D-mannose isomerase immobilized enzyme and preparation method and application thereof

文档序号:3299 发布日期:2021-09-17 浏览:63次 中文

1. A D-mannose isomerase (MIase for short) immobilized enzyme is characterized by comprising an enzyme carrier and MIase.

2. The MIase immobilized enzyme according to claim 1, wherein the enzyme activity mass ratio (U/g) of the MIase to the enzyme carrier is (800-: 1, preferably (1000-.

3. The MIase immobilized enzyme according to claim 1, wherein the enzyme carrier is a macroporous resin; the enzyme carrier and the MIase are combined together in a covalent bond or ion adsorption mode.

4. The MIase immobilized enzyme according to claim 1, wherein the enzyme carrier is a neutral or weakly basic macroporous resin, preferably a neutral macroporous resin.

5. The MIase immobilized enzyme according to claim 1, wherein the enzyme carrier is polyacrylic macroporous resin or polystyrene macroporous resin, preferably polyacrylic macroporous resin.

6. The MIase immobilized enzyme according to claim 1, wherein the enzyme carrier is polyacrylic macroporous resin or polystyrene macroporous resin with hydrophobic skeleton or hydrophilic skeleton, preferably polyacrylic macroporous resin with hydrophilic skeleton.

7. The MIase immobilized enzyme according to claim 1, wherein the enzyme carrier is selected from polyacrylic macroporous resin or polystyrene macroporous resin with epoxy group or amino group; the polyacrylic macroporous resin with epoxy group is preferred.

8. The MIase immobilized enzyme according to claim 1, wherein the enzyme activity of the MIase immobilized enzyme is 250-650U/g, preferably 500-650U/g.

9. A preparation method of an MIase immobilized enzyme is characterized by comprising the following steps:

mixing the MIase enzyme liquid with an enzyme carrier, oscillating by a shaking table to form a mixed liquid, discarding the upper layer liquid of the mixed liquid, and washing the lower layer solid of the mixed liquid, thereby obtaining the MIase immobilized enzyme.

10. The preparation method according to claim 9, wherein the shaking table is oscillated for 10 to 36 hours, preferably 12 to 20 hours;

after the MIase is mixed with an enzyme carrier, the shaking table oscillation reaction temperature is 15-35 ℃, and preferably 20-25 ℃.

11. The method according to claim 9, wherein the MIase immobilized enzyme is the MIase immobilized enzyme of any one of claims 1 to 8.

12. A preparation method of D-mannose is characterized by comprising the following steps:

continuously converting the fructose aqueous solution in a reactor filled with an MIase immobilized enzyme to obtain a solution containing D-mannose;

the MIase immobilized enzyme is the MIase immobilized enzyme of any one of claims 1-8;

preferably, the solution containing D-mannose is subjected to continuous chromatographic separation, concentration, decoloration and purification to obtain the D-mannose with the purity of more than 99%.

Background

D-mannose is an aldose isomer of D-fructose, and is also an epimer of the C-2 position of D-glucose. Its sweetness is about 57% -72% of sucrose and the calorie generated per gram is about half of glucose. In nature, D-mannose is often present in plant tissues in the form of mannans or other polymers.

In the human body, D-mannose is distributed in body fluid and tissues, is not easily metabolized, and is involved in immune regulation and glycoprotein synthesis in the human body. D-mannose has an important role in the health of the human body because it has various physiological effects such as promotion of wound healing, inhibition of cancer cell survival and tumor growth, prevention of bacterial infection of the urinary tract system, promotion of immune system regulation, anti-inflammatory action, and the like.

At present, the application of D-mannose is widely reported. In the food industry, D-mannose is often used as a sweet additive in health care products and beverages; in medicine, D-mannose can be used as a raw material to synthesize an immunostimulant and an anti-tumor related medicine with higher value; in the breeding industry, the D-mannose can be used in feed to prevent the broiler from being infected by salmonella so as to improve the egg yield; in chemical synthesis, D-mannose can be used as a raw material to synthesize various derivatives, such as mannose triflate, L-ribose and the like. The wide application of the D-mannose enables the D-mannose to have good economic value and market prospect.

The preparation method of D-mannose mainly comprises an extraction method, a chemical synthesis method and an enzyme conversion method. The extraction method usually uses D-mannose-rich polysaccharide such as palm seed, oligosaccharide, mannan, etc. as raw material, and the D-mannose is obtained by hydrolysis, separation and purification; the chemical synthesis method comprises epimerizing D-glucose to form D-mannose under the action of chemical catalyst of hexavalent molybdate, or increasing carbon chain with D-arabinose to obtain D-mannose, or oxidizing D-mannitol (byproduct of iodine production from herba Zosterae Marinae) with hydrogen peroxide in the presence of ferrous ion; the enzymatic conversion method converts glucose or fructose into D-mannose by using isomerase derived from a microorganism, and it has also been reported that formaldehyde, glycerol, maltodextrin, sucrose and the like can be converted into D-mannose by constructing a multi-enzyme cascade reaction system. The enzymatic conversion method has the advantages of mild reaction conditions, wide raw material sources, simple process steps and the like, and has great development value and application potential.

At present, four microbial enzymes are reported in the literature to be transformed to produce D-mannose, including D-lyxose isomerase (LIase, EC 5.3.1.15), D-mannose isomerase (MIase, EC5.3.1.7), cellobiose 2-isomerase (CEase, EC 5.1.3.11) and D-mannose 2-isomerase (MEAase, EC 5.1.3. -).

Enzymatic conversion processes are further classified into free enzyme (including whole cell, crude enzyme or purified enzyme) conversion processes and immobilized enzyme conversion processes. The free enzyme conversion method adopts a batch conversion mode, the enzyme can be used only once, and the utilization rate of the enzyme is extremely low; the immobilized enzyme conversion method usually adopts a continuous conversion mode and also can adopt a cyclic conversion mode of periodically updating a substrate, so that the utilization rate of the enzyme can be effectively improved, and the use cost of the enzyme can be greatly reduced.

The preparation of immobilized enzymes for preparing D-mannose reported in the literature at present mainly takes chitosan and anionic resin as immobilized carriers, or takes diatomite carriers for integral cell immobilization. US6897047 discloses a preparation method of immobilized MIase using chitosan as carrier, acetic acid and a large amount of NaOH are consumed in the carrier treatment process, the enzyme activity half-life period at 55 ℃ is about 10 months, and the conversion rate is stabilized at 23.1-24.0% within 25 days of continuous conversion under the conditions that the fructose substrate concentration is 20% and the flow rate is 0.067-0.01 BV/h; the half-life period of the enzyme activity at 60 ℃ is about 2-3 months, and the conversion rate is stabilized at 19.4-22.5% within 25 days of continuous conversion. Parkcs et al [ Biotechnol Lett (2010)32: 1305-. US5240717 discloses a method for preparing D-mannose by using diatomite (No. R-620) as a carrier and by using an integral cell immobilized enzyme, fructose substrate concentration is 30%, the fructose substrate is continuously converted at 45 ℃ under the condition of flow rate of 0.1BV/h, the concentration of the D-mannose in a conversion solution reaches 68mg/ml, the conversion rate is 22.67%, and after 7 days, the concentration of the D-mannose in the conversion solution is 65mg/ml, and the conversion rate is 21.67%. The methods have the problems of low substrate concentration (20-30%), low flow rate (0.067-0.1 BV/h), low conversion rate (19.4-24.0%) and the like during continuous conversion or cyclic conversion.

Disclosure of Invention

Aiming at the defects and shortcomings of the prior art, the MIase immobilized enzyme and the preparation method and application thereof are provided, the MIase immobilized enzyme adopts macroporous resin as a carrier, the MIase and the macroporous resin are combined together in a covalent bond or ion adsorption mode, the MIase immobilized enzyme has good stability and enzyme catalytic activity, is continuously converted for 90 days at 45 ℃ under the conditions of flow rate of 0.4BV/h and substrate concentration of 750g/L, the concentration of D-mannose in a conversion solution is stabilized to be more than 200g/L, and the conversion rate is stabilized to be more than 27%, and compared with the prior art, the enzyme conversion efficiency is greatly improved. In addition, the preparation process of the MIase immobilized enzyme is simple, and no toxic organic solvent is added in the process, so that the preparation method belongs to an environment-friendly green preparation process.

The present application provides the following technical solutions.

1. An MIase immobilized enzyme is characterized by comprising an enzyme carrier and MIase.

2. The MIase immobilized enzyme according to item 1, wherein the enzyme activity mass ratio (U/g) of the MIase to the enzyme carrier is (800-: 1, preferably (1000-.

3. The MIase immobilized enzyme of item 1, wherein the enzyme carrier is a macroporous resin; the enzyme carrier and the MIase are combined together in a covalent bond or ion adsorption mode.

4. The MIase immobilized enzyme according to item 1, wherein the enzyme carrier is a neutral or weakly basic macroporous resin, preferably a neutral macroporous resin.

5. The MIase immobilized enzyme according to the item 1, wherein the enzyme carrier is polyacrylic macroporous resin or polystyrene macroporous resin, preferably polyacrylic macroporous resin.

6. The MIase immobilized enzyme according to the item 1, wherein the enzyme carrier is polyacrylic macroporous resin or polystyrene macroporous resin with a hydrophobic skeleton or a hydrophilic skeleton, preferably polyacrylic macroporous resin with a hydrophilic skeleton.

7. The MIase immobilized enzyme according to claim 1, wherein the enzyme carrier is selected from polyacrylic macroporous resin or polystyrene macroporous resin with epoxy group or amino group; the polyacrylic macroporous resin with epoxy group is preferred.

8. The MIase immobilized enzyme according to the item 1, which is characterized in that the enzyme activity of the MIase immobilized enzyme is 250-650U/g, preferably 500-650U/g.

9. A preparation method of an MIase immobilized enzyme is characterized by comprising the following steps:

mixing the MIase enzyme liquid with an enzyme carrier, oscillating by a shaking table to form a mixed liquid, discarding the upper layer liquid of the mixed liquid, and washing the lower layer solid of the mixed liquid, thereby obtaining the MIase immobilized enzyme.

10. The method according to claim 9, wherein the shaking table is shaken for 10 to 36 hours, preferably 12 to 20 hours.

11. The method according to claim 9, wherein the temperature of the shaking table oscillation reaction after the MIase is mixed with the enzyme carrier is 15 to 35 ℃, preferably 20 to 25 ℃.

12. The method according to claim 9, wherein the MIase immobilized enzyme is the MIase immobilized enzyme according to any one of items 1 to 8.

13. A preparation method of D-mannose is characterized by comprising the following steps:

continuously converting the fructose aqueous solution in a reactor filled with an MIase immobilized enzyme to obtain a solution containing D-mannose;

the MIase immobilized enzyme is the MIase immobilized enzyme described in any one of items 1-8.

14. The process according to item 13, wherein the D-mannose-containing solution is subjected to continuous chromatographic separation, concentration, decolorization and purification to obtain D-mannose having a purity of 99% or more.

The application provides an MIase immobilized enzyme adopts macroporous resin as the carrier, and MIase combines together through covalent bond or ionic adsorption's mode with macroporous resin to form the MIase immobilized enzyme that has good stability and enzyme catalytic activity, and macroporous resin belongs to the material of manual synthesis moreover, and the physical properties and the chemical properties of this carrier are stable, and corrosion resistance is strong, and resin material intensity itself is great, is difficult for appearing warping or damaging, makes the MIase immobilized enzyme can continuous cycle use, has effectively improved the utilization ratio of enzyme, expects to reduce manufacturing cost by a wide margin.

Drawings

The drawings are included to provide a further understanding of the application and are not to be construed as limiting the application. Wherein:

FIG. 1 is a schematic diagram showing the change of fructose conversion rate in MIase conversion under different substrate concentrations;

FIG. 2 is a schematic diagram showing the change of fructose conversion rate in continuous conversion of the MIase immobilized enzyme prepared from the enzyme vector CZ01 under the condition of substrate concentration of 750 g/L.

Detailed Description

The following description of the exemplary embodiments of the present application, including various details of the embodiments of the present application to assist in understanding, should be taken as exemplary only. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the present application. Also, descriptions of well-known functions and constructions are omitted in the following description for clarity and conciseness.

The application provides an MIase immobilized enzyme, which comprises an enzyme carrier and an MIase.

In the application, the enzyme activity mass ratio (U/g) of the MIase to the enzyme carrier is (800-2500): 1, preferably (1000-;

the enzyme activity mass ratio (U/g) of the MIase to the enzyme carrier can be 800: 1. 850: 1. 900: 1. 950: 1. 1000: 1. 1100: 1. 1200: 1. 1300, and (2): 1. 1400: 1. 1500: 1. 1600: 1. 1700: 1. 1800: 1. 1900: 1. 2000: 1. 2100: 1. 2200: 1. 2300: 1. 2400: 1 or 2500: 1.

in the present application, the enzyme support is a neutral or weakly basic macroporous resin, preferably a neutral macroporous resin.

In the present application, the enzyme carrier is polyacrylic macroporous resin or polystyrene macroporous resin, preferably polyacrylic macroporous resin.

In the present application, the enzyme carrier may be a polyacrylic macroporous resin with a hydrophobic skeleton or a hydrophilic skeleton; the enzyme carrier can also be polystyrene macroporous resin with a hydrophobic skeleton or a hydrophilic skeleton, and preferably polyacrylic macroporous resin with a hydrophilic skeleton.

The enzyme carrier may have one or more of the following groups: carboxylic acid group, hydroxyl group, sulfonic acid group, phosphoric acid group, amino group, ammonium group, hydrocarbon group, ester group, polyoxypropylene group, long-chain perfluoroalkyl group, polysiloxane group.

In the present application, the enzyme carrier may be polyacrylic macroporous resin or polystyrene macroporous resin with epoxy group or amino group; the polyacrylic macroporous resin with epoxy group is preferred. Such as MC-150EP, ECR-1604, etc., from Hangzhou Chuangke Biotechnology Ltd.

Active functional groups on the surface of the epoxy macroporous resin can perform a multi-point covalent bond or ion adsorption combined reaction with amino, sulfydryl, phenolic hydroxyl and other unnecessary groups on an enzyme molecule under a mild condition, so that the enzyme is immobilized on a resin carrier, and the immobilized enzyme with good stability and enzyme catalytic activity is formed.

Before use, aldehyde groups can be formed on the surface of the amino macroporous resin by activating with glutaraldehyde, and the aldehyde groups react with amino groups on the surface of enzyme molecules to form Schiff base, so that firm multipoint covalent bonds or ion adsorption combined sites are generated, and the immobilized enzyme with good stability is formed.

In the application, the enzyme activity of the MIase immobilized enzyme is 250-650U/g, preferably 500-650U/g.

The enzyme activity of the MIase immobilized enzyme can be 250U/g, 300U/g, 350U/g, 400U/g, 450U/g, 500U/g, 550U/g, 600U/g and 650U/g.

The application also provides a preparation method of the MIase immobilized enzyme, which comprises the following steps:

the method comprises the following steps: preparation of the enzyme solution of MIase: fermenting and culturing a strain containing the MIase gene, centrifuging to collect thalli cells, resuspending the thalli by Phosphate Buffer Solution (PBS) with pH7.5, crushing the cells by a high-pressure homogenizer, and centrifuging to obtain supernatant, namely the MIase enzyme solution.

The strain can be any strain containing MIase gene of Escherichia coli, Pseudomonas, Agrobacterium, Streptomyces, Thermoactinomyces, Corynebacterium glutamicum, Bacillus licheniformis, Bifidobacterium thermophilum, Pseudomonas saccharophila, Salmonella, lactobacillus, Saccharomyces cerevisiae, etc.

Step two: mixing an MIase enzyme solution with an enzyme carrier, oscillating by a shaker to fix the MIase on the enzyme carrier, oscillating by the shaker to form a mixed solution, standing and layering, discarding the upper layer liquid of the mixed solution, slowly stirring and rinsing the solid at the lower layer of the mixed solution by deionized water with the volume of 1.5-2.0 times that of the enzyme carrier, repeatedly rinsing the solid for multiple times until the upper layer liquid is clarified, and removing the supernatant, wherein the solid at the lower layer is the MIase immobilized enzyme.

The enzyme activity of the MIase enzyme solution is 80U/ml-500U/m, preferably more than 200U/ml. Namely, the enzyme activity in 1ml of the MIase enzyme solution is 80U-500U, preferably more than 200U.

The enzyme activity of the MIase enzyme solution can be 80U/ml, 90U/ml, 100U/ml, 110U/ml, 120U/ml, 130U/ml, 140U/ml, 150U/ml, 160U/ml, 170U/ml, 180U/ml, 190U/ml, 200U/ml, 210U/ml, 220U/ml, 230U/ml, 240U/ml, 250U/ml, 260U/ml, 270U/ml, 280U/ml, 290U/ml, 300U/ml, 310U/ml, 320U/ml, 330U/ml, 340U/ml, 350U/ml, 360U/ml, 370U/ml, 380U/ml, 390U/ml, 400U/ml, 410U/ml, 420U/ml, 430U/ml, B/ml, C/ml, 440U/ml, 450U/ml, 460U/ml, 470U/ml, 480U/ml, 490U/ml, 500U/ml.

In the present application, the shaking table is oscillated for 10 to 36 hours, preferably 12 to 20 hours.

The shaking table may be oscillated for one of 10h, 12h, 15h, 20h, 25h, 30h, 35h and 36 h.

In the application, after the MIase enzyme solution is mixed with an enzyme carrier, the temperature of shaking table oscillation reaction is 15-35 ℃, and preferably 20-25 ℃.

The temperature of the shaking table oscillation reaction may be one of 15 ℃, 20 ℃, 25 ℃, 30 ℃ and 35 ℃.

In the present application, the number of times of washing the lower layer solid of the mixed solution is 2 to 8 times, preferably 3 to 5 times.

The number of times of washing the lower layer solid of the mixed solution may be one of 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, and 8 times.

The application also provides a preparation method of the D-mannose, which comprises the following steps:

the method comprises the following steps: and (3) continuously converting the fructose aqueous solution in a reactor filled with a MIase immobilized enzyme to obtain a solution containing D-mannose.

The concentration of the fructose solution can be 400g/L, 500g/L, 600g/L, 700g/L and 750 g/L.

Step two: and the solution containing the D-mannose is subjected to continuous chromatographic separation, concentration, decoloration and purification to obtain the D-mannose with the purity of more than 99 percent.

The steps of continuous chromatographic separation, concentration, decolorization, purification and the like are prior art and are not specifically limited in the application.

And (3) measuring the enzyme activity of the MIase enzyme solution: taking 0.9ml of fructose solution with the concentration of 400g/L, adding 0.1ml of 10-time diluted MIase enzyme solution, reacting for 5 minutes in a constant-temperature water bath at 45 ℃, boiling for 5 minutes to inactivate enzyme, centrifuging, taking supernate, diluting for 10 times, and detecting by using High Performance Liquid Chromatography (HPLC).

Enzyme activity determination of the MIase immobilized enzyme: taking 5ml of fructose solution with the concentration of 400g/L, adding 0.1-0.5 g of MIase immobilized enzyme filtered by filter paper, reacting for 5min at constant temperature of 45 ℃ in a water bath shaker at 120rpm, absorbing 1ml of supernatant solution, boiling in boiling water for 5min to inactivate enzyme, centrifuging, taking the supernatant, detecting by using HPLC, and calculating the enzyme activity.

The unit of enzyme activity U is defined as: the amount of enzyme required to produce 1. mu. mol of D-mannose per unit time at a constant temperature of 45 ℃.

The HPLC detection conditions were as follows: agilent 1260 high performance liquid chromatography system, Waters corporation; AminexHPX-87N chromatography columns, Waters; an Agilent differential refraction detector; column temperature: 80 ℃, flow rate: 1 mL/min; pure water was used as a mobile phase, which was filtered through a 0.22 μm pore size cellulose acetate membrane and degassed by ultrasound.

The MIase immobilized enzyme provided by the application adopts macroporous resin as a carrier, the MIase and the macroporous resin are combined together in a covalent bond or ion adsorption mode to form the MIase immobilized enzyme with good stability and enzyme catalytic activity, when the enzyme activity mass ratio of the MIase to the enzyme carrier is in the range of 800-2500:1, the prepared MIase immobilized enzyme has high enzyme activity and is continuously converted for 90 days, the concentration of D-mannose in a conversion solution is stabilized to be more than 200g/L, and the conversion rate is stabilized to be more than 27%.

Examples

The experimental methods used in the following examples are all conventional methods, unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of enzyme solution for MIase

Selecting recombinant Escherichia coli containing an MIase gene sequence to an LB liquid culture medium containing 50 mu g/ml kanamycin, and culturing at 37 ℃ and 200rpm for 8-10 h; then inoculating the activated bacterial liquid into a 5L fermentation tank filled with 3.5LLB culture medium in an inoculation amount of 3%, controlling the culture temperature to be 37 ℃, the stirring speed to be 300rpm, and the ventilation ratio to be 1: 0.5, when the OD value reaches 0.6-0.8, adding IPTG with final concentration of 0.4mmol/L, adjusting the rotating speed to 100rpm, and inducing and culturing for 15-16 h. Then, the cells were collected by centrifugation at 8000rpm, resuspended in Phosphate Buffer (PBS) having pH7.5, disrupted by a high pressure homogenizer, and centrifuged at 4 ℃ and 10000rpm to remove cell debris. And purifying the MIase in the supernatant by an ammonium sulfate fractional sedimentation method to obtain a primarily purified MIase enzyme solution, and sampling to determine the MIase enzyme activity.

Example 2 selection of substrate concentration for MIase conversion

Weighing 40g, 50g, 60g, 70g and 75g of fructose respectively, placing the fructose in a triangular flask, adding 3000U of purified MIase enzyme solution, adding water to a constant volume of 100ml, preparing enzyme conversion reaction solutions with substrate concentrations of 400g/L, 500g/L, 600g/L, 700g/L and 750g/L respectively, carrying out shaking reaction in a water bath at 45 ℃, and sampling at regular time to detect the D-mannose concentration, wherein the results show that (shown in figure 1) after the enzyme conversion reactions reach equilibrium, the average conversion rate of the D-mannose can reach over 27 percent, and comprehensively considering that 750g/L of fructose substrate concentration can be selected to carry out the enzyme conversion reaction of the MIase.

Example 3 preparation of MIase immobilized enzyme

Selecting the enzyme activity mass ratio (U/g) of the MIase to the enzyme carrier to be 1000: respectively weighing 5g of 6 enzyme carriers, placing the 6 enzyme carriers in a triangular flask, adding 5000U of MIase liquid according to the enzyme activity detection result of the MIase liquid, uniformly mixing, sealing a bottle mouth, slowly oscillating for 20 hours in a shaking table at 25 ℃, discarding the upper layer liquid, fully washing the lower layer solid by using about 20ml of deionized water, discarding a washing liquid, repeatedly washing for 3-5 times, thus obtaining the MIase immobilized enzyme, and sampling to detect the enzyme activity of the MIase immobilized enzyme. As shown in table 1, by comparing the enzyme activities of the MIase immobilized enzyme, the neutral hydrophilic epoxy polyacrylic acid macroporous resin is superior to the neutral hydrophobic epoxy polyacrylic acid macroporous resin, the weakly alkaline hydrophobic amino polystyrene macroporous resin, and the weakly alkaline hydrophilic amino polyacrylic acid macroporous resin.

TABLE 1 comparison of enzyme activities of MIase immobilized enzymes of different enzyme carriers

Example 4 enzyme Activity Mass ratio of MIase to enzyme Carrier is preferred

According to the result of the example 2, 5g of carriers CZ01, CZ02, CZ03 and CZ04 are respectively weighed and placed in a triangular flask, and according to the enzyme activity detection result of an MIase solution, the weight ratio (U/g) of enzyme activity carriers is 800: 1. 1000: 1. 1500: 1. 2000: 1. 2500:1, calculating and adding the MIase enzyme liquid of the corresponding enzyme activity unit, uniformly mixing, sealing a bottle opening, preparing the MIase immobilized enzyme, wherein the immobilization conditions, the washing method and the washing times are the same as those in the embodiment 3, and sampling to detect the enzyme activity of the MIase immobilized enzyme. As shown in Table 2, it is understood from Table 2 that, when the enzyme activity mass ratio of the MIase to the enzyme carrier is in the range of (1000-2000):1, the higher the ratio of the MIase to the enzyme carrier, the higher the enzyme activity of the MIase immobilized enzyme, but when the ratio of the MIase to the enzyme carrier is from 2000: 1 is increased to 2500:1, the enzyme activity of the MIase immobilized enzyme is not obviously improved any more, which shows that when the enzyme activity mass ratio of the MIase to an enzyme carrier is more than 2000U/g, the combination of active functional groups on the carrier and enzyme molecules tends to be saturated, the enzyme activity of the immobilized enzyme is not obviously increased any more, when the enzyme activity mass ratio of the MIase to the enzyme carrier is less than 1000U/g, the combination efficiency of the active functional groups on the carrier and the MIase enzyme molecules is low, the carrier is not fully utilized, the enzyme activity of the MIase immobilized enzyme is low, and the preferable enzyme activity mass ratio (U/g) of the MIase to the enzyme carrier is (1000-2000): 1.

TABLE 2 enzyme activity/mass ratio of MIase to enzyme carrier is preferred

Example 5 continuous transformation of the MIase immobilized enzyme

100ml of MIase immobilized enzyme prepared from an enzyme carrier CZ01 is taken and put into a glass reactor with a sleeve, the temperature of the sleeve is controlled to be 45 ℃, the fructose concentration of 750g/L is selected, the flow rate of 40ml/h is controlled, the MIase immobilized enzyme is continuously converted, after the reaction is balanced, the D-mannose concentration and the conversion rate are sampled and detected at regular time until the conversion rate is obviously reduced, and the continuous conversion reaction time is calculated.

As shown in FIG. 1, the MIase immobilized enzyme prepared from the enzyme vector CZ01 was continuously transformed for 90 days, the average fructose conversion rate reached 27.15%, and the concentration of D-mannose in the transformed solution reached 200g/L or more. The obtained conversion solution is subjected to continuous chromatographic separation, concentration, decoloration, purification and other steps to obtain the D-mannose with the purity of more than 99 percent.

While embodiments of the present application have been described above in connection with specific embodiments thereof, the present application is not limited to the above-described embodiments and fields of application, which are intended to be illustrative, instructive, and not restrictive. Those skilled in the art, having the benefit of this disclosure, may effect numerous modifications thereto and changes may be made without departing from the scope of the invention as defined by the appended claims.

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