CN114058611A - Immobilization of mannase and application in enzyme reactor - Google Patents

Immobilization of mannase and application in enzyme reactor Download PDF

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CN114058611A
CN114058611A CN202111222100.5A CN202111222100A CN114058611A CN 114058611 A CN114058611 A CN 114058611A CN 202111222100 A CN202111222100 A CN 202111222100A CN 114058611 A CN114058611 A CN 114058611A
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enzyme
immobilized
mannanase
immobilization
reaction
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刘力强
张伟
谷雅文
曹毅
武建
刘燕静
袁江宏
袁润
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Chengde Kangerrun Food Co ltd
Hebei University of Science and Technology
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Chengde Kangerrun Food Co ltd
Hebei University of Science and Technology
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/2488—Mannanases
    • C12N9/2491—Beta-mannosidase (3.2.1.25), i.e. mannanase
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    • C12P19/00—Preparation of compounds containing saccharide radicals
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01025—Beta-mannosidase (3.2.1.25), i.e. mannanase

Abstract

The invention discloses immobilization of mannase and application of the mannase in an enzyme reactor. Taking materials according to the mass ratio of the mannase to the carrier of 1:1, adding 0.1M phosphate buffer solution with pH value of 5, and carrying out immobilization reaction at 25 ℃ for 24 h. According to the invention, a resin immobilized carrier is selected to carry out immobilization treatment on mannase, and the optimal immobilization condition is inspected; the immobilized enzyme is used for preparing the enzyme reactor, and the reactor is used for preparing the mannan oligosaccharide, so that the enzyme is repeatedly utilized (more than 15 batches), the defect of high viscosity of reaction substrates is overcome, and a technical basis is laid for reducing the production cost of the mannan oligosaccharide.

Description

Immobilization of mannase and application in enzyme reactor
Technical Field
The invention belongs to the technical field of preparation of mannan-oligosaccharide, and particularly relates to immobilization of mannase and application of mannase in an enzyme reactor.
Background
The mannooligosaccharides are oligosaccharides formed by connecting 2-10 mannoses through beta-1, 4 glycosidic bonds, are novel antigen active substances extracted from yeast culture cell walls at the earliest, and are widely present in konjac flour, guar gum, sesbania gum and cell walls of various microorganisms. The mannan oligosaccharide has the characteristics of stable property, low calorie, no initiation of decayed tooth, blood sugar reduction and the like, so the mannan oligosaccharide is widely applied to the food industry as a new generation functional food ingredient.
At present, mannan oligosaccharide is prepared from mannan such as konjac flour, locust bean gum, coffee grounds and the like. The preparation method is an enzymatic decomposition method, a chemical method, a physical method and the like, and has already begun to be widely applied due to the advantages of low pollution, mild reaction and the like of the enzymatic preparation. However, since the liquid enzyme reaction can not be recycled, high-temperature treatment is mostly adopted when the liquid enzyme is removed, and the treatment affects the chromaticity of the product; meanwhile, the cost of liquid enzyme is high, and the factors limit the development of preparing the mannan oligosaccharide by an enzyme method. To solve this problem, researchers have conducted immobilized mannanase studies. Patent CN105274163A provides a method for preparing mannose by mixing black tea leaves residue with trichoderma reesei spores, which is complex to prepare and does not refer to reaction batches; the patent CN105524911A provides a method for preparing immobilized mannase by utilizing a nanofiber material for preparing mannan oligosaccharide, the method uses the immobilized enzyme to react with a substrate in a mixing way, the separation of the substrate and the enzyme is complex, and no actual use batch is mentioned, so that the advantages of the immobilized enzyme cannot be embodied; the patent CN101492667A provides a method for immobilizing enzyme-producing microbial cells by using a microporous filter membrane, the immobilized cells are used for carrying out 4-8 times of continuous reactions, and the relative enzyme activity is reduced from the initial 200% to 50%, which greatly limits the practical application; patent CN102586219A provides a method for preparing immobilized mannanase by using adsorption macroporous resin and applying to the degradation of aloe polysaccharide, but the method also does not mention the application in polysaccharide such as guar gum, and does not mention the use of batch.
Disclosure of Invention
The invention aims to provide the immobilization of mannanase and application of the mannanase in an enzyme reactor.
An immobilized mannanase is prepared by using LX-103B, LX-1000HA (A), LX-HFA001, LX1000HFA, LX-1000EA, LKZ126, LKZ618 ES-101 and ESR-1 as an immobilized carrier, preferably LX-HFA001 as an enzyme immobilized carrier.
According to the preparation method of the immobilized mannase, materials are taken according to the mass ratio of the mannase to the carrier of 1:1, 0.1M phosphate buffer solution with pH value of 5 is added, and immobilization reaction is carried out for 24h at 25 ℃.
The addition amount of the phosphate buffer solution is 20-30 times of the mass of the mannase and the carrier.
Preferably, the addition amount of the phosphate buffer is 25 times of the mass of the mannanase and the carrier.
The application of the immobilized mannanase in an enzyme reactor.
The mannan-oligosaccharide is prepared by performing a circulating reaction on guar gum with the concentration of 1 percent and the pH value of 5.5 at the temperature of 45 ℃ and the flow rate of 20mL/min of immobilized mannase liquid (reaction liquid) for 6 hours, wherein the reaction batch can reach more than 15 batches.
The invention has the beneficial effects that: according to the invention, an immobilized carrier LX-HFA001 is selected to carry out immobilization treatment on mannase, and the optimal immobilization condition is inspected; the immobilized enzyme is used for preparing the enzyme reactor, and the reactor is used for preparing the mannooligosaccharide, so that the enzyme is recycled, the defect of high viscosity of a reaction substrate is overcome, and a technical foundation is laid for reducing the production cost of the mannooligosaccharide.
Drawings
FIG. 1 is a standard curve of mannooligosaccharides.
FIG. 2 shows the effect of enzyme activity of immobilized enzyme mannanase on different carriers.
FIG. 3 shows the effect of different buffer pH on enzyme activity of immobilized enzyme mannanase.
FIG. 4 shows the effect of different temperatures on the enzymatic activity of immobilized enzyme mannanase.
FIG. 5 shows the effect of different enzyme dosages on the enzyme activity of immobilized enzyme mannanase.
FIG. 6 shows the effect of different immobilization times on the enzymatic activity of immobilized enzyme mannanase.
FIG. 7 is an immobilized enzyme reactor scheme.
FIG. 8 shows the concentration change of mannan oligosaccharide prepared by enzymolysis in an enzyme reactor.
FIG. 9 is a graph showing the effect of reaction temperature on the preparation of mannooligosaccharides.
FIG. 10 is a graph showing the effect of reaction pH on the preparation of mannooligosaccharides.
FIG. 11 is a graph showing the effect of substrate concentration on the production of mannooligosaccharides.
FIG. 12 shows the enzyme activity change of enzyme reactor reaction batches.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The solutions used in the following examples were prepared as follows:
acetic acid solution: accurately weighing 0.6mL of acetic acid solution, and adding deionized water to dilute to 100 mL.
Sodium acetate solution: 1.36g of sodium acetate trihydrate crystals are accurately weighed and dissolved to 100mL by adding deionized water.
Acetic acid-sodium acetate buffer solution: 23.14g of sodium acetate trihydrate crystals are accurately weighed, 1.7mL of glacial acetic acid solution is added, deionized water is added to dissolve the crystals to 2000mL, and the volume is determined by a measuring cylinder. The pH of the solution was measured, and when the pH deviated from 5.5, the pH was adjusted to 5.5 with the above acetic acid solution or sodium acetate solution.
Preparation of 1mg/mL mannose stock solution: 1g D-mannose is accurately weighed, and a small amount of acetic acid-sodium acetate buffer solution is added for dissolution to obtain a constant volume of 1L.
Preparation of a substrate: weighing 0.6000g of guar gum, accurately weighing to 0.001g, adding about 70mL of acetic acid-sodium acetate buffer solution, magnetically mixing and heating at intervals until the guar gum is completely dissolved, continuously stirring for 15 minutes, metering to 100mL in a measuring cylinder, putting into a beaker, wrapping with tinfoil all around, shading, and storing in a refrigerator at 4 ℃.
0.05M phosphate, weighing NaH2PO4，0.78g，Na2HPO41.79g, dissolved in 100mL of deionized water and adjusted to pH7 with phosphoric acid.
0.1M phosphate, weighing NaH2PO4，1.56g，Na2HPO43.58g of each of the two salts was dissolved in 100mL of deionized water and the pH was adjusted to 7.
Preparing 0.2M phosphate, weighing NaH2PO4，0.12g，Na2HPO40.44g, dissolved in 100mL of liquid enzyme, and adjusted to pH 7.
The enzyme activity determination method comprises the following steps:
determination of mannan standard: standard samples were typically assayed as D-mannose standard solutions diluted to 0.1-0.7mg/mL according to the following data.
TABLE 1 determination of the Standard Curve
Figure BDA0003312981240000041
Each D-mannose absorbs 200 mul, 200 mul of acetic acid-sodium acetate solution and 500 mul of LDNS reagent are added, the mixture is heated for 5min under the condition of boiling water bath, then the mixed solution is rapidly cooled to room temperature by flowing cold water, deionized water is added, the volume is determined to be 2.5mL, the mixture is shaken gently and mixed evenly, the control solution is used for zero setting under the condition of 540nm of a spectrophotometer, and the light absorption value is prepared to be measured. In the prepared data chart, a linear regression equation is obtained by taking the D-mannose concentration as a Y axis and the absorbance value as an X axis, a standard curve is drawn, the obtained standard curve is shown in figure 1, the absorbance value and the mannan concentration are linearly correlated, the area formula Y of the correlated mannan concentration and the absorbance value is 0.8026X +0.0946, and the variance is 0.9984, which indicates that the mannan standard curve is available, and the linear regression equation formula can be applied to the calculation of the final concentration determination of the mannan oligosaccharide solution of the sample.
60mL of substrate was taken and placed in a water bath at 37 ℃ for 10 minutes. Weighing about 0.5g of immobilized enzyme, adding 20mL of substrate balanced at 37 ℃, preserving heat at 37 ℃ and slightly vibrating by hand for 15 minutes, putting 200 mu L of supernatant into a 10mL centrifuge tube, adding 200 mu L of acetic acid-sodium acetate buffer solution, adding 500 mu L of DNS reagent, preparing to heat for five minutes under the condition of boiling water bath, taking out, rapidly cooling to room temperature under cold water impact, adding 1.6mL of deionized water, and uniformly mixing.
Preparing a blank solution: 200 mu L of substrate is taken and placed in a centrifuge tube with the capacity of 10mL, 200 mu L of acetic acid-sodium acetate buffer solution is firstly added, 500 mu L of DNS reagent is then added, the mixture is heated for five minutes in a boiling water bath, rapidly cooled to the room temperature and uniformly mixed with 1.6mL of deionized water.
And (3) measuring enzyme activity: and (3) adjusting the reagent blank solution to zero by using a spectrophotometer, measuring the absorbance of the solution by using a cuvette at 540nm, recording an accurate OD value, and calculating the enzyme activity according to the following formula.
XD＝[「(A-A0)*k]+b/(t*M)]*1000
m＝0.2×m0/20
X＝XD×1000×n/(15×m)
In the formula:
a, the absorbance of the sample tube; a. the0Absorbance of the sample blank tube; k is the slope of the standard curve; b intercept of standard curve(ii) a X is enzyme activity U/mL or U/g; xD: the corresponding concentration of mannan found by a standard curve is unit mg/mL; 1000: a unit conversion factor; n: dilution times; t: enzymolysis reaction time in unit of min; m: the volume of enzyme liquid corresponding to 200 mu L of reaction liquid, unit mL, or the mass of immobilized enzyme, unit g; m is0: the mass of the immobilized enzyme added in 25mL of the system, unit g; m: the molar mass of D-mannose, in grams per mole (g/moL) (M180.2 g/moL).
Example 1 vector screening
Selecting different carriers (Xian lan Heng Xiao science and technology New materials Co., Ltd.) LX-103B, LX-1000HA (A), LX-HFA001, LX1000HFA, LX-1000EA, (Aimexican China biological medicine Co., Ltd.) LKZ126, LKZ618, (Tianjin Nankai science and technology Co., Ltd.) ES-101, ESR-1, respectively 2g, subpackaging in 150 mL conical flasks, adding 2g of mannanase into each conical flask, adding 50mL of 0.1M pH7 phosphate buffer solution, sealing, placing on a shaking bed, keeping the temperature at 25 ℃, 15rpm, and immobilizing for 48 h. And (4) using a Buchner funnel to pump out the liquid, retaining the immobilized enzyme, and determining the activity of the immobilized enzyme.
The immobilized enzyme activity of the different carriers was determined and the results are shown in table 2 and fig. 2:
TABLE 2 determination of enzyme activity of immobilized enzyme mannanase with different vectors
Figure BDA0003312981240000061
The results show that the enzyme activity of the LX-HFA001 is highest, and the LX-HFA001 is finally selected as an enzyme immobilized carrier for subsequent experiments.
Example 2 optimal immobilization conditions
The optimum immobilization conditions were determined by a one-factor variable method.
(1) Effect of different concentrations of phosphate on the activity of the immobilized enzyme: selecting 0.05M, 0.1M and 0.2M phosphate buffer as buffer solution, adopting immobilization conditions: mannase (Beijing guar technologies, Inc.) 2g dry powder, LX-HFA001 carrier 2g, 25 ℃, pH7, 150rpm oscillation 48 h. The enzyme activity of the immobilized enzyme is measured to confirm the optimal buffer solution concentration.
The effect of different buffer concentrations on the activity of the immobilized enzyme, the results are shown in table 3.
TABLE 3 Effect of different phosphate concentrations on immobilization
Figure BDA0003312981240000062
As can be seen from the data in Table 3, different concentrations of phosphate have a greater effect on the enzymatic activity of the immobilized enzyme. The optimum concentration is 0.1M.
(2) Effect of different pH values on immobilized enzyme activity: the buffer conditions were chosen to be pH4.5, pH5, pH5.5, pH6, pH6.5, pH7, using immobilization conditions: mannase (Beijing guar technologies, Inc.) 2g dry powder, LX-HFA001 carrier 2g, 25 ℃, 0.1M phosphate concentration, 150rpm oscillation 48 h. And (4) measuring the enzyme activity of the immobilized enzyme and confirming the optimal buffering pH.
Because the enzyme is affected by acid and alkali, the immobilization effect varies greatly with the pH. The appropriate pH was determined by setting a gradient of pH and the results are shown in table 4, fig. 3.
TABLE 4 Effect of different pH values on immobilization
Figure BDA0003312981240000071
From the results, it can be seen that the activity of the immobilized enzyme increased with decreasing pH, and when it reached 4.5, precipitation occurred, and thus the activity of the enzyme decreased. The optimal buffer pH is 5.
(3) Effect of different temperatures on immobilized enzyme activity: different immobilization temperatures of 15 ℃, 20 ℃, 25 ℃ and 30 ℃ are selected, and the immobilization conditions are adopted: mannase (Beijing guar technologies, Inc.) 2g dry powder, LX-HFA001 carrier 2g, 0.1M phosphate concentration, pH5, 150rpm shaking for 48 h. And (3) measuring the enzyme activity of the immobilized enzyme and confirming the optimal immobilization temperature.
The temperature gradient was set, and the influence of temperature on the immobilized enzyme was investigated to investigate the immobilization conditions of the carrier, and the data results are shown in table 5 and fig. 4.
TABLE 5 Effect of temperature on immobilization
Figure BDA0003312981240000072
The result shows that the temperature has no obvious influence on the enzyme activity, and 25 ℃ is selected as the optimal immobilization temperature in comprehensive consideration of the cost.
(4) Influence of enzyme amount on immobilized enzyme activity: 1g, 2g, 4g, 6g and 8g of mannase dry powder are selected and added, and the immobilization conditions are adopted: LX-HFA001 Carrier 2g, 0.1M phosphate concentration, pH5, 25 ℃, 150rpm shaking for 48 h. And (4) measuring the enzyme activity of the immobilized enzyme and confirming the optimal enzyme adding amount.
The enzyme addition amount involved the problem of saturation of immobilization, and the optimum enzyme addition amount conditions were investigated by controlling different enzyme addition amounts and setting a certain enzyme amount gradient, and the data results are shown in table 6 and fig. 5.
TABLE 6 Effect of different enzyme dosages on immobilized enzyme Activity
Figure BDA0003312981240000081
From the results it can be concluded that: when the mass ratio of the enzyme to the carrier is more than 1:1, the activity of the immobilized enzyme is not changed greatly, and the optimal enzyme adding amount is selected from the mass ratio of the enzyme to the carrier of 1:1 in the comprehensive consideration of the cost.
(5) Effect of immobilization time on enzyme activity: with reference to the above optimal conditions for immobilization, samples were taken at 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h, and 48h, respectively, to determine the optimal immobilization time.
The optimum immobilization time was examined by measuring the enzyme activity at intervals. The data results are shown in table 7 and fig. 6.
TABLE 7 Effect of different immobilization times on immobilized enzymes
Figure BDA0003312981240000082
From the above results, it can be seen that the enzyme activity of the immobilized enzyme does not change much after 24h of immobilization, and therefore the optimal immobilization time is 24 h.
By combining the experimental results, the optimal immobilization conditions are as follows: the enzyme carrier mass ratio is 1:1, 0.1M phosphate buffer solution with pH5, and immobilization is carried out for 24h at 25 ℃.
EXAMPLE 3 preparation of mannooligosaccharides in an immobilized enzyme reactor
Assembling an enzyme reactor: filling an appropriate amount (selected according to the size of the chromatographic column) of immobilized mannanase prepared under optimal conditions into the chromatographic column (the filling amount is 2/3 of the volume of the column); the enzyme reactor was assembled as shown in FIG. 7.
Detection of mannan oligosaccharide: the reducing sugar production in the reaction solution was measured by the DNS method: respectively taking 200 mu L of the sample, placing the sample in a 10mL centrifuge tube, then placing 200 mu L of acetic acid-sodium acetate buffer solution into the centrifuge tube, then placing 500 mu L of DNS reagent, slightly vibrating to uniformly mix the reagents, plugging a cover of the centrifuge tube, placing the centrifuge tube in a boiling water bath to heat for 5 minutes, immediately taking out the centrifuge tube, rapidly cooling the centrifuge tube to room temperature by flowing cold water, then adding deionized water to reach 2.5mL of constant volume, uniformly mixing, measuring by using an ultraviolet spectrophotometer, adjusting the volume to zero by using a reagent blank sample solution, and measuring the absorbance value of the sample liquid in the sample tube by using a 10mm cuvette under the condition that the wavelength is 540 nm. The final concentration of the mannooligosaccharide solution was calculated by the linear regression equation.
Optimization of conditions for preparing mannan oligosaccharide in enzyme reactor
The optimal reaction time is as follows: weighing 0.5g of guar gum and acetic acid-sodium acetate buffer solution with the pH value adjusted to 5.5, fully mixing to prepare a substrate, circularly reacting in a reactor with the installed immobilized mannase at the flow rate of 20mL/min, setting the temperature of a water bath kettle to be 45 ℃, reacting for 8h, and taking a sample every 1 h. And (3) measuring the generation amount of reducing sugar in the reaction solution by using a DNS method for the sample after the reaction is finished for 6h, and calculating the final concentration of the obtained mannan oligosaccharide solution by using a formula of a linear regression equation of a mannose standard curve.
The results are shown in FIG. 8: when the reaction time is less than 5h, the concentration of the mannooligosaccharides gradually increases along with the increase of the reaction time; when the reaction time reaches 6h, the concentration of the mannooligosaccharides is in a stable state and does not increase any more, and the reaction reaches the end point. So 6h of reaction is the optimum condition.
Optimum reaction temperature: weighing 0.5g of guar gum and acetic acid-sodium acetate buffer solution with the pH value adjusted to 5.5, fully mixing to prepare a substrate, carrying out circular reaction in a reactor with the immobilized mannase at the flow rate of 20mL/min, setting the temperature through a water bath at 40 ℃, 45 ℃, 50 ℃ and 60 ℃ respectively, reacting for 6 hours, and taking a sample every 1 hour. The amount of reducing sugar produced in the reaction solution was measured by the DNS method.
The results are shown in FIG. 9: the concentration of mannooligosaccharides is highest at 45 ℃ and the reaction is best, and when the temperature is above 55 ℃, the early reaction is fast, but the final preparation concentration is not high, and when the activity of immobilized enzyme is measured afterwards, it is found that when the temperature is above 55 ℃, the enzyme is partially inactivated, so the reaction cannot be completely carried out. The optimum reaction temperature was therefore selected to be 45 ℃.
Optimum reaction pH: weighing 0.5g of guar gum and acetic acid-sodium acetate buffer solutions with pH values of 4.5, 5.5, 6.5 and 7.5 respectively, fully mixing to prepare a substrate, and carrying out circular reaction in an installed reactor for immobilized mannase at the flow rate of 20mL/min at the reaction temperature of 45 ℃ for 6h, wherein a sample is taken every 1 h. The amount of reducing sugar produced in the reaction solution was measured by the DNS method.
The results are shown in FIG. 10: the results of the immobilized mannanase reactor method for preparing the mannooligosaccharides under the conditions of different reaction pH values show that the concentration of the mannooligosaccharide solution is highest when the pH value is 5.5. Therefore, pH5.5 is the optimum condition.
Optimum substrate concentration: respectively weighing 0.5g, 1.0g, 1.5g and 2.0g of guar gum and acetic acid-sodium acetate buffer solution with the pH value adjusted to 5.5 under the optimal condition, fully mixing to prepare a substrate, and carrying out circular reaction in a reactor of the immobilized mannase at the flow rate of 20mL/min at the reaction temperature of 45 ℃ for 6h, wherein samples are taken every 1 h. The amount of reducing sugar produced in the reaction solution was measured by the DNS method.
The results are shown in FIG. 11: at 6 hours of reaction, 1% guar gum was used to prepare the most concentrated mannooligosaccharide solutions. Therefore a guar gum substrate concentration of 1% is the optimal substrate concentration under the current conditions. If the concentration of guar gum is further increased, the concentration of the prepared mannooligosaccharide solution is better, but the reaction time is prolonged due to the increased viscosity, and the reaction time can be prolonged or the flow rate can be increased to optimize the concentration.
Detection of immobilized enzyme reaction batch: performing circulating reaction for 6 hours at the guar gum concentration of 1 percent, the pH value of 5.5, the temperature of 45 ℃ and the flow rate of 20mL/min, recovering the immobilized enzyme to measure the enzyme activity, taking a proper amount of immobilized enzyme after each reaction, measuring the activity of the immobilized enzyme after washing by using deionized water, continuously measuring the activity of the immobilized enzyme for 15 batches, and inspecting the use batches of the immobilized enzyme.
The results are shown in FIG. 12: after 15 continuous batches of reactions, the immobilized enzyme keeps about 68% of enzyme activity, and the reactor can realize continuous reaction for preparing the mannooligosaccharides, and has excellent industrial application prospect.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (6)

1. An immobilized mannanase, which is characterized by being prepared by taking LX-103B, LX-1000HA (A), LX-HFA001, LX1000HFA, LX-1000EA, LKZ126, LKZ618 ES-101 and ESR-1 as immobilized carriers.
2. The process for producing the immobilized mannanase according to claim 1, wherein the mannanase is taken out at a mass ratio of mannanase to carrier of 1:1, and 0.1M phosphate buffer solution (pH 5) is added thereto, followed by immobilization at 25 ℃ for 24 hours.
3. The method for preparing the immobilized mannanase according to claim 2, wherein the phosphate buffer is added in an amount of 20-30 times the mass of the mannanase and the carrier.
4. The method for preparing the immobilized mannanase according to claim 3, wherein the phosphate buffer is added in an amount of 25 times the mass of the mannanase and the carrier.
5. Use of the immobilized mannanase of claim 1 in an enzyme reactor.
6. The application of the immobilized mannanase in an enzyme reactor according to claim 5, wherein the mannan oligosaccharide is prepared by performing a circulating reaction on the reaction solution at a flow rate of 20mL/min for 6h under the condition of a guar gum concentration of 1% and a pH value of 5.5 under a temperature condition of 45 ℃, and the reaction batch can reach more than 15 batches.
CN202111222100.5A 2021-10-20 2021-10-20 Immobilization of mannase and application in enzyme reactor Pending CN114058611A (en)

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