A method for increasing 2-deoxy-d-ribose-5-phosphate aldolase activity by immobilization

Abstract

The application provides a method for improving 2-deoxy-D-ribose-5-phosphate aldolase (DERA) activity through immobilization. The method can significantly activate the aldolase activity of DERA, and is simple, mild, simple and easy to operate, and can be applied to preparation of chiral side chains of statins.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis, and more specifically to a method for improving the activity of 2-deoxy-D-ribose-5-phosphoaldolase by immobilization. Background Technology

[0002] 2-Deoxy-D-ribose-5-phosphate aldolase (DERA, EC 4.1.2.4) is an acetaldehyde-dependent class I aldolase that mediates stereoselective C / C bond formation between acetaldehyde donors and various acceptor substrates, generating new chiral centers (J. Rouvinen, M. Andberg, Appl Microbiol Biotechnol, 2021, 105(16-17):6215-6228). DERA is widely distributed in various bacteria, fungi, plants, and mammalian cells, playing an important role in nucleic acid metabolism. The DERA-catalyzed aldol condensation reaction proceeds under mild conditions, forming a Schiff base between the amino group of a lysine residue at the enzyme's active site. Subsequently, the activated donor is stereoselectively added to the aldehyde acceptor via an enamine intermediate, generating multiple chiral centers. Based on this reactivity of DERA, it has been applied to the industrial production of various chemicals, including pentaerythritol, deoxyglucoses, fragrances, and complex drug molecules. Most notably, DERA has shown significant advantages in the synthesis of chiral side chains for lipid-lowering statins (atorvastatin and rosuvastatin) (W. Greenberg, A. Varvak. Proc Natl Acad Sci US A. 2004, 101(16):5788-93). However, practical applications have encountered problems such as enzyme sensitivity to substrates leading to inactivation, substrate inhibition, and the inability to recycle the enzyme. Therefore, immobilization of DERA not only improves its stability but also allows for reuse, effectively enhancing its industrial application value.

[0003] Enzyme immobilization refers to confining free enzymes within a specific space or onto a carrier, restricting their free movement, allowing them to exert their catalytic activity for an extended period and enabling recyclability. Immobilization can increase enzyme stability, organic solvent tolerance, recyclability, and storage stability, making it an excellent method for improving the physicochemical properties of enzymes. The main immobilization methods include adsorption, cross-linking, embedding, and covalent bonding. While these methods utilize readily available and mature materials, they also have several drawbacks. For example, after embedding, the enzyme's substrate and products are less likely to diffuse; covalent bonding can easily lead to enzyme inactivation; and cross-linked enzymes have relatively poor mechanical properties. Among enzyme immobilization methods, adsorption is simple to operate, low in cost, and has unique advantages. Adsorption immobilizes enzyme molecules on the carrier through non-specific physical adsorption, ion adsorption, biospecific adsorption, affinity adsorption, and hydrophobic interactions between the carrier and the enzyme molecule's own structure. Although the specificity of enzyme immobilization by adsorption is not very high, this method is simple to operate, does not involve chemical reactions, does not change the native structure of the enzyme, can prevent the function of the enzyme active site from being damaged, and keeps the enzyme in its native state, and has been widely used (T. Jesionowski; J. Zdarta, Adsorption. 2014, 20: 801–821).

[0004] Materials commonly used for enzyme immobilization via adsorption include silica, hydroxyapatite, diatomaceous earth, activated carbon, and nanomaterials. Diatomaceous earth, mineralogically classified as opal, is an important non-metallic mineral material. It possesses a unique diatom shell structure, with appearances including disc-shaped, linear, feather-like, and needle-like forms. Its characteristics include strong adsorption capacity, large specific surface area, and high porosity, leading to its applications in many industrial fields such as chemical, petroleum, and building materials. Among these, diatomaceous earth is most widely used in the preparation of filter aids, adsorbents, and carriers (Yuan Peng, Wu Daqing. Minerals and Rocks. 2000(01):101-104). Furthermore, diatomaceous earth exhibits acid and heat resistance, excellent biocompatibility, and no toxic side effects on the human body. Summary of the Invention

[0005] In overcoming the problems of easy inactivation, difficult recovery, poor environmental tolerance, and low recycling rate of 2-deoxy-D-ribose-5-phosphate aldolase in practical applications, this invention, while comparing the immobilization effects of various immobilization materials on DERA, accidentally discovered a material and method for improving the activity of 2-deoxy-D-ribose-5-phosphate aldolase through immobilization. This method can effectively improve the aldolase activity of DERA. Not only does this method achieve a better retention rate of DERA enzyme activity than other immobilization methods, but the immobilization and activation of DERA using diatomaceous earth is also simple, mild, and easy to operate.

[0006] The specific technical solution of this invention is as follows:

[0007] A method for improving the activity of 2-deoxy-D-ribose-5-phosphoaldolase involves adding diatomaceous earth to a solution containing 2-deoxy-D-ribose-5-phosphoaldolase for adsorption and fixation.

[0008] Preferably, the protein concentration of the solution containing 2-deoxy-D-ribose-5-phosphoaldolase is 5–50 mg / ml, and 20–200 mg of diatomaceous earth is added to each 1 ml of solution. More preferably, the protein concentration of the solution containing 2-deoxy-D-ribose-5-phosphoaldolase is 10–20 mg / ml, and 50–150 mg of diatomaceous earth is added to each 1 ml of solution.

[0009] Preferably, the solution containing 2-deoxy-D-ribose-5-phosphoaldolase is a buffer solution with a pH of 4.0 to 10.0. More preferably, the buffer solution has a pH of 7.0 and a concentration of 0.01 mol / L. A specific example of the present invention is a phosphate buffer solution.

[0010] Preferably, the fixed time is 1 to 24 hours. More preferably, it is 2 hours.

[0011] The 2-deoxy-D-ribose-5-phosphoaldolase described in this invention is derived from bacteria, archaea, fungi, environmental microorganisms, unculturable microorganisms, plants, animals, etc.

[0012] A specific example of the method described in this invention includes the following steps:

[0013] (1) Collect the bacterial cells of the recombinant engineered strain containing the 2-deoxy-D-ribose-5-phosphate aldolase gene obtained by fermentation culture, and resuspend them in buffer to obtain a bacterial suspension;

[0014] (2) After the bacterial cells are broken, the supernatant is collected by centrifugation to obtain crude enzyme solution;

[0015] (3) Add diatomaceous earth to the crude enzyme solution and shake to adsorb and fix it;

[0016] (4) After immobilization, the precipitate was collected by centrifugation, washed and freeze-dried to obtain the immobilized and activated 2-deoxy-D-ribose-5-phosphoaldolase.

[0017] Further, the pH of the buffer solution described in step (1) is 4.0 to 10.0, preferably 7.0, and the concentration is 0.01 mol / L.

[0018] Further, the resuspension ratio described in step (1) is 1g of bacterial cells resuspended in 10ml of pH 7.0 buffer.

[0019] Furthermore, the concentration of the crude enzyme solution in step (2) is 5–50 mg / ml.

[0020] Further, in step (3), the ratio of crude enzyme solution to immobilized diatomaceous earth is 40-400 mg of immobilized diatomaceous earth per 2 ml of crude enzyme solution, preferably 250 mg of immobilized diatomaceous earth per 2 ml of crude enzyme solution.

[0021] Furthermore, the fixed time mentioned in step (3) is 1 to 24 hours, preferably 2 hours.

[0022] Furthermore, the cleaning solution used in step (4) is a pH 7.0 buffer solution.

[0023] Another object of the present invention is an immobilized and activated 2-deoxy-D-ribose-5-phosphoaldolase, prepared by the method described in the present invention.

[0024] Another object of the present invention is to provide the application of the immobilized and activated 2-deoxy-D-ribose-5-phosphoaldolase in the field of enzyme catalysis.

[0025] As a specific example, the immobilized activated 2-deoxy-D-ribose-5-phosphoaldolase is used to prepare chiral side chains of statin drugs.

[0026] The beneficial effects of this invention are:

[0027] This invention provides an immobilization method that can effectively improve DERA activity. The method is simple to prepare, requiring only a one-pot process to successfully immobilize and activate DERA; the conditions are mild, requiring only room temperature and pressure for immobilization; compared with other immobilization materials, diatomaceous earth has superior activation efficiency, with the resulting immobilized DERA enzyme activity recovery rate >120%, and up to 240%. Attached Figure Description

[0028] Figure 1 The effect of different amounts of diatomaceous earth on the activation effect during immobilization.

[0029] Figure 2 To immobilize the effect of different time periods on activation.

[0030] Figure 3 The effect of using different pH buffer solutions during fixation on activation. Detailed Implementation

[0031] The present invention will be further illustrated by specific embodiments below, but the scope of the present invention is not limited thereto:

[0032] Example 1: The effect of different immobilization materials on immobilizing DERA enzyme

[0033] (1) A recombinant engineered strain containing the DERA gene (gene sequence: GenBank: AFY10769.1, amino acid sequence of which is listed in the following literature: W. Greenberg, A. Varvak. Proc Natl Acad Sci U SA. 2004, 101(16):5788-93) derived from an environmental biological sample (prepared according to the method in Example 2) was inoculated into LB liquid medium containing 50 mg / ml kanamycin and cultured at 37°C and 220 rpm for 9-12 h to obtain a seed culture. The seed culture was then inoculated into TB fermentation medium at a volume concentration of 1% and cultured at 37°C and 220 rpm until OD. 600 The concentration was increased to 0.6–0.8, and then IPTG was added to a final concentration of 0.2 mM for induction. The mixture was induced at 25 °C and 220 rpm for 18 h. The fermentation broth was then centrifuged to collect the wet cells.

[0034] (2) Resuspend the wet bacterial cells in pH 7.0 phosphate buffer at a ratio of 1g:10ml to obtain a bacterial suspension. Use an ultrasonic cell disruptor at 120W with a 3s-6s-pause cycle to disrupt the bacterial cells, and the disruption time should be at least half the volume of the bacterial suspension. After ultrasonic disruption, centrifuge at 12000r / min for 10min and collect the supernatant crude enzyme solution.

[0035] (3) Take 2ml of crude enzyme solution, add 200mg of diatomaceous earth, 200mg of hydroxyapatite and 200mg of activated carbon respectively, and fix in a shaker at 220rpm for 5h.

[0036] (4) After immobilization, centrifuge at 12000 r / min for 5 min, collect the precipitate, wash twice with pH 7.0 phosphate buffer, and centrifuge to collect the supernatant. Freeze-dry the precipitate at -50℃ for 12-24 h to obtain immobilized and activated DERA. Store at 4℃ for later use and determine its activity.

[0037] The immobilized enzyme activity is defined as the amount of enzyme required to catalyze a reaction at 37°C and 220 rpm with acetaldehyde and chloroacetaldehyde in a molar mass ratio of 2:1 as substrates for 2 hours, producing 1 μmol of product per minute.

[0038] The detection method for the product in this invention utilizes the dinitrosalicylic acid (DNS) method: An appropriate amount of immobilized enzyme is added to 800 μL of a 300 mM aldehyde mixture (200 mM acetaldehyde and 100 mM chloroacetaldehyde) and 200 μL of pH 7.0 phosphate buffer. After mixing, the mixture is briefly centrifuged and reacted on a shaker for 2 h. After the reaction, the mixture is centrifuged at 12000 rpm for 10 min. 50 μL of the supernatant is transferred to a 2 ml EP tube, and 100 μL of DNS reagent is added. The mixture is accurately boiled for 5 min, cooled with ice water, and diluted with 450 μL of water. 100 μL of the above solution is added to 900 μL of water, and 300 μL of the diluted solution is added to a 96-well plate. The absorbance is measured at 540 nm. The product amount is determined by comparing with a standard curve, and the enzyme activity is calculated. The enzyme activity recovery rate of immobilized DERA measured by the above method is as follows:

[0039]

[0040] The results in the table above show that the DERA enzyme activity recovery rate using diatomaceous earth fixation is twice that of the initial enzyme activity, while the DERA enzyme activity recovery rate obtained using hydroxyapatite and activated carbon fixation is much lower than that of diatomaceous earth-fixed DERA and the initial enzyme activity. These enzyme activity test results demonstrate that diatomaceous earth fixation of DERA has a significant activating effect on DERA.

[0041] Example 2: Effect of diatomaceous earth on the immobilization and activation of recombinant engineered bacterial enzyme products derived from the DRA gene of *Pyrobaculum aerophilum*.

[0042] (1) A recombinant engineered strain containing the DRA gene from *Pyrobaculum aerophilum* (gene sequence: Accession: Q8ZXK7) (refer to the method described in the literature: Haruhiko Sakuraba, Kazunari Yoneda. *Appl Environ Microbiol.* 2007, 73(22): 7427-34) was inoculated into LB liquid medium containing 50 mg / ml kanamycin and cultured at 37°C and 220 rpm for 9–12 h to obtain a seed culture. The seed culture was then inoculated into TB fermentation medium at a volume concentration of 1% and cultured at 37°C and 220 rpm until OD200. 600 The concentration was increased to 0.6–0.8, and then IPTG was added to a final concentration of 0.2 mM for induction. The mixture was induced at 25 °C and 220 rpm for 18 h. The fermentation broth was then centrifuged to collect the wet cells.

[0043] (2) Resuspend the wet bacterial cells in pH 7.0 phosphate buffer at a ratio of 1g:10ml to obtain a bacterial suspension. Use an ultrasonic cell disruptor at 120W with a 3s-6s-pause cycle to disrupt the bacterial cells, and the disruption time should be at least half the volume of the bacterial suspension. After ultrasonic disruption, centrifuge at 12000r / min for 10min and collect the supernatant crude enzyme solution.

[0044] (3) Take 2 ml of crude enzyme solution, add 200 mg of diatomaceous earth, and fix in a shaker at 220 rpm for 5 h.

[0045] (4) After immobilization, centrifuge at 12000 r / min for 5 min, collect the precipitate, wash twice with pH 7.0 phosphate buffer, and centrifuge to collect the supernatant. Freeze-dry the precipitate at -50℃ for 12-24 h to obtain immobilized and activated DERA. Store at 4℃ for later use and determine its activity. The enzyme activity recovery rate of DERA immobilized and activated using diatomaceous earth was 194.79% according to the above method.

[0046] Example 3: Effect of diatomaceous earth dosage on the activation of recombinant engineered bacterial enzyme products of the DERA gene

[0047] (1) A recombinant engineered strain containing the DERA gene from the thermophilic archaea *Aeropyrum pernix* (gene sequence: GenBank: BAA81452.2) (prepared according to the method in Example 2) was inoculated into LB liquid medium containing 50 mg / ml kanamycin and cultured at 37°C and 220 rpm for 9–12 h to obtain a seed culture. The seed culture was then inoculated into TB fermentation medium at a volume concentration of 1% and cultured at 37°C and 220 rpm until OD200 was reached. 600 The concentration was increased to 0.6–0.8, and then IPTG was added to a final concentration of 0.2 mM for induction. The mixture was induced at 25 °C and 220 rpm for 18 h. The fermentation broth was then centrifuged to collect the wet cells.

[0048] (2) Resuspend the wet bacterial cells in pH 7.0 phosphate buffer at a ratio of 1g:10ml to obtain a bacterial suspension. Use an ultrasonic cell disruptor at 120W with a 3s-6s-pause cycle to disrupt the bacterial cells, and the disruption time should be at least half the volume of the bacterial suspension. After ultrasonic disruption, centrifuge at 12000r / min for 10min and collect the supernatant crude enzyme solution.

[0049] (3) Take 2ml of crude enzyme solution and add 40mg, 100mg, 150mg, 200mg, 250mg, 300mg and 400mg of diatomaceous earth to each tube respectively, and fix in a shaker at 220rpm for 5h.

[0050] (4) After immobilization, centrifuge at 12000 r / min for 5 min, collect the precipitate, wash twice with pH 7.0 phosphate buffer, and centrifuge to collect the supernatant. Freeze-dry the precipitate at -50℃ for 12–24 h to obtain immobilized and activated DERA. Store at 4℃ for later use and determine its activity. According to the above method, the enzyme activity recovery rate of DERA immobilized and activated using diatomaceous earth was measured as follows:

[0051]

[0052] The results in the table above show that the amount of diatomaceous earth used in immobilization has different effects on the activation of DERA, but all of them have an activation effect.

[0053] Example 4: Activation effect of different fixed time on the enzyme product of recombinant engineered bacteria containing the DRA gene (1) A recombinant engineered strain containing the DRA gene from Lactobacillus brevis (gene sequence: WP_260145457) (prepared according to the method in Example 2) was inoculated into LB liquid medium containing 50 mg / ml kanamycin and cultured at 37°C and 220 rpm for 9-12 h to obtain seed culture. The seed culture was inoculated into TB fermentation medium at a volume concentration of 1% and cultured at 37°C and 220 rpm until OD 600 Reaching a concentration of 0.6–0.8, IPTG was added to a final concentration of 0.2 mM for induction. Induction was performed at 25°C and 220 rpm for 18 h. The fermentation broth was centrifuged to collect wet cells. (2) The wet cells were resuspended in pH 7.0 phosphate buffer at a ratio of 1 g: 10 ml to obtain a bacterial suspension. The cells were disrupted using an ultrasonic cell disruptor at 120 W with a 3-second disruption and 6-second pause program. The disruption time was at least half the volume of the bacterial suspension. After ultrasonic disruption, the cells were centrifuged at 12000 r / min for 10 min, and the supernatant crude enzyme solution was collected.

[0054] (3) Take 2ml of crude enzyme solution, add 250mg of diatomaceous earth, and place it in a shaker at 220rpm for 1h, 2h, 4h, 8h, 10h, 12h and 24h respectively.

[0055] (4) After immobilization, centrifuge at 12000 r / min for 5 min, collect the precipitate, wash twice with pH 7.0 phosphate buffer, and centrifuge to collect the supernatant. Freeze-dry the precipitate at -50℃ for 12–24 h to obtain immobilized and activated DERA. Store at 4℃ for later use and determine its activity. According to the above method, the enzyme activity recovery rate of DERA immobilized and activated using diatomaceous earth was measured as follows:

[0056]

[0057] The results in the table above show that the activation effect on DERA is strongest when the fixed time is 2 hours.

[0058] Example 5: The effect of the pH of the fixation buffer on the activation of recombinant engineered bacterial enzyme products of the DERA gene

[0059] (1) A recombinant engineered strain containing the DRA gene from *Pseudomonas syringae* pv. syringae B728a (gene sequence: Accession: Q5SJ28) (prepared according to the method in Example 2) was inoculated into LB liquid medium containing 50 mg / ml kanamycin and cultured at 37°C and 220 rpm for 9–12 h to obtain a seed culture. The seed culture was then inoculated into TB fermentation medium at a volume concentration of 1% and cultured at 37°C and 220 rpm until OD200. 600 The concentration was increased to 0.6–0.8, and then IPTG was added to a final concentration of 0.2 mM for induction. The mixture was induced at 25 °C and 220 rpm for 18 h. The fermentation broth was then centrifuged to collect the wet cells.

[0060] (2) The wet bacterial cells were resuspended in phosphate buffer solutions at pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0, and pH 10.0 at a ratio of 1g:10ml to obtain bacterial suspensions. The bacterial cells were disrupted using an ultrasonic cell disruptor at 120W with a 3s disruption time followed by a 6s pause. The disruption time was at least half the volume of the bacterial suspension. After ultrasonic disruption, the cells were centrifuged at 12000r / min for 10min, and the supernatant crude enzyme solution was collected.

[0061] (3) Take 2 ml of crude enzyme solution, add 250 mg of diatomaceous earth, and fix it in a shaker at 220 rpm for 2 h.

[0062] (4) After immobilization, centrifuge at 12000 r / min for 5 min, collect the precipitate, wash twice with pH 7.0 phosphate buffer, and centrifuge to collect the supernatant. Freeze-dry the precipitate at -50℃ for 12–24 h to obtain immobilized and activated DERA. Store at 4℃ for later use and determine its activity. According to the above method, the enzyme activity recovery rate of DERA immobilized and activated using diatomaceous earth was measured as follows:

[0063]

[0064] The results in the table above show that diatomaceous earth immobilized DERA has an activating effect under different pH conditions, with the best effect in a buffer solution at pH 7.0.

[0065] Example 6: Activation effect of diatomaceous earth immobilization on DERA from different bacterial or archaeal sources

[0066] (1) The following bacteria were found to be derived from the archaea Pyrobaculum calidifontis (GenBank: ABO08212.1), the red subterranean thermophile Meiothermus ruber (GenBank: WP_013014483), the marine thermophile Thermotoga maritima (GenBank: WP_004081965), Rhodococcus erythropolis (GenBank: JN665070.1), Thermusthermophilus HB8 (GenBank: Q5SJ28), Colwellia psychrerythraea (GenBank: WP_033095293), and Shewanella halifax. A recombinant engineered strain of *Halifaxensis* (gene sequence: WP_012278208) containing the DERA gene (prepared according to the method in Example 2) was inoculated into LB liquid medium containing 50 mg / ml kanamycin and cultured at 37°C and 220 rpm for 9–12 h to obtain a seed culture. The seed culture was then inoculated into TB fermentation medium at a volume concentration of 1% and cultured at 37°C and 220 rpm until OD200. 600 The concentration was increased to 0.6–0.8, and then IPTG was added to a final concentration of 0.2 mM for induction. The mixture was induced at 25 °C and 220 rpm for 18 h. The fermentation broth was then centrifuged to collect the wet cells.

[0067] (2) Resuspend the wet bacterial cells in pH 7.0 phosphate buffer at a ratio of 1g:10ml to obtain a bacterial suspension. Use an ultrasonic cell disruptor at 120W with a 3s-6s-pause cycle to disrupt the bacterial cells, and the disruption time should be at least half the volume of the bacterial suspension. After ultrasonic disruption, centrifuge at 12000r / min for 10min and collect the supernatant crude enzyme solution.

[0068] (3) Take 2 ml of crude enzyme solution, add 250 mg of diatomaceous earth, and fix it in a shaker at 220 rpm for 2 h.

[0069] (4) After immobilization, centrifuge at 12000 r / min for 5 min, collect the precipitate, wash twice with pH 7.0 phosphate buffer, and centrifuge to collect the supernatant. Freeze-dry the precipitate at -50℃ for 12–24 h to obtain immobilized and activated DERA. Store at 4℃ for later use and determine its activity. According to the above method, the enzyme activity recovery rate of DERA immobilized and activated using diatomaceous earth was measured as follows:

[0070]

[0071] The results in the table above indicate that diatomaceous earth immobilization has an activating effect on DERA from different bacterial or archaeal sources.

[0072] Example 7: Activation of DERA from fungi and animals by diatomaceous earth immobilization

[0073] (1) The following fungi were found to contain *Sepia pharaonis* (GenBank: CAE1330455.1), *Mus musculus* (GenBank: NP_766321XP_994490), *Lygus hesperus* (GenBank: JAQ17029.1), *Schistocephalus solidus* (GenBank: JAP52428.1), *Rhizoctonia solani* (GenBank: CUA77423.1), *Colletotrichum viniferum* (GenBank: KAF4900569.1), and *Cladophialophora*. A recombinant engineered strain of carrionii (gene sequence: GenBank: OCT53247.1) containing the DERA gene (prepared according to the method in Example 2) was inoculated into LB liquid medium containing 50 mg / ml kanamycin and cultured at 37°C and 220 rpm for 9–12 h to obtain seed culture. The seed culture was then inoculated into TB fermentation medium at a volume concentration of 1% and cultured at 37°C and 220 rpm until OD500 was reached. 600 The concentration was increased to 0.6–0.8, and then IPTG was added to a final concentration of 0.2 mM for induction. The mixture was induced at 25 °C and 220 rpm for 18 h. The fermentation broth was then centrifuged to collect the wet cells.

[0074] (2) Resuspend the wet bacterial cells in pH 7.0 phosphate buffer at a ratio of 1g:10ml to obtain a bacterial suspension. Use an ultrasonic cell disruptor at 120W with a 3s-6s-pause cycle to disrupt the bacterial cells, and the disruption time should be at least half the volume of the bacterial suspension. After ultrasonic disruption, centrifuge at 12000r / min for 10min and collect the supernatant crude enzyme solution.

[0075] (3) Take 2 ml of crude enzyme solution, add 250 mg of diatomaceous earth, and fix it in a shaker at 220 rpm for 2 h.

[0076] (4) After immobilization, centrifuge at 12000 r / min for 5 min, collect the precipitate, wash twice with pH 7.0 phosphate buffer, and centrifuge to collect the supernatant. Freeze-dry the precipitate at -50℃ for 12–24 h to obtain immobilized and activated DERA. Store at 4℃ for later use and determine its activity.

[0077] Based on the above method, the enzyme activity recovery rate of DERA immobilized and activated using diatomaceous earth was measured as follows:

[0078]

[0079] The results in the table above indicate that diatomaceous earth immobilization has an activating effect on DERA from both fungal and animal sources.

[0080] Example 8

[0081] (1) A recombinant engineered strain containing the DERA gene (gene sequence: GenBank: AFY10769.1, amino acid sequence of which is listed in the following literature: W. Greenberg, A. Varvak. Proc Natl Acad Sci U SA. 2004, 101(16):5788-93) derived from an environmental biological sample (prepared according to the method in Example 2) was inoculated into LB liquid medium containing 50 mg / ml kanamycin and cultured at 37°C and 220 rpm for 9-12 h to obtain a seed culture. The seed culture was then inoculated into TB fermentation medium at a volume concentration of 1% and cultured at 37°C and 220 rpm until OD. 600 The concentration was increased to 0.6–0.8, and then IPTG was added to a final concentration of 0.2 mM for induction. The mixture was induced at 25 °C and 220 rpm for 18 h. The fermentation broth was then centrifuged to collect the wet cells.

[0082] (2) Resuspend the wet bacterial cells in pH 7.0 phosphate buffer at a ratio of 1g:10ml to obtain a bacterial suspension. Use an ultrasonic cell disruptor at 120W with a 3s-6s-pause cycle to disrupt the bacterial cells, and the disruption time should be at least half the volume of the bacterial suspension. After ultrasonic disruption, centrifuge at 12000r / min for 10min and collect the supernatant crude enzyme solution.

[0083] (3) Take 2 ml of crude enzyme solution, add 250 mg of diatomaceous earth, and fix it in a shaker at 220 rpm for 2 h.

[0084] (4) After immobilization, centrifuge at 12000 r / min for 5 min, collect the precipitate, wash twice with phosphate buffer at pH 7.0, and centrifuge to collect the supernatant. Freeze-dry the precipitate at -50℃ for 12–24 h to obtain immobilized and activated DERA. Store at 4℃ for later use and determine its activity.

[0085] The above method shows that DERA, which is immobilized and activated by diatomaceous earth, still maintains its catalytic activity after 20 reaction cycles, and can be used to prepare chiral side chains of statin drugs.

Claims

1. A method for improving the activity of 2-deoxy-D-ribose-5-phosphate aldolase, characterized in that, Diatomaceous earth was added to a solution containing 2-deoxy-D-ribose-5-phosphoaldolase for adsorption and fixation for 1-4 hours. The protein concentration of the solution containing 2-deoxy-D-ribose-5-phosphoaldolase was 5-50 mg / ml, and 75-200 mg of diatomaceous earth was added to each 1 ml of solution. The solution containing 2-deoxy-D-ribose-5-phosphoaldolase was a buffer solution with a pH of 6.0-9.

0.

2. The method according to claim 1, characterized in that... The pH of the buffer solution is 7.

0.

3. The method according to claim 1 or 2, characterized in that... Includes the following steps: (1) Collect the bacterial cells of the recombinant engineered strain containing the 2-deoxy-D-ribose-5-phosphate aldolase gene obtained by fermentation culture, and resuspend them in buffer to obtain a bacterial suspension; (2) After the bacterial cells are broken, the supernatant is collected by centrifugation to obtain crude enzyme solution; (3) Add diatomaceous earth to the crude enzyme solution and shake to adsorb and fix it; (4) After immobilization, the precipitate was collected by centrifugation, washed and freeze-dried to obtain the immobilized and activated 2-deoxy-D-ribose-5-phosphoaldolase.

4. An immobilized and activated 2-deoxy-D-ribose-5-phosphoaldolase, characterized in that... It is prepared by the method described in any one of claims 1-3.

5. The application of the immobilized and activated 2-deoxy-D-ribose-5-phosphate aldolase according to claim 4 in the field of enzyme catalysis.

6. The application according to claim 5, characterized in that... The immobilized and activated 2-deoxy-D-ribose-5-phosphoaldolase is used to prepare chiral side chains for statin drugs.

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