A method for identifying the state of key saccharifying enzymes and liquefying enzymes in a large koji in a cell-free system

Abstract

The application provides a key saccharifying enzyme and liquefying enzyme state image recognition method in a cell-free system, which comprises the following steps: constructing a cell-free expression template according to the determined saccharifying enzyme and liquefying enzyme sequence in a Daqu or fermented grains sample, obtaining a target enzyme expression product through cell-free protein expression; making the expression product enter a saccharifying enzyme detection branch and a liquefying enzyme detection branch respectively, performing DNS coloration after soluble starch reaction in the saccharifying enzyme detection branch, and performing iodine coloration after soluble starch reaction in the liquefying enzyme detection branch; transferring the coloration liquid into a transparent well plate and collecting a well plate color image, and obtaining a saccharifying enzyme image equivalent activity and a liquefying enzyme image equivalent activity through well site recognition, interested region extraction, abnormal pixel elimination, color correction and image feature calculation, so that the application can realize rapid screening and state determination of the cell-free expression product of the key enzyme of Daqu.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and rapid enzyme activity detection technology, and in particular to an image recognition method for the state of key saccharifying enzymes and liquefying enzymes in a cell-free system. Background Technology

[0002] Daqu (a type of starter culture) is a crucial carrier providing saccharification, liquefaction, and fermentation motive power in the solid-state fermentation of Baijiu (Chinese liquor). Among these, saccharifying enzymes and liquefying enzymes directly influence starch degradation, reducing sugar production, and subsequent fermentation efficiency. Current methods for detecting Daqu enzyme activity primarily employ titration, spectrophotometry, or ELISA, mainly reflecting the overall saccharifying and liquefying power of the Daqu or fermentation mash extract. These methods are insufficient for quickly determining whether a specific candidate saccharifying or liquefying enzyme truly possesses detectable activity after expression. For key Daqu enzymes screened through metagenomics or proteomics, traditional cell expression, purification, and individual enzyme activity detection methods are time-consuming and unsuitable for batch comparisons of the reaction states of different target enzymes under varying temperature and pH conditions.

[0003] Cell-free protein expression systems can directly obtain target enzyme products from expression templates, making them suitable for rapid validation of key enzymes in Daqu (a type of Chinese liquor). However, cell-free expression products are complex in composition, and even if the target protein is expressed, it may not exhibit effective enzyme activity due to incomplete folding, precipitation, or background interference. Existing detection methods typically only provide a single absorbance value, making it difficult to simultaneously identify bubbles, precipitation, uneven edge development, and abnormalities in blank wells, which can easily lead to misjudgments.

[0004] Therefore, it is necessary to provide a new image recognition method for the state of key saccharifying enzymes and liquefying enzymes in Daqu (a type of Chinese liquor) in a cell-free system to solve the above-mentioned technical problems. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for image recognition of the state of key saccharifying enzymes and liquefying enzymes in a cell-free system of Daqu (a type of Chinese koji). This method solves the problems in the prior art, which makes it difficult to quickly, in batches, and visually identify the state of key saccharifying enzymes and liquefying enzymes expressed in a cell-free system of Daqu, and also addresses the inability to effectively identify abnormal states of reaction wells when relying solely on absorbance detection.

[0006] The method for image recognition of the state of key saccharifying enzymes and liquefying enzymes in a cell-free system provided by this invention has the following steps: S1. Based on the identified saccharifying enzyme and liquefying enzyme sequences in the Daqu sample or fermented mash sample, construct a cell-free expression template, wherein the cell-free expression template includes a promoter, a ribosome binding sequence, a target enzyme coding sequence, and a termination sequence; S2. The cell-free expression template is added to the cell-free protein expression system for expression reaction. After the reaction, cell-free expression product is obtained, and template-free control wells, substrate blank wells and positive enzyme control wells are set up. S3. The cell-free expression product is divided into a glycan detection branch and a liquefying enzyme detection branch; In the saccharifying enzyme detection pathway, the cell-free expression product is reacted with a soluble starch substrate, and then 3,5-dinitrosalicylic acid reagent is added for color development to obtain the saccharifying enzyme colorimetric solution. In the liquefaction enzyme detection pathway, the cell-free expression product is reacted with a soluble starch substrate, and then iodine colorimetric solution is added for color development to obtain the liquefaction enzyme colorimetric solution. S4. Add the saccharifying enzyme colorimetric solution and the liquefying enzyme colorimetric solution to the transparent well plate respectively, and collect color images of the well plate under a fixed light source, a fixed distance and a fixed angle. S5. Perform hole position recognition on the color image of the perforated plate, determine the center position of each reaction hole, and extract the central region of the reaction hole as the region of interest. S6. Perform color channel statistics on the region of interest, and perform color correction on each reaction well in combination with template-free control wells, substrate blank wells and positive enzyme control wells; S7. Based on the image features after color correction, calculate the image equivalent activity of saccharifying enzyme and the image equivalent activity of liquefying enzyme respectively. S8. Based on the equivalent activity of the saccharifying enzyme image and the equivalent activity of the liquefying enzyme image, output the state identification result of the target enzyme to be tested. The state identification result is: no detectable activity state, weak activity state, effective activity state, high activity state, or abnormal state.

[0007] Preferably, in step S1, the target enzyme includes a liquefying enzyme and a saccharifying enzyme; The liquefying enzyme is α-amylase P0C1B3 derived from Aspergillus oryzae. The saccharifying enzyme is Aspergillus niger-derived saccharifying enzyme P69327.

[0008] Preferably, in step S2, the cell-free protein expression system is a Rosetta2 Escherichia coli extract system; The single reaction volume of the expression reaction was 55 μL, the reaction temperature was 29 °C, and the reaction time was 8 h; The expression reaction system contains Rosetta2 extract, magnesium glutamate, potassium glutamate, amino acid solution, energy regeneration system, DTT, PEG-8000, T7 RNA polymerase, DNA template and maltose.

[0009] Preferably, in step S3, the saccharifying enzyme detection branch specifically includes: Mix the cell-free expression product, 20 g / L soluble starch solution, and pH 4.6 acetate-sodium acetate buffer, and react at 35°C for 30 min. After the reaction is complete, take an equal volume of the saccharification reaction solution and mix it with 3,5-dinitrosalicylic acid reagent, and heat it at 95°C for 30 min. After the color development is complete, the solution is cooled to room temperature and transferred to a transparent plate to obtain the saccharifying enzyme color development solution for image acquisition.

[0010] Preferably, in step S3, the liquefaction enzyme detection branch specifically includes: Mix the cell-free expression product, 4 g / L soluble starch solution, and pH 6.0 phosphate buffer, and react at 60°C for 10 min. After the reaction is complete, add 0.1 mol / L hydrochloric acid solution to terminate the reaction, and then add dilute iodine solution to develop color. After the color development is complete, the solution is transferred to a transparent plate to obtain the liquefied enzyme color development solution for image acquisition.

[0011] Preferably, in steps S4 and S5, the color image of the perforated plate is acquired in a light-shielding imaging box; The light-shielding imaging box is equipped with a white LED light source, a perforated plate positioning slot, and an image acquisition device. The lens of the image acquisition device is perpendicular to the plane of the transparent perforated plate; The region of interest is the circular region at the center of the reaction orifice, and the radius of the region of interest is 40%-70% of the radius of the reaction orifice. Before color channel statistics, remove bright reflective pixels, bubble edge pixels, sediment grain pixels, and hole wall shadow pixels from the region of interest.

[0012] Preferably, in step S7, the calculation of the image-equivalent activity of the saccharifying enzyme includes: Collect the green channel value of the corresponding reaction well of the saccharifying enzyme colorimetric solution; Using the green channel value of the template-free control well as the baseline value, the ratio of the green channel value of the reaction well to be tested to the baseline value is calculated, and the negative value is taken after taking the common logarithm of the obtained ratio to obtain the glucoamylase image feature value. A standard curve is established based on the glucose standard wells to determine the relationship between the glucoamylase image feature values ​​and glucose concentration, and the equivalent activity of the glucoamylase image is calculated from this standard curve.

[0013] Preferably, in step S7, the calculation of the equivalent activity of the liquefied enzyme image includes: Collect the brightness value of the corresponding reaction well of the liquefied enzyme colorimetric solution; Using the brightness value of the blank well in dilute iodine solution as the reference value, the brightness value of the reaction well to be tested is calculated as a ratio to the reference value, and the negative value is taken after taking the common logarithm of the obtained ratio to obtain the image feature value of liquefied enzyme. A standard curve was established based on the starch standard wells to compare the liquefaction enzyme image feature values ​​with the residual starch concentration, and the equivalent activity of the liquefaction enzyme image was calculated from the standard curve. During state recognition, the mean of the equivalent activity of the image without template control well plus three times the standard deviation is used as the detectable activity threshold. When the image equivalent activity of the reaction well to be tested is lower than the detectable activity threshold, it is determined that no detectable activity is observed. When the image equivalent activity of the reaction well to be tested is not lower than the detectable activity threshold and is lower than 30% of the image equivalent activity of the positive enzyme control well, it is determined to be a weak activity state. When the image equivalent activity of the reaction well to be tested reaches 30%-120% of the image equivalent activity of the positive enzyme control well, it is determined to be in an effective activity state. When the equivalent activity of the image of the reaction well to be tested is higher than 120% of the equivalent activity of the image of the positive enzyme control well, it is judged to be in a high activity state; An abnormal state is defined as the presence of bubbles, sediment, uneven color development at the edges of the reaction wells, or a coefficient of variation greater than 15% for repeatable wells.

[0014] The beneficial effects of this invention are: 1. This invention combines cell-free protein expression with colorimetric detection of key saccharifying and liquefying enzymes in Daqu (a type of Chinese liquor), enabling rapid determination of whether candidate enzymes have detectable activity without long-term cell culture, induction expression, and purification.

[0015] 2. Separately establish a DNS colorimetric pathway for saccharifying enzymes and an iodine colorimetric pathway for liquefying enzymes. This allows for the identification of the different reaction characteristics of saccharifying enzymes in producing reducing sugars and liquefying enzymes in degrading starch, avoiding the simple confusion of the activity states of the two types of enzymes.

[0016] 3. By acquiring images of the transparent plate, identifying the well location, and extracting the region of interest, a unified color statistical analysis is performed on each reaction well, reducing subjective errors caused by manual visual interpretation.

[0017] 4. Before color statistics, the present invention removes reflective pixels, bubble pixels, deposited pixels and hole wall edge areas, which can identify and eliminate abnormal conditions inside the hole and avoid misjudgment caused by simply relying on light absorption value.

[0018] 5. Color correction is performed using standard holes and control holes, and image features are converted into image equivalent activity to make the image recognition results have better intra-batch comparability.

[0019] 6. This invention can output no detectable activity, weak activity, effective activity, high activity and abnormal state, which is suitable for both the initial screening of candidate enzymes and the comparison of enzyme activity under different temperature and pH conditions. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the overall process of the method of the present invention; Figure 2 This is a flowchart illustrating the saccharifying enzyme detection branch and the liquefying enzyme detection branch in this invention. Figure 3 This is a schematic diagram of the hole arrangement of the transparent perforated plate in this invention; Figure 4 This is a schematic diagram of the extraction of the region of interest and the removal of abnormal pixels in the reaction hole in this invention; Figure 5 This is a schematic diagram of the process for calculating the equivalent activity of glycan images in this invention; Figure 6 This is a schematic diagram of the process for calculating the equivalent activity of liquefied enzyme images in this invention; Figure 7 This is a schematic diagram of the temperature-pH state diagram output in this invention. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] Please refer to the following: Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 as well as Figure 7 ,in Figure 1 This is a schematic diagram of the overall process of the method of the present invention; Figure 2 This is a flowchart illustrating the saccharifying enzyme detection branch and the liquefying enzyme detection branch in this invention. Figure 3 This is a schematic diagram of the hole arrangement of the transparent perforated plate in this invention; Figure 4 This is a schematic diagram of the extraction of the region of interest and the removal of abnormal pixels in the reaction hole in this invention; Figure 5 This is a schematic diagram of the process for calculating the equivalent activity of glycan images in this invention; Figure 6 This is a schematic diagram of the process for calculating the equivalent activity of liquefied enzyme images in this invention; Figure 7 This is a schematic diagram of the temperature-pH state diagram output in this invention.

[0023] In the specific implementation process, such as Figure 1As shown, the method of the present invention mainly includes target enzyme identification and cell-free expression template construction, cell-free protein expression, establishment of dual detection pathways for saccharifying enzyme and liquefying enzyme, transparent plate color development and image acquisition, well location identification and region of interest extraction, color correction and image feature extraction, image equivalent activity calculation, and output of enzyme activity status identification results.

[0024] In this embodiment, the target enzymes to be tested include saccharifying enzymes and liquefying enzymes. The saccharifying enzyme used is saccharifying enzyme P69327 from Aspergillus niger, and the liquefying enzyme used is α-amylase P0C1B3 from Aspergillus oryzae. Cell-free expression templates were constructed according to the coding sequences of the target enzymes. Each cell-free expression template sequentially includes a T7 promoter, a ribosome-binding sequence, the target enzyme coding sequence, and a termination sequence. To facilitate subsequent confirmation of expression, a His tag can be attached to the end of the target enzyme coding sequence; however, this tag is not a necessary condition for identifying enzyme activity in this method.

[0025] The constructed cell-free expression template was added to the Rosetta2 *E. coli* extract system for expression. Each expression reaction system consisted of 55 μL of Rosetta2 extract, magnesium glutamate, potassium glutamate, amino acid solution, energy regeneration system, DTT, PEG-8000, T7 RNA polymerase, DNA template, and maltose. The expression reaction was carried out at 29°C for 8 hours. After the reaction, the expression product was centrifuged at 4°C, and the supernatant was used as the cell-free expression product detection solution.

[0026] To ensure reliable image recognition results, each batch of reactions includes template-free control wells, substrate blank wells, and positive enzyme control wells. Template-free control wells are used to subtract the background of the cell-free system; substrate blank wells are used to determine if spontaneous color development occurs with the substrate and chromogenic reagent; and positive enzyme control wells are used to confirm whether the colorimetric reaction and image acquisition in this batch are normal.

[0027] like Figure 2 As shown, after obtaining the cell-free expression products, they were fed into the glucoamylase detection pathway and the liquefaction enzyme detection pathway, respectively. Both detection pathways used soluble starch as the substrate, but the color development methods differed. The glucoamylase detection pathway used DNS color development, while the liquefaction enzyme detection pathway used iodine color development. After color development in both pathways, the samples were transferred to clear plates for image acquisition and subsequent image analysis.

[0028] For the glycated enzyme detection pathway, 10 μL of cell-free expression product was added to 70 μL of 20 g / L soluble starch solution, followed by 20 μL of pH 4.6 acetate-sodium acetate buffer. The mixture was then incubated at 35°C for 30 min. After the reaction, 40 μL of the glycated reaction solution was mixed with 40 μL of 3,5-dinitrosalicylic acid reagent and heated at 95°C for 30 min. After color development, the reaction solution was cooled to room temperature and transferred to a 96-well transparent plate as the glycated enzyme color development solution.

[0029] For the liquefaction enzyme detection pathway, take 10 μL of cell-free expression product, add 80 μL of 4 g / L soluble starch solution, then add 10 μL of pH 6.0 phosphate buffer, mix well, and incubate at 60℃ for 10 min. After the reaction, take 30 μL of the liquefaction reaction solution, add 15 μL of 0.1 mol / L hydrochloric acid solution to terminate the reaction, then add 150 μL of dilute iodine solution for color development, mix well, and transfer to a transparent flat-bottomed 96-well plate as the liquefaction enzyme color development solution.

[0030] like Figure 3 As shown, the same transparent plate contains glucose standard wells, starch standard wells, template-free control wells, substrate blank wells, positive enzyme control wells, and sample wells. The glucose standard wells are used to establish the standard curve for the saccharifying enzyme detection pathway, and the starch standard wells are used to establish the standard curve for the liquefying enzyme detection pathway. The template-free control wells, substrate blank wells, and positive enzyme control wells are used for color correction, background subtraction, and state threshold setting. The sample wells are used to hold the reaction solution of the cell-free expression product after color development.

[0031] The concentrations of glucose standard wells were set sequentially to 0 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, and 0.07 mg / mL. Each glucose standard well was mixed with an equal volume of 3,5-dinitrosalicylic acid reagent and heated under the same conditions as the test samples for color development.

[0032] The concentrations of starch standard wells were set sequentially to 0 g / L, 0.075 g / L, 0.15 g / L, 0.30 g / L, 0.45 g / L, 0.60 g / L, and 0.90 g / L. Equal volumes of hydrochloric acid solution and dilute iodine solution were added to each starch standard well for color development.

[0033] After the well plates are developed, acquire color images of the transparent well plates under fixed light source, distance, and angle conditions. During image capture, maintain consistent well plate position, shooting distance, light source brightness, exposure time, and white balance to ensure comparability of images from the same batch of well plates. Acquire at least one clear color image for each batch of well plates, ensuring that the image includes the standard wells, control wells, and the sample wells to be tested.

[0034] like Figure 4 As shown, after acquiring color images of the orifice plate, well locations and regions of interest (ROIs) are identified and extracted. First, the outer boundary of the 96-well plate is identified, and the center position of each well is determined according to the row and column arrangement rules. Then, a central circular region is extracted as the ROI, centered on the center of each well. The radius of the ROI is set to 55% of the well radius. By extracting the central region, the influence of well walls, liquid surface edges, and plate reflections on color statistics can be avoided.

[0035] Within each region of interest, pixels are filtered. Pixels that are clearly highlighted (reflective), have bubble edges, contain deposits, or have shadows on the hole walls are excluded from color statistics. If the proportion of usable pixels in a given reaction hole is less than 80%, that hole is marked as an image anomaly and is not directly used for activity assessment.

[0036] After pixel selection, the red, green, and blue channel values ​​and brightness values ​​were statistically analyzed for each reaction well. For the saccharifying enzyme chromogenic solution, changes in the green channel were used as the primary image feature. Figure 5 As shown, after the image of the saccharifying enzyme chromogenic solution is extracted through the green channel, it is corrected with template-free control wells or blank wells to obtain the characteristic values ​​of the saccharifying enzyme image; then, a glucose standard curve is established based on the glucose standard wells, and the equivalent activity of the saccharifying enzyme image of the test well is calculated from the standard curve.

[0037] Specifically, using the green channel value of the template-free control well as a baseline, the ratio of the green channel value of the test well to this baseline value is calculated, and the negative value of the resulting ratio after taking the common logarithm is obtained to obtain the glucoamylase image feature value. Since the glucoamylase reaction produces reducing sugars, the color deepens after the reducing sugars react with DNS reagent, and the green channel value changes as the color deepens. Therefore, this image feature value can reflect the intensity of the glucoamylase reaction.

[0038] For liquefied enzyme chromogenic solutions, changes in brightness are used as the primary image feature. For example... Figure 6 As shown, after the brightness value of the liquefying enzyme colorimetric solution image is extracted, it is corrected with dilute iodine solution blank wells to obtain the liquefying enzyme image feature value; then, a starch standard curve is established based on the starch standard wells, and the equivalent activity of the liquefying enzyme image of the test well is calculated from the standard curve.

[0039] Specifically, using the brightness value of the blank well in dilute iodine solution as a reference value, the brightness value of the well to be tested is compared with this reference value. The resulting ratio is then divided by taking the common logarithm and taking the negative value to obtain the liquefying enzyme image feature value. Since residual starch turns blue-purple after reacting with iodine solution, the stronger the liquefying enzyme activity, the more complete the starch degradation, and the lighter the blue-purple color after development. Therefore, this image feature value can reflect the degree of starch degradation by the liquefying enzyme.

[0040] During state identification, the mean and standard deviation of the image equivalent activity of the template-free control wells are first calculated, and the mean of the image equivalent activity of the template-free control wells plus three times the standard deviation is used as the detectable activity threshold. When the image equivalent activity of the test reaction well is lower than this threshold, it is determined that no detectable activity is observed; when the image equivalent activity of the test reaction well is not lower than this threshold but is lower than 30% of the image equivalent activity of the positive enzyme control well, it is determined that the activity is weak; when the image equivalent activity of the test reaction well reaches 30% to 120% of the image equivalent activity of the positive enzyme control well, it is determined that the activity is effective; when the image equivalent activity of the test reaction well is higher than 120% of the image equivalent activity of the positive enzyme control well, it is determined that the activity is high.

[0041] When obvious bubbles, precipitates, uneven color development at the edges appear in the test reaction wells, or the coefficient of variation of the duplicate wells of the same sample is greater than 15%, the activity level of the sample is not directly judged. Instead, it is judged as an abnormal state, and a prompt is made to redevelop the color or reacquire the image.

[0042] like Figure 7 As shown, when comparing the adaptability of the same target enzyme to temperature and pH, the same cell-free expression product can be subjected to colorimetric reactions under different temperatures and pH conditions, and the state of the well plate images under each condition can be identified. Temperature conditions can be set to 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, and 60℃, and pH conditions can be set to 4.0, 4.6, 5.0, 5.5, 6.0, and 6.5. A state label is output for each temperature-pH combination, where A0 indicates no activity, A1 indicates weak activity, A2 indicates effective activity, A3 indicates high activity, and AX indicates an abnormal state. Filling these state labels into the temperature-pH matrix yields the temperature-pH state map of the target enzyme.

[0043] Through the above steps, this invention enables the identification of key saccharifying enzymes and liquefying enzymes in Daqu (a type of koji) through well plate image analysis after cell-free expression. This method utilizes the enzymatic basis of DNS colorimetry and starch-iodine colorimetry, and reduces errors caused by manual visual judgment through well location identification, region of interest extraction, abnormal pixel removal, and image-equivalent activity calculation. Compared with detection methods that rely solely on a single absorbance value, this method can simultaneously identify color intensity and abnormal states within the wells, making it suitable for rapid screening of cell-free expression products of key saccharifying enzymes and liquefying enzymes in Daqu.

[0044] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for image recognition of the states of key saccharifying enzymes and liquefying enzymes in a cell-free system of Daqu (a type of Chinese koji), characterized in that, Includes the following steps: S1. Based on the identified saccharifying enzyme and liquefying enzyme sequences in the Daqu sample or fermented mash sample, construct a cell-free expression template, wherein the cell-free expression template includes a promoter, a ribosome binding sequence, a target enzyme coding sequence, and a termination sequence; S2. The cell-free expression template is added to the cell-free protein expression system for expression reaction. After the reaction, cell-free expression product is obtained, and template-free control wells, substrate blank wells and positive enzyme control wells are set up. S3. The cell-free expression product is divided into a glycan detection branch and a liquefying enzyme detection branch; In the saccharifying enzyme detection pathway, the cell-free expression product is reacted with a soluble starch substrate, and then 3,5-dinitrosalicylic acid reagent is added for color development to obtain the saccharifying enzyme colorimetric solution. In the liquefaction enzyme detection pathway, the cell-free expression product is reacted with a soluble starch substrate, and then iodine colorimetric solution is added for color development to obtain the liquefaction enzyme colorimetric solution. S4. Add the saccharifying enzyme colorimetric solution and the liquefying enzyme colorimetric solution to the transparent well plate respectively, and collect color images of the well plate under a fixed light source, a fixed distance and a fixed angle. S5. Perform hole position recognition on the color image of the perforated plate, determine the center position of each reaction hole, and extract the central region of the reaction hole as the region of interest. S6. Perform color channel statistics on the region of interest, and perform color correction on each reaction well in combination with template-free control wells, substrate blank wells and positive enzyme control wells; S7. Based on the image features after color correction, calculate the image equivalent activity of saccharifying enzyme and the image equivalent activity of liquefying enzyme respectively. S8. Based on the equivalent activity of the saccharifying enzyme image and the equivalent activity of the liquefying enzyme image, output the state identification result of the target enzyme to be tested. The state identification result is: no detectable activity state, weak activity state, effective activity state, high activity state, or abnormal state.

2. The method for image recognition of the state of key saccharifying enzymes and liquefying enzymes in a cell-free system according to claim 1, characterized in that, In step S1, the target enzyme includes liquefying enzyme and saccharifying enzyme; The liquefying enzyme is α-amylase P0C1B3 derived from Aspergillus oryzae. The saccharifying enzyme is Aspergillus niger-derived saccharifying enzyme P69327.

3. The method for image recognition of the state of key saccharifying enzymes and liquefying enzymes in a cell-free system according to claim 1, characterized in that, In step S2, the cell-free protein expression system is the Rosetta2 Escherichia coli extract system; The single reaction volume of the expression reaction was 55 μL, the reaction temperature was 29 °C, and the reaction time was 8 h; The expression reaction system contains Rosetta2 extract, magnesium glutamate, potassium glutamate, amino acid solution, energy regeneration system, DTT, PEG-8000, T7 RNA polymerase, DNA template and maltose.

4. The method for image recognition of the state of key saccharifying enzymes and liquefying enzymes in a cell-free system according to claim 1, characterized in that, In step S3, the saccharifying enzyme detection branch specifically includes: Mix the cell-free expression product, 20 g / L soluble starch solution, and pH 4.6 acetate-sodium acetate buffer, and react at 35°C for 30 min. After the reaction is complete, take an equal volume of the saccharification reaction solution and mix it with 3,5-dinitrosalicylic acid reagent, and heat it at 95°C for 30 min. After the color development is complete, the solution is cooled to room temperature and transferred to a transparent plate to obtain the saccharifying enzyme color development solution for image acquisition.

5. The method for image recognition of the state of key saccharifying enzymes and liquefying enzymes in a cell-free system according to claim 1, characterized in that, In step S3, the liquefaction enzyme detection branch specifically includes: Mix the cell-free expression product, 4 g / L soluble starch solution, and pH 6.0 phosphate buffer, and react at 60°C for 10 min. After the reaction is complete, add 0.1 mol / L hydrochloric acid solution to terminate the reaction, and then add dilute iodine solution to develop color. After the color development is complete, the solution is transferred to a transparent plate to obtain the liquefied enzyme color development solution for image acquisition.

6. The method for image recognition of the state of key saccharifying enzymes and liquefying enzymes in a cell-free system according to claim 1, characterized in that, In steps S4 and S5, the color image of the perforated plate is acquired in a light-shielding imaging box. The light-shielding imaging box is equipped with a white LED light source, a perforated plate positioning slot, and an image acquisition device. The lens of the image acquisition device is perpendicular to the plane of the transparent perforated plate; The region of interest is the circular region at the center of the reaction orifice, and the radius of the region of interest is 40%-70% of the radius of the reaction orifice. Before color channel statistics, remove bright reflective pixels, bubble edge pixels, sediment grain pixels, and hole wall shadow pixels from the region of interest.

7. The method for image recognition of the state of key saccharifying enzymes and liquefying enzymes in a cell-free system according to claim 1, characterized in that, In step S7, the calculation of the equivalent activity of the glucoamylase image includes: Collect the green channel value of the corresponding reaction well of the saccharifying enzyme colorimetric solution; Using the green channel value of the template-free control well as the baseline value, the ratio of the green channel value of the reaction well to be tested to the baseline value is calculated, and the negative value is taken after taking the common logarithm of the obtained ratio to obtain the glucoamylase image feature value. A standard curve is established based on the glucose standard wells to determine the relationship between the glucoamylase image feature values ​​and glucose concentration, and the equivalent activity of the glucoamylase image is calculated from this standard curve.

8. The method for image recognition of the state of key saccharifying enzymes and liquefying enzymes in a cell-free system according to claim 1, characterized in that, In step S7, the calculation of the equivalent activity of the liquefied enzyme image includes: Collect the brightness value of the corresponding reaction well of the liquefied enzyme colorimetric solution; Using the brightness value of the blank well in dilute iodine solution as the reference value, the brightness value of the reaction well to be tested is calculated as a ratio to the reference value, and the negative value is taken after taking the common logarithm of the obtained ratio to obtain the image feature value of liquefied enzyme. A standard curve was established based on the starch standard wells to compare the liquefaction enzyme image feature values ​​with the residual starch concentration, and the equivalent activity of the liquefaction enzyme image was calculated from the standard curve. During state recognition, the mean of the equivalent activity of the image without template control well plus three times the standard deviation is used as the detectable activity threshold. When the image equivalent activity of the reaction well to be tested is lower than the detectable activity threshold, it is determined that no detectable activity is observed. When the image equivalent activity of the reaction well to be tested is not lower than the detectable activity threshold and is lower than 30% of the image equivalent activity of the positive enzyme control well, it is determined to be a weak activity state. When the image equivalent activity of the reaction well to be tested reaches 30%-120% of the image equivalent activity of the positive enzyme control well, it is determined to be in an effective activity state. When the equivalent activity of the image of the reaction well to be tested is higher than 120% of the equivalent activity of the image of the positive enzyme control well, it is judged to be in a high activity state; An abnormal state is defined as the presence of bubbles, sediment, uneven color development at the edges of the reaction wells, or a coefficient of variation greater than 15% for repeatable wells.

Images