CN109456955B

CN109456955B - Preparation method of immobilized dextranase

Info

Publication number: CN109456955B
Application number: CN201810987846.7A
Authority: CN
Inventors: 王雅洁; 王蔷
Original assignee: Anhui Medical College
Current assignee: Anhui Medical College
Priority date: 2018-08-28
Filing date: 2018-08-28
Publication date: 2022-01-07
Anticipated expiration: 2038-08-28
Also published as: CN109456955A

Abstract

The invention discloses a preparation method of immobilized dextranase, which comprises the following steps: will be provided withInoculating paecilomyces lilacinus to a fermentation culture medium for activation and then carrying out liquid fermentation; centrifuging the fermentation liquor, (NH)₄)₂SO₄Precipitating and ultrafiltering to obtain dextranase enzyme solution; activating the prepared magnetic carrier by glutaraldehyde, and obtaining dextranase enzyme solution to obtain immobilized dextranase; adding the immobilized dextranase into an acetate buffer solution containing dextran, and cracking the macromolecular dextran. The immobilized enzyme is recovered from the reaction liquid through magnetic separation and can be reused for 20 times. The preparation method takes the magnetic particles as the carrier, and the prepared immobilized dextranase has higher thermal stability and reaction; the immobilized dextranase can be quickly separated from an enzyme catalysis product through magnetic separation and can be repeatedly used. The characteristics are beneficial to the application of the enzyme in the sugar industry, and the cost is saved for industrial production.

Description

Preparation method of immobilized dextranase

Technical Field

The invention relates to the technical field of fermentation and immobilization preparation of heat-resistant dextranase, relates to the technical field of biology, and particularly relates to a preparation method of immobilized dextranase.

Background

Dextranase (dextran, e.c.3.2.1.11) is one of glycoside hydrolases, which can hydrolyze macromolecular dextran to form oligosaccharides and monosaccharides, and is mainly produced by bacteria, penicillium and actinomycetes at home and abroad. The application of dextranase is mainly in food industry and pharmaceutical industry. In the sugar industry, the dextranase can specifically hydrolyze macromolecular dextran, reduce macromolecules generated in the process of storing sugarcane, reduce the viscosity of sugarcane juice and optimize a sugar making process; in the medical industry, the dextranase can hydrolyze macromolecular dextran to prepare the dextran with low molecular weight required by medicine, and compared with the traditional hydrolysis method, the dextran hydrolysis method has the advantages of mild conditions, less introduced impurities and the like; in addition, the dextranase can be hydrolyzed to generate isomalto-oligosaccharide, the sweetness of the isomalto-oligosaccharide is about 30 percent of that of cane sugar, and the isomalto-oligosaccharide has the advantages of dental caries resistance, heat resistance, acid resistance, good heat stability, good safety and the like, and has important application in health care products, food processing and medicine industries.

There are some obvious problems with the use of free dextranase in the above mentioned areas: firstly, dextranase is costly and has a long production cycle, and especially in fungal production dextranase often requires fermentation for one week or more. The dextranase added into the feed liquid as the catalyst can not be recovered and can not be reused. Second, the separation of enzyme from substrate and product added to the feed solution is difficult, making it possible to detect residual enzyme in the product. Finally, in addition to thermostable dextranase, the activity of common free dextranase is susceptible to temperature, pH, and other agents.

Therefore, the preparation of the high-temperature resistant dextranase by adopting a proper method is beneficial to the application of the dextranase in various industries such as sugar production and the like; meanwhile, the stability, the product quality and the controllability of the catalytic process of the enzyme can be obviously improved through immobilization, and the application cost of the enzyme is saved through repeated utilization.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method of immobilized dextranase.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

a preparation method of immobilized dextranase comprises the following preparation steps:

(1) inoculating paecilomyces lilacinus to a fermentation culture medium, activating, and performing liquid fermentation under the conditions of 28-30 ℃ and 200r/min for 6-9 d;

(2) centrifuging the fermentation liquor, removing the precipitate, and using (NH) as supernatant₄)₂SO₄Precipitation is carried out. Collecting the precipitate with the saturation of 0-80% of ammonium sulfate, and using 0.1mol L^-1Dissolving the mixture in a buffer solution with the pH value of 5.5, and ultrafiltering the dissolved substance in a 10kDa ultrafiltration centrifugal tube for 30 minutes to obtain a dextranase enzyme solution;

(3) the immobilized enzyme magnetic carrier Fe₃O₄Activating the PDA GO-PEI with glutaraldehyde, and reacting with the dextranase solution obtained in the step 2 to obtain immobilized dextranase Fe₃O₄·PDA·GO-PEI-dextranase；

(4) Immobilized dextranase was added to dextran-containing T70(20g L)^-1) In the acetate buffer solution, the concentration of the acetate buffer solution is 0.1mol L^-1pH5.5 of acetate buffer solution, reaction at 50 ℃ and lysisMolecular dextran. The immobilized enzyme is recovered by magnetic separation in the reaction liquid and is reused for 20 times.

Further, the dextranase-producing strain is Paecilomyces lilacinus DG001(Paecilomyces lilacinus) screened in soil. The strain is preserved by the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.14797, the preservation date is 11 months and 17 days in 2017, and the preservation unit address is No. 3 of Xilu No.1 of Beijing, Chaoyang, North Chen.

Further, the dextranase free enzyme solution is heat-resistant enzyme, and the catalytic reaction is carried out at the temperature of 50 ℃.

Further, Fe₃O₄The conditions for the immobilization of PDA GO-PEI with free enzyme were: the enzyme concentration is 2.0mg/ml, and the reaction time is 6 h.

Further, the immobilized dextranase can rapidly realize product separation and recycling through magnetic separation.

Further, the immobilized dextranase is heat-resistant enzyme, and the temperature is 50-60 ℃ for catalytic reaction.

The invention has the technical characteristics and effects that:

(1) compared with the reported enzymes, the dextranase prepared by fermenting the enzyme producing strain paecilomyces lilacinus has improved heat resistance, can catalyze and degrade macromolecular dextran at 50 ℃, and is beneficial to the application of the dextranase in the sugar industry.

(2) Through immobilization, the thermal stability of the prepared immobilized dextranase is improved, the reaction temperature is increased from 50 ℃ of the free enzyme reaction to 55-60 ℃, and the temperature stability and the pH stability are improved.

(3) When the magnetic particles are used as a carrier, the prepared immobilized dextranase can be quickly separated from a substrate through magnetic separation when being used for degrading macromolecular dextran. Under the reaction conditions, the catalyst can be repeatedly used for 20 times.

Drawings

FIG. 1 is Fe of the present invention₃O₄Transmission electron microscopy of the material prepared from PDA GO-PEI-dextran.

FIG. 2 is a diagram of an infrared spectrum of Fe3O 4. PDA. GO-PEI-dextranase according to the present invention.

FIG. 3 shows the Zeta potential measurement results of the surface of the carrier matrix prepared by the present invention.

FIG. 4 shows the measurement results of the hysteresis curve of the present invention.

FIG. 5 is a digital photograph of the present invention.

FIG. 6 shows the results of pH stability tests of free dextranase, Fe3O4 PDA PEI-dextran and Fe3O4 PDA GO PEI-dextran of the present invention.

FIG. 7 shows the results of temperature stability tests for free dextranase, Fe3O4 PDA PEI-dextran and Fe3O4 PDA GO PEI-dextran of the present invention.

FIG. 8 shows the result of the recycling performance test of Fe3O4 PDA GO-PEI-dextran of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1: preparation of dextranase enzyme solution

Inoculating the preserved paecilomyces lilacinus strain into a fermentation culture medium, culturing and activating for 2-5 times at the temperature of 28-30 ℃ and at the speed of 200r/min, and then inoculating into a fresh fermentation culture medium; culturing at 28-30 ℃ and 200r/min for 6-9 days, then centrifuging at 4 ℃ for 10-15 min, wherein 8000-12000 g of culture solution is obtained. 80% (NH) of the obtained supernatant₄)₂SO₄Precipitating, and dissolving the obtained precipitate in 0.1mol L^-1pH5.5 buffer. Adding the solution into a 10kDa ultrafiltration centrifugal tube, and carrying out ultrafiltration for 30 minutes to obtain the dextranase solution.

The fermentation medium comprises the following components in percentage by mass and volume: 1.5% dextran-70 or dextran with larger molecular weight, 0.5% peptone or yeast extract, 0.1% K₂HPO₄·3H₂O、0.05％MgSO₄·7H₂O、0.05％KCl、0.001％FeSO₄·7H₂O; and the initial basal fermentation medium has a pH of 5.0.

Example 2:immobilized enzyme Fe₃O₄Preparation method of PDA, GO-PEI-dextranase

Weighing 400mg of Fe₃O₄Dispersing the particles in 200mL of aqueous solution of Tris (hydroxymethyl) aminomethane hydrochloride containing 400mg of dopamine hydrochloride, and stirring at room temperature for 6h to obtain Fe₃O₄PDA was washed 3 times with ethanol and water each. Subsequently, 400mg of Fe was weighed₃O₄PDA, dispersed in 400mL of graphene oxide aqueous solution (1mg mL)^-1) Mechanically stirred at room temperature for 8 h. After the reaction was completed, magnetic separation was performed, and washing with water was performed 3 times. Then 100mg of prepared Fe₃O₄PDA GO dispersed in 100mL of an aqueous polyethyleneimine solution (10mg mL)^-1) Mechanically stirred at room temperature for 12 h. After the reaction is finished, magnetic separation is carried out, and washing is carried out for 3 times by water, so as to obtain Fe₃O₄·PDA·GO-PEI。

20mg of the prepared magnetic carrier was weighed out and dispersed in 20mL of 5% (w/v) glutaraldehyde-containing phosphate buffer (0.1mol L)^-1pH6.0), mechanically stirred at 30 ℃ for 4h, magnetically separated, washed 3 times with phosphate buffer to remove excess glutaraldehyde. Glutaraldehyde-activated Fe₃O₄Dispersing PDA GO-PEI in 5mL of 2.0mg mL^-1Dextranase and 0.2mg mL^- ¹NaBH₄Phosphate buffer (0.1mol L)^-1pH6.0), shaking at 30 deg.C for 6 hr, magnetically separating, and adding phosphate buffer (0.1mol L)^-1pH6.0) to remove unreacted dextranase, and storing the prepared immobilized enzyme in acetate buffer (0.1mol L)^-1pH5.5), and storing at 4 ℃ for later use.

Example 3: immobilized enzyme Fe₃O₄Structural characterization of PDA GO-PEI-dextranase

Transmission Electron microscopy (TEM, FIG. 1a) shows the Fe prepared₃O₄Has spherical structure, rough outer surface and average size of about 90 nm. Coating with a Polydopamine layer (PDA) to obtain Fe₃O₄PDA surface was smooth and presented a typical core-shell structure with a peripheral poly-dopamine layer thickness of about 20nm (fig. 1 b). Graphene Oxide (GO) nanoplates used in the experiment exhibited a wrinkling phenomenon (fig. 1c), which was coated in Fe₃O₄The wrinkling phenomenon still remains behind on the surface of the PDA (fig. 1 d). Due to Fe₃O₄PDA nanoparticles are relatively small in size, GO nanosheets are relatively large in size, and multiple nanoparticles are encapsulated within one GO nanosheet at the same time. In addition, the edges of the GO nano-sheets present a spread carpet morphology without any agglomeration, the structure is favorable for maintaining the characteristic of high specific surface area of GO, and sufficient reaction sites are provided for fixing Polyethyleneimine (PEI). Further, after modification of PEI polymer and immobilization of dextranase, Fe in the dried state₃O₄·PDA·GO-PEI、Fe₃O₄PDA GO-PEI-dextranase and Fe₃O₄The morphology difference of PDA GO is not obvious.

Fourier transform infrared spectrometer for analyzing Fe₃O₄、Fe₃O₄·PDA、Fe₃O₄PDA GO and Fe₃O₄Chemical composition of PDA GO-PEI. As shown in FIG. 2a, characteristic absorption peak 588cm^-1Is Fe₃O₄Fe-O peak of (1); in FIG. 2b, characteristic absorption peaks 1506 and 1606cm^-11276cm corresponding to the vibration peak of benzene ring^-1Belongs to the C-OH vibration peak of the benzene ring of polydopamine, which proves that PDA is successfully coated on Fe₃O₄A surface. In FIG. 2c, characteristic absorption peaks 1051 and 1727cm^-1Respectively corresponding to a C-O vibration peak and a C ═ O vibration peak, which shows that GO liquid is successfully coated on Fe₃O₄PDA surface. In FIG. 2d, characteristic absorption peaks of 2935 and 2852cm appear^-1Indicating that PEI was successfully modified in Fe₃O₄PDA. GO surface. Reference is made to FIG. 1Fe₃O₄Transmission electron microscopy of the material prepared from PDA GO-PEI-dextran. (a) Fe₃O₄,(b)Fe₃O₄·PDA,(c)GO,(d)Fe₃O₄·PDA·GO,(e)Fe₃O₄PDA GO-PEI and (f) Fe₃O₄FIG. 2 shows an infrared spectrum of Fe3O 4. PDA GO PEI-dextran. (a) Fe3O4, (b) Fe3O 4. PDA, (c) Fe3O 4. PDA. GO and (d) Fe3O 4. PDA. GO-PEI.

Respectively measuring Fe by adopting Zeta potential meter₃O₄、Fe₃O₄·PDA、Fe₃O₄·PDA·GO、Fe₃O₄PDA GO-PEI and Fe₃O₄PDA. GO-PEI-dextran surface potential values, the results are shown in FIG. 3. In aqueous solution at pH 7.0, Fe₃O₄The surface potential of the particles was-18.87 mV. Modified polydopamine, then Fe₃O₄The surface potential of the PDA particles dropped to-25.3 mV. The GO has hydroxyl, carboxyl and other groups on the surface and is fixed on the Fe₃O₄After PDA surface, the material surface potential value further dropped to-39.78 mV. Importantly, after PEI polymer is modified and dextranase is immobilized in sequence, Fe₃O₄PDA GO-PEI and Fe₃O₄The surface potential of PDA GO-PEI-dextran was increased to +35.80mV and +10.68mV, indicating that PEI polymers and dextranase were successfully bound to the particle surface in sequence.

Determination of Fe by physical Property measurement System₃O₄And Fe₃O₄Magnetic properties of PDA. GO-PEI-dextranase. In FIG. 4, the two hysteresis curves are uniform and symmetrical, which shows that the prepared magnetic material has superparamagnetism and Fe₃O₄And Fe₃O₄The magnetic saturation measurements of PDA GO-PEI-dextran were 70.67 and 31.57emu g, respectively^-1The value can meet the requirement that the magnetic material is aggregated under the action of an external magnetic field and is quickly separated from the solution. FIG. 5a shows Fe prepared₃O₄PDA GO-PEI-dextranase dispersed in acetic acid buffer (0.1mol L^-1Ph5.5) shows excellent dispersibility. When the magnet is placed on the outer wall of the glass bottle, Fe₃O₄PDA. GO-PEI-dextran was rapidly adsorbed on the inner glass wall (FIG. 5 b). After the magnet is removed, the glass bottle is gently shaken, Fe₃O₄PDA. GO-PEI-dextran redispersed homogeneously in solution (FIG. 5 c). Figure 5 digital photograph: (a) fe3O4 PDA GO PEI dextranase is evenly dispersed in acetate buffer solution; (b) fe3O4 PDA GO PEI dextranase is attracted by an external magnet to be rapidly gathered on the inner wall of the glass bottle; (c) the magnet was removed and the glass bottle gently shaken and then the Fe3O 4. PDA. GO-PEI-dextranase was again dispersed uniformly. (6) Fixing devicePerforming catalytic reaction of fixed dextranase, measuring 0.4mL of free dextranase solution or magnetic separation nanoparticle fixed dextranase suspension, and adding 1.6mL of dextran-containing T70(20g L)^-1) Acetate buffer (0.1mol L)^-1pH5.5), reaction at 50 ℃. The produced reducing sugar is quantitatively determined by a 3, 5-dinitrosalicylic acid method. The mass of protease required to produce 1. mu. mol maltose within 1min is defined as 1 unit of active dextranase.

Example 4: immobilized enzyme Fe₃O₄PDA GO-PEI-dextranase stability test (in Fe)₃O₄PDA-PEI-dextranase as a control

Free dextranase and immobilized dextranase were treated with buffer solutions at different pH values (3.0-8.0), respectively, and the enzyme activity was measured after treatment, the results are shown in FIG. 6. The pH value corresponding to the highest activity of free dextran is 5.5, while Fe₃O₄PDA-PEI-dextranase and Fe₃O₄The pH at which the activity of PDA GO-PEI-dextran reached the maximum value corresponds to a pH of 6.0. In addition, Fe compared to free dextranase₃O₄PDA-PEI-dextranase and Fe₃O₄The stability of PDA GO-PEI-dextranase is significantly improved under acidic or alkaline pH conditions.

FIG. 7 shows free dextranase, Fe₃O₄PDA-PEI-dextranase and Fe₃O₄Temperature stability test results for PDA. GO-PEI-dextranase. The temperature at which the free dextranase activity reaches a maximum is 50 ℃ and Fe₃O₄PDA-PEI-dextranase and Fe₃O₄The temperature value corresponding to the time when the activity of PDA GO-PEI-dextran reached the maximum value was 55 ℃. In addition, Fe compared to free dextranase₃O₄PDA-PEI-dextranase and Fe₃O₄The stability of PDA GO-PEI-dextranase is significantly improved at higher or lower temperature conditions.

Example 5: immobilized enzyme Fe₃O₄PDA GO-PEI-dextranase reusability test

Wherein, FIG. 8 shows the result of the recycling performance test of Fe3O4 PDA GO-PEI-dextran. As shown in FIG. 8, the enzyme activity of Fe3O4 PDA GO-PEI-dextran remained 85.78% after 20 consecutive reuses. Fe3O4 PDA GO PEI-dextran enzyme shows excellent recycling performance mainly benefited by its excellent magnetic response performance, fast and mild separation process. In addition, the stability of the immobilized dextranase in a complex changing environment is improved by the flexible graphene oxide and the polyethyleneimine in the carrier matrix.

Claims

1. A preparation method of immobilized dextranase is characterized by comprising the following steps: the preparation method comprises the following preparation steps:

(1) inoculating paecilomyces lilacinus to a fermentation culture medium, activating, and performing liquid fermentation under the conditions of 28-30 ℃ and 200r/min for 6-9 d; the paecilomyces lilacinus is screened paecilomyces lilacinus DG001 in soil; the strain is preserved by the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC No. 14797;

(2) centrifuging the fermentation liquor, removing the precipitate, and using (NH) as supernatant₄)₂SO₄Carrying out precipitation; adding (NH) to the supernatant₄)₂SO₄To (NH)₄)₂SO₄The saturation degree is 80 percent, the precipitate is collected by centrifugation and is added with 0.1mol L^-1Dissolving the solution in a buffer solution with pH of 5.5, performing centrifugal ultrafiltration on the dissolved substance by using an ultrafiltration centrifugal tube with the molecular weight of 10KDa, and performing ultrafiltration for 30 minutes to obtain a dextranase enzyme solution;

(3) the immobilized enzyme magnetic carrier Fe₃O₄Activating PDA-GO-PEI with glutaraldehyde, reacting with the dextranase solution obtained in step (2) to obtain immobilized dextranase Fe₃O₄·PDA·GO-PEI-dextranase；

(4) Adding immobilized dextranase into acetate buffer solution containing dextran T70, wherein the concentration of dextran T70 in the acetate buffer solution is 20g L^-1The concentration of acetate buffer solution is 0.1mol L^-1Reacting at 50 ℃ with the pH value of acetate buffer solution to crack the macromolecular dextran; reaction solutionThe magnetic adsorption of the magnetic functional immobilized dextranase is realized by using a magnet, so that the immobilized enzyme is recovered and reused for 20 times.

2. The method for preparing immobilized dextranase according to claim 1, wherein the dextranase enzyme solution is a thermostable enzyme and the temperature is 50 ℃ for catalytic reaction.

3. The method of claim 1, wherein the immobilized dextranase is Fe₃O₄The conditions for immobilizing the PDA-GO-PEI and the dextranase enzyme solution are as follows: the enzyme concentration is 2.0mg/ml, and the reaction time is 6 h.

4. The method for preparing immobilized dextranase according to claim 1, wherein the immobilized dextranase is used for rapid product separation and recycling by magnetic separation.

5. The method for preparing immobilized dextranase according to claim 1, wherein the immobilized dextranase is thermostable enzyme and the temperature is 50 ℃ to 60 ℃.