CN113025605A

CN113025605A - Method for fixing D-glucose isomerase and D-psicose 3-epimerase

Info

Publication number: CN113025605A
Application number: CN202110266275.XA
Authority: CN
Inventors: 江波; 卜一凡; 张涛; 李梦丽
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-03-11
Filing date: 2021-03-11
Publication date: 2021-06-25
Anticipated expiration: 2041-03-11
Also published as: CN113025605B

Abstract

The invention discloses a method for fixing D-glucose isomerase and D-psicose 3-epimerase, belonging to the technical field of food processing. The invention specifically discloses an immobilized double-enzyme method for forming nanoflowers by hybridization of a macroporous carrier, a cross-linking agent, double enzymes and inorganic metal ions, which comprises the following specific steps: a, adsorbing D-glucose isomerase and D-psicose 3-epimerase by a macroporous carrier with an adsorption function; b, adding a cross-linking agent to cross-link the enzyme; c, assembling the enzyme and inorganic metal ions to form double-enzyme-inorganic hybrid nanoflowers; wherein a, b and c can be carried out in any order or simultaneously to prepare the immobilized enzyme. The immobilized double-enzyme has higher enzyme activity, can also keep better stability and mechanical strength, not only improves the problem that the macroporous carrier immobilized enzyme is easy to desorb, but also solves the defect that the organic-inorganic hybrid nano flower is difficult to be packed into a column for continuous production, and has important significance for the immobilized application of double-enzyme and multi-enzyme.

Description

Method for fixing D-glucose isomerase and D-psicose 3-epimerase

Technical Field

The invention relates to a method for fixing D-glucose isomerase and D-psicose 3-epimerase, belonging to the technical field of food processing.

Background

The development of the functional sweetener becomes a research hotspot in the food biotechnology field, and the D-psicose has lower calorie and lower blood sugar reaction, has high sweetness and tastes similar to sucrose, and becomes a good functional sweetener. In the method for producing D-psicose, the psicose produced by biological enzyme method can reach high product concentration, and the product extraction process is relatively simple and has considerable development potential. At present, D-psicose is produced by a biological method, mainly by using D-psicose 3-epimerase to catalyze the epimerization of D-fructose, but the price of the D-fructose is relatively high, so that D-fructose is generated by using relatively low-price glucose under the catalysis of the D-glucose isomerase, and then the D-psicose is prepared by the catalysis of the D-psicose 3-epimerase.

When the D-psicose is prepared by a free enzyme biological method, the free enzyme has poor stability and cannot be recycled, so the cost for industrially producing the D-psicose by using the free enzyme is high. The enzyme immobilization technology overcomes the defects of poor stability, incapability of recycling and the like of free enzyme, and has high technical value. The mode of jointly fixing the double enzymes promotes the automation and the continuous reaction, weakens the industrial production cost and is beneficial to enlarging the production scale.

The prior immobilized enzyme technology mainly comprises the following steps: adsorption method, covalent binding method, embedding method, etc., but the prior immobilized enzyme technology also has the following defects: although the adsorption method has high recovery rate of enzyme activity, the enzyme is easy to desorb; the immobilized enzyme prepared by the covalent bonding method has high stability, but the recovery rate of enzyme activity is low; the gel formed by the embedding method has low mechanical strength and poor thermal stability.

In recent years, organic-inorganic hybrid nanoflowers formed by self-assembly of enzymes with inorganic metal ions have received attention, which significantly improve enzyme activity and stability, but have low mechanical strength, and separation from a substrate can only rely on centrifugation, so that it has limited industrial applications. Based on this, many researchers embed the enzyme-inorganic nanoflower in the gel microsphere, but the leakage of the enzyme from the gel occurs during the use process, and the carrier cannot be recovered.

Therefore, an enzyme immobilization method capable of improving enzyme activity and maintaining stability is found, and the improved immobilized enzyme method is used for producing functional sweeteners such as D-psicose, and has important significance for further application of immobilized enzymes.

Disclosure of Invention

The technical problem is as follows:

provides an enzyme immobilization method which can improve the enzyme activity and maintain the stability, and utilizes the improved immobilized enzyme method to produce the D-psicose.

The technical scheme is as follows:

in order to solve the problems, the invention provides a method for jointly immobilizing D-glucose isomerase and D-psicose 3-epimerase, which is characterized in that a macroporous carrier, a cross-linking agent, D-glucose isomerase, D-psicose 3-epimerase and inorganic metal ions form hybrid nanoflowers to obtain immobilized double enzymes.

The present invention provides a method for co-immobilizing D-glucose isomerase and D-psicose 3-epimerase, comprising the steps of:

a. adding D-glucose isomerase and D-psicose 3-epimerase into a reaction system containing a macroporous carrier for adsorption;

b. adding a cross-linking agent to cross-link the enzyme;

c. adding inorganic metal ions, and assembling with enzyme to form double-enzyme-inorganic hybrid nanoflower;

the immobilized enzyme is prepared by carrying out the steps of the method in an unlimited order.

In one embodiment of the present invention, the inorganic metal ion is any one of cobalt chloride, magnesium chloride, copper sulfate, cobalt sulfate, and zinc chloride.

In one embodiment of the present invention, the macroporous carrier is any one of macroporous adsorption resin, epoxy resin, amino resin, diatomaceous earth, and macroporous cryogel.

In one embodiment of the present invention, the D-glucose isomerase and the D-psicose 3-epimerase are added to the reaction system in the ratio of (2:9) to (9: 2).

In one embodiment of the present invention, the amount of D-glucose isomerase added is: 200U-900U; the addition amount of the D-psicose 3-epimerase is as follows: 200U to 900U.

In one embodiment of the invention, the amino acid sequence of the D-glucose isomerase is shown as SEQ ID NO. 1.

In one embodiment of the invention, the nucleotide sequence encoding the D-glucose isomerase is shown as SEQ ID NO. 2.

In one embodiment of the present invention, the amino acid sequence of the D-psicose 3-epimerase is shown in SEQ ID NO. 3.

In one embodiment of the present invention, the nucleotide sequence encoding the D-psicose 3-epimerase is represented by SEQ ID NO. 4.

In one embodiment of the present invention, the adsorption time of the macroporous carrier is 2 to 72 hours.

In one embodiment of the invention, the crosslinking agent is glutaraldehyde or polyethylene glycol diglycidyl ether, the crosslinking time is 2-72 hours, and the concentration is 0.01-2% (v/v).

In one embodiment of the present invention, the method for preparing the double-enzyme-inorganic hybrid nanoflower comprises the following steps:

(1) adding a phosphate buffer solution and an inorganic metal ion solution into a reaction system containing D-glucose isomerase and D-psicose 3-epimerase in a ratio of (300: 1) - (300: 4), and standing at 10-35 ℃ for 12-72 h to obtain a reaction solution;

(2) and (2) centrifuging the reaction solution obtained in the step (1), collecting the precipitate, and drying the precipitate at 25 ℃ to obtain the double-enzyme-inorganic hybrid nano flower.

In one embodiment of the present invention, the drying mode is normal temperature drying at 25 ℃.

In one embodiment of the present invention, the pH of the phosphate buffer is 6.5 to 7.5, and the concentration of the phosphate buffer is 20 to 100 mmol/L.

The invention also provides the immobilized D-glucose isomerase and the D-psicose 3-epimerase prepared by the method.

The invention also provides the method or the application of the immobilized D-glucose isomerase and D-psicose 3-epimerase in preparing D-psicose or a product containing D-psicose.

Advantageous effects

(1) According to the invention, a macroporous carrier, D-glucose isomerase, D-psicose 3-epimerase and inorganic metal ions are formed into a hybrid nanoflower in any order to obtain the immobilized double enzyme. By adopting the preparation method, the immobilized double-enzyme has the highest enzyme activity which can reach 387U/g carrier; after the compound enzyme is repeatedly used for 10 times, the enzyme activity can be kept at 66%.

(2) The immobilized double enzymes prepared by the method have high enzyme activity, can keep good stability and mechanical strength, and is suitable for application in industrial production.

Detailed Description

The macroporous adsorbent resins referred to in the following examples were purchased from Shanghai lan De Biotech Ltd.

The construction of the macrogel referred to in the following examples is as follows:

preparing 2% (w/v) chitosan solution by using 1% acetic acid, putting 5mL of the prepared chitosan solution into a 10mL centrifuge tube, adding 25 mu L of glutaraldehyde solution with the volume fraction of 25%, uniformly swirling, and immediately freezing in a refrigerator at-80 ℃ for 12 h. Taking out and unfreezing at 4 ℃ to obtain the macroporous gel.

The detection methods referred to in the following examples are as follows:

enzyme activity determination of immobilized enzyme:

(1) preparing 500g/L glucose solution by adopting a phosphate buffer solution, wherein the phosphate buffer solution is 50mmol/L, pH 6.5.5;

(2) 0.1g of immobilized enzyme is added into 10mL of 500g/L glucose solution, the mixture is subjected to heat preservation reaction in a water bath at 60 ℃ for 10min, and then the mixture is immediately transferred into boiling hot water and is heated to inactivate the enzyme for 5min to terminate the reaction.

Definition of enzyme activity: under the conditions, the enzyme amount required for generating 1 mu mol of D-psicose in 1min is one enzyme activity unit.

Operational stability of immobilized enzyme:

(1) firstly, preparing 500g/L glucose solution by adopting a phosphate buffer solution, wherein the phosphate buffer solution is 50mmol/L and has the pH value of 6.5;

(2) adding 0.1g of immobilized enzyme into 10mL of 500g/L glucose solution, carrying out heat preservation reaction in a water bath at 60 ℃ for 10min, separating the immobilized enzyme from the supernatant, transferring the supernatant into boiling hot water, and heating for 5min to inactivate the enzyme to terminate the reaction. The immobilized enzyme was washed with phosphate buffer and centrifuged three times for the next reaction.

Determination of D-psicose content:

high Performance Liquid Chromatography (HPLC) is adopted for determination, and the sample is centrifuged at 12000rpm for 10min before being injected, and is filtered by a 0.22 mu m filter membrane. HPLC conditions: waters e2695 model hplc; a chromatographic column: carbomix Ca-NP; mobile phase: ultrapure water; flow rate: 0.4 mL/min; column temperature: 85 ℃; a detector: a differential refractive detector; detector temperature: 30 ℃; sample introduction amount: 10 μ L.

The method of activating the pretreated macroporous support referred to in the examples below is as follows:

washing the macroporous carrier with ultrapure water to remove attached impurities, soaking with 95% ethanol, and removing ethanol with excessive ultrapure water.

The solutions referred to in the following examples were prepared as follows:

magnesium chloride solution: 2.44g of MgCl were weighed₂·7H₂And O, dissolving in ultrapure water, and then diluting to 100mL to obtain a 120mM magnesium chloride solution.

Cobalt sulfate solution: 1.86g of CoSO were weighed₄And dissolving the ultrapure water and then fixing the volume to 100mL to obtain a 120mM cobalt sulfate solution.

Zinc chloride solution: 1.64g of ZnCl2 is weighed, and the volume is adjusted to 100mL after the dissolution of ultrapure water, namely the zinc chloride solution with the concentration of 120mM is obtained.

The media involved in the following examples are as follows:

LB liquid Medium (g/L): NaCl 10, peptone 10 and yeast extract 5, and adjusting the pH value to 7.0.

Fermentation medium (g/L): glucose 15, yeast extract 20, NaCl 8 and MgSO₄·7H₂O 1.0、Na₂HPO₄·12H₂O 1.0。

Example 1: production of crude enzyme solution containing D-glucose isomerase and crude enzyme solution containing D-psicose 3-epimerase

(1) Preparation of recombinant bacterium for expressing D-glucose isomerase

A PCR fragment of D-glucose isomerase (GI:1750868775) is obtained by using Bacillus megaterium genome as a template and primers P1 and P2 for amplification, the nucleotide sequence of the PCR fragment is SEQ ID NO.2, and a target fragment is recovered from gel after nucleic acid electrophoresis verification. The vector backbone sequence was amplified using P3 and P4 using pUB-P43-dpe-dal (the construction method is described in the Chinese patent document CN 104894047B) plasmid as template, the circular plasmid template was digested with Dpn I, and the vector fragment was purified and recovered.

The two fragments were ligated and transformed into Bacillus subtilis 1A751 (dal) according to In-Fusion cloning technique^-) (construction method is described in the Chinese patent application of CN 104894047B.) competent cells were plated on an antibody-free LB solid plate, and positive clones that were successfully transformed could grow on a normal antibody-free LB plate, whereas plasmid-free alanine racemase (dal) -deficient mutants could not grow in normal medium. Screening positive transformants and carrying out plasmid sequencing to obtain a successfully constructed recombinant strain Bacillus subtilis 1A751 (dal) for producing D-glucose isomerase^-)/pUB-P43-xylA-dal。

TABLE 1 primer sequences of pUB-P43-xylA-dal plasmid

(2) Preparation of recombinant bacterium for producing D-psicose 3-epimerase

According to HE W, MU W, JIANG B, et al.constraction of a food grade recombinant Bacillus subtilis based on regenerative plasmids with an auto-matic marker for biological formation of D-dependent to D-dependent [ J]J agricultural Food Chem,2016,64(16):3243-3250. recombinant bacterium Bacillus subtilis 1A751 (dal) for producing D-psicose 3-epimerase^-)/pUB-P43-dpe-dal。

(3) Preparation of crude enzyme solution

Respectively inoculating the recombinant bacteria producing the D-glucose isomerase and the recombinant bacteria producing the D-psicose 3-epimerase, which are respectively prepared in the step (1) and the step (2), into an LB liquid culture medium, and culturing at 37 ℃ and 200rpm for 12h to obtain seed liquid.

Transferring the prepared seed solution into 10% fermentation medium according to the inoculation amount of 3% (v/v), and fermenting to OD₆₀₀10 to 14; stopping fermentation, collecting thallus, re-suspending thallus, crushing, centrifuging, and filtering to obtain coarse enzyme liquid.

The detection proves that the enzyme activity of the crude enzyme solution of the D-glucose isomerase is as follows: 40U/mL, the enzyme activity of the crude enzyme solution of the D-psicose 3-epimerase is as follows: 300U/mL.

Example 2: method for immobilizing D-glucose isomerase and D-psicose 3-epimerase

The method comprises the following specific steps:

(1) adsorbing double enzymes by using a macroporous carrier:

adding the crude enzyme solution of the D-glucose isomerase and the crude enzyme solution of the D-psicose 3-epimerase prepared in example 1 into 5g of the activated pretreated macroporous cryogel, wherein the enzyme adding amount of the D-glucose isomerase and the enzyme adding amount of the D-psicose 3-epimerase are 800U and 900U respectively to obtain a reaction system, and placing the reaction system at 20 ℃ and the rotating speed of 100rpm in a constant temperature shaking table for oscillation and immobilization for 6 hours.

(2) And (3) crosslinking:

adding glutaraldehyde with the final concentration of 0.01% (v/v) into the immobilized reaction system obtained in the step (1), standing for crosslinking for 4h, centrifuging at 6000rpm and 20 ℃ for 10min, and collecting precipitates to obtain the immobilized double enzyme.

(3) Preparing double-enzyme-inorganic hybrid nano flowers:

5g of the above immobilized double enzyme was prepared in a 250mL Erlenmeyer flask, and 30mL of phosphate buffer (50mM, pH 7.5) was added thereto, followed by 200. mu.L of 120mM magnesium chloride solution to obtain a reaction solution;

and standing the reaction solution at 25 ℃ for 60h, centrifuging at 6000rpm at 20 ℃ for 10min, collecting the precipitate, and drying at the normal temperature of 25 ℃ to obtain the immobilized double-enzyme-inorganic hybrid nano flower.

The result shows that the activity of the immobilized double-enzyme-inorganic hybrid nano-anther is measured as follows: 360U/g carrier;

the operation stability of the immobilized double-enzyme-inorganic hybrid nano flower is detected, and the result shows that after the immobilized double-enzyme-inorganic hybrid nano flower is repeatedly used for 10 times, the enzyme activity is as follows: 212U/g carrier, decrease by 41%.

Example 3: method for immobilizing D-glucose isomerase and D-psicose 3-epimerase

The method comprises the following specific steps:

(1) and (3) crosslinking:

30mL of the crude enzyme solution of D-glucose isomerase and the crude enzyme solution of D-psicose 3-epimerase prepared in example 1 having enzyme activities of 800U and 800U, respectively, were added with a glutaraldehyde solution having a final concentration of 0.6% (v/v) and allowed to stand for crosslinking for 6 hours.

(2) Preparation of resin immobilized double enzymes:

adding 5g of macroporous adsorption resin subjected to activation pretreatment into the crosslinked solution, and carrying out oscillation immobilization for 5h in a constant-temperature shaking table at the rotating speed of 100rpm at the temperature of 20 ℃.

(3) Preparing double-enzyme-inorganic hybrid nano flowers:

and centrifuging the solution after the fixation for 10min at 6000rpm and 20 ℃, collecting the precipitate, adding 30mL of phosphate buffer (50mM and pH 7.0) into the precipitate, then adding 200 mu L of 120mM magnesium chloride solution to obtain a reaction solution, standing the reaction solution for 72h at 25 ℃, then centrifuging for 10min at 6000rpm and 20 ℃, collecting the precipitate, and drying at 25 ℃ to obtain the immobilized double-enzyme-inorganic hybrid nano flower.

The result shows that the activity of the immobilized double-enzyme-inorganic hybrid nano-amylase is 387U/g carrier;

the operation stability of the immobilized double-enzyme-inorganic hybrid nano flower is detected, and the result shows that after the immobilized double-enzyme-inorganic hybrid nano flower is repeatedly used for 10 times, the enzyme activity is as follows: 255U/g carrier, the enzyme activity is reduced by 34 percent.

Example 4: method for immobilizing D-glucose isomerase and D-psicose 3-epimerase

The method comprises the following specific steps:

(1) preparing double-enzyme-inorganic hybrid nano flowers:

to 30mL of phosphate buffer (50mM, pH 7.0), 100. mu.L of 120mM cobalt sulfate solution, and the crude enzyme solutions of D-glucose isomerase and D-psicose 3-epimerase prepared in example 1 were added, wherein the enzyme amounts of D-glucose isomerase and D-psicose 3-epimerase were 900U and 900U, respectively, and the mixture was allowed to stand at 25 ℃ for 60 hours to obtain a double-enzyme-inorganic hybrid nanoflower.

(2) And (3) crosslinking:

glutaraldehyde with the final concentration of 0.2% (v/v) is added into the double-enzyme-inorganic hybrid nano flower solution, and the solution is kept stand for crosslinking for 2 hours.

(3) Adsorbing double enzymes by using a macroporous carrier:

adding the above cross-linked solution into 5g of macroporous adsorbent resin, and adsorbing at 25 deg.C for 10 hr. Centrifuging at 6000rpm and 20 deg.C for 10min, collecting precipitate, and drying at 25 deg.C to obtain double-enzyme-inorganic hybrid nanometer flower.

The result shows that the activity of the immobilized double-enzyme-inorganic hybrid nano-amylase is 306U/g carrier;

the operation stability of the immobilized double-enzyme-inorganic hybrid nano flower is detected, and the result shows that after the immobilized double-enzyme-inorganic hybrid nano flower is repeatedly used for 10 times, the enzyme activity is as follows: 214U/g carrier, decrease by 30%.

Example 5: method for immobilizing D-glucose isomerase and D-psicose 3-epimerase

The method comprises the following specific steps:

(1) adding 5g of macroporous cryogel, the crude enzyme solution of D-glucose isomerase prepared in example 1 with the enzyme activities of 800U and 800U respectively, the crude enzyme solution of D-psicose 3-epimerase, glutaraldehyde with the final concentration of 0.1% (v/v) and 200 μ L of 120mM cobalt sulfate solution into 30mL of phosphate buffer solution (50mM, pH 7.4) to obtain a reaction system;

(2) and standing the reaction system at 25 ℃ for 60h, centrifuging at 6000rpm at 20 ℃ for 10min, collecting precipitate, and drying at 25 ℃ to obtain the immobilized double-enzyme-inorganic hybrid nano flower.

The result shows that the activity of the immobilized double-enzyme-inorganic hybrid nano-anther is measured to be 265U/g carrier;

the operation stability of the immobilized double-enzyme-inorganic hybrid nano flower is detected, and the result shows that after the immobilized double-enzyme-inorganic hybrid nano flower is repeatedly used for 10 times, the enzyme activity is as follows: 188U/g carrier, 29% reduction.

Example 6: method for immobilizing D-glucose isomerase and D-psicose 3-epimerase

The method comprises the following specific steps:

(1) adsorbing double enzymes by using a macroporous carrier:

adding the crude enzyme solution of D-glucose isomerase and the crude enzyme solution of D-psicose 3-epimerase prepared in example 1 to 5g of macroporous adsorption resin subjected to activation pretreatment; the enzyme adding amount of the D-glucose isomerase and the D-psicose 3-epimerase is 700U and 800U respectively, and the immobilized double enzymes are obtained by oscillating and immobilizing for 8 hours in a constant temperature shaking table at the temperature of 20 ℃ and the rotating speed of 100 rpm.

(2) Preparing double-enzyme-inorganic hybrid nano flowers:

5g of the above-described immobilized double enzyme was added to a 250mL Erlenmeyer flask, 30mL of phosphate buffer (50mM, pH 7.5) was added, and 200. mu.L of a 120mM cobalt sulfate solution was added to obtain a reaction system, which was left to stand at 25 ℃ for 72 hours.

(3) And (3) crosslinking:

adding glutaraldehyde with the final concentration of 0.4% (v/v) into the solution after standing in the step (2), standing and crosslinking for 5h at 25 ℃, centrifuging for 10min at 6000rpm and 20 ℃, collecting the precipitate, and drying at normal temperature at 25 ℃ to obtain the immobilized double-enzyme-inorganic hybrid nano flower.

The result shows that the activity of the immobilized double-enzyme-inorganic hybrid nano-anther is 210U/g carrier;

the operation stability of the immobilized double-enzyme-inorganic hybrid nano flower is detected, and the result shows that after the immobilized double-enzyme-inorganic hybrid nano flower is repeatedly used for 10 times, the enzyme activity is as follows: 122U/g carrier, and 42% reduction.

Comparative example 1: immobilization method without adopting double-enzyme-inorganic hybrid nanoflower

The method comprises the following specific steps:

(1) adsorbing double enzymes by using a macroporous carrier:

adding the crude enzyme solution of the D-glucose isomerase and the crude enzyme solution of the D-psicose 3-epimerase prepared in example 1 into 5g of macroporous adsorption resin subjected to activation pretreatment, wherein the enzyme adding amount of the D-glucose isomerase and the enzyme adding amount of the D-psicose 3-epimerase are 700U and 800U respectively, and carrying out oscillation immobilization for 8 hours in a constant temperature shaking table at the rotating speed of 100rpm at the temperature of 20 ℃.

(2) And (3) crosslinking:

adding glutaraldehyde with final concentration of 0.4% (v/v) into the immobilized product, standing at 25 deg.C for crosslinking for 5 hr, centrifuging at 6000rpm and 20 deg.C for 10min, collecting precipitate, and drying at 25 deg.C.

The result shows that the activity of the immobilized double-enzyme-inorganic hybrid nano-anther is 47U/g carrier;

the operation stability of the immobilized double-enzyme-inorganic hybrid nano flower is detected, and the result shows that after the immobilized double-enzyme-inorganic hybrid nano flower is repeatedly used for 10 times, the enzyme activity is as follows: 15U/g carrier, decrease by 68%.

Comparative example 2: preparation of double-enzyme-inorganic hybrid nano flower

The method comprises the following specific steps:

(1) adding the crude enzyme solution of D-glucose isomerase and the crude enzyme solution of D-psicose 3-epimerase prepared in example 1, which have enzyme activities of 700U and 800U, respectively, to a flask containing 50mM phosphate buffer solution with pH of 7.4 to obtain a mixed solution;

(2) and (2) adding 200 mu L of 120mM cobalt sulfate solution into the mixed solution prepared in the step (1), standing at 25 ℃ for 72h, centrifuging at 6000rpm at 20 ℃ for 10min, collecting precipitate, and drying at 25 ℃ to obtain the double-enzyme-inorganic hybrid nano flower.

The result shows that the activity of the immobilized double-enzyme-inorganic hybrid nano-amylase is 180U/g carrier;

the operation stability of the immobilized double-enzyme-inorganic hybrid nano flower is detected, and the result shows that after the immobilized double-enzyme-inorganic hybrid nano flower is repeatedly used for 10 times, the enzyme activity is as follows: 86U/g carrier, decrease 52%.

Comparative example 3: effect of different vectors on immobilized enzymes

(1) Adding 5g of macroporous resin XAD761, the D-glucose isomerase crude enzyme solution prepared in the example 1 with the enzyme activity of 800U and 800U respectively, the D-psicose 3-epimerase crude enzyme solution, glutaraldehyde with the final concentration of 0.1% (v/v) and 200 microliter of 120mM cobalt sulfate solution into 30mL of phosphate buffer solution (50mM, pH 7.4) to obtain a reaction system;

The result shows that the activity of the immobilized double-enzyme-inorganic hybrid nano-amylase is measured to be 120U/g carrier;

the operation stability of the immobilized double-enzyme-inorganic hybrid nano flower is detected, and the result shows that the enzyme activity is 84U/g carrier and is reduced by 30 percent after the immobilized double-enzyme-inorganic hybrid nano flower is repeatedly used for 10 times.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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gctgggcata catttgaaca cgaattacga gtagctagag tacaaggctt cttagggtcc 900

gtggatgcaa accaaggtga ccctctttta ggctgggata cagatgaatt cccaacggat 960

ttatattcta caacgctagc aatgtatgaa attctgcaaa atggagggct gggcagcgga 1020

ggattaaact ttgatgctaa agtacgcaga ggttcttttg aacaagatga tttactctat 1080

gctcatgtag caggaatgga tgcgtttgct cgagggttaa aagtagcaca taaattagta 1140

gaagatcgtg ttttcgaaaa tgttatcaac gagcgctaca gcagctttaa agaaggaatt 1200

gggcttgaga ttgtggaagg aaaagctaac ttccatacac tggagcagta cgcgtttaaa 1260

aatccgaata ttgcgaataa atcaggaaga caagaacgtt taaagtccat tttaaatcaa 1320

tatattttag aagtttaa 1338

<210> 3

<211> 289

<212> PRT

<213> Artificial sequence

<400> 3

Met Lys His Gly Ile Tyr Tyr Ala Tyr Trp Glu Gln Glu Trp Ala Ala

1 5 10 15

Asp Tyr Lys Arg Tyr Val Glu Lys Ala Ala Lys Leu Gly Phe Asp Ile

20 25 30

Leu Glu Val Gly Ala Ala Pro Leu Pro Asp Tyr Ser Ala Gln Glu Val

35 40 45

Lys Glu Leu Lys Lys Cys Ala Asp Asp Asn Gly Ile Gln Leu Thr Ala

50 55 60

Gly Tyr Gly Pro Ala Phe Asn His Asn Met Gly Ser Ser Asp Pro Lys

65 70 75 80

Ile Arg Glu Glu Ala Leu Gln Trp Tyr Lys Arg Leu Phe Glu Val Met

85 90 95

Ala Gly Leu Asp Ile His Leu Ile Gly Gly Ala Leu Tyr Ser Tyr Trp

100 105 110

Pro Val Asp Phe Ala Thr Ala Asn Lys Glu Glu Asp Trp Lys His Ser

115 120 125

Val Glu Gly Met Gln Ile Leu Ala Pro Ile Ala Ser Gln Tyr Gly Ile

130 135 140

Asn Leu Gly Met Glu Val Leu Asn Arg Phe Glu Ser His Ile Leu Asn

145 150 155 160

Thr Ser Glu Glu Gly Val Lys Phe Val Thr Glu Val Gly Met Asp Asn

165 170 175

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

180 185 190

Ile Gly Asp Ala Ile Arg His Ala Gly Lys Leu Leu Gly His Phe His

195 200 205

Thr Gly Glu Cys Asn Arg Met Val Pro Gly Lys Gly Arg Thr Pro Trp

210 215 220

Arg Glu Ile Gly Asp Ala Leu Arg Glu Ile Glu Tyr Asp Gly Thr Val

225 230 235 240

Val Met Glu Pro Phe Val Arg Met Gly Gly Gln Val Gly Ser Asp Ile

245 250 255

Lys Val Trp Arg Asp Ile Ser Lys Gly Ala Gly Glu Asp Arg Leu Asp

260 265 270

Glu Asp Ala Arg Arg Ala Val Glu Phe Gln Arg Tyr Met Leu Glu Trp

275 280 285

Lys

<210> 4

<211> 870

<212> DNA

<213> Artificial sequence

<400> 4

atgaagcatg gtatttatta cgcgtactgg gaacaggaat gggcagcaga ttacaagcgg 60

tatgtagaga aggcggcaaa gcttggattc gatatactgg aggttggcgc ggcgccactg 120

ccggactatt ctgcgcagga ggtaaaggaa ctgaaaaaat gcgccgatga taacggtatc 180

cagctgaccg cgggatatgg tcccgccttc aatcataata tgggttcctc agatccgaag 240

atcagggaag aggcgcttca atggtataaa cgcctgttcg aggtgatggc aggccttgat 300

attcatctga ttggcggagc gctttattca tactggccgg tggactttgc cacagccaat 360

aaggaagagg actggaagca cagcgtggag ggaatgcaga ttctggcgcc catcgccagc 420

cagtatggca tcaatctggg aatggaagtc ctgaaccgct ttgagagcca tatcttaaat 480

acttcggaag aaggcgtgaa gttcgtgacg gaagtaggca tggataatgt gaaagtcatg 540

ctggatacgt tccatatgaa catcgaggaa tcgagcattg gcgacgcgat ccgccatgcc 600

gggaaacttc ttggacactt ccacaccggc gagtgcaacc gcatggtacc cggaaagggc 660

cgcaccccat ggagggagat cggggatgcc ttgcgcgaga ttgagtatga cggaaccgtg 720

gttatggagc catttgtacg catgggcgga caggtaggct ctgatatcaa ggtctggaga 780

gacatcagca agggcgcggg agaggaccgg ctggatgagg atgcaaggcg cgcggtagag 840

ttccagagat acatgcttga atggaagtaa 870

<210> 5

<211> 44

<212> DNA

<213> Artificial sequence

<400> 5

gagaggaatg tacacatggt ccaaactagt actaataaaa ttaa 44

<210> 6

<211> 46

<212> DNA

<213> Artificial sequence

<400> 6

gttccttaag gaattcttaa acttctaaaa tatattgatt taaaat 46

<210> 7

<211> 31

<212> DNA

<213> Artificial sequence

<400> 7

gtgtacattc ctctcttacc tataatggta c 31

<210> 8

<211> 30

<212> DNA

<213> Artificial sequence

<400> 8

gaattcctta aggaacgtac agacggctta 30

Claims

1. A method for immobilizing D-glucose isomerase and D-psicose 3-epimerase, comprising the steps of:

b. adding a cross-linking agent to cross-link the enzyme;

c. adding inorganic metal ions, and assembling with enzyme to form double-enzyme-inorganic hybrid nanoflower.

2. The method of claim 1, wherein the inorganic metal ion is any one of cobalt chloride, magnesium chloride, copper sulfate, cobalt sulfate, and zinc chloride.

3. The method of claim 1 or 2, wherein the macroporous carrier is any one of macroporous adsorption resin, epoxy resin, amino resin, diatomaceous earth, and macroporous cryogel.

4. The method according to any one of claims 1 to 3, wherein the D-glucose isomerase and the D-psicose 3-epimerase are added to the reaction system in the ratio of (2:9) to (9: 2).

5. The method according to any one of claims 1 to 4, wherein the D-glucose isomerase is added in an amount of: 200U-900U; the addition amount of the D-psicose 3-epimerase is as follows: 200U to 900U.

6. The method according to any one of claims 1 to 5, wherein the adsorption time in step a is 2 to 72 hours.

7. The method of any one of claims 1 to 6, wherein the cross-linking agent is glutaraldehyde or polyethylene glycol diglycidyl ether.

8. The method according to any one of claims 1 to 7, wherein in step b, the concentration of the crosslinking agent is 0.01 to 2% and the crosslinking time is 2 to 72 hours.

9. An immobilized D-glucose isomerase and D-psicose 3-epimerase produced by the method according to any one of claims 1 to 8.

10. The method according to any one of claims 1 to 8, or the use of the immobilized D-glucose isomerase according to claim 9 and a D-psicose 3-epimerase for the preparation of a D-psicose-containing or D-psicose-containing product.