CN112048501A - Method for immobilizing D-xylose dehydrogenase - Google Patents

Method for immobilizing D-xylose dehydrogenase Download PDF

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CN112048501A
CN112048501A CN202011021626.2A CN202011021626A CN112048501A CN 112048501 A CN112048501 A CN 112048501A CN 202011021626 A CN202011021626 A CN 202011021626A CN 112048501 A CN112048501 A CN 112048501A
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chitin
xylose dehydrogenase
magnesium
enzyme
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陈可泉
王昕�
许晟
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Nanjing Kainuo Biotechnology Co ltd
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
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    • C01F—COMPOUNDS OF THE METALS BERYLLIUM, MAGNESIUM, ALUMINIUM, CALCIUM, STRONTIUM, BARIUM, RADIUM, THORIUM, OR OF THE RARE-EARTH METALS
    • C01F7/00—Compounds of aluminium
    • C01F7/78—Compounds containing aluminium and two or more other elements, with the exception of oxygen and hydrogen
    • C01F7/784—Layered double hydroxide, e.g. comprising nitrate, sulfate or carbonate ions as intercalating anions
    • C01F7/785—Hydrotalcite
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01175—D-Xylose 1-dehydrogenase (1.1.1.175)

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Abstract

The invention discloses a method for immobilizing D-xylose dehydrogenase, which comprises the steps of immobilizing the D-xylose dehydrogenase by using magnesium-aluminum hydrotalcite doped with chitin; adding an alkali solution into a salt solution until the pH value of the salt solution is 9-10, adding chitin into the obtained solution, stirring, heating for crystallization, filtering, washing the solid to be neutral, and drying to obtain the chitin-doped magnesium-aluminum hydrotalcite; wherein the salt solution is a mixed aqueous solution of magnesium nitrate and aluminum nitrate. According to the invention, the immobilized packaging of the D-xylose dehydrogenase is realized by using the chitin-doped magnesium-aluminum hydrotalcite, so that the tolerance and the reutilization of the immobilized D-xylose dehydrogenase are effectively improved, and the enzyme activity loss of the D-xylose dehydrogenase in practical application is reduced. Meanwhile, the preparation cost of the immobilized enzyme is reduced by using environment-friendly renewable materials from the aspect of process.

Description

Method for immobilizing D-xylose dehydrogenase
Technical Field
The invention belongs to the technical field of immobilized enzymes, and particularly relates to a method for immobilizing D-xylose dehydrogenase.
Background
D-xylonic acid is a high value-added functional sugar, and can be used as a precursor of various compounds, such as copolyamide, polyester, hydrogel, 1,2, 4-butanetriol and the like. In addition, D-xylonic acid has effects of regulating intestinal flora ecological balance, lowering blood pressure, serum cholesterol and blood sugar. However, the production process of synthesizing D-xylonic acid by a chemical method is complicated, the reaction conditions are harsh, and the ecological environment is polluted. And chemical synthesis methodCompared with the biological enzyme method, the process conditions are milder. NAD in the cytoplasm+D-xylose is oxidized into D-xylonolactone by dependent D-xylose dehydrogenase, and then the D-xylonolactone is cleaved under the action of lactonase to generate D-xylonic acid. However, with the increasing temperature, the conformation of the enzyme is destroyed and gradually denatured and inactivated under the condition of peracid or overbase, thereby causing the reduction of the reaction rate and limiting the further popularization of the D-xylose dehydrogenase.
The immobilized carrier can bind the free enzyme in a limited space, the structural stability of the enzyme protein is enhanced, and the sensitivity to the surrounding environment is reduced. In addition, the immobilized material enables the enzyme to meet continuous catalytic reaction, and the active conformation of enzyme molecules can be still maintained after the reaction, so that the enzyme is convenient to recover and recycle. The immobilized enzyme preparation can be recycled for a plurality of times, so that the loss of the substrate can be reduced, the separation of the immobilized enzyme preparation and the product is facilitated, and the whole using process is more economical. The hydrotalcite has a nano structure with multilayer space, and is an ideal material for immobilizing enzyme in a limited space. However, the structural characteristics of hydrotalcite materials remain insufficient for immobilization and protection of enzymes, and therefore, the use of intercalation materials for structural modification of hydrotalcite is a desirable approach. Chitin is a natural linear aminopolysaccharide, contains a large amount of hydroxyl and amino and is an excellent modified material. Therefore, the chitin-hydrotalcite composite material is constructed, the advantages of the chitin-hydrotalcite composite material and the hydrotalcite composite material are integrated, and the immobilized carrier is simple to manufacture, high in tolerance and strong in enzyme binding property. The composite material is utilized to make up the deficiency of the D-xylose dehydrogenase in the catalytic process and improve the service cycle and the catalytic performance of the D-xylose dehydrogenase.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a method for immobilizing D-xylose dehydrogenase aiming at the defects of the prior art.
In order to solve the technical problem, the invention discloses a method for immobilizing D-xylose dehydrogenase, namely immobilizing the D-xylose dehydrogenase by using magnesium-aluminum hydrotalcite doped with chitin.
The preparation method of the chitin-doped magnesium-aluminum hydrotalcite comprises the steps of slowly adding an alkali solution into a salt solution until the pH value of the salt solution is 9-10 (preferably 9.5), adding chitin into the obtained solution, stirring, heating for crystallization, filtering, washing a solid to be neutral, drying, and grinding to obtain the chitin-doped magnesium-aluminum hydrotalcite with the particle size of about 50 microns; wherein the salt solution is a mixed aqueous solution of magnesium nitrate and aluminum nitrate.
Wherein the alkali solution is a mixed aqueous solution of sodium hydroxide and sodium carbonate; wherein the concentration of the sodium hydroxide is 9-10 mmol/mL, and the concentration of the sodium carbonate is 18-20 mmol/mL; preferably, the concentration of sodium hydroxide is 500mmol/55mL and the concentration of sodium carbonate is 1000mmol/55 mL.
Wherein in the mixed aqueous solution of magnesium nitrate and aluminum nitrate, the concentration of magnesium nitrate is 2-3 mmol/mL, and the concentration of aluminum nitrate is 0.6-0.8 mmol/mL; preferably, the concentration of magnesium nitrate is 100mmol/45mL and the concentration of aluminum nitrate is 30mmol/45 mL.
Wherein the slow addition is completed within 5-60 min.
Wherein the addition amount of the chitin is 45-60 mg/mL, preferably 50 mg/mL.
Wherein the stirring is carried out at 500-1300 rpm for 1.5-2.5 h, preferably at 1000rpm for 2 h.
Wherein the temperature-rising crystallization is crystallization for 10-14 h when the temperature rises to 60-70 ℃, and preferably crystallization for 12h when the temperature rises to 65 ℃.
The method comprises the following steps of adding the chitin-doped magnesium-aluminum hydrotalcite into PBS buffer solution with the pH value of 7.450 mM, ultrasonically mixing the mixture uniformly, adding glutaraldehyde aqueous solution, ultrasonically treating the mixture for 10min, standing the mixture for 30min, adding D-xylose dehydrogenase, stirring the mixture, centrifuging the mixture, and washing precipitates to obtain the immobilized D-xylose dehydrogenase.
Wherein the dosage ratio of the chitin-doped magnesium-aluminum hydrotalcite to the buffer solution is 0.1-0.2 mg/mL, and preferably 0.15 mg/mL.
Wherein the concentration of the glutaraldehyde aqueous solution is 2.5% w/w, and the volume ratio of the glutaraldehyde aqueous solution to the buffer solution is 1: 9-11, preferably 1: 10.
Wherein the dosage of the D-xylose dehydrogenase is controlled to ensure that the protein concentration is 0.1mg/mL, the enzyme activity is 80U/mg, and the enzyme activity is defined as that the enzyme quantity required by consuming xylose per minute to generate 1 mu M D-xylonic acid is 1U.
Wherein the stirring is carried out at 500-1300 rpm for 1.5-2.5 h, preferably at 1000rpm for 2 h.
Wherein the centrifugation is low-temperature high-speed centrifugation at 4 ℃ and 6000rpm for 10 min; preferably, it is repeated three times.
Has the advantages that: compared with the prior art, the invention has the following advantages:
according to the invention, the immobilized packaging of the D-xylose dehydrogenase is realized by using the chitin-doped magnesium-aluminum hydrotalcite, so that the tolerance and the reutilization of the immobilized D-xylose dehydrogenase are effectively improved, and the enzyme activity loss of the D-xylose dehydrogenase in practical application is reduced. Meanwhile, the preparation cost of the immobilized enzyme is reduced by using environment-friendly renewable materials from the aspect of process.
Detailed Description
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
EXAMPLE 1 construction of recombinant plasmid
The XylB gene and PRSFDuet-1 were digested simultaneously by introducing BamHI and HindIII cleavage sites into the 5 '-and 3' -ends of the D-xylose dehydrogenase xylB gene, respectively, and then ligated to the PRSFDuet-1 vector (see example 1 in "A method for synthesizing 1,2, 4-butanetriol by enzymatic reaction").
Example 2 construction of recombinant strains
The constructed recombinant plasmid is transformed into a competent cell, and is coated on LB solid medium of kanamycin (Kana) with the final concentration of 50mg/L, colony PCR verification is carried out after the culture at 37 ℃, and the nucleotide sequence is shown as SEQ ID NO.1 in the method for synthesizing 1,2, 4-butanetriol by CN201711190972.1 enzyme reaction. And (3) carrying out sequencing verification on the correct strain after colony PCR verification, and finally preserving the correct strain in 25 vt% glycerol at the ultralow temperature of-80 ℃.
Example 3 cultivation method
3.1 plate culture
The glycerol bacteria preserved in a refrigerator at-80 ℃ are taken out, streaked on a plate containing 50mg/L Kana, and the plate is cultured in an incubator at 37 ℃ for 12-14 h.
3.2 seed culture
Single colonies on the plate were picked and inoculated into 10mL of medium containing 50mg/L Kana, and cultured for 8-10 h at 37 ℃ on a shaker at 200 rpm.
3.3 Shake flask fermentation culture
The seed solution was inoculated into 100mL of LB liquid medium containing 50mg/L Kana at an inoculum size of 1 vt% and cultured at 200rpm at 37 ℃. When the OD600 is 0.6-0.8, adding IPTG with the final concentration of 1mmol/L for induction, cooling to 30 ℃, culturing for 12h, and centrifuging to collect thalli.
EXAMPLE 4 preparation of pure enzyme solution
The thalli collected by centrifugation is washed twice by pure water to remove the culture medium, the thalli is suspended by pure water through vibration, and the thalli is crushed by an ultrasonic crusher (the time is 10min, the time is 2s and 3s are stopped, and the power is 30%). Centrifuging the obtained cell lysate for 20min at 8000rpm and 4 ℃ in a centrifuge, removing precipitate, and collecting supernatant running protein gel. Protein concentration was determined by Coomassie Brilliant blue method.
The obtained crude enzyme solution was filtered through a 0.2 μm filter to remove solid impurities. By using
Figure BDA0002700825800000041
And (4) carrying out protein purification by using a protein purifier. Phase A: the loading buffer was Tris 20mM, NaCl 0.5M, imidazole 50mM, pH adjusted to 7.0. Phase B: elution buffer was Tris 20mM, NaCl 0.5M, imidazole 500mM, pH adjusted to 7.0. Firstly, the phase A is used for balancing the nickel column, then the crude enzyme solution is loaded (1mL/min), finally the target protein bound on the nickel column is eluted by 100 percent of phase B (2mL/min), and the enzyme solution is collected at the moment, namely the pure enzyme solution. Because the obtained pure enzyme solution contains a large amount of NaCl and imidazole and has low protein concentration, the pure enzyme solution is desalted and concentrated by using a Millipore ultrafiltration tube, the enzyme activity is 80U/mg, and the enzyme activity is defined as the enzyme amount required by consuming xylose per minute to generate 1 mu M D-xylonic acidIs 1U.
Example 5 method for detecting enzyme Activity of D-xylose dehydrogenase
D-xylose dehydrogenase can be detected by NAD+The enzyme activity was measured by the consumption, i.e., the amount of NADH produced. As NADH has an absorbance value at 340nm, the enzyme activity is determined by monitoring the change value of OD340 by using a microplate reader. The enzyme activity determination system is as follows: to 200. mu.L of PBS (50mM, pH 7.0) buffer was added the appropriate amount of enzyme protein, xylose at a final concentration of 5mM, NAD + at 1 mM. Molar absorptivity of 6.22mM-1cm-1。
The enzyme activity was defined as the amount of 1U of enzyme required to produce 1. mu.M of D-xylonic acid per minute from xylose consumed.
EXAMPLE 6 preparation of chitin-doped magnesium aluminum hydrotalcite
0.1mol Mg (NO) was added to 45mL of water at room temperature3)2、0.03mol Al(NO3)3A solution was prepared by adding 0.5mol of NaOH and 1mol of Na to 55mL of water2CO3And preparing a solution B, dropwise adding the solution A into the solution B at the speed of one drop per 2s until the pH value is 9.5, after dropwise adding (the total volume is 100mL), adding 50mg of chitin, continuously stirring at 1000rpm for 2h, heating to 65 ℃, crystallizing for 12h, then carrying out suction filtration on the solution, washing a solid precipitate to be neutral, carrying out vacuum drying on the solid precipitate at 90 ℃, grinding, crushing and sieving to obtain the magnesium-aluminum hydrotalcite (the size is about 50 micrometers).
Example 7 immobilization of D-xylose dehydrogenase
Weighing 15mg of the chitin-doped magnesium aluminum hydrotalcite prepared in example 6, adding the chitin-doped magnesium aluminum hydrotalcite into 100mL of PBS buffer solution with pH 7.450 mM, uniformly mixing by ultrasonic, adding 10mL of glutaraldehyde aqueous solution with the concentration of 2.5% w/w, standing for 30min after ultrasonic treatment for 10min, continuously adding 1mL of D-xylose dehydrogenase solution with the protein concentration of 0.1mg/mL prepared in example 4, stirring at 1000rpm for 2h at 4 ℃, washing precipitates by 3 times of high-speed low-temperature centrifugation (6000rpm,10min), washing the precipitates by distilled water, and determining the protein concentration in supernatant. And finally drying the precipitate in a vacuum freeze dryer to obtain the immobilized enzyme A.
Comparative example 1
1.1 preparation of magnesium aluminum hydrotalcite
At room temperature, with 0.1mol concentration of Mg (NO)3)2And 0.03mol of Al (NO)3)3Preparing salt solution, weighing NaOH with 0.5mol concentration and Na with 1mol concentration2CO3Preparing an aqueous alkali by using ultrapure water, dropwise adding the aqueous alkali into a salt solution at a speed of one drop per 2s until the pH value is 9.5 (the total volume is 100mL), stirring at the rotating speed of 1000rpm for 2h after dropwise adding, heating to 65 ℃ and crystallizing for 12h, then carrying out suction filtration on the solution, washing a solid precipitate to be neutral, drying the solid precipitate at 90 ℃ in vacuum, grinding, crushing and sieving to obtain the magnesium-aluminum hydrotalcite (the size is about 50 micrometers).
1.2 Magnalium hydrotalcite immobilized D-xylose dehydrogenase
Weighing 10mg of magnesium aluminum hydrotalcite, adding the magnesium aluminum hydrotalcite into 100mL of PBS buffer solution with the pH value of 7.450 mM, uniformly mixing by ultrasonic, adding 10mL of glutaraldehyde water solution with the concentration of 2.5% w/w, standing for 30min after ultrasonic treatment for 10min, continuously adding 1mL of D-xylose dehydrogenase solution with the protein concentration of 0.1mg/mL, stirring for 2h at 1000rpm at 4 ℃, carrying out high-speed low-temperature centrifugation (6000rpm,10min) for 3 times, washing precipitates by distilled water, and measuring the protein concentration in supernatant. And finally drying the precipitate in a vacuum freeze dryer to obtain the immobilized enzyme B.
Example 8 catalytic Performance
10mg (wherein the enzyme content is 0.05mg) of the immobilized enzyme A is taken for enzyme activity verification according to the embodiment 5, and the control group comprises free enzyme solution and immobilized enzyme B with the same protein concentration, and the reaction time is 2 hours.
(1) Temperature of
Sample (I) Reaction at 37 deg.C Reaction at 40 deg.C Reaction at 45 deg.C
Free enzyme 480U 379U 312U
Immobilized enzyme A 462U 430U 381U
Immobilized enzyme B 450U 392U 347U
Within the range of 20-37 ℃, the activities of the immobilized enzyme A, the immobilized enzyme B and the free enzyme are increased along with the increase of the temperature, the activities of the immobilized enzyme A, the immobilized enzyme B and the free enzyme are simultaneously kept to be the highest at 37 ℃, when the temperature reaches 40 ℃, the activity of the immobilized enzyme A is 93 percent of the highest activity, the activity of the immobilized enzyme B is 87 percent of the highest activity, and the activity of the free enzyme is 79 percent of the highest activity. At 45 ℃, the activity of the immobilized enzyme A is about 82 percent of the highest activity, the activity of the immobilized enzyme B is 77 percent of the highest activity, and the activity of the free enzyme is 65 percent of the highest activity.
(2)pH
Sample (I) pH 5.5 pH 7.5 pH 9.0
Free enzyme 0U 480U 432U
Immobilized enzyme A 277U 462U 453U
Immobilized enzyme B 246U 450U 428U
When the pH value is 7.5, the activity of the immobilized enzyme and the free enzyme can simultaneously keep the maximum enzyme activity. Under the conditions of peracid (pH 5.5) and alkali (pH 9.0), the free enzyme is completely inactivated (peracid), and 90% of activity is maintained, while the immobilized enzyme A can maintain 60% and 98% of activity, and the immobilized enzyme B can maintain 55% and 92% of activity.
(3) Cycle test
And (3) carrying out repeated circulation to verify the reusability of the immobilized enzyme, wherein after each circulation is 2 hours, the temperature is 37 ℃, the pH value is 7.5, and 5 times of circulation, the immobilized enzyme A can still keep 80% of the initial enzyme activity, and the immobilized enzyme B can only keep 60% of the initial enzyme activity.
The present invention provides a method and a concept of immobilizing D-xylose dehydrogenase, and a method and a way for implementing the method and the concept are numerous, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should be considered as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.

Claims (10)

1. A method for immobilizing D-xylose dehydrogenase is characterized in that the D-xylose dehydrogenase is immobilized by using magnesium-aluminum hydrotalcite doped with chitin.
2. The method of claim 1, wherein the chitin-doped magnesium-aluminum hydrotalcite is prepared by adding an alkali solution to a salt solution until the pH of the salt solution is 9-10, adding chitin to the obtained solution, stirring, heating for crystallization, filtering, washing the solid to be neutral, and drying; wherein the salt solution is a mixed aqueous solution of magnesium nitrate and aluminum nitrate.
3. The method according to claim 2, wherein the alkali solution is a mixed aqueous solution of sodium hydroxide and sodium carbonate; wherein the concentration of the sodium hydroxide is 9-10 mmol/mL, and the concentration of the sodium carbonate is 18-20 mmol/mL.
4. The method according to claim 2, wherein the concentration of magnesium nitrate is 2 to 3mmol/mL and the concentration of aluminum nitrate is 0.6 to 0.8mmol/mL in the mixed aqueous solution of magnesium nitrate and aluminum nitrate.
5. The method of claim 2, wherein the chitin is added at a final concentration of 45-60 mg/mL.
6. The method according to claim 2, wherein the temperature-rising crystallization is performed for 10-14 hours at a temperature of 60-70 ℃.
7. The method of claim 1, wherein the immobilized D-xylose dehydrogenase is obtained by adding the chitin-doped magnesium-aluminum hydrotalcite into a buffer solution, uniformly mixing, adding a glutaraldehyde aqueous solution, performing ultrasonic standing, adding D-xylose dehydrogenase, stirring, centrifuging, and washing a precipitate.
8. The method of claim 7, wherein the amount ratio of the chitin-doped magnesium aluminum hydrotalcite to the buffer is 0.1-0.2 mg/mL.
9. The method according to claim 7, wherein the concentration of the glutaraldehyde aqueous solution is 2.5% w/w, and the volume ratio of the glutaraldehyde aqueous solution to the buffer solution is 1: 9-11.
10. The method according to claim 7, wherein the amount of D-xylose dehydrogenase is controlled so that the protein concentration is 0.1mg/mL and the enzyme activity is 80U/mg.
CN202011021626.2A 2020-09-25 2020-09-25 Method for immobilizing D-xylose dehydrogenase Withdrawn CN112048501A (en)

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