaction to reach the equilibrium within 2h under the current enzyme adding amount, and the conversion rate is about 30%; simultaneously, Mn is added into the reaction system²⁺The stability of the psicose 3-epimerase immobilized enzyme can be greatly improved, the reaction is repeated for 50 times, and the conversion rate from fructose to psicose is 28-29%.

Example 4 reuse efficiency of psicose 3-epimerase-immobilized enzyme

This example was modified based on example 2 in order to further increase the reaction conversion and shorten the reaction time.

The number of times of reuse of the immobilized enzyme was measured by batch reaction, 50% (w/v) fructose solution was used as a substrate, the immobilized enzyme was added in an amount of 10% (w/w) of the enzyme, the reaction temperature was 60 ℃, a sample was taken after 1 hour of reaction to measure the conversion rate, and the rest of the procedure was the same as in example 2. The result shows that the psicose 3-epimerase immobilized enzyme can catalyze the epimerization reaction to reach the balance within 1h under the current enzyme adding amount, the conversion rate exceeds 30 percent, the reaction is repeated for 50 times, and the conversion rate of fructose to psicose is 30 to 31 percent.

Example 5 reuse efficiency of psicose 3-epimerase-immobilized enzyme

The embodiment is adjusted on the basis of the embodiment 2, and aims to reduce the enzyme adding amount, improve the reuse rate of the immobilized enzyme and further reduce the cost.

The repeated use times of the immobilized enzyme is determined by batch reaction, 50 percent (w/v) fructose solution is taken as a substrate, the immobilized enzyme is added, the enzyme adding amount is 5 percent (w/w), the reaction temperature is 50 ℃, a sample is taken after 4 hours of reaction to determine the conversion rate, and the rest steps are the same as the step 2. The result shows that the psicose 3-epimerase immobilized enzyme can catalyze the epimerization reaction to reach the balance within 4h under the current enzyme adding amount, the repeated reaction times of the immobilized enzyme reach 80 times, and the conversion rate of fructose to psicose reaches 27-28%.

Example 6 reuse efficiency of psicose 3-epimerase-immobilized enzyme

This example was modified on the basis of example 4 in order to further increase the reuse rate of the immobilized enzyme and reduce the reaction energy consumption by lowering the reaction temperature.

The repeated use times of the immobilized enzyme is determined by batch reaction, 50 percent (w/v) fructose solution is taken as a substrate, the immobilized enzyme is added, the enzyme adding amount is 5 percent (w/w), the reaction temperature is 40 ℃, a sample is taken after 4 hours of reaction to determine the conversion rate, and the rest steps are the same as the step 2. The result shows that the psicose 3-epimerase immobilized enzyme can catalyze the epimerization reaction to reach the balance within 4h under the current enzyme adding amount, the repeated reaction times of the immobilized enzyme reach 100 times, and the conversion rate of fructose to psicose is 26-27%.

EXAMPLE 7 continuous production of psicose by psicose 3-epimerase immobilized enzyme reactor

The psicose 3-epimerase immobilized enzyme is filled into a column (phi 26 x 200) with a heat-preservation jacket, namely the immobilized enzyme reactor. Through optimization of reaction conditions and parameters, it was found that the temperature of the jacket was controlled at 50 ℃ and 70% (w/v) fructose solution (containing 1mM Mn) was added²⁺) When the solution passes through the immobilization reactor at the flow rate of 2.5ml/min, the ratio of the psicose to the fructose in the effluent of the reactor is basically balanced, the equilibrium conversion rate is 28-29%, and the half-life period of the immobilization reactor is more than 100 days.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> psicose 3-epimerase immobilized enzyme, and immobilization method and application thereof

<130> 2020.09.18

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Met Lys Tyr Gly Ile Tyr Tyr Ala Tyr Trp Glu Lys Glu Trp Asn Gly

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Asp Tyr Lys Tyr Tyr Ile Asp Lys Ile Ser Lys Leu Gly Phe Asp Ile

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Leu Glu Ile Ser Cys Gly Ala Phe Ser Asp Tyr Tyr Thr Lys Asp Gln

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Tyr Trp Pro Val Asp Tyr Ser Lys Pro Phe Asp Lys Lys Arg Asp Leu

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Glu Asn Ser Ile Lys Asn Met Lys Ile Ile Ser Gln Tyr Ala Glu Glu

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Ser Ser Asn Val Gly Val Met Leu Asp Thr Phe His Met Asn Ile Glu

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His Phe His Ile Gly Glu Gly Asn Arg Lys Val Pro Gly Lys Gly Met

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His Ala Ala Val Met Glu Pro Phe Val Met Gln Gly Gly Thr Val Gly

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His Asp Ile Lys Ile Trp Arg Asp Ile Ile Gly Asn Cys Ser Glu Val

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Thr Leu Asp Met Asp Ala Gln Ser Ala Leu His Phe Val Lys His Val

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Phe Glu Val

290