CN106701699B - Biocatalyst and preparation method and application thereof - Google Patents

Abstract

The present invention provides a biocatalyst comprising an NADH dependent specific 3-quinuclidinone reductase (QNR), a Glucose Dehydrogenase (GDH), Ni²⁺Functionalized agarose microspheres, the catalyst having a diffraction angle of 2θDiffraction peaks are found at 27.5 + -0.2 deg., 31.7 + -0.2 deg., 45.5 + -0.2 deg., 52.1 + -0.2 deg., 66.4 + -0.2 deg., and 75.8 + -0.2 deg.. The preparation method of the biocatalyst has the advantages of simple operation, no need of special equipment, mild process conditions, low cost and suitability for industrialization. The method is used for the biocatalytic synthesis of (R) -3-quinuclidinol, can simultaneously realize carbonyl reduction and coenzyme in-situ regeneration, and does not need to add expensive coenzyme NADH. Meanwhile, the biological catalyst can be recovered and recycled, and has long-term operation stability.

Description

Biocatalyst and preparation method and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a biocatalyst, and a preparation method and application thereof.

Background

(R) -3-quininol (formula C)₇H₁₃NO, molecular weight 127.18, CAS number: 25333-42-0) is a key intermediate for synthesizing such drugs as aclidinium bromide, solifenacin, revaprepitant and tasalidine. At present, the industry mainly utilizes chiral catalysts to asymmetrically reduce 3-quinuclidinone to synthesize (R) -3-quinuclidinol, such as: XylSnewphos/PICA-Ruthenium (II) complex or BINAP/IPHAN-Ru (II) complex, etc. However, the chemical synthesis method requires screening of chiral ligands; the transition metal used is expensive, highly toxic and difficult to remove from the product; and the prepared product has low optical purity and needs further purification. Another method is the racemic resolution, which has the disadvantage that the theoretical yield is only 50% at the maximum.

Compared with a chemical synthesis method, the biological synthesis method using enzyme as a catalyst has substrate specificity; highly chemo-, regio-and stereoselective; the reaction condition is mild; the catalytic activity is high; good atom economy and the like. Biological method for synthesizing (R) -3-quininol involves two processes: one is the reduction of 3-quinuclidinone to (R) -3-quinuclidinol by NADH or NADPH with the aid of carbonyl reductase; secondly, oxidized NAD by coenzyme regeneration enzyme⁺Or NADP⁺Converted into reduced NADH or NADPH to realize the asymmetric reduction synthesis of (R) -3-quininol by coupling of double enzymes. The specific process is as follows:

a process for synthesizing (R) -3-quininol by biocatalysis asymmetric reduction.

No matter wild microorganisms or recombinant Escherichia coli are used as biocatalysts, substrate/product and coenzyme exchange inside and outside cells is hindered due to the structural limitation of cells, so that the biotransformation time is long and the catalysis efficiency is low. Free enzyme as a catalyst, enzyme stability and high cost due to enzyme purification are problems that must be solved for enzyme as a biocatalyst. In addition, both of these biocatalysts are difficult to recover and recycle.

In practice, in order to improve the stability, catalytic activity and selectivity of the enzyme, the enzyme may be bound to a specific support to form an immobilized enzyme, and the immobilized enzyme biocatalyst may be easily separated from the reaction mixture, recycled and used for continuous reactions (Robert Dicosimo, Joseph Mcoauliffe, Ayrookara J. Pouloseband GregoryBohlmann, Industrial use of immobilized enzymes, chem. Soc. Rev.,2013,42, 6437-type 6474; Joshua Britton, Colin L. Raston and Gregory A. Weiss, Rapid protein catalysis for protein filtration consistent with protein synthesis flow biological, chem. Commun, 2016,52, 10159. 10162). Conventional enzyme immobilization methods include physical adsorption, chemical binding, entrapment, and formation of cross-linked enzyme aggregates. The physical method is simple and rapid, enzyme inactivation is not easy to cause, but the enzyme is easy to leak, and the carrier is easy to shield the enzyme active site to prevent substrate combination. The chemical method is advantageous for increasing the enzyme stability, but is susceptible to enzyme inactivation (horizontal healthy and Y. -H. Percific Zhang, cyclic cellulose-stabilizing semiconducting peptides: immobilization of cellulose-binding module-labeled protein and a synthetic surfactant binding, J. Mater. chem. B,2013,1, 4419. eye 4427; WenWang, Daniel I. C. Wang and Zhi Li, facility binding of reactive biocompatible, purification and immobilization of cellulose binding, adsorption and hydrolysis of cellulose binding and Ni-zeyla functional, digestion and digestion of cellulose binding, adsorption and digestion of cellulose binding of Ni-zeylactic reaction, hydrolysis of cellulose binding of cellulose, hydrolysis of cellulose binding, hydrolysis of cellulose binding of cellulose, hydrolysis of cellulose.

Disclosure of Invention

In order to solve the problems of the prior art, according to a first aspect of the present invention, it is an object of the present invention to provide a biocatalyst which is good in stability, good in catalytic activity, stable in long-term operation and recyclable.

The purpose of the invention is realized as follows:

a biocatalyst comprising an NADH dependent specific 3-quinuclidinone reductase (QNR), a Glucose Dehydrogenase (GDH), Ni²⁺The functionalized agarose microspheres are characterized in that: diffraction peaks appear at diffraction angles 2 theta of 27.5 +/-0.2 degrees, 31.7 +/-0.2 degrees, 45.5 +/-0.2 degrees, 52.1 +/-0.2 degrees, 66.4 +/-0.2 degrees and 75.8 +/-0.2 degrees.

According to an embodiment of the present invention, the above biocatalyst further comprises sodium chloride and sodium dihydrogen phosphate.

According to one embodiment of the present invention, the above biocatalyst has a foam-like porous structure with a pore size of 10nm to 2.5. mu.m.

According to one embodiment of the invention, the above biocatalyst has an enzyme loading of 5-15%.

According to a second aspect of the present invention, it is another object of the present invention to provide a method for preparing the above biocatalyst.

According to an embodiment of the present invention, the above-mentioned method for preparing a biocatalyst is characterized by comprising the steps of:

step (1)

Balancing Ni with buffer solution A²⁺Functionalized agarose microspheres, adding cell lysis mixed supernatant containing histidine-tagged 3-quinuclidinone reductase (His-QNR) and histidine-tagged glucose dehydrogenase (His-GDH), and diluting with buffer solution A containing 5-15mM imidazole to obtain suspension;

step (2)

Continuously stirring the suspension prepared in the step (1) for 1-3 hours at the rotating speed of 30-100 rpm under the conditions of the temperature of 4-30 ℃ and the pH value of 5.0-10.0; then centrifuging, collecting enzyme-loaded agarose microspheres, and washing with the buffer solution A in the step (1);

step (3)

Dispersing the washed enzyme-loaded agarose microspheres in the buffer solution A in the step (1), and incubating for 1-5 days in a refrigerator at the temperature of 2-8 ℃; obtaining;

the pH value of the buffer solution A is 7.8-8.2.

According to one embodiment of the present invention, the above buffer solution A contains 50mM sodium dihydrogenphosphate and 300mM sodium chloride.

According to a third aspect of the present invention, it is still another object of the present invention to provide the use of the above biocatalyst for the synthesis of (R) -3-quinuclidinol.

According to a fourth aspect of the present invention, it is still another object of the present invention to provide a method for synthesizing (R) -3-quinuclidinol by performing an enzymatic reaction using the above biocatalyst.

According to one embodiment of the present invention, a method for synthesizing (R) -3-quinuclidinol is characterized in that:

dispersing the biocatalyst in the buffer solution B, adding 3-quininone, glucose and NAD⁺And NADH, oscillating at the rotating speed of 50-150 rpm under the condition of room temperature and the pH value of 7.5-8.0, wherein the biotransformation time is 1-12 h; the pH value of the buffer solution B is 7.2-7.4.

According to one embodiment of the invention, the buffer solution B is 10mM Phosphate Buffered Saline (PBS) containing 137mM NaCl, 2.7mM KCl, Na₂HPO₄10mM，KH₂PO₄2mM。

Specifically, the biocatalytic synthesis method of (R) -3-quininol comprises the following steps:

step (1)

Balancing Ni with buffer solution A²⁺Functionalized agarose microspheres, adding cell lysis mixed supernatant containing histidine-tagged 3-quinuclidinone reductase (His-QNR) and histidine-tagged glucose dehydrogenase (His-GDH), and diluting with buffer solution A containing 5-15mM imidazole to obtain suspension;

step (2)

Continuously stirring the suspension prepared in the step (1) for 1-3 hours at the rotating speed of 30-100 rpm under the conditions of the temperature of 4-30 ℃ and the pH value of 5.0-10.0; then centrifuging, collecting enzyme-loaded agarose microspheres, and washing with the buffer solution A in the step (1);

step (3)

Dispersing the washed enzyme-loaded agarose microspheres in the buffer solution A in the step (1), and incubating for 1-5 days in a refrigerator at the temperature of 2-8 ℃; preparing a biocatalyst;

step (4)

Dispersing the biocatalyst in the buffer solution B, adding 3-quininone, glucose and NAD⁺And NADH, oscillating at the rotating speed of 50-150 rpm under the condition of room temperature and the pH value of 7.5-8.0, wherein the biotransformation time is 1-12 h;

the buffer solution A contains 50mM sodium dihydrogen phosphate and 300mM sodium chloride, and has a pH value of 7.8-8.2;

the buffer solution B is 10mM phosphate buffer saline solution (PBS) containing 137mM NaCl, 2.7mM KCl and Na₂HPO₄10mM，KH₂PO₄2mM, pH 7.2-7.4.

Has the advantages that:

1. the invention is based on Ni²⁺And the strong affinity effect between the poly-histidine tag directly purifies and immobilizes His-QNR and His-GDH from cell lysate at the same time, and the prepared biocatalyst has a diffraction angle 2 theta of 27.5 +/-0.Diffraction peaks are arranged at 2 degrees, 31.7 +/-0.2 degrees, 45.5 +/-0.2 degrees, 52.1 +/-0.2 degrees, 66.4 +/-0.2 degrees and 75.8 +/-0.2 degrees. The biological catalyst has a foam-like porous structure, the aperture is 10nm-2.5 mu m, the enzyme loading is up to 5-15%, and the catalytic activity is good.

2. The biocatalyst of the invention is used as an immobilized enzyme catalyst for the biocatalytic synthesis of (R) -3-quinuclidinol, can simultaneously realize carbonyl reduction and coenzyme in-situ regeneration, and does not need to add expensive coenzyme NADH. Meanwhile, the biocatalyst can be recycled and reused, and has long-term operation stability.

3. The enzyme immobilization method of the invention does not need to pre-purify the target protein, and can simultaneously complete the purification and immobilization of the target protein; meanwhile, the activity of the target protein can be specifically enriched; the prepared immobilized enzyme has good stability, the method is simple to operate, special equipment is not needed, the process condition is mild, the cost is low, and the method is suitable for industrialization.

4. The (R) -3-quinine alcohol is asymmetrically synthesized by using the biocatalyst, the yield is up to 70-85%, the conversion rate is 100%, the enantiomer value is 100%, and the biocatalyst can be recycled for more than 15 times, so that the economic benefit is great. The medium for the biocatalytic asymmetric synthesis of (R) -3-quininol is water, so that resources can be saved, the environment is not polluted, and the method meets the requirement of green chemistry.

Drawings

FIG. 1 is an SDS-PAGE analysis of biocatalysts;

FIG. 2 is temperature vs. Ni²⁺Influence of enzyme load of the functionalized agarose microspheres;

FIG. 3 shows pH value versus Ni²⁺Influence of enzyme load of the functionalized agarose microspheres;

FIG. 4 is Ni²⁺The influence of the mass ratio of the functionalized agarose microspheres and the total protein on the specific activity and activity recovery rate of the enzyme;

FIG. 5 is a scanning electron microscope analysis of biocatalysts;

FIG. 6 is a powder X-ray diffraction analysis of a biocatalyst;

FIG. 7 is a thermogravimetric analysis of a biocatalyst;

FIG. 8 is an infrared spectroscopic analysis of 3-quininone and (R) -3-quininol;

FIG. 9 is a recycle of biocatalyst;

FIG. 10 is an SDS-PAGE analysis of different cycle batches of biocatalysts.

Detailed Description

The present invention is described in detail below with reference to specific examples, which are given for the purpose of further illustrating the invention and are not to be construed as limiting the scope of the invention, and the invention may be modified and adapted by those skilled in the art in light of the above disclosure. All the raw materials and reagents of the invention are commercial products. The carbonyl reductase used in the invention is specific 3-quininone reductase (QNR, access No: AB733448) depending on NADH, and the coenzyme regeneration enzyme is glucose dehydrogenase (GDH, access No: AY 930464).

Example 1

Enzyme Activity assay

QNR enzyme Activity assay:

standard reaction mix system: buffer B (PBS, pH7.2-7.4), 3. mu. mol 3-quinuclidinone, 0.3. mu. mol NADH, appropriate amount of enzyme QNR, total volume 1 mL. The change in absorbance was measured at λ 340 nm. Definition of enzyme activity units: the amount of enzyme required to convert 1. mu. mol NADH at 25 ℃ in 1 min.

GDH enzyme Activity measurement:

standard reaction mix system: buffer B (PBS, pH7.2-7.4), 10. mu. mol glucose, 1. mu. mol NAD⁺Appropriate amount of enzyme GDH, 1mL in total volume. The change in absorbance was measured at λ 340 nm. Definition of enzyme activity units: conversion of 1. mu. mol NAD within 1min at 25 ℃⁺The amount of enzyme required.

Example 2

Co-immobilization of His-QNR and His-GDH

Taking 50mg of Ni²⁺Functionalized agarose microspheres equilibrated with buffer solution A (containing 50mM sodium dihydrogen phosphate and 300mM sodium chloride, pH 7.8-8.2); then 2mL of the cell lysis mix supernatant containing quinuclidinone reductase (His-QNR) and glucose dehydrogenase (His-GDH) was added and diluted to 5mL with buffer solution A containing 10mM imidazole. At a temperature ofThe suspension was stirred continuously at 25 ℃ and pH8.0 at 50rpm for 2 h. And (3) centrifuging the suspension, collecting the enzyme-loaded agarose microspheres, and washing the enzyme-loaded agarose microspheres twice by using a buffer solution A. The washed enzyme-loaded agarose microspheres were redispersed in buffer solution A and incubated in a refrigerator at 4 ℃ for 4 days.

Polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the biocatalyst prepared in example 2

The results of SDS-PAGE analysis are shown in FIG. 1. In the figure, M is the standard protein molecular weight; l is₁Recombinant Escherichia coli expressing His-QNR and recombinant Escherichia coli expressing His-GDH; l is₂Cell lysis mixed supernatants of two recombinant escherichia coli; l is₃、L₄、L₅And L₆Collections of biocatalyst were eluted with 10mM, 250mM, 500mM and 1M. Between 35-25kDa in molecular weight, two obvious protein bands appear, corresponding to QNR (25.74kDa) and GDH (28.25kDa), respectively, and the purity of the target protein is above 90%, thus confirming that the affinity immobilization method provided by the invention can simultaneously purify and immobilize His-QNR and His-GDH directly from cell lysate.

With reference to the preparation method of example 2, the temperature and pH values were examined for Ni²⁺Influence of enzyme load and enzyme load efficiency of the functionalized agarose microspheres; investigation of Ni²⁺Effect of functionalized agarose microspheres and total protein mass ratio on enzyme specific activity and activity recovery.

Temperature to Ni²⁺The results of the effect of the loading capacity and loading efficiency of the functionalized agarose microballons enzyme are shown in the attached figure 2. When the temperature was increased from 4 ℃ to 25 ℃, the enzyme loading increased from 27.5 to 59.6mg/g (Ni)²⁺Functionalized agarose microspheres), the loading efficiency increased from 51.6% to 89.7%. When the temperature exceeded 30 ℃, the enzyme loading and loading efficiency did not increase, indicating that the optimum temperature for immobilization was 25 ℃.

pH to Ni²⁺The results of the effect of the loading capacity and loading efficiency of the functionalized agarose microballons enzyme are shown in the attached figure 3. At a temperature of 25 ℃ and a pH of 8.0, the enzyme loading reached a maximum of 60.8mg/g (Ni)²⁺Functionalized agarose microspheres), whereas the enzyme loading efficiency reaches a maximum of 92.3%. Therefore, it is fixedThe optimum pH for the quantification was 8.0.

Ni²⁺The effect of the mass ratio of functionalized agarose microspheres and total protein on the specific activity and activity recovery of the enzyme is shown in figure 4. Ni²⁺When the mass ratio of the functionalized agarose microspheres to the total protein was increased from 4/1 to 8/1, the specific activities of QNR and GDH were increased from 5.3U/mg to 8.4U/mg and 5.6U/mg to 11.5U/mg, respectively, and the activity recovery was increased from 61.2% to 94.6% and 64.2% to 98.6%, respectively. However, when the mass ratio was increased from 8/1 to 10/1, the specific activity and the activity recovery rate did not change much. When the enzyme is immobilized, Ni²⁺The mass ratio of the functionalized agarose microspheres to the total protein is preferably 8/1.

Scanning Electron microscopy analysis of the biocatalyst prepared in example 2

The sample solution was dropped on a clean cover glass, vacuum dried at 40 ℃, gold-sprayed to cover the sample, imaged with a scanning electron microscope (SEM, S-3000N type) and a field emission scanning electron microscope (FE-SEM, FEI NOVA NanoSEM 400), and the analysis results are shown in FIG. 5. FIG. 5a shows Ni²⁺Functionalized agarose microspheres with smooth surface. FIG. 5b shows Ni after enzyme immobilization²⁺The surface morphology of functionalized agarose microspheres, i.e., biocatalysts, is completely different from that shown in FIG. 5a, with the appearance of small spheres that are irregular in size and shape and tightly connected to each other. The biocatalyst is further analyzed by FE-SEM to find that the surface of the biocatalyst is a foam-like porous structure with the aperture between 10nm and 2.5 μm (figures 5c and d), and the porous structure is beneficial to the rapid exchange of locally enriched high-concentration substrates/coenzymes and substances, thereby improving the reaction speed and the catalytic efficiency.

Powder X-ray diffraction (XRD) analysis of the biocatalyst prepared in example 2

The crystal diffraction peaks of the samples were measured by using a prohibition XD-2X-ray diffractometer under the following test conditions: cu target

Voltage of 30kV, current of 15mA, scanning speed of 2 degree/min, step length of 0.02 degree, and scanningThe range is 5 to 50, and the analysis result is shown in figure 6. It can be seen from the figure that: in the XRD spectrum of the biocatalyst, characteristic peaks were observed at 27.5. + -. 0.2 °, 31.7. + -. 0.2 °, 45.5. + -. 0.2 °, 52.1. + -. 0.2 °, 66.4. + -. 0.2 ° and 75.8. + -. 0.2 ° for 2. theta. and the intensity distributions were 845, 1450, 1005, 850, 800 and 830. And protein and Ni²⁺In the XRD spectrogram of the functionalized agarose microspheres, no corresponding characteristic peak appears.

Thermogravimetric analysis (TGA) of the biocatalyst prepared in example 2

The enzyme loading of the biocatalyst was determined using a Mettler 1100SF system. About 2mg of the sample was placed in an aluminum pot, and nitrogen was introduced at 20mL/min with a heating rate of 15 ℃/min and a scanning range of 30-600 ℃. The TGA analysis results are shown in FIG. 7. The biocatalyst enzyme loading was 5.94% calculated on weight loss.

Example 3

The biocatalyst catalyzes the asymmetric synthesis of (R) -3-quininol:

reaction system: 50mg biocatalyst, 5mM 3-quininone, 9mM glucose, 0.05mM NAD⁺And 0.05mM ADH, buffer B (10mM phosphate buffered saline, PBS) containing 137mM NaCl, 2.7mM KCl, Na₂HPO₄10mM，KH₂PO₄2mM, pH7.2-7.4), total volume 10 mL. The reaction temperature is 25 ℃, the stirring is continuously carried out at 100rpm, the pH is controlled to be 7.5 to 8.0 in the reaction process, the reaction is monitored by TLC, and the mobile phase is V_{Methylene dichloride}/V _Methanol9/1. After completion of the biotransformation, the reaction mixture was centrifuged to separate the biocatalyst. The supernatant was adjusted to a pH greater than 12 with high concentration NaOH and then concentrated at 80 ℃ under reduced pressure to 1/4 of total volume. Adding n-butanol of the same volume, extracting for 3 times, collecting organic phase, concentrating under reduced pressure, and evaporating to dryness. Dissolving the solid in toluene at 90 deg.C, filtering the insoluble substance while it is hot, cooling the filtrate to obtain white needle-like crystals, and filtering to obtain white solid: (¹H-NMR (400MHz, DMSO): 4.946(s, 1H), 3.879(q, 1H), 2.531-3.164(m, 6H), 1.392-2.014(m, 5H), the theoretical molecular weight of the product being: 127.18, mass spectrometry: 127).

Infrared spectroscopic analysis and analysis of the configuration of the white solid obtained in example 3

Infrared spectrum analysis: preparing a sample by adopting a KBr tabletting method, wherein the normal scanning wavelength is 4000-400 cm^-1The infrared spectrum of the substrate and the product is shown in figure 8. The results show that 3-quinuclidinone is at 1747cm^-1The characteristic peaks of the left and right carbonyl groups disappear and are 3103cm^-1The appearance of a characteristic peak for hydroxyl groups indicates that 3-quinuclidinone is reduced to 3-quinuclidinol.

Analysis of configuration

The enantiomeric values were determined using a Clarus580GC system (Perkin Elmer, USA) on a chiral column (HYDRODEX-. beta. -6-TBDM,25 m.times.0.25 mm.times.0.25 μm, Macherey-Nagel) using a flame ionization detector. The temperature is programmed from 60 ℃ to 180 ℃, the temperature rise speed is 5 ℃/min, the temperature is kept for 3min, and the temperatures of the sample injector and the checker are 220 ℃ and 250 ℃ respectively. The enantiomer value of the product is 100 percent and the R form of the configuration is determined by taking the standard substance (R) and the (S) -quininol as the control.

The influence of the amounts of substrate and catalyst on the conversion time, conversion and enantiomeric value was examined with reference to example 3, and is shown in Table 1 below.

When the amount of the biocatalyst was 6.12% of the substrate, the biotransformation time increased from 1.0h to 4.5h with increasing substrate amount, both conversion and enantiomeric values being 100%. When the amount of the substrate is 204g/L and the catalyst is halved, the conversion time is 9.5h, the catalytic activity is obviously reduced, the conversion rate is less than 100 percent, and the enantiomer value is still 100 percent. The substrate dosage is shown to influence the activity of the biocatalyst, the conversion time is prolonged, but the stereoselectivity is not influenced.

Example 4

And (4) recovering and recycling the biocatalyst.

Reaction system: 100mg biocatalyst, 10mM quininone, 18mM glucose, 0.05mM NAD⁺And 0.05mM ADH, buffer solution B (PBS, pH7.2-7.4)) Total volume 10 mL. The reaction temperature is 25 ℃, the stirring is continuously carried out at 100rpm, the pH is controlled to be 7.5-8.0 in the reaction process, and after the reaction is monitored by TLC, the catalyst is centrifugally collected and directly used for the next conversion. The biocatalyst provided by the invention was found to be recyclable 15 times, with 100% conversion and enantiomeric value for a single cycle, and the results are shown in FIG. 9. After 15 cycles, the catalytic efficiency decreased significantly. In the recycling process of the biocatalyst, after the 2 nd conversion, the 4 th conversion and the 6 th conversion, respectively, 20 th conversion, a small amount of the catalyst is taken for SDS-PAGE analysis, and the result is shown in figure 10. The immobilized enzyme amount is not obviously reduced in the recycling process of the biocatalyst, which shows that the biocatalyst provided by the invention has long-term operation stability.

Example 5 (comparative example)

Experiments were treated directly with recombinant E.coli expressing His-QNR and expressing His-GDH without treatment with the biocatalyst of the present invention for comparison. Reaction system: 0.1g of mixed wet cells as biocatalyst, 5mM 3-quininone, 9mM glucose, 0.05mM NAD⁺And 0.05mM NADH, buffer solution B (PBS, pH7.2-7.4), in a total volume of 10 mL. The reaction temperature is 25 ℃, the stirring is continuously carried out at 100rpm, the pH is controlled to be 7.5 to 8.0 in the reaction process, the reaction is monitored by TLC, and the mobile phase is V_{Methylene dichloride}/V _Methanol9/1. The conversion time is obviously prolonged (5.5h), the conversion rate is 95 percent, the enantiomer value is 100 percent, and the recovery and the recycling can not be realized.

Claims (8)

1. A biocatalyst comprising an NADH dependent specific 3-quinuclidinone reductase QNR, a glucose dehydrogenase GDH, Ni^{2 +}The functionalized agarose microspheres are characterized in that: at diffraction angle 2θDiffraction peaks are found at 27.5 + -0.2 deg., 31.7 + -0.2 deg., 45.5 + -0.2 deg., 52.1 + -0.2 deg., 66.4 + -0.2 deg., and 75.8 + -0.2 deg..

2. The biocatalyst of claim 1, wherein: the biocatalyst also contains sodium chloride and sodium dihydrogen phosphate.

3. The biocatalyst of claim 1 or 2, wherein: the biocatalyst has a foam-like porous structure with a pore size of 10nm-2.5 μm.

4. The biocatalyst of claim 3, wherein: the enzyme loading amount of the biocatalyst is 5-15% by mass percentage.

5. A process for the preparation of the biocatalyst as claimed in any one of claims 1 to 4, characterized in that the following steps are used:

step (1)

Balancing Ni with buffer solution A²⁺Adding cell lysis mixed supernatant containing histidine-tagged 3-quininone reductase His-QNR and histidine-tagged glucose dehydrogenase His-GDH into functionalized agarose microspheres, and diluting the supernatant with buffer solution A containing 5-15mM imidazole to form suspension;

step (2)

Continuously stirring the suspension prepared in the step (1) for 1-3 hours at the rotating speed of 30-100 rpm under the conditions of the temperature of 4-30 ℃ and the pH value of 5.0-10.0; then centrifuging, collecting enzyme-loaded agarose microspheres, and washing with the buffer solution A in the step (1);

step (3)

Dispersing the washed enzyme-loaded agarose microspheres in the buffer solution A in the step (1), and incubating for 1-5 days in a refrigerator at the temperature of 2-8 ℃; obtaining the product;

the pH value of the buffer solution A is 7.8-8.2; the buffer solution A contained 50mM sodium dihydrogen phosphate and 300mM sodium chloride.

6. Use of the biocatalyst of any one of claims 1 to 4 for the synthesis of (R) -3-quinuclidinol.

7. A method for synthesizing (R) -3-quinuclidinol using the biocatalyst of any one of claims 1-4, wherein:

dispersing the biocatalyst in the buffer solution B, adding 3-quininone, glucose and NAD⁺And NADH at pH 7.5-8.0 at room temperature in a range of 50 ℃ toOscillating at the rotating speed of 150rpm, and performing biotransformation for 1-12 h; the pH value of the buffer solution B is 7.2-7.4; the buffer solution B is 10mM phosphate buffer saline solution PBS containing 137mM NaCl, 2.7mM KCl and Na₂HPO₄10mM，KH₂PO₄2mM。

8. A biocatalytic synthesis method of (R) -3-quininol comprises the following steps:

step (1)

Balancing Ni with buffer solution A²⁺Adding cell lysis mixed supernatant containing histidine-tagged 3-quininone reductase His-QNR and histidine-tagged glucose dehydrogenase His-GDH into functionalized agarose microspheres, and diluting the supernatant with buffer solution A containing 5-15mM imidazole to form suspension;

step (2)

Continuously stirring the suspension prepared in the step (1) for 1-3 hours at the rotating speed of 30-100 rpm under the conditions of the temperature of 4-30 ℃ and the pH value of 5.0-10.0; then centrifuging, collecting enzyme-loaded agarose microspheres, and washing with the buffer solution A in the step (1);

step (3)

Dispersing the washed enzyme-loaded agarose microspheres in the buffer solution A in the step (1), and incubating for 1-5 days in a refrigerator at the temperature of 2-8 ℃; preparing a biocatalyst;

step (4)

Dispersing the biocatalyst in the buffer solution B, adding 3-quininone, glucose and NAD⁺And NADH, oscillating at the rotating speed of 50-150 rpm under the condition of room temperature and the pH value of 7.5-8.0, wherein the biotransformation time is 1-12 h;

the buffer solution A contains 50mM sodium dihydrogen phosphate and 300mM sodium chloride, and has a pH value of 7.8-8.2;

the buffer solution B is 10mM phosphate buffer saline solution PBS containing 137mM NaCl, 2.7mM KCl and Na₂HPO₄10mM，KH₂PO₄2mM, pH 7.2-7.4.

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