Composite catalyst containing rhodococcus fermenting broth and use thereof

Abstract

The application provides a complex catalyst containing rhodococcus fermentation liquor and application thereof. The complex catalyst contains rhodococcus fermentation liquor and A component, the A component is selected from one or more than one of glycerol, glutaraldehyde, gelatin, nipagin methyl ester and polyethylene glycol, and the complex catalyst can significantly improve the storage stability of single rhodococcus whole cell catalyst, so that the catalyst can maintain high catalytic activity after long-time storage, and effectively solves the problem of increased production cost caused by catalyst deactivation.

Description

Technical Field

[0001] This application relates to the field of biocatalysis technology, and in particular to a composite catalyst containing Rhodococcus cells and its uses. Background Technology

[0002] Biocatalysis is widely used in industrial production due to its mild reaction conditions, high specificity, high catalytic efficiency, and avoidance of problems such as heavy metal residues and environmental pollution. Currently, whole-cell catalysts are commonly used in biocatalysis. However, in actual industrial production, whole-cell catalysts suffer from poor storage stability, leading to cell autolysis and reduced catalytic performance over long periods. Furthermore, most cell catalysts have poor organic tolerance, failing to meet the demands of industrial production.

[0003] Existing methods for improving the stability of biocatalysts in industry mainly involve altering catalyst composition, designing nanocatalysts, and surface modification techniques. However, while these methods have improved the stability of biocatalysts to some extent, they still suffer from high costs, complex preparation processes, and environmental hazards, failing to meet the requirements of sustainable industrial development. Therefore, it is necessary to choose green, low-cost, and sustainable composite methods for industrial applications. Rhodococcus bacteria exhibit strong organic tolerance and can adapt to various extreme environments, but its whole-cell catalysts still face problems of poor storage stability and insufficient catalytic stability during large-scale industrial production. Therefore, developing novel composite catalysts to further improve the stability of Rhodococcus whole-cell catalysts is a key technical problem that urgently needs to be solved. Summary of the Invention

[0004] To address the technical problems existing in the prior art, this application provides a composite catalyst containing Rhodococcus cells and its uses. Compared with a single Rhodococcus cell catalyst, the composite catalyst is more stable and efficient, and can realize the large-scale production of high-concentration acrylamide, exhibiting higher catalytic activity and stability in the production of acrylamide.

[0005] The specific technical solution of this application is as follows:

[0006] 1. A composite catalyst comprising Rhodococcus ferment broth and component A, wherein component A is selected from one or more of glycerol, glutaraldehyde, gelatin, methylparaben, and polyethylene glycol.

[0007] 2. The composite catalyst according to claim 1, wherein when component A contains glycerol, component A is 1-50% by mass percentage in the composite catalyst, preferably 5-50%, more preferably 10-30%.

[0008] 3. The composite catalyst according to claim 1, wherein when component A contains glutaraldehyde, component A is 0.01-3% by mass percentage in the composite catalyst, preferably 0.01-1%, more preferably 0.03-0.07%.

[0009] 4. The composite catalyst according to claim 1, wherein when component A contains gelatin, component A is 1-20% by mass percentage in the composite catalyst, preferably 2-10%, more preferably 2-8%.

[0010] 5. The composite catalyst according to claim 1, wherein when component A contains methylparaben, component A is 1-50% by mass percentage in the composite catalyst, preferably 5-50%, more preferably 5-30%.

[0011] 6. The composite catalyst according to claim 1, wherein when component A contains polyethylene glycol, component A is 1-20% by mass percentage in the composite catalyst, preferably 2-10%, more preferably 2-8%.

[0012] 7. The composite catalyst according to any one of items 1-6, wherein the concentration of Rhodococcus in the Rhodococcus fermentation broth in the composite catalyst is 100-200 g / L.

[0013] 8. Use of the composite catalyst described in any one of items 1-7 in catalyzing nitrile compounds to prepare amide compounds.

[0014] 9. The use according to item 8, wherein the amide compound is selected from acrylamide, 2,6-difluorobenzamide, p-hydroxyphenylacetamide, p-hydroxybenzamide, pyrazinamide and nicotinamide.

[0015] 10. The use of component A in improving the stability of Rhodococcus ferment broth, wherein component A is selected from one or more of glycerol, glutaraldehyde, gelatin, methylparaben, and polyethylene glycol.

[0016] 11. The use of component A in improving the catalytic performance of Rhodococcus ferment broth, wherein component A is selected from one or more of glycerol, glutaraldehyde, gelatin, methylparaben, and polyethylene glycol.

[0017] 12. The use of Rhodococcus fermentation broth and component A in the catalytic preparation of amide compounds from nitrile compounds, wherein component A is selected from one or more of glycerol, glutaraldehyde, gelatin, methylparaben, and polyethylene glycol.

[0018] 13. The use according to any one of items 10-12, wherein when component A contains glycerin, the concentration of component A is 1-50 wt%, preferably 5-50 wt%, more preferably 10-30 wt%.

[0019] 14. The use according to any one of items 10-12, wherein when component A contains glutaraldehyde, the concentration of component A is 0.01-3 wt%, preferably 0.01-1 wt%, more preferably 0.03-0.07 wt%.

[0020] 15. The use according to any one of items 10-12, wherein when component A contains gelatin, the concentration of component A is 1-20 wt%, preferably 2-10 wt%, more preferably 2-8 wt%.

[0021] 16. The use according to any one of items 10-12, wherein when component A contains methylparaben, the concentration of component A is 1-50 wt%, preferably 5-50 wt%, more preferably 5-30 wt%.

[0022] 17. The use according to any one of items 10-12, wherein when component A contains polyethylene glycol, the concentration of component A is 1-20 wt%, preferably 2-10 wt%, more preferably 2-8 wt%.

[0023] 18. The use according to any one of items 10-17, wherein the concentration of Rhodococcus in the Rhodococcus fermentation broth is 100-200 g / L.

[0024] 19. A method for preparing amide compounds from nitrile compounds by catalysis, comprising:

[0025] The composite catalyst described in any one of items 1-7 is contacted with a nitrile compound to undergo a catalytic reaction to obtain an amide compound.

[0026] 20. The method according to claim 19, wherein the amide compound is selected from acrylamide, 2,6-difluorobenzamide, p-hydroxyphenylacetamide, p-hydroxybenzamide, pyrazinamide, and nicotinamide.

[0027] The effects of the invention

[0028] The composite catalyst described in this application can significantly improve the storage stability of whole-cell Rhodococcus moschata catalysts, enabling them to maintain high catalytic activity even after long-term storage, effectively solving the problem of increased production costs caused by catalyst deactivation. Furthermore, the composite catalyst described in this application has mild application conditions, is easy to operate, low in cost, and environmentally friendly, aligning with the development trend of green chemistry. More importantly, this composite catalyst has broad applicability, not only suitable for the production of amide compounds but also for the synthesis of other fine chemicals. Its high catalytic activity and stability ensure that the catalytic reaction can be completed in a short time, thereby reducing energy consumption and demonstrating high efficiency and energy saving advantages, making the composite catalyst of this application extremely valuable for large-scale industrial production. Attached Figure Description

[0029] Figure 1 This is a schematic diagram showing the enzyme activity loss after 7 days of storage following the mixing of different concentrations of glycerol with Rhodococcus ferment broth.

[0030] Figure 2 This is a schematic diagram showing the enzyme activity loss after 7 days of storage following the mixing of different concentrations of glutaraldehyde with Rhodococcus ferment broth.

[0031] Figure 3 This is a schematic diagram showing the enzyme activity loss after 7 days of storage following the mixing of gelatin with Rhodococcus ferment broth at different concentrations.

[0032] Figure 4 This is a schematic diagram showing the enzyme activity loss after 7 days of storage following the mixing of different concentrations of methylparaben (PVA) with Rhodococcus ferment broth.

[0033] Figure 5 This is a schematic diagram showing the enzyme activity loss after 7 days of storage following the mixing of different concentrations of polyethylene glycol (PEG 4000) with Rhodococcus ferment broth.

[0034] Figure 6 This is a schematic diagram showing the enzyme activity loss after mixing 10% glycerol + 0.07% glutaraldehyde, 10% glycerol + 5% PVA, 0.07% glutaraldehyde + 2% PEG with Rhodococcus ferment broth and storing it at 25°C for 7 days.

[0035] Figure 7 This diagram illustrates the enzyme activity loss after storing a mixture of 10% glycerol + 0.07% glutaraldehyde + 4% gelatin, 10% glycerol + 5% PVA + 2% PEG, and 0.07% glutaraldehyde + 2% PEG + 5% PVA with Rhodococcus ferment broth at 25°C for 7 days.

[0036] Figure 8This diagram illustrates the enzyme activity loss after mixing 10% glycerol + 0.07% glutaraldehyde + 4% gelatin + 2% PEG, 10% glycerol + 5% PVA + 2% PEG + 4% gelatin, or 0.07% glutaraldehyde + 2% PEG + 5% PVA + 4% gelatin with Rhodococcus ferment broth and storing the mixture at 25°C for 7 days.

[0037] Figure 9 This is a schematic diagram showing the enzyme activity loss after storing a mixture of 10% glycerol, 0.07% glutaraldehyde, 4% gelatin, 2% PEG, and 5% PVA with Rhodococcus ferment broth at 25°C for 7 days.

[0038] Figure 10 This is a schematic diagram illustrating the catalytic production of acrylamide using different composite catalysts. Detailed Implementation

[0039] The present application will now be described in detail with reference to the described embodiments. Although specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this application will be thorough and complete, and will fully convey the scope of the present application to those skilled in the art.

[0040] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0041] This application provides a composite catalyst comprising Rhodococcus ferment broth and component A, wherein component A is selected from one or more of glycerol, glutaraldehyde, gelatin, methylparaben, and polyethylene glycol.

[0042] This application combines Rhodococcus ferment broth with component A, which can significantly improve the storage stability of Rhodococcus ferment broth.

[0043] In this application, the Rhodococcus ferment broth can be obtained by commercial purchase, fermentation using conventional methods with Rhodococcus commonly used in the art, or by conventional genetic modification of Rhodococcus and fermentation using conventional methods in the art.

[0044] In this application, polyethylene glycol (PEG) can be polyethylene glycol with different degrees of polymerization, such as PEG2000, PEG4000, PEG6000, PEG8000, etc.

[0045] In some embodiments, when component A contains glycerol, the component A is 1-50% by mass percentage in the composite catalyst, preferably 5-50%, and more preferably 10-30%.

[0046] For example, the A component can be 1%, 5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, etc., based on its mass percentage in the composite catalyst.

[0047] In some embodiments, when component A contains glutaraldehyde, the component A is 0.01-3% by mass percentage in the composite catalyst, preferably 0.01-1%, and more preferably 0.03-0.07%.

[0048] For example, the A component can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, etc., based on its mass percentage in the composite catalyst.

[0049] In some embodiments, when component A contains gelatin, the mass percentage of component A in the composite catalyst is 1-20%, preferably 2-10%, and more preferably 2-8%.

[0050] For example, the A component can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc., based on its mass percentage in the composite catalyst.

[0051] In some embodiments, when component A contains methylparaben (PVA), the component A is 1-50% by mass percentage in the composite catalyst, preferably 5-50%, and more preferably 5-30%.

[0052] For example, the A component can be 1%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, etc., based on its mass percentage in the composite catalyst.

[0053] In some embodiments, when component A contains polyethylene glycol, the component A is 1-20% by mass percentage in the composite catalyst, preferably 2-10%, and more preferably 2-8%.

[0054] For example, the A component can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc., based on its mass percentage in the composite catalyst.

[0055] In this application, component A can be one, two, three, four or five of them. For example, when component A contains two components, it can include glycerin and glutaraldehyde, glycerin and gelatin, glycerin and PVA, glycerin and PEG, glutaraldehyde and gelatin, glutaraldehyde and PVA, glutaraldehyde and PEG, gelatin and PVA, gelatin and PEG or PVA and PEG.

[0056] When ingredient A contains three components, it can include glycerin, glutaraldehyde and gelatin; glycerin, glutaraldehyde and PVA; glycerin, glutaraldehyde and PEG; glycerin, gelatin and PVA; glycerin, gelatin and PEG; glycerin, PVA and PEG; glutaraldehyde, gelatin and PVA; glutaraldehyde, PVA and PEG; or gelatin, PVA and PEG.

[0057] When ingredient A contains four components, it can include glycerin, glutaraldehyde, gelatin and PVA; glycerin, glutaraldehyde, gelatin and PEG; or glutaraldehyde, gelatin, PVA and PEG.

[0058] When ingredient A contains five components, it may include glycerin, glutaraldehyde, gelatin, PVA, and PEG.

[0059] In some embodiments, when component A comprises glycerol and glutaraldehyde, the glycerol comprises 1-50%, preferably 5-50%, more preferably 10-30%, by mass percentage in the composite catalyst, and the glutaraldehyde comprises 0.01-3%, preferably 0.01-1%, more preferably 0.03-0.07%; preferably, the composite catalyst comprises 10% glycerol and 0.07% glutaraldehyde, which can be expressed as 10% glycerol + 0.07% glutaraldehyde.

[0060] In some embodiments, when component A contains glycerol and PVA, the glycerol is 1-50%, preferably 5-50%, more preferably 10-30%, and the PVA is 1-50%, preferably 5-50%, more preferably 5-30%, by mass percentage in the composite catalyst; preferably, the composite catalyst contains 10% glycerol and 5% PVA, which can be expressed as 10% glycerol + 5% PVA.

[0061] In some embodiments, when component A contains glutaraldehyde and PEG, the glutaraldehyde is 0.01-3%, preferably 0.01-1%, more preferably 0.03-0.07% by mass percentage in the composite catalyst, and the PEG is 1-20%, preferably 2-10%, more preferably 2-8%; preferably, the composite catalyst contains 0.07% glutaraldehyde and 2% PEG, which can be expressed as 0.07% glutaraldehyde + 2% PEG.

[0062] In some embodiments, when component A comprises glycerol, glutaraldehyde, and gelatin, the glycerol comprises 1-50%, preferably 5-50%, more preferably 10-30%, by mass percentage in the composite catalyst; the glutaraldehyde comprises 0.01-3%, preferably 0.01-1%, more preferably 0.03-0.07%; and the gelatin comprises 1-20%, preferably 2-10%, more preferably 2-8%. Preferably, the composite catalyst comprises 10% glycerol, 0.07% glutaraldehyde, and 4% gelatin, which can be expressed as 10% glycerol + 0.07% glutaraldehyde + 4% gelatin.

[0063] In some embodiments, when component A comprises glycerol, PVA, and PEG, the glycerol is 1-50%, preferably 5-50%, more preferably 10-30% by mass percentage in the composite catalyst; the PVA is 1-50%, preferably 5-50%, more preferably 5-30%; and the PEG is 1-20%, preferably 2-10%, more preferably 2-8%. Preferably, the composite catalyst comprises 10% glycerol, 5% PVA, and 2% PEG, which can be expressed as 10% glycerol + 5% PVA + 2% PEG.

[0064] In some embodiments, when component A comprises glutaraldehyde, PEG, and PVA, the glutaraldehyde is 0.01-3%, preferably 0.01-1%, more preferably 0.03-0.07% by mass percentage in the composite catalyst; the PEG is 1-20%, preferably 2-10%, more preferably 2-8%; and the PVA is 1-50%, preferably 5-50%, more preferably 5-30%. Preferably, the composite catalyst comprises 0.07% glutaraldehyde, 2% PEG, and 5% PVA, which can be expressed as 0.07% glutaraldehyde + 2% PEG + 5% PVA.

[0065] In some embodiments, when component A comprises glycerol, glutaraldehyde, gelatin, and PEG, the glycerol comprises 1-50%, preferably 5-50%, more preferably 10-30% by mass percentage in the composite catalyst; the glutaraldehyde comprises 0.01-3%, preferably 0.01-1%, more preferably 0.03-0.07%; the gelatin comprises 1-20%, preferably 2-10%, more preferably 2-8%; and the PEG comprises 1-20%, preferably 2-10%, more preferably 2-8%. Preferably, the composite catalyst comprises 10% glycerol, 0.07% glutaraldehyde, 4% gelatin, and 2% PEG, which can be expressed as 10% glycerol + 0.07% glutaraldehyde + 4% gelatin + 2% PEG.

[0066] In some embodiments, when component A comprises glycerol, PVA, PEG, and gelatin, the glycerol is 1-50%, preferably 5-50%, more preferably 10-30% by mass percentage in the composite catalyst; the PVA is 1-50%, preferably 5-50%, more preferably 5-30%; the PEG is 1-20%, preferably 2-10%, more preferably 2-8%; and the gelatin is 1-20%, preferably 2-10%, more preferably 2-8%. Preferably, the composite catalyst comprises 10% glycerol, 5% PVA, 2% PEG, and 4% gelatin, which can be expressed as 10% glycerol + 5% PVA + 2% PEG + 4% gelatin.

[0067] In some embodiments, when component A comprises glutaraldehyde, PEG, PVA, and gelatin, the glutaraldehyde is 0.01-3%, preferably 0.01-1%, more preferably 0.03-0.07% by mass percentage in the composite catalyst; the PEG is 1-20%, preferably 2-10%, more preferably 2-8%; the PVA is 1-50%, preferably 5-50%, more preferably 5-30%; and the gelatin is 1-20%, preferably 2-10%, more preferably 2-8%. Preferably, the composite catalyst comprises 0.07% glutaraldehyde, 2% PEG, 5% PVA, and 4% gelatin, which can be expressed as 0.07% glutaraldehyde + 2% PEG + 5% PVA + 4% gelatin.

[0068] In some embodiments, when component A comprises glycerol, glutaraldehyde, gelatin, PEG, and PVA, the glycerol comprises 1-50%, preferably 5-50%, more preferably 10-30% by mass percentage in the composite catalyst; the glutaraldehyde comprises 0.01-3%, preferably 0.01-1%, more preferably 0.03-0.07%; the gelatin comprises 1-20%, preferably 2-10%, more preferably 2-8%; the PEG comprises 1-20%, preferably 2-10%, more preferably 2-8%; and the PVA comprises 1-50%, preferably 5-50%, more preferably 5-30%. Preferably, the composite catalyst comprises 10% glycerol, 0.07% glutaraldehyde, 4% gelatin, 2% PEG, and 5% PVA, which can be expressed as 10% glycerol + 0.07% glutaraldehyde + 4% gelatin + 2% PEG + 5% PVA.

[0069] In some embodiments, the concentration of Rhodococcus in the Rhodococcus fermentation broth in the composite catalyst is 100-200 g / L. In some embodiments, the OD of the Rhodococcus in the Rhodococcus fermentation broth is... 460 It is 40-50.

[0070] For example, the concentration of Rhodococcus in the Rhodococcus fermentation broth in the composite catalyst can be 100 g / L, 110 g / L, 120 g / L, 130 g / L, 140 g / L, 150 g / L, 160 g / L, 170 g / L, 180 g / L, 190 g / L, 200 g / L, etc.

[0071] For example, the OD of Rhodococcus in the Rhodococcus ferment broth 460 It can be 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, etc.

[0072] This application does not impose any restrictions on the method for obtaining OD460. Those skilled in the art can make conventional choices as needed, such as using a spectrophotometer for measurement.

[0073] Within this community, this application does not impose any restrictions on the method for determining the concentration of Rhodococcus in the composite catalyst, and conventional methods in the art can be used for determination.

[0074] The composite catalyst of this application can be stored at temperatures ranging from -80 to 20°C.

[0075] The composite catalyst described in this application can improve the storage stability of Rhodococcus cells compared to single Rhodococcus cells, and the composite catalyst can be used to catalyze nitriles to prepare amide compounds with high catalytic performance.

[0076] This application provides the use of the composite catalyst described in any of the foregoing claims in catalyzing nitrile compounds to prepare amide compounds. In some embodiments, the amide compound is selected from acrylamide, 2,6-difluorobenzamide, p-hydroxyphenylacetamide, p-hydroxybenzamide, pyrazinamide, and nicotinamide.

[0077] This application provides the use of component A in the stability of Rhodococcus ferment broth, wherein component A is selected from one or more of glycerol, glutaraldehyde, gelatin, methylparaben, and polyethylene glycol.

[0078] This application adds component A to the Rhodococcus ferment broth, which remains stable at, for example, 4°C, 25°C or 37°C, and exhibits minimal loss of enzyme activity after 7 days of storage.

[0079] This application provides the use of component A in improving the catalytic performance of Rhodococcus ferment broth, wherein component A is selected from one or more of glycerol, glutaraldehyde, gelatin, methylparaben, and polyethylene glycol.

[0080] This application adds component A to the Rhodococcus ferment broth, which not only improves the storage stability of the Rhodococcus ferment broth, but also enhances its catalytic performance.

[0081] This application provides the use of Rhodococcus fermentation broth and component A in catalyzing nitrile compounds to prepare amide compounds, wherein component A is selected from one or more of glycerol, glutaraldehyde, gelatin, methylparaben, and polyethylene glycol.

[0082] As mentioned above, since component A can improve the storage stability of Rhodococcus ferment broth and enhance its catalytic properties, this application uses component A mixed with Rhodococcus ferment broth to catalyze the preparation of amide compounds from nitrile compounds.

[0083] In some embodiments, when component A contains glycerin, the concentration of component A is 1-50 wt%, preferably 5-50 wt%, and more preferably 10-30 wt%.

[0084] For example, the concentration of component A can be 1 wt%, 5 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25 wt%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, etc.

[0085] In some embodiments, when component A contains glutaraldehyde, the concentration of component A is 0.01-3 wt%, preferably 0.01-1 wt%, and more preferably 0.03-0.07 wt%.

[0086] For example, the concentration of component A can be 0.01wt%, 0.02wt%, 0.03wt%, 0.04wt%, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, etc.

[0087] In some embodiments, when component A contains gelatin, the concentration of component A is 1-20 wt%, preferably 2-10 wt%, and more preferably 2-8 wt%.

[0088] For example, the concentration of component A can be 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 15wt%, 16wt%, 17wt%, 18wt%, 19wt%, 20wt%, etc.

[0089] In some embodiments, when component A contains methylparaben, the concentration of component A is 1-50 wt%, preferably 5-50 wt%, and more preferably 5-30 wt%.

[0090] For example, the concentration of component A can be 1 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25 wt%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, etc.

[0091] In some embodiments, when component A contains polyethylene glycol, the concentration of component A is 1-20 wt%, preferably 2-10 wt%, and more preferably 2-8 wt%.

[0092] For example, the concentration of component A can be 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 15wt%, 16wt%, 17wt%, 18wt%, 19wt%, 20wt%, etc.

[0093] In some embodiments, the concentration of Rhodococcus in the Rhodococcus fermentation broth is 100-200 g / L.

[0094] For example, the concentration of Rhodococcus in the fermentation broth can be 100 g / L, 110 g / L, 120 g / L, 130 g / L, 140 g / L, 150 g / L, 160 g / L, 170 g / L, 180 g / L, 190 g / L, 200 g / L, etc.

[0095] This application provides a method for preparing amide compounds from nitriles by catalysis, comprising:

[0096] The composite catalyst described in any of the above embodiments is contacted with a nitrile compound to undergo a catalytic reaction to obtain an amide compound. In some embodiments, the amide compound is selected from acrylamide, 2,6-difluorobenzamide, p-hydroxyphenylacetamide, p-hydroxybenzamide, pyrazinamide, and nicotinamide.

[0097] In this application, no restrictions are placed on the process conditions for the preparation of amide compounds from catalytic nitrile compounds. Those skilled in the art can make conventional selections as needed, such as carrying out the reaction at room temperature.

[0098] Example

[0099] This application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0100] Example 1

[0101] The strain was prepared and fermented using the method described in the embodiment of CN 110499274 A1 to obtain the fermentation stock solution.

[0102] A composite catalyst was prepared by combining glycerol (5%, 10%, 25%, 30%, and 50% in the composite catalyst), glutaraldehyde (0.01%, 0.03%, 0.05%, 0.07%, and 1% in the composite catalyst), gelatin (2%, 4%, 6%, 8%, and 10% in the composite catalyst), PVA (5%, 10%, 30%, and 50% in the composite catalyst), and polyethylene glycol (PEG 4000) (2%, 4%, 6%, 8%, and 10% in the composite catalyst) with the prepared erythroid cell fermentation stock solution. After storage at different temperatures (4℃, 25℃, and 37℃) for 7 days, the catalytic reaction was carried out using the composite catalyst and the fermentation stock solution as a control. The enzyme activity of the reacted samples was measured by gas chromatography. The percentage of enzyme activity loss after 7 days was calculated with the enzyme activity at 0 days of storage as 100%. The results are shown below. Figures 1 to 6 As shown.

[0103] The catalyst activity determination involved dissolving 20-100 μL of the composite catalyst stored for 7 days in water and incubating it in a constant temperature water bath shaker for 10 minutes. After incubation, 200 μL of acrylonitrile was added to the activation system and shaken rapidly several times to ensure uniform dispersion of acrylonitrile. The activation system was then placed in a water bath shaker and reacted for 5 minutes. Finally, acid was added to terminate the reaction and obtain the reaction solution.

[0104] Sample preparation: Take 500 μL of the above reaction solution and filter it through a 0.22 μm filter membrane to obtain the filtrate. Take 300 μL of the filtrate and mix it with an equal volume of internal standard acetamide to obtain the treated sample. Perform gas chromatography to detect enzyme activity in the treated sample.

[0105] The gas chromatography detection conditions were as follows: column temperature, injection port temperature, and detector temperature were 190℃, 240℃, and 280℃, respectively; the carrier gas was nitrogen; constant pressure mode was used with a partial pressure of 110.3 kPa; the injection volume was 0.5-2 μl, and the split ratio was 20:1. After the detection was completed, the acrylonitrile and acrylamide contents in the reaction solution were calculated using a pre-prepared standard curve.

[0106] Enzyme activity calculation:

[0107] Where t refers to time, and the unit is min;

[0108] v 总 This refers to the total volume of the reaction system, which is the total volume of acrylonitrile, Rhodococcus ferment broth, and water.

[0109] v 菌 This refers to the volume of the Rhodococcus fermentation broth.

[0110] from Figures 1 to 6 It can be seen that after storing the fermentation broth and the composite catalyst for 7 days, the enzyme activity loss of the composite catalyst was significantly reduced, indicating that adding component A to the Rhodococcus fermentation broth can significantly improve the storage stability of the Rhodococcus fermentation broth. Among them, the enzyme activity loss was lowest when the content of glycerol in the composite catalyst was 30%, glutaraldehyde was 0.07%, gelatin was 6%, methylparaben (PVA) was 10%, and polyethylene glycol (PEG 4000) was 8%. This indicates that the enzyme activity loss was relatively low when the content of glycerol in the composite catalyst was 10-30%, glutaraldehyde was 0.03-0.07%, gelatin was 2-8%, methylparaben (PVA) was 5-30%, and polyethylene glycol (PEG 4000) was 2-8%.

[0111] Example 2

[0112] A composite catalyst was prepared by combining 10% glycerol + 0.07% glutaraldehyde, 10% glycerol + 5% PVA, 0.07% glutaraldehyde + 2% PEG with the erythroid cell fermentation stock broth prepared in Example 1. After storage at 25°C for 7 days, the catalytic reaction was carried out using the composite catalyst and the fermentation stock broth as a control. The enzyme activity of the reacted samples was detected by gas chromatography. The percentage of enzyme activity loss after 7 days was calculated with the enzyme activity after 0 days of storage as 100%. The results are as follows: Figure 6 As shown.

[0113] Example 3

[0114] A composite catalyst was prepared by combining 10% glycerol + 0.07% glutaraldehyde + 4% gelatin, 4% gelatin + 5% PVA + 2% PEG, and 0.07% glutaraldehyde + 2% PEG + 5% PVA with the erythroid cell fermentation stock solution prepared in Example 1. After storage at 25°C for 7 days, the catalytic reaction was carried out using the composite catalyst and the fermentation stock solution as a control. The enzyme activity of the reacted samples was detected by gas chromatography. The percentage of enzyme activity loss after 7 days was calculated with the enzyme activity after 0 days of storage as 100%. The results are as follows: Figure 7 As shown.

[0115] Example 4

[0116] A composite catalyst was prepared by combining 10% glycerol + 0.07% glutaraldehyde + 4% gelatin + 2% PEG, 10% glycerol + 5% PVA + 2% PEG + 4% gelatin, 0.07% glutaraldehyde + 2% PEG + 5% PVA + 4% gelatin with the erythroid cell fermentation stock solution prepared in Example 1. After storage at 25°C for 7 days, the catalytic reaction was carried out using the composite catalyst and the fermentation stock solution as a control. The enzyme activity of the reacted samples was detected by gas chromatography. The percentage of enzyme activity loss after 7 days was calculated with the enzyme activity after 0 days of storage as 100%. The results are as follows: Figure 8 As shown.

[0117] Example 5

[0118] A composite catalyst was prepared by combining 10% glycerol, 0.07% glutaraldehyde, 4% gelatin, 2% PEG, and 5% PVA with the erythroid cell fermentation stock solution obtained in Example 1. After storage at 25°C for 7 days, the erythroid cell fermentation stock solution was used as a control. Catalytic reactions were carried out using the composite catalyst and the fermentation stock solution respectively. The enzyme activity of the reacted samples was detected by gas chromatography. The percentage of enzyme activity loss after 7 days was calculated with the enzyme activity after 0 days of storage as 100%. The results are as follows: Figure 9 As shown.

[0119] Example 6

[0120] This embodiment uses a composite catalyst to simulate the catalytic performance of the industrial catalytic reaction of acrylonitrile to acrylamide.

[0121] The reaction system consisted of 500 mL of a composite catalyst (stored at 20°C for 7 days) at a rate of 25 mL. 475 mL of acrylonitrile aqueous solution (30%) was added to initiate the reaction. The reactor was stirred at 200 rpm, and the temperature was maintained at 20-25°C. Reaction progress was measured at intervals. The catalytic results are as follows. Figure 7 As shown, where

[0122] When the composite catalyst A contains gelatin, the gelatin content is 6%;

[0123] When composite catalyst B contains PVA, the PVA content is 10%.

[0124] When the composite catalyst C contains glycerol, the glycerol content is 30%;

[0125] When the composite catalyst D contains PEG, the PEG content is 10%.

[0126] When composite catalyst E contains glutaraldehyde, the content of glutaraldehyde is 0.07%.

[0127] When the composite catalyst F contains glycerol and glutaraldehyde, the content of glycerol is 10% and the content of glutaraldehyde is 0.07%.

[0128] When the composite catalyst G contains glycerol and PVA, the content of glycerol is 10% and the content of PVA is 5%.

[0129] When the composite catalyst H contains glutaraldehyde and PEG, the content of glutaraldehyde is 0.07% and the content of PEG is 2%.

[0130] When composite catalyst I contains glycerol, glutaraldehyde and gelatin, the content of glycerol is 10%, the content of glutaraldehyde is 0.07%, and the content of gelatin is 4%.

[0131] When the composite catalyst J contains gelatin, PVA and PEG, the content of gelatin is 4%, the content of PVA is 5%, and the content of PEG is 2%.

[0132] When the composite catalyst K contains glutaraldehyde, PEG and PVA, the content of glutaraldehyde is 0.07%, the content of PEG is 2% and the content of PVA is 5%.

[0133] When the composite catalyst L contains glycerol, glutaraldehyde, gelatin and PEG, the content of glycerol is 10%, the content of glutaraldehyde is 0.07%, the content of gelatin is 4%, and the content of PEG is 2%.

[0134] When the composite catalyst M contains glycerol, PVA, PEG and gelatin, the content of glycerol is 10%, the content of PVA is 5%, the content of PEG is 2%, and the content of gelatin is 4%.

[0135] When the composite catalyst N contains glutaraldehyde, PEG, PVA and gelatin, the content of glutaraldehyde is 0.07%, the content of PEG is 2%, the content of PVA is 5%, and the content of gelatin is 4%.

[0136] When the composite catalyst O contains glycerol, glutaraldehyde, gelatin, PEG and PVA, the content of glycerol is 10%, the content of glutaraldehyde is 0.07%, the content of gelatin is 5%, the content of PEG is 2%, and the content of PVA is 5%.

[0137] from Figure 10 It can be seen that different composite catalysts can all catalyze the formation of acrylonitrile into acrylamide. Compared with the original fermentation broth, the composite catalyst has a higher catalytic efficiency. This indicates that when component A is added to the composite catalyst, it can not only improve the storage stability of the Rhodococcus fermentation broth, but also greatly improve the catalytic performance of the Rhodococcus fermentation broth.

[0138] For example, when using a composite catalyst containing glutaraldehyde and Rhodococcus ferment broth, the amount of acrylamide (AM) detected in the reaction solution after 42 min of catalytic reaction was 30%, and no acrylonitrile was detected. However, when using the fermentation stock broth prepared in Example 1 for catalytic reaction, it took 51 min of catalytic reaction with the fermentation stock broth to detect 30% acrylamide. This shows that the composite catalyst has higher catalytic efficiency compared with the fermentation stock broth.

[0139] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A composite catalyst comprising Rhodococcus ferment broth and component A, wherein component A comprises glycerol and glutaraldehyde; wherein the glycerol comprises 5-30% by mass percentage in the composite catalyst and the glutaraldehyde comprises 0.01-1%.

2. The composite catalyst according to claim 1, wherein the glycerol is 10-30% and the glutaraldehyde is 0.03-0.07% by mass percentage in the composite catalyst.

3. The composite catalyst according to claim 1, wherein component A further comprises gelatin, wherein the gelatin accounts for 1-20% by mass percentage in the composite catalyst.

4. The composite catalyst according to claim 3, wherein component A further comprises polyethylene glycol, wherein the polyethylene glycol accounts for 1-20% by mass percentage in the composite catalyst.

5. The composite catalyst according to claim 4, wherein component A further comprises methylparaben, wherein the methylparaben is 1-50% by mass percentage in the composite catalyst.

6. The composite catalyst according to claim 1, wherein component A further comprises gelatin, wherein the gelatin accounts for 2-10% by mass percentage in the composite catalyst.

7. The composite catalyst according to claim 6, wherein component A further comprises polyethylene glycol, wherein the polyethylene glycol accounts for 2-10% by mass percentage in the composite catalyst.

8. The composite catalyst according to claim 7, wherein component A further comprises methylparaben, wherein the methylparaben is 5-50% by mass percentage in the composite catalyst.

9. The composite catalyst according to claim 1, wherein component A further comprises gelatin, wherein the gelatin accounts for 2-8% by mass percentage in the composite catalyst.

10. The composite catalyst according to claim 9, wherein component A further comprises polyethylene glycol, wherein the polyethylene glycol is 2-8% by mass percentage in the composite catalyst.

11. The composite catalyst according to claim 10, wherein component A further comprises methylparaben, wherein the methylparaben is 5-30% by mass percentage in the composite catalyst.

12. The composite catalyst according to any one of claims 1-11, wherein, The concentration of Rhodococcus in the Rhodococcus fermentation broth in the composite catalyst is 100-200 g / L.

13. The use of component A in improving the stability of Rhodococcus ferment broth, wherein component A comprises glycerol and glutaraldehyde; wherein, The concentration of glycerol is 5-30 wt%, and the concentration of glutaraldehyde is 0.01-1 wt%.

14. The use of component A in improving the catalytic performance of Rhodococcus ferment broth, wherein component A comprises glycerol and glutaraldehyde; wherein, The concentration of glycerol is 5-30 wt%, and the concentration of glutaraldehyde is 0.01-1 wt%.

15. Use of Rhodococcus ferment broth and component A in the catalytic preparation of amide compounds from nitrile compounds, wherein component A comprises glycerol and glutaraldehyde; wherein, The concentration of glycerol is 5-30 wt%, and the concentration of glutaraldehyde is 0.01-1 wt%.

16. The use according to any one of claims 13-15, wherein, The concentration of glycerol is 10-30 wt%, and the concentration of glutaraldehyde is 0.03-0.07 wt%.

17. The use according to any one of claims 13-15, wherein component A further comprises gelatin, said gelatin having a concentration of 1-20 wt%.

18. The use according to claim 17, wherein component A further comprises polyethylene glycol, the concentration of which is 1-20 wt%.

19. The use according to claim 18, wherein component A further comprises methylparaben, said methylparaben having a concentration of 1-50 wt%.

20. The use according to any one of claims 13-15, wherein component A further comprises gelatin, said gelatin having a concentration of 2-10 wt%.

21. The use according to claim 20, wherein component A further comprises polyethylene glycol, said polyethylene glycol having a concentration of 2-10 wt%.

22. The use according to claim 21, wherein component A further comprises methylparaben, said methylparaben having a concentration of 5-50 wt%.

23. The use according to any one of claims 13-15, wherein component A further comprises gelatin, said gelatin having a concentration of 2-8 wt%.

24. The use according to claim 23, wherein component A further comprises polyethylene glycol, said polyethylene glycol having a concentration of 2-8 wt%.

25. The use according to claim 24, wherein component A further comprises methylparaben, said methylparaben having a concentration of 5-30 wt%.

26. The use according to any one of claims 13-15, wherein the concentration of Rhodococcus in the Rhodococcus fermentation broth is 100-200 g / L.

27. The use according to claim 15, wherein the amide compound is selected from acrylamide, 2,6-difluorobenzamide, p-hydroxyphenylacetamide, p-hydroxybenzamide, pyrazinamide, and nicotinamide.

28. A method for preparing amide compounds from nitrile compounds by catalysis, comprising: The composite catalyst according to any one of claims 1-12 is contacted with a nitrile compound to undergo a catalytic reaction to obtain an amide compound.

29. The method according to claim 28, wherein the amide compound is selected from acrylamide, 2,6-difluorobenzamide, p-hydroxyphenylacetamide, p-hydroxybenzamide, pyrazinamide, and nicotinamide.

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