A carrier for enzyme fixation and method for preparing trehalose

Abstract

The invention discloses a method for preparing a carrier for enzyme fixation, comprising the following steps: S11, adding an intercalating agent, magnesium chloride and montmorillonite into deionized water, ultrasonically dispersing and uniformly mixing; S12, placing the uniformly mixed materials in S11 in a microwave reactor, and under the action of microwaves, the intercalating agent and magnesium chloride enter the interlayer of the montmorillonite; S13, evaporating the deionized water to obtain a solid; S14, calcining the solid obtained in S13 to obtain a modified montmorillonite mixture; quenching; and ball milling to obtain a modified montmorillonite carrier powder with a particle size of 600nm to 800nm. The invention also discloses a method for preparing starch into trehalose by using the modified montmorillonite carrier powder for enzyme fixation; the invention utilizes magnesium chloride to improve the morphology of montmorillonite, increase the specific surface area of ​​the enzyme fixation carrier, increase the contact area between the enzyme fixed on the carrier and the reaction solution, improve the reaction efficiency, and shorten the production time.

Description

Technical Field

[0001] The present invention relates to the technical field of trehalose preparation, in particular to a carrier for enzyme fixation and a method for preparing trehalose. Background Art

[0002] Trehalose has a protective effect on cells under harsh environmental conditions and effectively protects protein molecules from denaturation and inactivation, so it has broad application prospects in the field of biomedicine.

[0003] Chinese invention patent CN108130351B discloses a method for preparing large-particle trehalose, and CN103981233B discloses a method for preparing trehalose using cassava starch as raw material. Among the current trehalose preparation methods, the most prominent one is the enzyme conversion method, which is based on the principle of using glucose, maltose or starch as substrates and converting them into trehalose through the action of enzymes related to trehalose synthesis. The disadvantage of this method is that the solubility of the enzyme is not good, resulting in a large amount of enzyme used and a long reaction time, which increases the cost of preparing trehalose and is not conducive to the industrial production of trehalose. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing an enzyme-immobilized carrier. The present invention utilizes magnesium chloride to improve the morphology of montmorillonite, increase the specific surface area of ​​the enzyme-immobilized carrier, increase the contact area between the enzyme fixed on the carrier and the reaction solution, and improve the reaction efficiency.

[0005] To solve this technical problem, the technical solution of the present invention is: a method for preparing an enzyme immobilization carrier, comprising the following steps:

[0006] S11, adding intercalation agent, magnesium chloride and montmorillonite into deionized water, dispersing by ultrasonic, and mixing evenly;

[0007] S12, placing the uniformly mixed materials in S11 into a microwave reactor, and under the action of microwaves, the intercalation agent and magnesium chloride enter between the montmorillonite layers;

[0008] S13, evaporating deionized water to obtain a solid;

[0009] S14, calcining the solid obtained in S13 to obtain a modified montmorillonite mixture;

[0010] Quenching;

[0011] The powder is crushed by ball milling to obtain a modified montmorillonite carrier powder with a particle size of 600nm to 800nm.

[0012] Preferably, the intercalation agent is dioctadecyltetrahydroxyethyldibromopropanediammonium. In the present invention, dioctadecyltetrahydroxyethyldibromopropanediammonium undergoes a cation exchange reaction with montmorillonite to be adsorbed and inserted into the interlayer of the montmorillonite, thereby causing changes on the surface of the montmorillonite and increasing the interlayer spacing. As the long molecular chain of dioctadecyltetrahydroxyethyldibromopropanediammonium enters, magnesium chloride also enters the interlayer of the montmorillonite, increasing the interlayer spacing and introducing magnesium chloride.

[0013] Preferably, in step S11, the mass ratio of magnesium chloride to montmorillonite is 1: (10 to 16);

[0014] The mass ratio of the intercalant to magnesium chloride is (0.5 to 0.8):1.

[0015] The preferred process conditions of the microwave in step S12 are: microwave power 800 W, microwave time 45 min to 60 min.

[0016] Preferably, the calcination temperature in step S14 is 600° C. to 700° C., and the calcination time is 2 h to 3 h. In the present invention, the intercalation agent between montmorillonite layers is removed by calcination, and magnesium chloride is calcined to produce Mg-O, and Mg-O is mixed with Si, Al, and Mg between montmorillonite layers, and the radius of Mg is the smallest, which causes the original crystal to deform, the bond length at the Mg site is short, and the formed crystal is smaller. At the same time, the departure of the intercalation agent and the chlorine element will form pores, and the modified montmorillonite carrier has a larger specific surface area.

[0017] Another object of the present invention is to provide a method for preparing trehalose using the prepared enzyme immobilization carrier. The present invention utilizes the high specific surface area of ​​modified montmorillonite and the adsorption characteristics of montmorillonite to effectively adsorb the enzyme, thereby increasing the contact area between the enzyme and the reaction solution. The modified montmorillonite is used to promote the dispersion rate and degree of the enzyme in the reaction solution, thereby improving the reaction efficiency and shortening the reaction time.

[0018] To solve this technical problem, the technical solution of the present invention is: an application of a modified montmorillonite carrier prepared by the method for preparing an enzyme immobilization carrier proposed in the present invention, and the modified montmorillonite carrier is used to immobilize enzymes to prepare trehalose.

[0019] To solve this technical problem, the technical solution of the present invention is: a method for preparing trehalose using a modified montmorillonite carrier, comprising the following steps:

[0020] S1. Using a phosphate buffer solution, the pH of the starch dispersion is adjusted to 5.5 to 6.0, the temperature is adjusted to 45° C. to 55° C., and the α-amylase immobilized by the modified montmorillonite carrier is added under constant temperature stirring;

[0021] S2, filtering the material of S1 to obtain α-amylase conversion liquid;

[0022] S3, adjusting the pH of the filtrate obtained in S2 to 6.5 to 7.0, and maintaining the temperature at 30° C. to 40° C.;

[0023] S4, adding maltooligosaccharide-based trehalose hydrolase fixed by a modified montmorillonite carrier to the liquid phase after S3, stirring, maintaining a constant temperature of 30° C. to 40° C., and converting for 12 hours to obtain a maltooligosaccharide-based trehalose hydrolase conversion liquid;

[0024] S5, filter the material after S4, add activated carbon to the liquid phase for decolorization, stir; filter the activated carbon to obtain a decolorized liquid;

[0025] S6, desalting the filtrate obtained in S5 by ion exchange until the conductivity is less than 50 μs / cm to obtain a desalted solution;

[0026] S7. Dilute the desalted liquid obtained in S6, and perform chromatographic separation at a separation temperature of 65° C. to 75° C. to obtain a trehalose solution.

[0027] Preferably, the conversion time of the α-amylase fixed by the modified montmorillonite carrier in step S1 is 10 to 30 minutes. Since the α-amylase disperses quickly in the solution along with the modified montmorillonite carrier, the dissolution time of the enzyme is effectively shortened.

[0028] Preferably, the mass ratio of α-amylase to starch in step S1 is 1:(20 to 30).

[0029] Preferably, the mass ratio of maltooligosaccharide-trehalose hydrolase in step S4 to starch in step S1 is 1:(23 to 31).

[0030] The mass ratio of activated carbon in step S5 to starch in S1 is (0.1 to 0.3):1.

[0031] By adopting the above technical solution, the beneficial effects of the present invention are:

[0032] In the present invention, magnesium chloride enters the interlayer of montmorillonite along with an intercalating agent, and montmorillonite mixed with the intercalating agent and magnesium chloride is calcined. Under high temperature, the calcination of magnesium chloride generates Mg-O, which is mixed with Si, Al and Mg in the original montmorillonite. The radius of Mg is the smallest, which causes the original crystal to deform. The bond length at the site of Mg is short, and the formed crystal is smaller. At the same time, the departure of chlorine element and the intercalating agent forms pores, and the modified carrier has a larger specific surface area. The montmorillonite has a layered structure and is insoluble in water. When alpha-amylase or maltooligosaccharide-based trehalose hydrolase is adsorbed on the surface of the modified montmorillonite, the enzyme is dispersed in the reaction liquid along with the montmorillonite, and the contact area between the enzyme fixed on the carrier and the liquid phase is increased, and the dispersion of the particles is faster than the dispersion of the enzyme itself. Further, the present invention uses the obtained modified montmorillonite carrier as the fixed phase of the enzyme to produce trehalose, which can greatly shorten the reaction time and reduce the amount of the enzyme. At the same time, the enzyme carrier can be reused many times after use. In addition, during the enzymatic hydrolysis process, the contact between the enzyme dispersed by the modified montmorillonite and the substrate is greater, which increases the activity of the enzyme and reduces the reaction time. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the infrared spectrum of trehalose prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0034] In order to further explain the technical solution of the present invention, the present invention is described in detail below through specific embodiments.

[0035] Example 1

[0036] This embodiment discloses a method for preparing an enzyme immobilization carrier, comprising the following steps:

[0037] S11, adding dioctadecyl tetrahydroxyethyl dibromopropane diammonium as an intercalation agent, magnesium chloride and montmorillonite into deionized water, and performing ultrasonic dispersion and uniform mixing;

[0038] The mass ratio of magnesium chloride to montmorillonite and the mass ratio of intercalating agent to magnesium chloride are shown in Table 1.

[0039] S12, placing the uniformly mixed materials in S11 into a microwave reactor, and under the action of microwaves, the intercalation agent and magnesium chloride enter between the montmorillonite layers;

[0040] The process conditions of microwave in step S12 are shown in Table 1.

[0041] S13, evaporating deionized water to obtain a solid;

[0042] S14, calcining the solid obtained in S13 to obtain a modified montmorillonite mixture;

[0043] The calcination temperature and calcination time in step S14 are shown in Table 1.

[0044] Quenching;

[0045] The modified montmorillonite carrier powder is obtained by ball milling.

[0046] This embodiment also discloses a method for preparing trehalose using the prepared enzyme-immobilized carrier, comprising the following steps:

[0047] S1. Using phosphate buffer to adjust the pH of the starch dispersion to 5.5, adjusting the temperature to 45° C., adding the α-amylase fixed by the modified montmorillonite carrier under constant temperature stirring; the mass ratio of α-amylase to starch in step S1 is 1:20; the conversion time of the α-amylase fixed by the modified montmorillonite carrier added in step S1 is 30 minutes.

[0048] Preparation method of α-amylase fixed by modified montmorillonite carrier:

[0049] A modified montmorillonite carrier with a particle size of 600 nm was dispersed in a neutral phosphate buffer, stirred, and α-amylase was added, wherein the mass ratio of the modified montmorillonite carrier to the α-amylase was 1:1. After the addition, ultrasonic vibration was performed for 12 hours, and the product was centrifuged to obtain α-amylase fixed by the modified montmorillonite carrier.

[0050] S2, filtering the material of S1, filtering the liquid phase using a 0.45 μm filter membrane to obtain an α-amylase conversion liquid;

[0051] S3, adjusting the pH of the filtrate obtained in S2 to 6.5 and maintaining the temperature at 30°C;

[0052] S4, adding maltooligosaccharide-based trehalose hydrolase immobilized by a modified montmorillonite carrier to the liquid phase after S3, stirring, maintaining a constant temperature of 30° C., and converting for 12 hours to obtain a maltooligosaccharide-based trehalose hydrolase conversion liquid;

[0053] The mass ratio of the maltooligosaccharide-trehalose hydrolase in step S4 to the starch in step S1 is 1:23.

[0054] Preparation method of maltooligosaccharide-based trehalose hydrolase immobilized on a modified montmorillonite carrier:

[0055] A modified montmorillonite carrier with a particle size of 600 nm was dispersed in a neutral phosphate buffer, stirred, and maltooligosaccharide-based trehalose hydrolase was added, wherein the mass ratio of the modified montmorillonite carrier to the maltooligosaccharide-based trehalose hydrolase was 1:1. After the addition, ultrasonic vibration was performed for 12 hours, and the product was centrifuged to obtain the maltooligosaccharide-based trehalose hydrolase fixed by the modified montmorillonite carrier.

[0056] S5, filter the material after S4, add activated carbon to the liquid phase for decolorization, and stir; filter the activated carbon through a 0.25 μm filter membrane to obtain a decolorized liquid; the mass ratio of the activated carbon in step S5 to the starch in S1 is 0.1:1.

[0057] S6, desalting the filtrate obtained in S5 by ion exchange until the conductivity is less than 50 μs / cm to obtain a desalted solution;

[0058] S7. Dilute the desalted liquid obtained in S6, and perform chromatographic separation at a separation temperature of 65° C. to obtain a trehalose solution.

[0059] Example 2

[0060] The differences between this embodiment and embodiment 1 regarding the preparation method of the modified montmorillonite carrier are shown in Table 1.

[0061] The method for preparing trehalose in this example is the same as that in Example 1.

[0062] Example 3

[0063] The differences between this embodiment and embodiment 1 regarding the preparation method of the modified montmorillonite carrier are shown in Table 1.

[0064] The method for preparing trehalose in this example is the same as that in Example 1.

[0065] Example 4

[0066] The differences between this embodiment and embodiment 1 regarding the preparation method of the modified montmorillonite carrier are shown in Table 1.

[0067] The method for preparing trehalose in this example is the same as that in Example 1.

[0068] Example 5

[0069] The differences between this embodiment and embodiment 1 regarding the preparation method of the modified montmorillonite carrier are shown in Table 1.

[0070] The method for preparing trehalose in this example is the same as that in Example 1.

[0071] Example 6

[0072] The differences between this embodiment and embodiment 1 regarding the preparation method of the modified montmorillonite carrier are shown in Table 1.

[0073] This embodiment also discloses a method for preparing trehalose using the prepared enzyme-immobilized carrier, comprising the following steps:

[0074] S1. Using phosphate buffer to adjust the pH of starch dispersion to 5.5, adjusting the temperature to 45° C., adding α-amylase fixed by the modified montmorillonite carrier with constant temperature stirring at 40 r / min; the mass ratio of α-amylase to starch in step S1 is 1:23; the conversion time of α-amylase fixed by the modified montmorillonite carrier added in step S1 is 30 min.

[0075] Preparation method of α-amylase fixed by modified montmorillonite carrier:

[0076] A modified montmorillonite carrier with a particle size of 600 nm was dispersed in a neutral phosphate buffer, stirred, and α-amylase was added, wherein the mass ratio of the modified montmorillonite carrier to the α-amylase was 1:1. After the addition, ultrasonic vibration was performed for 12 hours, and the product was centrifuged to obtain α-amylase fixed by the modified montmorillonite carrier.

[0077] S2, filtering the material of S1, filtering the liquid phase using a 0.45 μm filter membrane to obtain an α-amylase conversion liquid;

[0078] S3, adjusting the pH of the filtrate obtained in S2 to 6.5 and maintaining the temperature at 30°C;

[0079] S4, adding maltooligosaccharide-based trehalose hydrolase immobilized by a modified montmorillonite carrier to the liquid phase after S3, stirring, maintaining a constant temperature of 30° C., and converting for 12 hours to obtain a maltooligosaccharide-based trehalose hydrolase conversion liquid;

[0080] The mass ratio of the maltooligosaccharide-trehalose hydrolase in step S4 to the starch in step S1 is 1:27.

[0081] Preparation method of maltooligosaccharide-based trehalose hydrolase immobilized on a modified montmorillonite carrier:

[0082] A modified montmorillonite carrier with a particle size of 600 nm was dispersed in a neutral phosphate buffer, stirred, and maltooligosaccharide-based trehalose hydrolase was added, wherein the mass ratio of the modified montmorillonite carrier to the maltooligosaccharide-based trehalose hydrolase was 1:1. After the addition, ultrasonic vibration was performed for 12 hours, and the product was centrifuged to obtain the maltooligosaccharide-based trehalose hydrolase fixed by the modified montmorillonite carrier.

[0083] S5, filter the material after S4, add activated carbon to the liquid phase for decolorization, and stir; filter the activated carbon through a 0.25 μm filter membrane to obtain a decolorized liquid; the mass ratio of the activated carbon in step S5 to the starch in S1 is 0.1:1.

[0084] S6, desalting the filtrate obtained in S5 by ion exchange until the conductivity is less than 50 μs / cm to obtain a desalted solution;

[0085] S7. Dilute the desalted liquid obtained in S6, and perform chromatographic separation at a separation temperature of 65° C. to obtain a trehalose solution.

[0086] Example 7

[0087] The differences between this embodiment and embodiment 1 regarding the preparation method of the modified montmorillonite carrier are shown in Table 1.

[0088] This embodiment also discloses a method for preparing trehalose using the prepared enzyme-immobilized carrier, comprising the following steps:

[0089] S1. Using phosphate buffer to adjust the pH of starch dispersion to 5.5, adjusting the temperature to 45° C., adding α-amylase fixed by the modified montmorillonite carrier with constant temperature stirring at 40 r / min; the mass ratio of α-amylase to starch in step S1 is 1:30; the conversion time of α-amylase fixed by the modified montmorillonite carrier added in step S1 is 30 min.

[0090] Preparation method of α-amylase fixed by modified montmorillonite carrier:

[0091] A modified montmorillonite carrier with a particle size of 600 nm was dispersed in a neutral phosphate buffer, stirred, and α-amylase was added, wherein the mass ratio of the modified montmorillonite carrier to the α-amylase was 1:1. After the addition, ultrasonic vibration was performed for 12 hours, and the product was centrifuged to obtain α-amylase fixed by the modified montmorillonite carrier.

[0092] S2, filtering the material of S1, filtering the liquid phase using a 0.45 μm filter membrane to obtain an α-amylase conversion liquid;

[0093] S3, adjusting the pH of the filtrate obtained in S2 to 7.0 and maintaining the temperature at 30°C;

[0094] S4, adding maltooligosaccharide-based trehalose hydrolase immobilized by a modified montmorillonite carrier to the liquid phase after S3, stirring, maintaining a constant temperature of 30° C., and converting for 12 hours to obtain a maltooligosaccharide-based trehalose hydrolase conversion liquid;

[0095] The mass ratio of the maltooligosaccharide-trehalose hydrolase in step S4 to the starch in step S1 is 1:31.

[0096] Preparation method of maltooligosaccharide-based trehalose hydrolase immobilized on a modified montmorillonite carrier:

[0097] A modified montmorillonite carrier with a particle size of 600 nm was dispersed in a neutral phosphate buffer, stirred, and maltooligosaccharide-based trehalose hydrolase was added, wherein the mass ratio of the modified montmorillonite carrier to the maltooligosaccharide-based trehalose hydrolase was 1:1. After the addition, ultrasonic vibration was performed for 12 hours, and the product was centrifuged to obtain the maltooligosaccharide-based trehalose hydrolase fixed by the modified montmorillonite carrier.

[0098] S5, filter the material after S4, add activated carbon to the liquid phase for decolorization, and stir; filter the activated carbon through a 0.25 μm filter membrane to obtain a decolorized liquid; the mass ratio of the activated carbon in step S5 to the starch in S1 is 0.1:1.

[0099] S6, desalting the filtrate obtained in S5 by ion exchange until the conductivity is less than 50 μs / cm to obtain a desalted solution;

[0100] S7. Dilute the desalted liquid obtained in S6, and perform chromatographic separation at a separation temperature of 65° C. to obtain a trehalose solution.

[0101] Comparative Example 1

[0102] This comparative example discloses a method for preparing trehalose, comprising the following steps:

[0103] S1. Adjust the pH of starch dispersion to 5.5 with phosphate buffer and keep the temperature to 45°C. Add α-amylase with a mass ratio of α-amylase to starch of 1:20. Stir at a constant temperature of 40 r / min and enzymolysis for 60 h.

[0104] S2, filter the solution of S1 converted by α-amylase, with the filter membrane specification of 0.45 μm;

[0105] The filtrate was adjusted to pH 6.5 with alkali and the temperature was maintained at 30°C;

[0106] S3, adding maltooligosaccharide trehalose hydrolase to the solution of S2 while maintaining the temperature, with the mass ratio of enzyme to starch being 1:23, stirring slowly at 10 r / min, and converting at a constant temperature of 30°C for 60 hours;

[0107] S4, filtering the solution converted by constant temperature in S3 through a 0.45 μm filter membrane, adding activated carbon for decolorization, wherein the mass ratio of activated carbon to starch is 0.1:1, stirring for 30 minutes, and then filtering through a 0.25 μm filter membrane;

[0108] S5, desalting the filtrate obtained in S4 by ion exchange, wherein the conductivity of the desalted solution is less than 50 μs / cm;

[0109] S6. Dilute the desalted liquid obtained in S6 with pure water to obtain a diluted liquid; perform chromatographic separation on the diluted liquid at a chromatographic separation temperature of 65° C. to obtain a purified liquid, which is a trehalose solution.

[0110] Comparative Example 2

[0111] This comparative example discloses a method for preparing trehalose using the modified montmorillonite carrier prepared in Example 1, comprising the following steps:

[0112] S1. Adjust the pH of starch dispersion to 5.5 with phosphate buffer, adjust the temperature to 45°C, add α-amylase fixed by modified montmorillonite under constant temperature stirring at 40r / min, the mass ratio of α-amylase to starch is 1:20, react for 30min, and obtain α-amylase conversion solution;

[0113] The preparation method of α-amylase immobilized by modified montmorillonite is as follows: modified montmorillonite carrier powder with a particle size of 600 nm is dispersed with phosphate buffer, stirred, and α-amylase is added to the solution, wherein the mass ratio of the modified montmorillonite carrier powder to the α-amylase is 1:1, after addition, ultrasonic vibration is performed for 12 hours, and the product is centrifuged to obtain α-amylase immobilized by modified montmorillonite;

[0114] S2, filtering the α-amylase conversion liquid, the filter membrane specification is 0.45 μm, adjusting the pH of the filtrate to 6.5 with alkali, and maintaining the temperature at 30°C;

[0115] S3, adding maltooligosaccharide trehalose hydrolase to the filtrate after S2 under stirring, the mass ratio of maltooligosaccharide trehalose hydrolase to starch is 1:23, stirring slowly at 10r / min, constant temperature at 30°C, and conversion time is 60h;

[0116] S4, filtering the maltooligosaccharide trehalose hydrolase conversion solution, the filter membrane specification is 0.45 μm, and then adding activated carbon for decolorization, the mass ratio of activated carbon to starch is 0.1:1, stirring for 30 minutes, and then filtering through a 0.25 μm filter membrane to obtain a decolorized solution;

[0117] S5, desalting the ion exchange decolorizing solution, the conductivity of the desalting solution is less than 50μs / cm;

[0118] S6. diluting the desalted liquid with pure water to obtain a diluted liquid; performing chromatographic separation on the diluted liquid at a chromatographic separation temperature of 65° C. to obtain a trehalose solution.

[0119] Comparative Example 3

[0120] This comparative example discloses a method for preparing trehalose using the modified montmorillonite carrier prepared in Example 1, comprising the following steps:

[0121] S1. Adjust the pH of starch dispersion to 5.5 with phosphate buffer and the temperature to 45°C, add α-amylase, the mass ratio of α-amylase to starch is 1:20; stir at a constant temperature of 40 r / min, and the enzymolysis time is 60 h to obtain α-amylase conversion liquid;

[0122] S2, filtering the α-amylase conversion liquid, the filter membrane specification is 0.45 μm, adjusting the pH of the filtrate to 6.5 with alkali, and maintaining the temperature at 30°C;

[0123] S3, adding maltooligosaccharide-based trehalose hydrolase fixed by modified montmorillonite to the heat-insulated α-amylase conversion liquid under stirring, the mass ratio of maltooligosaccharide-based trehalose hydrolase to starch is 1:23, stirring slowly at 10 r / min, constant temperature at 30°C, enzymolysis time is 12 h, and obtaining maltooligosaccharide-based trehalose hydrolase conversion liquid;

[0124] The preparation method of maltooligosaccharide-based trehalose hydrolase immobilized by modified montmorillonite is as follows: a modified montmorillonite carrier with a particle size of 600 nm is dispersed in a neutral phosphate buffer, maltooligosaccharide-based trehalose hydrolase is added to the solution under stirring, the mass ratio of the modified montmorillonite carrier to the maltooligosaccharide-based trehalose hydrolase is 1:1, after the addition, ultrasonic vibration is performed for 12 hours, and the product is centrifuged to obtain the maltooligosaccharide-based trehalose hydrolase immobilized by the modified montmorillonite;

[0125] S4, the maltooligosaccharide trehalose hydrolase conversion liquid was filtered through a 0.45 μm filter membrane, and then activated carbon was added for decolorization, and the mass ratio of activated carbon to starch was 0.1:1; stirred for 30 minutes, and then filtered through a 0.25 μm filter membrane to obtain a decolorized liquid;

[0126] S5, decolorizing solution is desalted by ion exchange, and the conductivity of the desalted solution is less than 50μs / cm;

[0127] S6. Dilute the desalted liquid with pure water to obtain a diluted liquid; perform chromatographic separation on the diluted liquid at a chromatographic separation temperature of 65° C. to obtain a trehalose solution.

[0128] Comparative Example 4

[0129] This comparative example discloses a method for preparing trehalose by using a montmorillonite carrier to immobilize an enzyme, comprising the following steps:

[0130] S1. Use phosphate buffer to adjust the pH of starch dispersion to 5.5, adjust the temperature to 45°C, add α-amylase fixed by montmorillonite under constant temperature stirring at 40r / min, the mass ratio of α-amylase to starch is 1:20, react for 30min, and obtain α-amylase conversion solution;

[0131] The preparation method of α-amylase immobilized by montmorillonite is as follows:

[0132] The montmorillonite carrier powder with a particle size of 600 nm is obtained by ball milling, the montmorillonite carrier is dispersed in a neutral phosphate buffer, and α-amylase is added to the solution with stirring, and the mass ratio of the montmorillonite carrier to the α-amylase is 1:1. After the addition, ultrasonic vibration is performed for 12 hours, and the product is centrifuged to obtain a solid α-amylase fixed by montmorillonite;

[0133] S2, filtering the α-amylase conversion liquid, the filter membrane specification is 0.45 μm, adjusting the pH of the filtrate to 6.5 with alkali, and maintaining the temperature at 30°C;

[0134] S3, adding maltooligosaccharide-based trehalose hydrolase fixed by montmorillonite to the heat-insulated filtrate under stirring, the mass ratio of maltooligosaccharide-based trehalose hydrolase to starch is 1:23, stirring slowly at 10 r / min, and converting at a constant temperature of 30°C for 12 hours to obtain maltooligosaccharide-based trehalose hydrolase conversion liquid;

[0135] The preparation method of maltooligosaccharide-based trehalose hydrolase fixed by montmorillonite is as follows: ball milling montmorillonite to obtain a montmorillonite carrier with a particle size of 600 nm, dispersing the montmorillonite carrier with a neutral phosphate buffer, adding maltooligosaccharide-based trehalose hydrolase to the solution under stirring, the mass ratio of the montmorillonite carrier to the maltooligosaccharide-based trehalose hydrolase is 1:1, after adding, ultrasonically vibrating for 12 hours, and centrifuging the product to obtain a solid maltooligosaccharide-based trehalose hydrolase fixed by montmorillonite;

[0136] S4, filtering the maltooligosaccharide trehalose hydrolase conversion liquid, the filter membrane specification is 0.45 μm, and then adding activated carbon for decolorization, the mass ratio of activated carbon to starch is 0.1:1, stirring for 30 minutes, and then filtering through a 0.25 μm filter membrane to obtain a decolorized liquid;

[0137] S5, decolorizing the solution by ion exchange, and the conductivity of the decolorizing solution after desalination is less than 50 μs / cm, thereby obtaining a desalted solution;

[0138] S6. Dilute the desalted liquid with pure water to obtain a diluted liquid; perform chromatographic separation on the diluted liquid at a chromatographic separation temperature of 65° C. to obtain a trehalose solution.

[0139] Table 1 Process parameters for preparing modified montmorillonite carriers in Examples 1 to 7

[0140]

[0141] The production time, yield and purity of trehalose prepared in Examples 1 to 7 and Comparative Examples 1 to 4 were respectively statistically shown in Table 2. The production time refers to the total time from the addition of starch stock solution to the acquisition of trehalose, wherein the carrier preparation and enzyme fixation are carried out simultaneously with the preparation of trehalose, and the actual process time is recorded; yield = product trehalose mass / input starch mass × 100%; wherein the trehalose mass is calculated by drying the obtained trehalose solution, weighing it, and taking a sample; and the purity is tested by chromatography, and the trehalose mass = crude product mass × purity.

[0142] Table 2 Production time, yield and purity of trehalose

[0143]

[0144] Combining Table 1, the process parameters for preparing trehalose and the performance indicators of the trehalose prepared in Table 2, it can be seen that compared with Comparative Example 1, the production time of Example 1 is greatly shortened, and the amount of enzyme added is the same. In Example 1, the distribution form of the enzyme in the solution is adjusted by fixing the enzyme on the modified montmorillonite carrier. The enzyme fixed on the carrier is in full contact with the solution and dispersed more evenly, thereby increasing the efficiency of enzyme use. Therefore, the yield of trehalose is improved to a certain extent; Compared with Example 1, the yield of α-amylase and maltooligosaccharide trehalose hydrolase fixed by montmorillonite modified with magnesium chloride as a carrier is higher than that of Comparative Example 4. The effect of using unmodified montmorillonite loaded enzyme on the conversion effect of trehalase is shown in the above figure, indicating that the simple layered structure of unmodified montmorillonite has limited fixation effect of enzyme by adsorption, and the overly dense layered structure is difficult to effectively disperse the enzyme; the enzyme fixed carrier used in Comparative Example 4 and Example 1 was further tested for specific surface area determination, wherein the specific surface area of ​​the montmorillonite carrier without intercalation and modified with magnesium chloride in Comparative Example 4 is 737 m 2 / g, the specific surface area of ​​the modified montmorillonite carrier prepared in Example 1 is 921m 2 / g, the above specific surface area differences come from:

[0145] In the present invention, magnesium chloride enters the interlayer of montmorillonite along with the intercalating agent, and the montmorillonite mixed with the intercalating agent and magnesium chloride is calcined. Under high temperature, the calcination of magnesium chloride generates Mg-O, which is mixed with the original Si, Al and Mg in the montmorillonite. The radius of Mg is the smallest, which causes the original crystal to be deformed. The bond length at the site of Mg is short, and the formed crystal is smaller. At the same time, the departure of chlorine element and the intercalating agent forms pores, and the modified carrier has a larger specific surface area.

[0146] From the change in specific surface area of ​​the enzyme fixed carrier of Comparative Example 4 and Example 1, it can be seen that the specific surface area of ​​the product obtained by mixing and calcining the magnesium chloride intercalated into the montmorillonite and the montmorillonite is 25% higher than the specific surface area of ​​the montmorillonite after the same ball milling, which can effectively increase the enzyme loading, provide more enzyme reaction conditions, and increase the yield. Compared with Example 1, Example 2, and Example 3, when other conditions remain unchanged, the size of the carrier particles is changed. The smaller the carrier particles, the higher the reaction efficiency; compared with Example 1 and Example 4 and Example 5, the mass ratio of magnesium chloride to montmorillonite is changed, and the yield first increases and then decreases. Therefore, when the mass ratio of magnesium chloride to montmorillonite is 1:13, its loading effect is the best; compared with the data of Example 1, Example 6 and Example 7, when the ratio of enzyme to starch gradually increases, the yield of Example 1 is the same as that of Example 6, and the yield of Example 7 is significantly reduced. Therefore, the yield is affected by the amount of enzyme added. The current yield of the present invention is as high as 50% or more. The present invention is insoluble in water due to the layered structure of montmorillonite. When alpha-amylase or maltooligosaccharide trehalose hydrolase is adsorbed on the surface of the modified montmorillonite, the enzyme is dispersed in the reaction solution along with the montmorillonite, increasing the contact area of ​​the fixed carrier reaction, and the dispersion of the particles is faster than the dispersion of the enzyme itself. Further, the present invention uses the obtained modified montmorillonite carrier as the fixed phase of the enzyme to produce trehalose, which can greatly shorten the reaction time and reduce the amount of enzyme used. At the same time, the enzyme carrier can be reused many times after use. In addition, during the enzymolysis process, as the enzyme dispersed by the modified montmorillonite contacts more with the substrate, the activity of the enzyme is improved and the reaction time is reduced.

[0147] The infrared spectrum of trehalose prepared in Example 1 is as follows: Figure 1 As shown, compared with the standard spectrum of trehalose, 3490cm -1 The characteristic peak of hydroxyl group appears at 2932cm -1 The peak of CH stretching vibration is 1650cm -1 The hydroxyl bending vibration absorption peak is at 1472 cm -1 =CH2 deformation absorption peak appears at 1163cm -1 The CO absorption peak on the ring is 1009cm -1 The position is the alcohol hydroxyl deformation vibration absorption peak, confirming that the trehalose prepared by the present invention.

Claims

1. A method for preparing an enzyme-immobilized carrier, characterized in that: The following steps are involved: S11, adding intercalation agent, magnesium chloride and montmorillonite into deionized water, dispersing by ultrasonic, and mixing evenly; S12, placing the uniformly mixed materials in S11 into a microwave reactor, and under the action of microwaves, the intercalation agent and magnesium chloride enter between the montmorillonite layers; The intercalant is dioctadecyl tetrahydroxyethyl dibromopropane diammonium; S13, evaporating deionized water to obtain a solid; S14, calcining the solid obtained in S13 to remove the intercalant between the montmorillonite layers, the intercalant and the chlorine element will form pores, and a modified montmorillonite mixture is obtained; The calcination temperature is 600°C to 700°C, and the calcination time is 2h to 3h; Quenching; The powder is crushed by ball milling to obtain a modified montmorillonite carrier powder with a particle size of 600nm to 800nm.

2. The method for preparing an enzyme-immobilized carrier according to claim 1, characterized in that: In step S11, the mass ratio of magnesium chloride to montmorillonite is 1: (10 to 16); The mass ratio of the intercalant to magnesium chloride is (0.5 to 0.8):

1.

3. The method for preparing an enzyme-immobilized carrier according to claim 1, characterized in that: The process conditions of the microwave in step S12 are: microwave power 800 W, microwave time 45 min to 60 min.

4. A method for preparing trehalose by using the modified montmorillonite carrier prepared by the method for preparing an enzyme immobilized carrier according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Using a phosphate buffer solution, the pH of the starch dispersion is adjusted to 5.5 to 6.0, the temperature is adjusted to 45° C. to 55° C., and the α-amylase immobilized by the modified montmorillonite carrier is added under constant temperature stirring; S2, filtering the material of S1 to obtain α-amylase conversion liquid; S3, adjusting the pH of the filtrate obtained in S2 to 6.5 to 7.0, and maintaining the temperature at 30° C. to 40° C.; S4, adding maltooligosaccharide-based trehalose hydrolase fixed by a modified montmorillonite carrier to the liquid phase after S3, stirring, maintaining a constant temperature of 30° C. to 40° C., and converting for 12 hours to obtain a maltooligosaccharide-based trehalose hydrolase conversion liquid; S5, filter the material passed through S4, add activated carbon to the liquid phase for decolorization, stir; filter the activated carbon to obtain a decolorized liquid; S6, desalting the filtrate obtained in S5 by ion exchange until the conductivity is 50 μs / cm to obtain a desalted solution; S7. Dilute the desalted liquid obtained in S6, and perform chromatographic separation to obtain a trehalose solution.

5. The method for preparing trehalose according to claim 4, characterized in that: The conversion time of the α-amylase immobilized on the modified montmorillonite carrier in step S1 is 30 minutes.

6. The method for preparing trehalose according to claim 4, characterized in that: The mass ratio of α-amylase to starch in step S1 is 1:(20 to 30).

7. The method for preparing trehalose according to claim 4, characterized in that: The mass ratio of maltooligosaccharide trehalose hydrolase in step S4 to starch in step S1 is 1:(23 to 31).

8. The method for preparing trehalose according to claim 4, characterized in that: The mass ratio of activated carbon in step S5 to starch in S1 is (0.1 to 0.3):1.

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