CN116121312A - Method for preparing chiral amine by emulsion microreactor continuous reaction - Google Patents

Method for preparing chiral amine by emulsion microreactor continuous reaction Download PDF

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CN116121312A
CN116121312A CN202310048184.8A CN202310048184A CN116121312A CN 116121312 A CN116121312 A CN 116121312A CN 202310048184 A CN202310048184 A CN 202310048184A CN 116121312 A CN116121312 A CN 116121312A
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chiral amine
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杨恒权
樊敏
卫伟
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Shanxi University
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    • C12Y206/01—Transaminases (2.6.1)

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Abstract

The invention discloses a method for preparing chiral amine by emulsion microreactor continuous reaction, belonging to the technical field of enzyme catalysis. Aiming at the defects of relatively low stability of Pickering emulsion, limited selection of organic solvent, poor conversion of high substrate concentration, pressure resistance, enzyme activity loss, incapability of efficient cyclic regeneration of cofactors, difficulty in continuous and industrialized preparation process and the like, the invention realizes the synergic catalysis and long-time continuous conversion of transaminase and cofactors in a limited space by crosslinking the water-in-oil Pickering emulsion. The preparation method mainly comprises the steps of taking transaminase and cofactor dispersed in PBS buffer solution as water phase, taking reactant dispersed in organic solvent as oil phase, taking silicon dioxide nano particles with interfacial activity as emulsifier to stabilize an oil-water interface, forming water-in-oil Pickering emulsion for encapsulating enzyme and cofactor, and then adding cross-linking agent by taking Pickering emulsion as template to prepare the microcapsule with certain strength and organic solvent tolerance.

Description

Method for preparing chiral amine by emulsion microreactor continuous reaction
Technical Field
The invention belongs to the technical field of enzyme catalysis, and particularly relates to a method for preparing chiral amine by continuous reaction of an emulsion microreactor.
Background
Chiral amine compounds are key intermediates for synthesizing a plurality of medicaments, occupy important positions in the pharmaceutical industry and the agricultural chemical industry, and have higher economic value. The transaminase biocatalysis provides an effective strategy for preparing chiral amine, and can catalyze amino groups on an amino donor to transfer to carbonyl groups of prochiral ketone to obtain chiral amine compounds. The chiral amine prepared by the transaminase biocatalysis has the advantages of high catalytic efficiency, strong specificity, mild reaction conditions, environmental friendliness and the like, and has a huge application prospect.
Pickering emulsion is emulsion with oil-water two phases stabilized by solid nano particles, and has been widely used because of its advantages of good stability, large immiscible interface, etc. The stable liquid drop of Pickering emulsion can be used as an excellent carrier of immobilized enzyme, on one hand, biological enzyme is encapsulated in a disperse phase, so that the immobilization of enzyme is realized, on the other hand, reactants are catalyzed into products in a mobile phase, and a large interface is favorable for mass transfer and energy exchange between an oil phase and an aqueous phase of the reactants and the products. The use of Pickering emulsions offers unique advantages for cofactor-dependent enzyme-catalyzed reactions. Enzyme and cofactor are co-encapsulated in the disperse phase of Pickering emulsion, compared with the physical adsorption of immobilized enzyme and cofactor, the enzyme and cofactor loss in the continuous reaction process is greatly reduced; compared with covalent co-immobilization of enzymes and cofactors, it reduces the loss of enzyme activity while increasing the freedom of cofactors; in addition, the dispersed phase of the Pickering emulsion can provide a microenvironment for the biological enzyme that facilitates retention of the enzyme activity, thereby improving the stability of the enzyme. Although Pickering emulsions have some stability, for chemical reactions at higher substrate concentrations and higher temperatures and pressures, pickering emulsions tend to deform and coalesce during the reaction. Therefore, on the basis of retaining the basic advantages of Pickering emulsion, the further strengthening of the Pickering emulsion interface to prepare the microcapsule is imperative. Compared with other crosslinking methods, the chemical crosslinking method can realize the regulation and control of the thickness and permeability of the outer layer by changing the type and the dosage of the crosslinking agent and the length of the crosslinking time, thereby achieving the shape selecting effect, and the crosslinking method can generate covalent crosslinking at room temperature by only selecting proper solid particles and the crosslinking agent to stabilize Pickering emulsion to prepare the microcapsule, and has simple operation and strong operability.
Disclosure of Invention
Aiming at the defects of relatively low stability of Pickering emulsion, limited selection of organic solvent, poor conversion of high substrate concentration, pressure resistance, enzyme activity loss, difficult continuous and industrialized preparation process and the like, the novel method for encapsulating transaminase and cofactor for continuous fixed bed based on cross-linked Pickering emulsion is provided, the improvement of the stability of Pickering emulsion, the expansion of the selection range of organic solvent, the improvement of pressure resistance, better conversion of high substrate concentration, and efficient regeneration and utilization of transaminase and cofactor encapsulation and cofactor are realized, and the foundation is laid for industrialization by continuous stable and efficient operation for a long time.
The invention provides a method for preparing chiral amine by emulsion microreactor continuous reaction.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for preparing chiral amine by emulsion microreactor continuous reaction, which comprises the following steps:
step 1, siO with interfacial activity 2 Is prepared from the following steps: will commercialize SiO 2 The nano particles are dispersed in an organic solvent by ultrasonic, and then hydrophobic silane and organic amine are added; stirring and refluxing for 3-6 h under the protection of nitrogen at 60-120 ℃ to obtain a mixed system, cooling, centrifugally separating the mixed system, washing the analyzed solid with toluene for 3-5 times, and drying to obtain SiO with interfacial activity 2 A nanoparticle;
step 2, preparing Pickering emulsion for immobilizing enzyme and cofactor: adding transaminase and cofactor into phosphate buffer solution with pH value of 7-9, and uniformly mixing to obtain a water phase system; siO with interfacial activity 2 Dispersing the nano particles in an organic solvent by ultrasonic waves to obtain an oil phase system; mixing the oil phase system and the water phase system, and stirring at high speed to obtain Pickering with immobilized enzyme and cofactor having uniform particle diameterg, emulsion;
step 3, adding an organic solvent and a cross-linking agent into the Pickering emulsion obtained in the step 2, and placing the mixture on a rotary evaporator for cross-linking to prepare microcapsules;
and 4, placing the microcapsule obtained in the step 3 in a fixed bed reactor, and then introducing an organic solution containing cofactor regenerant for reaction to obtain chiral amine.
Further, the organic solvent is any one of toluene, heptane, hexane, ethyl acetate, methyl tertiary butyl ether or octane.
Further, the hydrophobic silane is any one of methyltrimethoxysilane, dichlorodimethylsilane and octyltrimethoxysilane.
Further, the transaminase includes one or more of transaminases ATA-101 to ATA-165 or mutants of transaminases ATA-101 to ATA-165.
Further, the cofactor is any one of pyridoxal 5' -phosphate, nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate.
Further, the cross-linking agent is any one of methyltrimethoxysilane, octyltrimethoxysilane, tetraethyl silicate, tetramethoxysilane, trimethoxy silane, dichlorodimethylsilane, dimethyldimethoxysilane and hyperbranched polyethoxysilane.
Further, the phosphate buffer solution and SiO with interfacial activity 2 The mass ratio of the nano particles is 1:0.02-1:0.10; the volume ratio of the phosphate buffer solution to the organic solvent is 1:0.5-1:10.
Further, the diameter of the Pickering emulsion microdroplet in the step 2 is 5-300 mu m, and the phosphate buffer solution is phosphate buffer solution with the enzyme content of 10-500 mu L/mL.
Further, the rotating speed of high-speed stirring in the step 2 is 5000-10000 rpm; in the step 3, the temperature for preparing the microcapsule through crosslinking by a rotary evaporator is 30-45 ℃; the temperature of the reactor in the step 4 is 25-50 ℃; the flow rate of the reactor is 0.2-10 mL/h.
Further, the cofactor regenerant in the step 4 is any one of ammonium formate-ammonium formate dehydrogenase, isopropylamine, alpha-phenethylamine or 2-octylamine.
Compared with the prior art, the invention has the following advantages:
1. in the traditional method for co-immobilizing transaminase and cofactor, schiff base is usually formed by electrostatic action or utilizing aldehyde group of PLP and amino group of immobilization material to immobilize PLP, so that PLP is not firmly immobilized and is easy to run off or PLP is prevented from contacting with lysine amino residue of main active site of transaminase, and the degree of freedom of cofactor is reduced. The invention utilizes Pickering emulsion to realize in-situ encapsulation of aminotransferase and cofactor PLP, improves the degree of freedom of PLP, effectively avoids the cofactor loss along with the mobile phase, and runs 1000h with little PLP loss.
2. The substances required by the cofactor PLP circulation regeneration flow into the fixed bed along with the continuous phase, and flow out of the fixed bed along with the continuous phase after the PLP is reacted and regenerated, thereby realizing the circulation and regeneration utilization in the cofactor finite space.
3. The invention utilizes the silane cross-linking agent to hydrolyze and cross-link at the emulsion interface to form the shell on the basis of Pickering emulsion, effectively combines the advantages of homogeneous catalysis and heterogeneous catalysis, on one hand, the disperse phase can provide a homogeneous catalysis environment for transaminase and PLP, and can improve the stability of enzyme by changing the components of the disperse phase to adjust the microenvironment of the enzyme, on the other hand, the cross-linked silicon shell has good stability, so that the cross-linked silicon shell can be directly filled in a fixed bed reactor for continuous reaction, and in addition, the firm silicon shell effectively avoids the toxic and side effects of external environment on the activity of internal enzyme and PLP.
4. In the continuous flow process of the traditional Pickering emulsion, the problem of demulsification and the like can be caused by the excessive concentration of the substrate, so that the conversion with higher substrate concentration can not be realized. On the basis of retaining the basic advantages of Pickering emulsion, the invention adds the cross-linking agent to crosslink and fix the emulsion surface, which can be used for converting high concentration substrate, the substrate concentration can reach 300mM or above, the continuous reaction is carried out for more than 1000 hours, the conversion rate is more than 90%, and the ee value of the generated chiral compound is more than 99%.
5. In the conventional Pickering emulsion continuous flow process, the selection of a continuous phase is very limited, and the use of a polar solvent can lead to emulsion breaking. According to the invention, the cross-linked Pickering emulsion is utilized to fix the surface of the emulsion, the microcapsule can resist polar solvents such as ethyl acetate and the like, and the selection range of a continuous phase is wider, so that the applicable substrate range of the method is enlarged.
6. In the continuous preparation process of the traditional Pickering emulsion method, the emulsion pressure resistance is poor. The microcapsule is prepared by using the cross-linked Pickering emulsion, the organic phase can smoothly pass through the microcapsule under the flushing of the organic reagent with the hydraulic pressure of 1.0MPa and the flow rate of 50mL/h, and the microcapsule can be maintained for 48h without deformation, so that the microcapsule has better pressure resistance.
7. According to the invention, covalent crosslinking can be carried out at room temperature to stabilize Pickering emulsion to prepare the microcapsule only by selecting proper solid particles and a crosslinking agent, the operation is simple, the regulation and control on the thickness and permeability of the outer layer can be realized by changing the type and the dosage of the crosslinking agent and the length of the crosslinking time, and the controllability is strong.
Drawings
FIG. 1 is a scanning electron microscope image of the emulsifier prepared in example 1 of the present invention;
FIG. 2 is a Pickering morphology graph prepared in example 1 of the present invention;
FIG. 3 is a morphology diagram of the microcapsules prepared in example 1 of the present invention;
FIG. 4 shows the application of Pickering emulsion encapsulating transaminase and cofactor in example 2 of the present invention to asymmetric catalytic reduction of p-nitronitroacetophenone after crosslinking; reaction conversion and selectivity versus time.
Detailed Description
The following examples are given by taking the technical scheme of the invention as a premise, and detailed implementation modes and specific operation processes are given, but the protection scope of the patent of the invention is not limited, and all technical schemes obtained by adopting equivalent substitution or equivalent transformation are within the protection scope of the invention.
Example 1
A method for preparing chiral amine by emulsion microreactor continuous reaction, which comprises the following steps:
step 1: 1.0g of commercial SiO with particle size of 20nm after drying 2 The nanoparticles were dispersed in 40mL of toluene, 0.4g of dichlorodimethylsilane and 0.6g of n-hexylamine were added, and the mixture was refluxed for 4 hours under stirring at 60℃and under nitrogen protection. Then the interfacial active nano SiO can be obtained after centrifugation, washing and drying 2 (the morphology is shown in FIG. 1);
step 2: 0.25mL of aminotransferase ATA-117 solution and 3.3mg of PLP are added into 4mL of 100mM PBS buffer solution, and the mixture is stirred and mixed evenly by magnetic force; then 0.08g of interfacial active SiO 2 Dispersing in 12mL toluene by ultrasonic wave and adding into the mixed solution of the buffer solution and enzyme; finally, stirring at a high speed of 5000rpm to form Pickering emulsion (the morphology is shown in figure 2) of the immobilized enzyme catalyst;
step 3: the prepared Pickering emulsion with the immobilized transaminase catalyst was transferred to a 100mL round bottom flask, toluene, 0.8g methyltrimethoxysilane and 0.3g n-hexylamine were added, and the mixture was placed on a rotary evaporator and crosslinked at 40℃to prepare microcapsules (morphology is shown in FIG. 3).
Step 4: the Pickering emulsion of the prepared immobilized enzyme catalyst is transferred to a fixed bed reactor with the inner diameter of 2.8cm after being crosslinked, and the temperature of the reactor is set to be 45 ℃; introducing toluene solution of p-nitroacetophenone and alpha-phenethylamine with the concentration of 0.3mol/L at the flow rate of 0.3mL/h through a constant flow pump to react; the reaction is continuously carried out for 1000 hours, the conversion rate is more than 90%, and the ee values of the generated chiral amine are all more than 99%.
Example 2
A method for preparing chiral amine by emulsion microreactor continuous reaction, which comprises the following steps:
step 1: 1g of SiO with a particle size of 60nm 2 Dispersing nano particles into 20mL of toluene by ultrasonic, adding 4.5mmol of dichlorodimethylsilane and 2.25mmol of n-hexylamine, stirring and refluxing for 6h at 60 ℃ under the protection of nitrogen, cooling, centrifugally separating the mixed system, washing the obtained solid with toluene for 3-5 times, and dryingObtaining the interfacial active nano SiO 2 ；
Step 2: 0.72mL of aminotransferase ATA-113 solution and 40mgNADP+ are added into 3.2mL of 100mM PBS buffer solution, and the mixture is uniformly mixed by magnetic force; then 0.16g of interfacial active SiO 2 Dispersing in 8mL of n-heptane by ultrasonic wave and adding into the mixed solution of the buffer solution and the enzyme; finally, the mixture is stirred at a high speed of 5000rpm to form Pickering emulsion of the immobilized enzyme catalyst (the morphology is shown in figure 4).
Step 3: the prepared Pickering emulsion of the immobilized enzyme catalyst was transferred to a 100mL round bottom flask, n-heptane and 1.2g of hyperbranched polyethoxy silane were added, and the mixture was placed on a rotary evaporator and crosslinked at 30℃to prepare microcapsules.
Step 4: transferring the water-in-oil Pickering emulsion obtained in the step 2 into a fixed bed reactor with the inner diameter of 2.0cm, and setting the temperature of the reactor to be 30 ℃; introducing n-heptane solution with the concentration of 0.1mol/L acetophenone, 0.4mol/L ammonium formate and 80mg/L ammonium formate dehydrogenase into the reactor at the flow rate of 4mL/h through a constant flow pump, and carrying out reduction reaction, wherein the reaction is continuously carried out for 300 hours, the conversion rate is maintained to be more than 90%, and the ee value of the generated chiral amine is more than 99%.
Example 3
A method for preparing chiral amine by emulsion microreactor continuous reaction, which comprises the following steps:
step 1: 1g of SiO with a particle size of 80nm 2 Dispersing nano particles into 20mL of toluene by ultrasonic, adding 1.5mmol of dichlorodimethylsilane and 4.5mmol of n-hexylamine, stirring and refluxing for 5h at 60 ℃ under the protection of nitrogen, cooling, centrifugally separating the mixed system, washing the obtained solid with toluene for 3-5 times, and drying to obtain the interfacial active nano SiO 2 ；
Step 2: adding 0.06mL of aminotransferase ATA-119 solution and 10mgPLP into 3.2mL of 100mM PBS buffer solution, and uniformly mixing by magnetic stirring to obtain a water phase system; 0.158g of interfacial active SiO 2 Dispersing the mixture in 20mL of ethyl acetate by ultrasonic waves, and uniformly mixing to obtain an oil phase system; mixing an oil-water two-phase system, and stirring at a high speed of 10000rpm to form a water-in-oil Pickering emulsion for encapsulating transaminase and cofactor;
step 3: the prepared Pickering emulsion of the immobilized enzyme catalyst is transferred to a 100mL round bottom flask, n-heptane and octyl trimethoxy silane are added, and the mixture is placed on a rotary evaporator and crosslinked at 45 ℃ to prepare microcapsules.
Step 4: transferring the water-in-oil Pickering emulsion obtained in the step 2 into a fixed bed reactor with the inner diameter of 1.8cm, and setting the temperature of the reactor to be 50 ℃; and (3) introducing ethyl acetate solution with the concentration of 0.1mol/L of p-methoxyacetophenone and 0.4mol/L of 2-octylamine into the reactor at the flow rate of 0.5mL/h through a constant flow pump, performing reduction reaction, continuously performing the reaction for 400 hours, maintaining the conversion rate to be more than 80%, and ensuring the ee value of the generated chiral amine to be more than 99%.
Example 4:
a method for preparing chiral amine by emulsion microreactor continuous reaction, which comprises the following steps:
step 1: 1.2g of SiO with a particle size of 60nm 2 Dispersing nano particles into 12mL of toluene by ultrasonic, adding 1.5mmol of methyltrimethoxysilane and 4.5mmol of triethylamine, stirring and refluxing for 3 hours under the protection of nitrogen at 120 ℃, cooling, centrifugally separating the mixed system, washing the obtained solid with toluene for 3-5 times, and drying to obtain the interfacial active nano SiO 2 ；
Step 2: 1.6mL of aminotransferase ATA-142 solution and 500mgPLP are added into 3.2mL of 100mM PBS buffer solution, and the mixture is stirred and mixed evenly by magnetic force to obtain a water phase system; 0.316g of interfacial active SiO 2 Dispersing the mixture in 12mL of methyl tertiary butyl ether by ultrasonic waves, and uniformly mixing to obtain an oil phase system; mixing an oil-water two-phase system, and stirring at a high speed of 5000rpm to form a water-in-oil Pickering emulsion for encapsulating transaminase and cofactor;
step 3: the prepared Pickering emulsion of the immobilized enzyme catalyst was transferred to a 100mL round bottom flask, n-heptane and 0.9g of dichlorodimethylsilane were added, and the mixture was placed on a rotary evaporator and crosslinked at 40℃to prepare microcapsules.
Step 4: transferring the water-in-oil Pickering emulsion obtained in the step 2 into a fixed bed reactor with the inner diameter of 2.8cm, and setting the temperature of the reactor to be 40 ℃; methyl tertiary butyl ether solution with concentration of 0.1mol/L methyl benzoylformate and 0.2mol/L isopropylamine is introduced into the reactor through a constant flow pump at a flow rate of 0.5mL/h to react, the reaction is continuously carried out for 600 hours, the conversion rate is maintained above 90%, and the ee value of the generated chiral amine is greater than 99%.
Example 5
The chiral amine preparation method of this example is basically the same as that of example 1, except that: in step 3 toluene was replaced with equal amounts of hexane and in step 4 alpha-phenylethylamine was replaced with equal concentrations of isopropylamine. The reaction is continuously carried out for 400 hours, the conversion rate is maintained above 90%, and the ee value of the generated chiral amine is more than 99%.
Example 6
The chiral amine preparation method of this example is basically the same as that of example 4, except that: methyl tertiary butyl ether was replaced with an equivalent amount of octane in step 4. The reaction is continuously carried out for 400 hours, the conversion rate is maintained above 90%, and the ee value of the generated chiral amine is more than 99%.
What is not described in detail in the present specification belongs to the prior art known to those skilled in the art. While the foregoing describes illustrative embodiments of the present invention to facilitate an understanding of the present invention by those skilled in the art, it should be understood that the present invention is not limited to the scope of the embodiments, but is to be construed as protected by the accompanying claims insofar as various changes are within the spirit and scope of the present invention as defined and defined by the appended claims.

Claims (10)

1. A method for preparing chiral amine by emulsion microreactor continuous reaction is characterized in that: the method comprises the following steps:
step 1, siO with interfacial activity 2 Is prepared from the following steps: will commercialize SiO 2 The nano particles are dispersed in an organic solvent by ultrasonic, and then hydrophobic silane and organic amine are added; stirring and refluxing for 3-6 h under the protection of nitrogen at 60-120 ℃ to obtain a mixed system, cooling, centrifugally separating the mixed system, washing the analyzed solid with toluene for 3-5 times, and drying to obtain SiO with interfacial activity 2 A nanoparticle;
step 2, preparing Pickering emulsion for immobilizing enzyme and cofactor: adding transaminase and cofactor into phosphate buffer solution with pH value of 7-9, and uniformly mixing to obtain a water phase system; siO with interfacial activity 2 Dispersing the nano particles in an organic solvent by ultrasonic waves to obtain an oil phase system; mixing the oil phase system and the water phase system, and stirring at high speed to form Pickering emulsion of immobilized enzyme and cofactor with uniform particle size;
step 3, adding an organic solvent and a cross-linking agent into the Pickering emulsion obtained in the step 2, and placing the mixture on a rotary evaporator for cross-linking to prepare microcapsules;
and 4, placing the microcapsule obtained in the step 3 in a fixed bed reactor, and then introducing an organic solution containing cofactor regenerant for reaction to obtain chiral amine.
2. The method for preparing chiral amine by continuous reaction in an emulsion microreactor according to claim 1, wherein the method comprises the following steps: the organic solvent is any one of toluene, heptane, hexane, ethyl acetate, methyl tertiary butyl ether or octane.
3. The method for preparing chiral amine by continuous reaction in an emulsion microreactor according to claim 1, wherein the method comprises the following steps: the hydrophobic silane is any one of methyltrimethoxysilane, dichlorodimethylsilane and octyltrimethoxysilane.
4. The method for preparing chiral amine by continuous reaction in an emulsion microreactor according to claim 1, wherein the method comprises the following steps: the aminotransferase includes one or more of aminotransferase ATA-101-ATA-165 or mutants of aminotransferase ATA-101-ATA-165.
5. The method for preparing chiral amine by continuous reaction in an emulsion microreactor according to claim 1, wherein the method comprises the following steps: the cofactor is any one of pyridoxal 5' -phosphate, nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate.
6. The method for preparing chiral amine by continuous reaction in an emulsion microreactor according to claim 1, wherein the method comprises the following steps: the cross-linking agent is any one of methyltrimethoxysilane, octyltrimethoxysilane, tetraethyl silicate, tetramethoxysilane, trimethoxy silane, dichloro-dimethyl-silane, dimethyl-dimethoxy-silane and hyperbranched polyethoxy-silane.
7. The method for preparing chiral amine by continuous reaction in an emulsion microreactor according to claim 1, wherein the method comprises the following steps: the phosphate buffer solution and SiO with interfacial activity 2 The mass ratio of the nano particles is 1:0.02-1:0.10; the volume ratio of the phosphate buffer solution to the organic solvent is 1:0.5-1:10.
8. The method for preparing chiral amine by continuous reaction in an emulsion microreactor according to claim 1, wherein the method comprises the following steps: the diameter of the Pickering emulsion microdroplet in the step 2 is 5-300 mu m, and the phosphate buffer solution is phosphate buffer solution with the enzyme content of 10-500 mu L/mL.
9. The method for preparing chiral amine by continuous reaction in an emulsion microreactor according to claim 1, wherein the method comprises the following steps: the rotating speed of high-speed stirring in the step 2 is 5000-10000 rpm; in the step 3, the temperature for preparing the microcapsule through crosslinking by a rotary evaporator is 30-45 ℃; the temperature of the reactor in the step 4 is 25-50 ℃; the flow rate of the reactor is 0.2-10 mL/h.
10. The method for preparing chiral amine by continuous reaction in an emulsion microreactor according to claim 1, wherein the method comprises the following steps: the cofactor regenerated substance in the step 4 is any one of ammonium formate-ammonium formate dehydrogenase, isopropylamine, alpha-phenethylamine or 2-octylamine.
CN202310048184.8A 2023-01-31 2023-01-31 Method for preparing chiral amine by emulsion microreactor continuous reaction Pending CN116121312A (en)

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