Method for producing indoxacarb intermediate by using nano-ferrous carbonate to immobilize hydroxylase

Abstract

The present invention belongs to the field of biochemical engineering, and specifically relates to a method for producing an indoxacarb intermediate by using nano-ferrous carbonate immobilized hydroxylase. It includes preparing an indoxacarb intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone by preparing nano-ferrous carbonate immobilized hydroxylase and polydopamine-modified nano-ferrous carbonate immobilized hydroxylase. The nano-ferrous carbonate immobilized hydroxylase of the present invention has the advantages of small particle size, good dispersibility, high reuse efficiency, etc., and its surface is covered with a polydopamine film, which further improves the stability and reuse rate of the biocatalyst, and the nano-ferrous carbonate immobilized hydroxylase does not rely on expensive cofactors. The process route of the present invention is simple, no expensive catalyst is required, the production cost is low, the by-products are less, and the production efficiency is high.

Description

Technical Field

[0001] The invention belongs to the field of biochemical engineering, and particularly relates to a method for producing an indoxacarb intermediate by utilizing nano-ferrous carbonate to immobilize hydroxylase. Background Art

[0002] In recent years, with the increase in population, the reduction in cultivated land, the adjustment of agricultural structure and the demand of agricultural market, high-efficiency, high-selectivity and low-toxicity residual pesticides have become a research hotspot. The new insecticide indoxacarb belongs to a broad-spectrum oxadiazine insecticide. It blocks the sodium ion channels in insect nerve cells, making them lose their function. It has a contact and stomach poisoning effect and can effectively prevent and control various pests on crops such as grain, cotton, fruits and vegetables.

[0003] (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone is the key chiral intermediate of indoxacarb, which is prepared by hydroxylation of 5-chloro-2-methoxycarbonyl-1-indanone:

[0004]

[0005] Patent CN 109485568A reports a method for preparing an optical indoxacarb intermediate, in which 5-chloro-2-methoxycarbonyl-1-indanone reacts with an oxidant in the presence of a chiral Zr-salen polymer to obtain an indoxacarb intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone. Using this preparation method, the yield can reach 90% and the S-isomer content can be as high as 99%. However, the synthesis process of the catalyst chiral Zr-salen polymer used is relatively cumbersome and the synthesis cost is high.

[0006] Patent CN 115807042A reports a method for preparing an indoxacarb intermediate, wherein 5-chloro-2-methoxycarbonyl-1-indanone undergoes an enzymatic reaction in the presence of monooxygenase, glucose dehydrogenase, and a cofactor to obtain the indoxacarb intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone. The preparation method can achieve a good yield and stereoselectivity. However, monooxygenase, glucose dehydrogenase, and cofactor are used in the preparation process, and the monooxygenase and glucose dehydrogenase cannot be reused, and the price of the cofactor is relatively expensive. Summary of the invention

[0007] The object of the present invention is to provide a method for producing an indoxacarb intermediate by using nano-ferrous carbonate immobilized hydroxylase, which has low preparation cost, high product optical purity and reusable catalyst.

[0008] In order to solve the problems existing in the prior art, the present invention adopts the following technical solutions:

[0009] The method for producing an indoxacarb intermediate by using nano-ferrous carbonate to immobilize hydroxylase comprises the following steps:

[0010] (1) Adding soluble carbonate, surfactant, hydroxylase and water into the reactor in sequence, stirring and mixing until completely dissolved, wherein the soluble carbonate includes any one of sodium carbonate, potassium carbonate and ammonium carbonate; the surfactant includes at least one of sodium dodecylbenzene sulfonate, Tween-80, Span-80, Pluronic F-108 and Pluronic F-127; the hydroxylase is derived from the fermentation product of Escherichia coli; then adding an aqueous solution of a divalent iron salt, wherein the divalent iron salt includes any one of ferrous sulfate heptahydrate, ferrous chloride and ferrous nitrate, stirring and mixing, and obtaining nano-ferrous carbonate immobilized hydroxylase after the reaction is completed at a reaction temperature of 20-40° C.; the prepared nano-ferrous carbonate immobilized hydroxylase has the advantages of small particle size, good dispersibility and high reuse efficiency, and the hydroxylase is a ferrous ion activated enzyme, and the ferrous ions can effectively activate the hydroxylase during the enzyme immobilization process, and the synthesized carrier nano-ferrous carbonate can improve the catalytic performance of the hydroxylase;

[0011] (2) adding dopamine to the reaction solution of the nano-ferrous carbonate immobilized hydroxylase obtained in the above step, stirring evenly, and standing for a period of time at a standing temperature of 20-40° C., and after standing, centrifuging, washing, and drying to obtain polydopamine-modified nano-ferrous carbonate immobilized hydroxylase; dopamine will generate polydopamine through intramolecular ring rearrangement and intermolecular cross-linking under the presence of oxygen and alkaline conditions, and polydopamine can be deposited on the surface of the nano-ferrous carbonate immobilized hydroxylase to form a dense polymer film, further improving the stability and reuse rate of the nano-ferrous carbonate immobilized hydroxylase;

[0012] (3) 5-chloro-2-methoxycarbonyl-1-indanone, an organic solvent, nano-ferrous carbonate immobilized hydroxylase, α-ketoglutaric acid and water, wherein the organic solvent is selected from at least one of dichloromethane, methanol, ethanol, dimethyl sulfoxide and toluene, and the mixture is stirred and mixed. After the reaction is completed at a reaction temperature of 20-40° C., an indoxacarb intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone is obtained; the nano-ferrous carbonate immobilized hydroxylase does not rely on expensive cofactors and can catalyze the reaction of 5-chloro-2-methoxycarbonyl-1-indanone with α-ketoglutaric acid in the presence of oxygen to produce (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone, succinic acid and carbon dioxide;

[0013] (4) The reaction solution after the reaction in the above step (3) is centrifuged, washed and dried to obtain nano-ferrous carbonate immobilized hydroxylase, which is ready for the next batch of feed.

[0014] In the above-mentioned method for producing an indoxacarb intermediate by immobilizing hydroxylase with nano-ferrous carbonate, the mass ratio of the soluble carbonate, surfactant, hydroxylase and water in step (1) is (1-10): (0.1-1): (0.1-1): 100, and the mass ratio of the divalent iron salt and water is (1-20): 100.

[0015] In the above method for producing an indoxacarb intermediate by using nano-ferrous carbonate to immobilize hydroxylase, the concentration of dopamine in step (2) is 0.09-0.9 g / L.

[0016] In the above-mentioned method for producing an indoxacarb intermediate using nano-ferrous carbonate immobilized hydroxylase, the mass ratio of 5-chloro-2-methoxycarbonyl-1-indanone, organic solvent, nano-ferrous carbonate immobilized hydroxylase, α-ketoglutaric acid and water in step (3) is (0.1-1):(1-5):(0.01-0.1):(0.1-1):100.

[0017] In the above method for producing an indoxacarb intermediate by using nano-ferrous carbonate to immobilize hydroxylase, the reaction time in step (1) is 1-6 hours, the standing time in step (2) is 12-48 hours, and the reaction time in step (3) is 12-48 hours.

[0018] In the above method for producing an indoxacarb intermediate by using nano-ferrous carbonate to immobilize hydroxylase, the centrifugal speed of the centrifugal separation in step (2) and step (4) is 4000-12000 rpm.

[0019] In the above method for producing an indoxacarb intermediate by using nano-ferrous carbonate to immobilize hydroxylase, the washing liquid in step (2) and step (4) is selected from water, methanol or ethanol.

[0020] In the above method for producing an indoxacarb intermediate by using nano-ferrous carbonate to immobilize hydroxylase, the drying method of step (2) and step (4) is selected from at least one of natural air drying, freeze drying and vacuum drying.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The nano-ferrous carbonate immobilized hydroxylase prepared by the present invention has the advantages of small particle size, good dispersibility, high reuse efficiency, etc.

[0023] (2) The surface of the nano-ferrous carbonate immobilized hydroxylase prepared by the present invention is covered with a polydopamine film, which further improves the stability and reusability of the biocatalyst;

[0024] (3) The nano-ferrous carbonate immobilized hydroxylase prepared by the present invention does not rely on expensive cofactors, only needs to add α-ketoglutarate, and has high selectivity;

[0025] (4) The process route for synthesizing (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone of the present invention is simple, does not require expensive catalysts, has low production costs, produces fewer by-products, and has high production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0027] Figure 1 This is the particle size distribution diagram of the nano-ferrous carbonate immobilized hydroxylase prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0028] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0029] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0030] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0031] Unless otherwise specified, the raw materials used in the examples were purchased commercially.

[0032] Example 1

[0033] Reference Figure 1 This embodiment provides a method for producing an indoxacarb intermediate by using nano-ferrous carbonate to immobilize hydroxylase:

[0034] (1) Add 1g sodium carbonate, 0.2g sodium dodecylbenzene sulfonate, 0.2g hydroxylase and 50mL water in a 100mL beaker in turn. Hydroxylase is derived from the fermentation product of Escherichia coli. Stir and mix to completely dissolve it. Dissolve 0.2g ferrous sulfate heptahydrate in 5mL water and add it to the above mixture and stir and mix. After stirring at 25°C for 2h, nano-ferrous carbonate immobilized hydroxylase is obtained. Nano-ferrous carbonate immobilized hydroxylase has the advantages of small particle size, good dispersion performance, and high reuse efficiency. Hydroxylase is a ferrous ion activated enzyme. Ferrous ions can effectively activate hydroxylase during the enzyme immobilization process. The synthesized carrier nano-ferrous carbonate can improve the catalytic performance of hydroxylase.

[0035] (2) Add 0.02g dopamine to the reaction solution of the nano-ferrous carbonate immobilized hydroxylase obtained in the above step, stir evenly, stand at a standing temperature of 25°C for 24h, centrifuge after standing, centrifuge at a speed of 8000rpm, wash with water, and naturally air-dry to obtain polydopamine-modified nano-ferrous carbonate immobilized hydroxylase, the particle size of which is 645nm. Dopamine will generate polydopamine through intramolecular ring rearrangement and intermolecular cross-linking under the presence of oxygen and alkaline conditions. Polydopamine can be deposited on the surface of the nano-ferrous carbonate immobilized hydroxylase to form a dense polymer film, further improving the stability and reuse rate of the nano-ferrous carbonate immobilized hydroxylase.

[0036] (3) Add 0.05g 5-chloro-2-methoxycarbonyl-1-indanone, 1g dichloromethane, 0.02g nano-ferrous carbonate immobilized hydroxylase, 0.04g α-ketoglutaric acid and 25mL water into a 50mL beaker, stir and mix, and stir and react at a reaction temperature of 25°C for 24h. After the reaction, the intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone of indoxacarb is obtained. The purity of the product (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone is 96%, and the ee value is 99%. Nano-ferrous carbonate immobilized hydroxylase does not rely on expensive cofactors and can catalyze the reaction of 5-chloro-2-methoxycarbonyl-1-indanone with α-ketoglutaric acid in the presence of oxygen to produce (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone, succinic acid and carbon dioxide.

[0037] (4) The reaction solution after the reaction in the above step was centrifuged at a centrifugal speed of 8000 rpm, washed with water, and naturally air-dried to obtain polydopamine-modified nano-ferrous carbonate immobilized hydroxylase, which was prepared for the next batch of feed.

[0038] Example 2

[0039] This example discloses a method for producing an indoxacarb intermediate by using nano-ferrous carbonate to immobilize hydroxylase:

[0040] (1) Add 0.5 g potassium carbonate, 0.05 g Tween-80, 0.05 g hydroxylase and 50 mL water in a 100 mL beaker in sequence. The hydroxylase is derived from the fermentation product of Escherichia coli and stirred to completely dissolve. Dissolve 0.05 g ferrous nitrate in 5 mL water and add it to the above mixture. After stirring at 20°C for 1 hour, nano-ferrous carbonate-immobilized hydroxylase is obtained.

[0041] (2) Add 0.005 g of dopamine to the reaction solution of the nano-ferrous carbonate immobilized hydroxylase obtained in the above step, stir evenly, and let it stand at a standing temperature of 20° C. for 12 h. After standing, centrifuge at a centrifugal speed of 4000 rpm, wash with methanol, and freeze-dry to obtain polydopamine-modified nano-ferrous carbonate immobilized hydroxylase with a particle size of 819 nm.

[0042] (3) In a 50 mL beaker, 0.025 g of 5-chloro-2-methoxycarbonyl-1-indanone, 0.25 g of methanol, 0.0025 g of nano-ferrous immobilized hydroxylase, 0.025 g of α-ketoglutaric acid and 25 mL of water were added and stirred. The mixture was stirred and reacted at a reaction temperature of 20° C. for 12 h. After the reaction, the indoxacarb intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone was obtained. The purity of the product (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone was 92%, and the ee value was 98%.

[0043] (4) The reaction solution after the reaction in the above step was centrifuged at a speed of 4000 rpm, washed with methanol, and freeze-dried to obtain polydopamine-modified nano-ferrous carbonate immobilized hydroxylase, which was prepared for the next batch of feed.

[0044] Example 3

[0045] This example discloses a method for producing an indoxacarb intermediate by using nano-ferrous carbonate to immobilize hydroxylase:

[0046] (1) Add 5 g of ammonium carbonate, 0.5 g of Pluronic F-127, 0.5 g of hydroxylase and 50 mL of water in a 100 mL beaker in sequence. The hydroxylase is derived from the fermentation product of Escherichia coli and stirred to completely dissolve. Dissolve 1 g of ferrous chloride in 5 mL of water and add it to the above mixture. After stirring at 40°C for 6 h, nano-ferrous carbonate-immobilized hydroxylase is obtained.

[0047] (2) 0.05 g of dopamine was added to the reaction solution of the nano-ferrous carbonate immobilized hydroxylase obtained in the above step, and the mixture was stirred evenly. The mixture was allowed to stand at a temperature of 40° C. for 48 h. After the standing period, the mixture was centrifuged at a speed of 12,000 rpm. The mixture was washed with ethanol and vacuum dried to obtain polydopamine-modified nano-ferrous carbonate immobilized hydroxylase with a particle size of 576 nm.

[0048] (3) In a 50 mL beaker, 0.25 g of 5-chloro-2-methoxycarbonyl-1-indanone, 1.25 g of dimethyl sulfoxide, 0.025 g of nano-ferrous immobilized hydroxylase, 0.25 g of α-ketoglutaric acid and 25 mL of water were added and stirred. The mixture was stirred and reacted at a reaction temperature of 40° C. for 48 h. After the reaction, the indoxacarb intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone was obtained. The purity of the product (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone was 96%, and the ee value was 98%.

[0049] (4) The reaction solution after the reaction in the above step was centrifuged at a speed of 12000 rpm, washed with ethanol, and vacuum dried to obtain polydopamine-modified nano-ferrous carbonate immobilized hydroxylase, which was prepared for the next batch of feed.

[0050] Example 4

[0051] This example discloses a method for producing an indoxacarb intermediate by using nano-ferrous carbonate to immobilize hydroxylase:

[0052] (1) Add 1 g of sodium carbonate, 0.2 g of Span-80, 0.2 g of Pluronic F-108, 0.2 g of hydroxylase and 50 mL of water in a 100 mL beaker in sequence. The hydroxylase is derived from the fermentation product of Escherichia coli and stirred to completely dissolve. Dissolve 0.2 g of ferrous sulfate heptahydrate in 5 mL of water and add it to the above mixture. After stirring at 25°C for 2 h, nano-ferrous carbonate-immobilized hydroxylase is obtained.

[0053] (2) 0.02 g of dopamine was added to the reaction solution of the nano-ferrous carbonate immobilized hydroxylase obtained in the above step, and the mixture was stirred evenly. The mixture was allowed to stand at a temperature of 25° C. for 24 h. After the standing period, the mixture was centrifuged at a speed of 6000 rpm. The mixture was washed with water and naturally air-dried to obtain polydopamine-modified nano-ferrous carbonate immobilized hydroxylase with a particle size of 612 nm.

[0054] (3) In a 50 mL beaker, 0.05 g of 5-chloro-2-methoxycarbonyl-1-indanone, 1 g of dichloromethane, 0.02 g of nano-ferrous immobilized hydroxylase, 0.04 g of α-ketoglutaric acid and 25 mL of water were added and stirred. The mixture was stirred and reacted at a reaction temperature of 25° C. for 24 h. After the reaction, the indoxacarb intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone was obtained. The purity of the product (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone was 97%, and the ee value was 98%.

[0055] (4) The reaction solution after the reaction in the above step was centrifuged at a speed of 6000 rpm, washed with water, and naturally air-dried to obtain polydopamine-modified nano-ferrous carbonate immobilized hydroxylase, which was prepared for the next batch of feed.

[0056] Example 5

[0057] Reference Figure 1 This embodiment provides a method for producing an indoxacarb intermediate by using nano-ferrous carbonate to immobilize hydroxylase:

[0058] (1) Add 1g sodium carbonate, 0.2g sodium dodecylbenzene sulfonate, 0.2g hydroxylase and 50mL water in a 100mL beaker in sequence. The hydroxylase is derived from the fermentation product of Escherichia coli and stirred to completely dissolve. Dissolve 0.2g ferrous sulfate heptahydrate in 5mL water and add it to the above mixture. After stirring at 30°C for 2h, nano-ferrous carbonate-immobilized hydroxylase is obtained.

[0059] (2) 0.02 g of dopamine was added to the reaction solution of the nano-ferrous carbonate immobilized hydroxylase obtained in the above step, and the mixture was stirred evenly. The mixture was allowed to stand at 30° C. for 24 h, and then centrifuged at 8000 rpm. The mixture was washed with water and naturally air-dried to obtain polydopamine-modified nano-ferrous carbonate immobilized hydroxylase with a particle size of 690 nm.

[0060] (3) In a 50 mL beaker, 0.05 g of 5-chloro-2-methoxycarbonyl-1-indanone, 0.5 g of ethanol, 0.5 g of toluene, 0.02 g of nano-ferrous immobilized hydroxylase, 0.04 g of α-ketoglutaric acid and 25 mL of water were added and stirred. The mixture was stirred and reacted at a reaction temperature of 25° C. for 24 h. After the reaction, the indoxacarb intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone was obtained. The purity of the product (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone was 96%, and the ee value was 97%.

[0061] (4) The reaction solution after the reaction in the above step was centrifuged at a speed of 8000 rpm, washed with water, and naturally air-dried to obtain polydopamine-modified nano-ferrous carbonate immobilized hydroxylase, which was prepared for the next batch of feed.

[0062] Example 6

[0063] Reference Figure 1 This embodiment provides a method for producing an indoxacarb intermediate by using nano-ferrous carbonate to immobilize hydroxylase:

[0064] (1) Add 1 g of ammonium carbonate, 0.2 g of Span-80, 0.2 g of hydroxylase and 50 mL of water in a 100 mL beaker in sequence. The hydroxylase is derived from the fermentation product of Escherichia coli and stirred to completely dissolve. Dissolve 0.2 g of ferrous sulfate heptahydrate in 5 mL of water and add it to the above mixture. After stirring at 25°C for 2 h, nano-ferrous carbonate-immobilized hydroxylase is obtained.

[0065] (2) 0.02 g of dopamine was added to the reaction solution of the nano-ferrous carbonate immobilized hydroxylase obtained in the above step, and the mixture was stirred evenly, and then allowed to stand at a standing temperature of 25° C. for 24 h. After the standing period, the mixture was centrifuged (the centrifugal speed was 8000 rpm), washed with water, naturally air-dried, and then freeze-dried to obtain polydopamine-modified nano-ferrous carbonate immobilized hydroxylase with a particle size of 723 nm.

[0066] (3) In a 50 mL beaker, 0.05 g of 5-chloro-2-methoxycarbonyl-1-indanone, 1 g of dichloromethane, 0.02 g of nano-ferrous immobilized hydroxylase, 0.04 g of α-ketoglutaric acid and 25 mL of water were added and stirred. The mixture was stirred and reacted at a reaction temperature of 25° C. for 24 h. After the reaction, the indoxacarb intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone was obtained. The purity of the product (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone was 98%, and the ee value was 99%.

[0067] (4) The reaction solution after the reaction in the above step was centrifuged (the centrifugal speed was 8000 rpm), washed with water, naturally air-dried and then freeze-dried to obtain polydopamine-modified nano-ferrous carbonate immobilized hydroxylase, which was prepared for the next batch of feed.

[0068] Example 7

[0069] This example discloses a method for producing an indoxacarb intermediate by using nano-ferrous carbonate to immobilize hydroxylase:

[0070] (1) Add 10g of sodium carbonate, 10g of sodium dodecylbenzene sulfonate, 10g of hydroxylase and 2500mL of water in a 5000mL beaker in sequence. The hydroxylase is derived from the fermentation product of Escherichia coli and stirred to completely dissolve. Dissolve 10g of ferrous sulfate heptahydrate in 250mL of water and add it to the above mixture. After stirring at 25°C for 2h, nano-ferrous carbonate-immobilized hydroxylase is obtained.

[0071] (2) Add 1 g of dopamine to the reaction solution of the nano-ferrous carbonate immobilized hydroxylase obtained in the above step, stir evenly, and let it stand at a standing temperature of 25° C. for 24 h. After the standing period, centrifuge (centrifugal speed is 8000 rpm), wash with water, and naturally air-dry to obtain polydopamine-modified nano-ferrous carbonate immobilized hydroxylase with a particle size of 715 nm.

[0072] (3) In a 2500 mL beaker, 2.5 g of 5-chloro-2-methoxycarbonyl-1-indanone, 50 g of dichloromethane, 1 g of nano-ferrous immobilized hydroxylase, 2 g of α-ketoglutaric acid and 1250 mL of water were added and stirred. The mixture was stirred and reacted at a reaction temperature of 25° C. for 24 h. After the reaction, an indoxacarb intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone was obtained. The purity of the product (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone was 95%, and the ee value was 97%.

[0073] (4) The reaction solution after the reaction in the above step was centrifuged (the centrifugal speed was 8000 rpm), washed with water, and naturally air-dried to obtain polydopamine-modified nano-ferrous carbonate immobilized hydroxylase, which was prepared for the next batch of feed.

[0074] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for producing an indoxacarb intermediate by using nano-ferrous carbonate to immobilize hydroxylase, characterized in that: The following steps are involved: (1) Adding soluble carbonate, surfactant, hydroxylase and water into a reactor in sequence, stirring and mixing until completely dissolved, wherein the soluble carbonate includes any one of sodium carbonate, potassium carbonate and ammonium carbonate, the surfactant includes at least one of sodium dodecylbenzene sulfonate, Tween-80, Span-80, Pluronic F-108 and Pluronic F-127, and the hydroxylase is derived from the fermentation product of Escherichia coli; then adding an aqueous solution of a divalent iron salt, wherein the divalent iron salt includes any one of ferrous sulfate heptahydrate, ferrous chloride and ferrous nitrate, stirring and mixing, and after the reaction is completed at a reaction temperature of 20-40°C, obtaining nano-ferrous carbonate immobilized hydroxylase; (2) adding dopamine to the reaction solution of the nano-ferrous carbonate immobilized hydroxylase obtained in the above step, stirring evenly, and standing at a temperature of 20-40° C. for a period of time. After standing, centrifuging, washing, and drying to obtain the polydopamine-modified nano-ferrous carbonate immobilized hydroxylase; (3) 5-chloro-2-methoxycarbonyl-1-indanone, an organic solvent, nano-ferrous carbonate immobilized hydroxylase, α-ketoglutaric acid and water, wherein the organic solvent is at least one selected from dichloromethane, methanol, ethanol, dimethyl sulfoxide and toluene, are stirred and mixed, and the reaction is completed at a reaction temperature of 20-40° C. to obtain an indoxacarb intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone; (4) After the reaction in step (3), the reaction liquid is centrifuged, washed and dried to obtain nano-ferrous carbonate immobilized hydroxylase, which is ready for the next batch of feed.

2. The method for producing an indoxacarb intermediate by using nano-ferrous carbonate immobilized hydroxylase according to claim 1, characterized in that: The mass ratio of the soluble carbonate, surfactant, hydroxylase and water in step (1) is (1-10): (0.1-1): (0.1-1): 100, and the mass ratio of the divalent iron salt and water is (1-20):

100.

3. The method for producing an indoxacarb intermediate by using nano-ferrous carbonate immobilized hydroxylase according to claim 1, characterized in that: The concentration of dopamine in step (2) is 0.09-0.9 g / L.

4. The method for producing an indoxacarb intermediate by using nano-ferrous carbonate immobilized hydroxylase according to claim 1, characterized in that: The mass ratio of 5-chloro-2-methoxycarbonyl-1-indanone, organic solvent, nano-ferrous carbonate immobilized hydroxylase, α-ketoglutaric acid and water in step (3) is (0.1-1):(1-5):(0.01-0.1):(0.1-1):

100.

5. The method for producing an indoxacarb intermediate by using nano-ferrous carbonate immobilized hydroxylase according to claim 1, characterized in that: The reaction time in step (1) is 1-6 h, the standing time in step (2) is 12-48 h, and the reaction time in step (3) is 12-48 h.

6. The method for producing an indoxacarb intermediate by using nano-ferrous carbonate immobilized hydroxylase according to claim 1, characterized in that: The centrifugal speed of the centrifugal separation in step (2) and step (4) is 4000-12000 rpm.

7. The method for producing an indoxacarb intermediate by using nano-ferrous carbonate immobilized hydroxylase according to claim 1, characterized in that: The washing liquid in step (2) and step (4) is selected from water, methanol or ethanol.

8. The method for producing an indoxacarb intermediate by using nano-ferrous carbonate immobilized hydroxylase according to claim 1, characterized in that: The drying method of step (2) and step (4) is selected from at least one of natural air drying, freeze drying and vacuum drying.

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