CN114636682A - A high-throughput assay method and application of PET hydrolase activity based on fluorescence detection - Google Patents

Abstract

The invention relates to the technical field of enzyme activity determination, in particular to a high-throughput determination method for activity of polyethylene terephthalate (PET) hydrolase based on fluorescence detection and application thereof. A high-throughput determination method of PET hydrolase activity based on fluorescence detection comprises (1) performing fusion expression of PET hydrolase and fluorescent protein, and determining fluorescence value in cell lysate before reaction to determine enzyme concentration; (2) preparing a PET-FDL film; (3) reacting the cell lysate with a PET-FDL membrane in a glycine buffer solution; after the reaction is finished, measuring the fluorescence value in the product to determine the concentration of the product; (4) and (3) calculating the ratio of the fluorescence value in the product to the fluorescence value in the cell lysate before reaction, namely the specific enzyme activity of the PET hydrolase. The PET hydrolase activity high-throughput determination method based on fluorescence detection and the application thereof provided by the invention have the advantages of high throughput, low cost and convenience in operation, provide a new method for screening high-efficiency PET plastic degrading enzymes, and have wide application prospects.

Description

A high-throughput assay method and application of PET hydrolase activity based on fluorescence detection

technical field

The invention relates to the technical field of enzyme activity determination, in particular to a high-throughput determination method and application of PET hydrolase activity based on fluorescence detection.

Background technique

Plastic products are widely used in the manufacturing industry due to their excellent properties such as good acid and alkali resistance, but at the same time, this makes it difficult to effectively degrade them. Only by thermal-mechanical means to reduce their mechanical properties and further incinerate and landfill can not achieve a real meaning. of recycling. The accumulation of plastic waste generated by this process can lead to environmental pollution and further threaten the ecosystem. According to Eurostat, the total global plastic production in 2019 reached almost 370 million tons, of which China’s production accounted for 31% of the world’s total. Although there are existing methods to dispose of plastic waste through chemical and physical technologies, the by-products produced by these methods also pollute the environment. Therefore, the use of microorganisms to degrade plastics has become a current research hotspot. Ethylene terephthalate (PET) is one of the widely used plastic materials in the market. As a polyester plastic, the key step of its degradation is the hydrolysis of ester bonds catalyzed by PET hydrolase. In view of the important research and application value of PET hydrolase, the artificial modification of this enzyme has become a hot spot in recent years. The current PET hydrolase transformation is mainly based on the structure of the enzyme to mutate the target protein at single or multi-point amino acids, purify all the mutated target proteins separately, and detect the production of the product by HPLC after the enzymatic reaction. This method not only works Due to the large amount and low throughput, the number of mutants that can be detected and screened is limited. When designing PET hydrolase mutations rationally, a large number of mutation selections are discarded because of the need to compromise the detection throughput.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a high-throughput screening method, which can perform high-throughput quantitative activity detection of PET hydrolase based on fluorescence detection, and can be widely used in PET hydrolase mutant library and PET plastic hydrolase in the environment screening identification.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows: a high-throughput assay method for PET hydrolase activity based on fluorescence detection, comprising:

(1) The PET hydrolase and fluorescent protein are fused and expressed, and the fluorescence value in the cell lysate is measured before the reaction to determine the concentration of the enzyme;

(2) preparing a PET-FDL film, the PET-FDL film is a PET film embedded with fluorescein dilaurate;

(3) react the cell lysate with the PET-FDL membrane in a glycine buffer; after the reaction, measure the fluorescence value in the product to determine the concentration of the product;

(4) Calculate the ratio of the fluorescence value in the product to the fluorescence value in the cell lysate before the reaction, which is the specific enzyme activity of the PET hydrolase.

As a preferred mode of the present invention, in the step (1), the maximum emission wavelength of fluorescence of the fluorescent protein should be different from the maximum emission wavelength of fluorescein by more than 100 nm.

Further, in the step (2), the preparation method of the PET-FDL film is as follows: FDL and PET plastic are dissolved in hexafluoroisopropanol according to a mass ratio of 0.25-1 μg/mg, and then 40 μL is added to 96 wells In the plate, a film is formed after volatilization at 30°C to 50°C.

Further, the excitation wavelength range of the fluorescent protein is 485nm-670nm, the maximum excitation wavelength is 603nm, the emission wavelength range is 610nm-805nm, and the maximum emission wavelength is 675nm.

Further, the excitation wavelength range of fluorescein is 420nm-530nm, the maximum excitation wavelength is 494nm, the emission wavelength range is 485nm-615nm, and the maximum emission wavelength is 525nm.

The invention also provides the application of a high-throughput assay method for PET hydrolase activity based on fluorescence detection, which can be applied to the screening of PET hydrolase mutants.

The high-throughput assay method for PET hydrolase activity based on fluorescence detection and its application provided by the invention have good activity screening effect on PET hydrolase. By using the method of the invention to degrade the PET film embedded with fluorescein, high-throughput screening of the mutant library can be performed to obtain mutants with high activity. The high-throughput screening method provided by the invention has high throughput, low cost and convenient operation, provides a new method for screening high-efficiency plastic degrading enzymes, and has broad application prospects.

Description of drawings

Fig. 1 is a graph showing the relationship between fusion protein concentration and red fluorescence value;

Figure 2 shows the release of fluorescence during the reaction of PET-FDL films with different proportions volatilized at 30°C;

Figure 3 shows the release of fluorescence during the reaction of PET-FDL films with different proportions volatilized at 40°C;

Figure 4 shows the release of fluorescence during the reaction of PET-FDL films of different proportions prepared by volatilization at 50°C;

Figure 5 shows the correlation between the fluorescence value and the product yield after the reaction of the self-made membrane with FDL/PET of 1 μg/mg volatilized at 50 °C;

Figure 6 shows the correlation between the fluorescence value and the product yield after the reaction of the self-made membrane with FDL/PET of 0.5μg/mg volatilized at 50°C;

Figure 7 shows the correlation between the fluorescence value and the product yield after the reaction of the self-made membrane with FDL/PET of 0.33 μg/mg volatilized at 50 °C;

Figure 8 shows the correlation between the fluorescence value and the product yield after the reaction of the self-made membrane with FDL/PET of 0.25μg/mg volatilized at 50°C;

Fig. 9 Evaluation of the stability parameter Z factor of high-throughput screening experiments under different conditions in the embodiment of the present invention.

Detailed ways

In order to facilitate understanding of the present invention, the present invention will be described in more detail below with reference to the accompanying drawings and specific embodiments. Preferred embodiments of the invention are shown in the accompanying drawings. However, the present invention may be embodied in many different forms and is not limited to the embodiments described in this specification. Rather, these embodiments are provided so that a thorough and complete understanding of the present disclosure will be provided.

The raw materials and reagents used in the examples of the present invention are all commercially available commodities unless otherwise specified. The experimental methods and instruments used are all routine laboratory techniques. In the present invention, green fluorescein and red fluorescent protein are respectively introduced to quantify the yield of PET products and the amount of enzymes. As the enzymatic hydrolysis reaction proceeds, the substrate is decomposed, and the green fluorescein in the substrate is gradually released. The ratio of the green fluorescence value to the red fluorescence value can represent the relative activity of the enzyme. Using enzyme activity as the screening condition, high-throughput screening of PET hydrolase mutants can be achieved.

Embodiment 1 This embodiment provides a high-throughput assay method for PET hydrolase activity based on fluorescence detection, which includes the following detailed steps:

1. Quantification of enzyme amount by red fluorescence value

In order to quantify the expression level of the target protein in the mutant strain, the present invention uses the red fluorescent protein mCarmine (Fabritius, A.; Ng, D.; Kist, AM; Erdogan, M.; Portugal, R.; Griesbeck, O. Imaging -Based Screening Platform Assists Protein Engineering. Cell Chemical Biology 2018, 25, 1554-1561.e8.) and PET-degrading enzyme were fused and expressed to obtain a fusion protein. First, in order to prove that the red fluorescence value can reflect the amount of the fusion protein involved in the enzymatic reaction to a certain extent, in this example, the concentration of the fusion protein was diluted to 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, and tested at different concentrations. The relationship between the red fluorescence value and the protein concentration. The results are shown in Figure 1, indicating that the correlation R ² between the two is greater than 0.99, indicating that the red fluorescence value can accurately reflect the concentration of the enzyme.

2. Quantification of the product by green fluorescein

(1) Film making conditions for fusion of green fluorescein and PET

The determination of enzyme activity involves the quantification of the enzyme and the quantification of the product. In order to quantify the amount of products generated after PET decomposition, green fluorescein dilaurate (Fluorescein dilaurate, FDL) was introduced into the substrate PET in this example.

Dilauryl FDL 5μg/ml, according to different mass ratios: 1μg/mg, 0.5μg/mg, 0.33μg/mg, 0.25μg/mg, were added to 5mg/ml, 10mg/ml, 15mg/ml, 20mg /ml of PET, dissolved in hexafluoroisopropanol together, and then added 40 μL to a 96-well plate, and formed membranes at three temperatures of 30°C, 40°C, and 50°C.

After the prepared film was reacted with PET degrading enzyme for 48 hours, the release amount of green fluorescence was measured by a microplate reader, and the production amount of the product during the enzyme reaction was detected by HPLC, as shown in Figure 2-4. Therefore, it can be concluded that the PET-FDL prepared according to the above different ratios reacts with PET hydrolase at 30°C, 40°C and 50°C, and the fluorescence value of the product increases with time. Among them, the optimum film-forming temperature is 50°C.

(2) Correlation between green fluorescence value and product yield

The reaction solution after the above enzymatic reaction was detected by HPLC, and the concentration of the relevant product was measured, as shown in Figure 5-8. It was calculated that R ² was greater than 0.96, and the green fluorescence released by the PET-FDL membrane with the four mass ratios was related to the product. The concentrations are all linear, indicating that the green fluorescence value can accurately reflect the amount of products generated after PET decomposition.

3. Determination of PET-degrading enzyme activity

(1) Detect the red fluorescence value in the cell lysate before the reaction, and quantify the enzyme (fusion protein) concentration in the cell lysate by the red fluorescence value of mCarmine.

(2) The fusion protein is hydrolyzed with the PET-FDL membrane.

The cell lysate was reacted with the PET-FDL membrane in 50 mM glycine buffer at 40 °C for 24 h to carry out the hydrolysis reaction.

FDL is released with the hydrolysis of PET and is immediately hydrolyzed to Fluorescein by PET hydrolase.

(3) Determination of the green fluorescence value in the product after the reaction

The release of fluorescein is proportional to the PET hydrolyzate, and the PET hydrolyzate can be quantitatively detected by green fluorescence.

(4) The activity of PET hydrolase can be determined by the ratio of the measured green fluorescence value to the red fluorescence value.

The above detection methods can be quickly implemented on a microplate reader. The excitation wavelength range of red fluorescent protein mCarmine is 485nm-670nm, the maximum excitation wavelength is 603nm, the emission wavelength range is 610nm-805nm, and the maximum emission wavelength is 675nm; The excitation wavelength range is 420nm-530nm, the maximum excitation wavelength is 494nm, the emission wavelength range is 485nm-615nm, and the maximum emission wavelength is 525nm.

Example 2 Evaluation of high-throughput screening of PET-degrading enzymes using the method of Example 1

Z factor (Z factor) is a widely used parameter in the evaluation and verification of high-throughput screening experiments. ). The calculation formula and application method of Z value are as follows:

When 0.5≤Z<1, the separation of the sample signal distribution and the control signal distribution in the detection system is good, which indicates that the analysis method is very good; when 0<Z<0.5, it indicates a moderate degree of distribution separation, indicating that the analysis can be carried out ; When Z<0, it means that the system is basically impossible for screening.

In this example, the Z value of the ratio of the green fluorescence value and the red fluorescence value obtained by the enzymatic reaction with different amounts of cell disrupting liquid at three times, as shown in FIG. 9 .

The amount of cell disrupting solution selected in the experiment was 2 μL, 10 μL and 15 μL, and the enzyme reaction time was 17 h, 24 h and 62 h. According to the size of Z value, it can be concluded that the optimal amount of cell disrupting solution was 10 μL, and the Z value was under different reaction times. The changes were the most stable and were all greater than 0.5, indicating that the high-throughput screening under this condition was validated as effective.

Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention, but not to limit it. Although the present invention has been described in detail with reference to the foregoing steps, those of ordinary skill in the art should understand that the technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features thereof can be equivalently replaced. , and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims (7)

1. a PET hydrolase activity high-throughput assay method based on fluorescence detection, is characterized in that, comprises: (1) The PET hydrolase was fused and expressed with fluorescent protein, and the fluorescence value in the cell lysate was measured before the reaction to determine the concentration of the enzyme; (2) preparing a PET-FDL film, wherein the PET-FDL film is a PET film embedded with fluorescein dilaurate; (3) The cell lysate was reacted with the PET-FDL membrane in glycine buffer; after the reaction, the fluorescence value in the product was measured to determine the product concentration; (4) Calculate the ratio of the fluorescence value in the product to the fluorescence value in the cell lysate before the reaction, which is the specific enzyme activity of the PET hydrolase. 2. The high-throughput assay method for PET hydrolase activity based on fluorescence detection according to claim 1, wherein in the step (1), the maximum emission wavelength of fluorescence of fluorescent protein should be different from the maximum emission wavelength of fluorescein greater than 100nm. 3. The high-throughput assay method for PET hydrolase activity based on fluorescence detection according to claim 1, wherein in the step (2), the preparation method of the PET-FDL film is: the FDL and the PET plastic are according to the following steps. The mass ratio is 0.25~1 μg/mg, dissolved in hexafluoroisopropanol, and 40 μL was added to a 96-well plate, and evaporated at 30°C to 50°C to form a film. 4. the PET hydrolase activity high-throughput assay method based on fluorescence detection according to claim 1, is characterized in that, the excitation wavelength range of fluorescent protein is 485 nm～670 nm, and the emission wavelength range is 610 nm～805 nm. 5. the PET hydrolase activity high-throughput assay method based on fluorescence detection according to claim 1, is characterized in that, the excitation wavelength range of fluorescein is 420 nm～530 nm, and the emission wavelength range is 485 nm～615 nm, . 6. the application of the PET hydrolase activity high-throughput assay method based on fluorescence detection as described in any one of claim 1-5, it is characterized in that: this method can be used for the high-throughput screening of PET hydrolase mutant . 7. the application of the PET hydrolase activity high-throughput assay method based on fluorescence detection according to claim 6, is characterized in that: in the high-throughput screening of PET hydrolase mutant, the film-making ratio of FDL:PET is 0.25~1 μg/mg; the volatilization temperature during membrane formation is 30℃~50℃; the amount of cell disruption solution is 2-15 μL.

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