Immobilization method of inositol-1-phosphate synthetase and application thereof in inositol production

Abstract

The invention discloses a high-activity IPS immobilized enzyme based on a functional design MOF carrier as well as a preparation method and application of the high-activity IPS immobilized enzyme. According to the method, a methyl (-CH3) functional group is introduced into MIL-101 (Cr), a carrier-enzyme interface microenvironment is accurately regulated and controlled, and IPS is fixed by adopting a physical adsorption method. The immobilized enzyme prepared from the methyl-modified carrier has excellent performance, and the catalytic efficiency and operation stability of the immobilized enzyme are far superior to those of free enzyme. Molecular mechanism research shows that the hydrophobic microenvironment created by methyl modification can stabilize enzyme conformation and optimize mass transfer. The composite material can be used for constructing an efficient, stable and reusable inositol biosynthesis system, and has great industrial application value.

Description

Technical Field

[0001] This invention relates to the fields of biocatalysis and enzyme engineering, specifically to a method for immobilizing inositol-1-phosphate synthase (IPS) using metal-organic framework (MOF) material MIL-101(Cr)-CH3 as a carrier, and its application in inositol production.

[0002] This invention also relates to the application of the above-described method. Background Technology

[0003] Inositol is an important bioactive substance widely used in the pharmaceutical, food, and feed industries. To meet the growing market demand, it is necessary to optimize and improve inositol production methods. Traditional chemical hydrolysis and synthesis methods suffer from high costs, low yields, environmental unfriendliness, and difficulties in product separation. Green bioprocesses, such as microbial fermentation and enzymatic catalysis, have attracted much attention due to their low cost and environmental friendliness. Enzymatic synthesis of inositol typically involves two steps: inositol-1-phosphate synthase catalyzes the cyclization of glucose-6-phosphate to inositol-3-phosphate (the rate-limiting step), followed by dephosphorylation by inositol monophosphatase (IMP) to generate free inositol. To improve efficiency, studies have constructed multi-enzyme cascade systems starting from starch or glucose and have achieved industrial-scale trials. However, industrial applications are still limited by high enzyme production costs, inherent enzyme instability, and difficulties in recycling and reusing the enzymes.

[0004] Enzyme immobilization technology is considered a crucial strategy for overcoming the aforementioned bottlenecks. Immobilized enzymes can significantly improve the stability of biocatalysts, facilitate recovery, and enable reuse. For example, multi-enzyme co-immobilization systems based on porous microspheres have been developed for the production of inositol from starch, exhibiting superior thermal stability and operational reproducibility compared to free enzymes. However, such traditional supports often lead to significant loss of enzyme activity. In recent years, metal-organic frameworks (MOFs) have become highly promising advanced enzyme immobilization supports due to their extremely high specific surface area, tunable pore structure, good biocompatibility, and structural stability. Among them, MIL-101(Cr), with its ultra-high specific surface area, large porosity, and ease of functionalization, has been successfully used to immobilize various enzymes and significantly improve their catalytic performance.

[0005] Despite significant progress in enzyme immobilization technology in recent years, MOF materials have not yet been used as immobilization supports for iPS. More importantly, current research on MOF-immobilized enzymes largely focuses on the supports themselves, generally lacking rational design of supports for specific enzymes and a deep understanding of the molecular-level interactions and microenvironmental influences at the MOF-enzyme interface. This results in poor predictability of immobilization effects and difficulty in achieving performance optimization. Therefore, there is an urgent need to develop an immobilization method capable of precisely regulating the microenvironment at the support-enzyme interface, thereby significantly improving the catalytic efficiency and stability of iPS.

[0006] Based on the above research background, this invention prepared methyl-modified MIL-101(Cr), namely MIL-101(Cr)-CH3, and used it as a support for loading IPS to prepare the IPS@MIL-101(Cr)-CH3 complex, which was then used for the synthesis of inositol. No research in this area has been reported in the literature. Summary of the Invention

[0007] The purpose of this invention is to provide a method and application for IPS enzyme immobilization.

[0008] The present invention provides a method for immobilizing IPS using organically modified MIL-101(Cr). Using MIL-101(Cr) as a platform, different organic functional groups are introduced into the ligand to prepare organically modified MIL-101(Cr). IPS is then immobilized by physical adsorption to obtain a series of IPS immobilization complexes, which can be used to synthesize inositol.

[0009] The IPS@MIL-101(Cr)-CH3 complex was analyzed by XRD ( Figure 1 ), FT-IR ( Figure 2 TGA ( Figure 3 Nitrogen adsorption-desorption ( Figure 4 The test confirmed the successful preparation of the IPS@MIL-101(Cr)-CH3 complex.

[0010] The immobilized enzyme IPS@MIL-101(Cr)-CH3 complex was shown by scanning electron microscopy to have a nanoparticle morphology (see [link]). Figure 5 ); The adsorption of IPS enzyme on the surface of MOF materials was demonstrated by labeling with 5-carboxyfluorescein succinimide (5-FAM SE) and laser confocal microscopy (see [link to study]). Figure 6 ).

[0011] Catalytic performance tests showed that the immobilized enzyme IPS@MIL-101(Cr)-CH3 complex can be used for the synthesis of inositol, with a yield 1.27 times that of the free enzyme IPS.

[0012] Activity assays of the immobilized enzyme IPS@MIL-101(Cr)-CH3 complex showed that the specific activity of IPS@MIL-101(Cr)-CH3 was significantly increased (see [link to relevant documentation]). Figure 7 Stability tests showed that its activity was higher than that of the free enzyme after treatment with organic reagents and trypsin (see...). Figure 8 a) Its activity retention rate exceeded 70% during five consecutive cycles of repeated use (see Figure 8 b) Storage stability tests show that at 4 o After storage for 5 days, C still retains 50% of its initial activity (see...). Figure 8 c).

[0013] Compared with other immobilization methods, the physical adsorption method of this invention has the advantages of simple synthesis, mild reaction conditions, minimal impact on enzyme conformation, and high catalytic efficiency. Attached Figure Description

[0014] Figure 1 These are the XRD patterns of MIL-101(Cr)-CH3 and IPS@MIL-101(Cr)-CH3.

[0015] Figure 2 These are the FT-IR spectra of MIL-101(Cr)-CH3 and IPS@MIL-101(Cr)-CH3.

[0016] Figure 3 These are the TGA curves of MIL-101(Cr)-CH3 and IPS@MIL-101(Cr)-CH3.

[0017] Figure 4 These are the nitrogen adsorption-desorption curves of MIL-101(Cr)-CH3 and IPS@MIL-101(Cr)-CH3.

[0018] Figure 5 These are scanning electron microscope images of MIL-101(Cr)-CH3 and IPS@MIL-101(Cr)-CH3.

[0019] Figure 6 These are laser confocal images of IPS@MIL-101(Cr)-CH3.

[0020] Figure 7 It represents the relative catalytic activity of free IPS and IPS@MIL-101(Cr)-CH3.

[0021] Figure 8This is a comparison of the stability of free IPS and IPS@MIL-101(Cr)-CH3 in organic solvents, trypsin (A), reusability (B), and storage stability (C).

[0022] Figure 9 The HPLC analysis of the inositol synthesis products (A) and the comparison of relative yields (B) are shown. Detailed Implementation

[0023] The primary objective of this invention is to overcome the problems of blind carrier design and insufficient interfacial microenvironment control in existing IPS immobilization technologies, and to provide an IPS enzyme immobilization method based on a rationally functionalized MIL-101(Cr) carrier. A further objective of this invention is to provide an immobilized IPS enzyme composite material with ultra-high catalytic efficiency and excellent operational stability prepared by this method, and to apply it to the efficient production of inositol, thereby solving the industrial bottlenecks of high enzyme cost, poor stability, and difficulty in reusability in existing biological methods for inositol production.

[0024] To achieve the above objectives, this invention proposes a rational design strategy of "carrier functionalization-microenvironment adaptation". The core of the technical solution is: by introducing a specific functional group methyl (-CH3) into the MIL-101(Cr) backbone during synthesis modification, the physicochemical properties (such as hydrophobicity / hydrophilicity) of the carrier surface are precisely controlled, thereby creating the most suitable local microenvironment for the IPS enzyme. Through optimized multiple non-covalent interactions, the enzyme is immobilized efficiently, stably and conformationally friendly.

[0025] The immobilization method is a simple physical adsorption method with minimal impact on enzyme conformation. Specific steps include: a) carrier functionalization: preparing MIL-101(Cr) and its derivatives with -CH3 functional groups; b) adsorption immobilization: dispersing the MOF carrier in buffer solution, adding the IPS enzyme solution, and adsorbing under gentle shaking conditions; c) post-treatment: separating and washing the solid, and freeze-drying to obtain the immobilized IPS enzyme composite material. The specific embodiments of the present invention are further illustrated below.

[0026] The specific implementation of the present invention will be further illustrated below with reference to specific embodiments.

[0027] Example 1 Preparation of immobilized enzyme: MIL-101(Cr)-CH3 (3.0 mg) and IPS (0.5 mg) were stirred at room temperature for 12 hours in a buffer containing 50 mM HEPES and 50 mM NaCl. After the reaction was complete, the precipitate was collected by centrifugation at 15000 rpm for 1 minute. The precipitate was washed three times with HEPES buffer and then freeze-dried under vacuum. The resulting product was named IPS@MIL-101(Cr)-CH3.

[0028] Enzyme activity assay: Enzyme activity was quantitatively determined by monitoring changes in absorbance at 850 nm. The enzyme activity assay showed that the specific activity of IPS@MIL-101(Cr)-CH3 was increased. Stability assay showed that its activity was higher than that of the free enzyme after treatment with organic reagents and trypsin. In five consecutive batches of repeated use, its activity retention rate exceeded 70%.

[0029] As a catalyst: The enzymatic reaction was carried out in a 100 mL Erlenmeyer flask with a total reaction volume of 20 mL, containing 50 mM Tris-HCl buffer (pH 8.0) and 0.4 mM NAD⁺ as a cofactor. The substrate was a mixture of glucose and sodium hexametaphosphate in a 4:1 molar ratio, catalyzed by a ternary enzyme system with a stoichiometric ratio of PPGK:IPS / IPS@MOF:IMP = 0.5:1:2. The reaction was carried out at 30°C and 200 rpm for 12 h with shaking, followed by incubation at 70°C for 24 h. After the reaction, the product was analyzed by high-performance liquid chromatography (HPLC). The results showed that the yield was 1.27 times that of the free enzyme IPS, enabling efficient and stable production of inositol. The system is capable of long-term operation and is easily regenerated.

[0030] Example 2 Preparation of immobilized enzyme: MIL-101(Cr)-CH3 (5.0 mg) and IPS (1.0 mg) were stirred at room temperature for 12 hours in a buffer containing 50 mM HEPES and 50 mM NaCl. After the reaction was complete, the precipitate was collected by centrifugation at 15000 rpm for 1 minute. The precipitate was washed three times with HEPES buffer and then freeze-dried under vacuum. The resulting product was named IPS@MIL-101(Cr)-CH3.

[0031] Enzyme activity assay: Enzyme activity was quantitatively determined by monitoring changes in absorbance at 850 nm. The enzyme activity assay showed that the specific activity of IPS@MIL-101(Cr)-CH3 was increased. Stability assay showed that its activity was higher than that of the free enzyme after treatment with organic reagents and trypsin. In five consecutive batches of repeated use, its activity retention rate exceeded 72%.

[0032] As a catalyst: The enzymatic reaction was carried out in a 100 mL Erlenmeyer flask with a total reaction volume of 20 mL, containing 50 mM Tris-HCl buffer (pH 8.0) and 0.4 mM NAD⁺ as a cofactor. The substrate was a mixture of glucose and sodium hexametaphosphate in a 4:1 molar ratio, catalyzed by a ternary enzyme system with a stoichiometric ratio of PPGK:IPS / IPS@MOF:IMP = 0.5:1:2. The reaction was carried out at 35°C with shaking at 200 rpm for 12 h, followed by incubation at 70°C for 24 h. After the reaction, the product was analyzed by high-performance liquid chromatography (HPLC). The results showed that the yield was 1.30 times that of the free enzyme IPS, enabling efficient and stable production of inositol. The system can operate for a long time and is easily regenerated.

Claims

1. An immobilized enzyme composite material for the production of inositol, characterized in that... Its chemical formula is: IPS@MIL-101(Cr)-CH3, where IPS is inositol-1-phosphate synthase and MIL-101(Cr)-CH3 is MIL-101(Cr) modified with methyl (-CH3).

2. The method for synthesizing the immobilized enzyme as described in claim 1, characterized in that... The process includes the following steps: using a methyl-functionalized MOF carrier MIL-101(Cr)-CH3, the carrier is dispersed in a buffer solution (50 mM HEPES and 50 mM NaCl) to obtain a carrier dispersion. Then, an enzyme solution containing IPS is mixed with the carrier dispersion, and an adsorption reaction is carried out at room temperature. After the reaction is completed, the solid is separated, washed, and freeze-dried to obtain the immobilized enzyme composite material.

3. The method for preparing immobilized enzyme as described in claim 1, characterized in that: Using MIL-101(Cr)-CH3 as a carrier, its space group is F d-3m , a = b = c ≈8.9 Å, cell volume: approximately 700 Å 3 The topology is: mtn.

4. The method for preparing immobilized enzyme as described in claim 1, characterized in that: It is fixed to the surface of the material by physical adsorption.

5. The method for preparing immobilized enzyme as described in claim 1, characterized in that: The immobilized physical adsorption reaction time was 12 hours.

6. The method for preparing immobilized enzyme as described in claim 1, characterized in that: In the immobilized physical adsorption reaction, the mass ratio of the carrier MIL-101(Cr)-CH3 to the IPS enzyme was between 5:1 and 6:

1.

7. The use of the immobilized enzyme as described in claim 1, characterized in that: The immobilized enzyme IPS@MIL-101(Cr)-CH3 complex can be used to catalyze the synthesis of inositol.

8. The use of the immobilized enzyme as described in claim 1, characterized in that: The catalytic reaction temperature is 70 °C and the reaction pH is 8.0.

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