Method for detecting guanosine deaminase activity

Abstract

The present invention relates to the technical field of enzyme activity determination, and discloses a guanosine deaminase activity detection method, which comprises: carrying out centrifugation on a guanosine deaminase production fermentation broth, collecting wet thalli, carrying out resuspension by using a PBS phosphate buffer solution, carrying out centrifugation again to collect the wet thalli, and recording the mass of the wet thalli, re-suspending the wet thalli by adopting a Tris-HCl buffer solution, adding lysozyme for cracking, ultrasonically crushing at low temperature and centrifuging to obtain a crude enzyme solution, and recording the total volume of the crude enzyme solution; preparing a reaction system, adding the crude enzyme solution into the reaction system to serve as an experimental group, adding a Tris-HCl buffer solution with the same amount as the crude enzyme solution into the reaction system to serve as a blank control group, carrying out oscillation reaction, adding NaOH to terminate the reaction after the reaction is finished, and recording the reaction time; respectively measuring the peak areas of the xanthosine in the experimental group and the blank control group by using high performance liquid chromatography, and calculating the content of the xanthosine by using an external standard method; and finally, calculating to obtain the enzyme activity of each gram of wet thalli. The detection method is simple, efficient, few in impurities and high in accuracy.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme activity determination, and particularly relates to a method for detecting guanosine deaminase activity. Background Art

[0002] Guanosine deaminase is a highly specific purine metabolic enzyme that exists in plants and algae. It can convert guanosine into xanthosine through deamination for nitrogen recovery and reuse. Guanosine deaminase mutants can show delayed germination, yellowing, etc. Guanosine deaminase plays an important role in the normal growth of plants. In recent years, researchers have made new progress in the research field of guanosine deaminase, successfully analyzed the crystal structure of the enzyme, and further revealed the enzymatic reaction mechanism between the enzyme and the substrate. The catalytic effect of guanosine deaminase is affected by many factors. On the one hand, there are multiple conformations at the C-terminus of the enzyme, and the lack of functional groups at the active site or improper interaction with the ligand hinders the reaction; on the other hand, the enzyme and the substrate will undergo complex conformational changes during the enzymatic reaction, and the reaction environment may affect the conformational changes. Therefore, determining suitable reaction conditions is crucial for the smooth progress of enzyme-catalyzed deamination.

[0003] Heterologously expressed guanosine deaminase can be obtained by microbial modification, but there are few reports on how to quickly and accurately detect the activity of guanosine deaminase. At the same time, the enzymatic reaction conditions suitable for guanosine deaminase have not yet been fully clarified, including key factors such as the appropriate reaction system, pH value and temperature. Summary of the invention

[0004] In order to solve the deficiencies of the prior art, the present invention provides a method for detecting the activity of guanosine deaminase, which comprises adding lysozyme to a heavy suspension of wet cells for lysis, performing low-temperature ultrasonic crushing after the lysis to obtain a crude enzyme solution, configuring a buffer system in proportion and adding the crude enzyme solution to the buffer system, carrying out an enzymatic reaction in a suitable buffer system to ensure that the catalytic action proceeds smoothly, and calculating the conversion rate of the substrate to the product by using a high-performance liquid phase external standard method after the reaction, thereby greatly improving the accuracy of the detection of the enzyme activity of guanosine deaminase, and the detection method is simple and rapid, and the result is close to reality, which provides convenience for the subsequent research and application of the properties of such enzymes.

[0005] The technical effects to be achieved by the present invention are achieved through the following technical aspects:

[0006] The present invention provides a method for detecting guanosine deaminase activity, comprising the following steps:

[0007] S1. Centrifuge the fermentation broth producing guanosine deaminase, collect the wet cells, discard the supernatant, resuspend with PBS phosphate buffer, centrifuge again, collect the wet cells, and record the mass of the wet cells;

[0008] S2, resuspend the wet cells in Tris-HCl buffer, add lysozyme to lyse, then ultrasonically disrupt under low temperature, centrifuge, retain the supernatant to obtain a crude enzyme solution, and record the total volume of the crude enzyme solution;

[0009] S3, preparing a reaction system, wherein the reaction system includes Tris-HCl, NaCl and guanosine, adding a crude enzyme solution to the reaction system as an experimental group, adding a Tris-HCl buffer solution equal to the crude enzyme solution to the reaction system as a blank control group, both the experimental group and the blank control group are subjected to oscillation reaction, and after the reaction is completed, NaOH is added to both the experimental group and the blank control group to terminate the reaction, and the reaction time is recorded;

[0010] S4, diluting the reaction solution of the experimental group and the blank control group respectively, measuring the peak area of ​​the conversion product xanthoglobin in the experimental group and the blank control group respectively by high performance liquid chromatography, and calculating the xanthoglobin content in the experimental group and the blank control group by external standard method;

[0011] S5. The amount of guanosine deaminase required to convert 1 μmol of substrate guanosine into product xanthosine per minute is defined as 1 activity unit (IU), and the enzyme activity per gram of wet bacteria is calculated.

[0012] As a further description of the technical solution of the present invention, the wet bacteria are Escherichia coli that recombinantly express guanosine deaminase.

[0013] As a further description of the technical solution of the present invention, in S1, the centrifugal speed is 7000-8000 rpm, the centrifugal temperature is 3-5°C, and the centrifugal time is 10-20 min.

[0014] As a further description of the technical solution of the present invention, in S1, the concentration of PBS phosphate buffer is 0.01-0.02 mol / L and the pH is 7.2-7.4.

[0015] As a further description of the technical solution of the present invention, in S2, the concentration of Tris-HCl buffer is 0.03-0.06 mol / L, the pH is 7.9-8.1, and the amount of Tris-HCl buffer added is 5-10 times the mass of the wet bacteria.

[0016] As a further description of the technical solution of the present invention, in S2, the added amount of lysozyme is 0.1-2 mg per gram of wet bacteria, the lysis temperature is 3-5°C, and the lysis time is 20-60 min.

[0017] As a further description of the technical solution of the present invention, in S2, the temperature of ultrasonic crushing is 3-5°C, the ultrasonic power of ultrasonic crushing is 250-350W, the ultrasonic time is 8-12s, the interval time is 10-20s, and the total time is 10-20min.

[0018] As a further description of the technical solution of the present invention, in S3, the amount of crude enzyme solution added is 0.5-10% of the volume of the reaction system.

[0019] As a further description of the technical solution of the present invention, in S3, the reaction system includes 15-25 mmol / L Tris-HCl, 130-160 mmol / L NaCl and 5-50 mmol / L guanosine, the reaction temperature is 37±1°C, and the reaction time is 30-150 min.

[0020] As a further description of the technical solution of the present invention, the enzyme activity calculation formula per gram of wet bacteria is:

[0021]

[0022] Among them, C1 is the xanthoglobin concentration of the experimental group (μmol / L);

[0023] C2 is the xanthoglobin concentration of the blank control group (μmol / L);

[0024] V1 is the volume of the reaction system (L);

[0025] V2 is the total volume of crude enzyme solution (mL);

[0026] V3 is the volume of crude enzyme solution added to the reaction system (mL);

[0027] T is the reaction time (min);

[0028] M is the wet cell mass (g).

[0029] In summary, the present invention has at least the following benefits:

[0030] The method for detecting guanosine deaminase activity provided by the present invention comprises the following steps: adding lysozyme to a wet bacterial resuspension to lyse bacterial cells, and performing ultrasonic crushing under low temperature conditions to obtain a crude enzyme solution after the lysis is completed, so that guanosine deaminase can be completely released from bacterial cells, ensuring that guanosine deaminase is completely separated from bacterial cells, thereby reducing impurities in the crude enzyme solution and ensuring the adequacy of the reaction between guanosine deaminase and substrate guanosine; ultrasonic crushing under low temperature can better maintain the activity of guanosine deaminase; performing an enzymatic reaction in a suitable buffer system to ensure that the catalytic action proceeds smoothly; and using a high-performance liquid phase external standard method to calculate the conversion rate of substrate to product after the reaction is completed, thereby greatly improving the accuracy and reliability of guanosine deaminase enzyme activity detection; and the detection method has simple and rapid steps and high detection efficiency. DETAILED DESCRIPTION

[0031] For ease of understanding of the present invention, the present invention will be described more fully below in conjunction with specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0033] The present invention provides a method for detecting guanosine deaminase activity, comprising the following steps:

[0034] S1. Centrifuge the fermentation broth producing guanosine deaminase, collect the wet cells, discard the supernatant, resuspend with PBS phosphate buffer, centrifuge again, collect the wet cells, and record the mass of the wet cells;

[0035] S2, resuspend the wet cells in Tris-HCl buffer, add lysozyme to lyse, then ultrasonically disrupt under low temperature, centrifuge, retain the supernatant to obtain a crude enzyme solution, and record the total volume of the crude enzyme solution;

[0036] S3, preparing a reaction system, the reaction system includes Tris-HCl, NaCl and guanosine, adding crude enzyme solution to the reaction system as an experimental group, adding Tris-HCl buffer equal to the crude enzyme solution to the reaction system as a blank control group, the experimental group and the blank control group are both shaken for reaction, after the reaction is completed, NaOH is added to both the experimental group and the blank control group to terminate the reaction, and the reaction time is recorded;

[0037] S4, diluting the reaction solution of the experimental group and the blank control group respectively, measuring the peak area of ​​the conversion product xanthoglobin in the experimental group and the blank control group respectively by high performance liquid chromatography, and calculating the xanthoglobin content in the experimental group and the blank control group by external standard method;

[0038] S5. The amount of guanosine deaminase required to convert 1 μmol of substrate guanosine into product xanthosine per minute is defined as 1 activity unit (IU), and the enzyme activity per gram of wet bacteria is calculated.

[0039] By adding lysozyme to the wet bacterial resuspension to lyse the bacterial cells, and then performing ultrasonic disruption under low temperature conditions after the lysis is completed, guanosine deaminase can be completely released from the bacterial cells, ensuring the complete separation of guanosine deaminase from the bacterial cells, thereby reducing impurities in the crude enzyme solution and ensuring the adequacy of the reaction between guanosine deaminase and the substrate guanosine. At the same time, ultrasonic disruption at low temperature can better maintain the activity of guanosine deaminase, thereby improving the accuracy and reliability of enzyme activity detection; and ultrasonic disruption at low temperature can make cell disruption more uniform and thorough, with high disruption efficiency, thereby improving the efficiency of enzyme activity detection.

[0040] The above detection method is simple, fast, easy to operate, and time-saving, which greatly improves the detection efficiency, and does not require purification of the target protease. While simplifying the steps, it reduces the impact on the properties of the target protease itself, and there are few impurities in the reaction system, which has little impact on the detection results of enzyme activity, and the detection results are true and reliable. In addition, the reagents used in the detection method are low-toxic and harmless, and the pollutants produced are few, which is environmentally friendly.

[0041] Specifically, the wet bacteria are Escherichia coli. In some embodiments, the Escherichia coli used is an engineered bacterium that recombinantly expresses guanosine deaminase.

[0042] In some embodiments, in S1, the centrifugal speed is 7000-8000 rpm, preferably 8000 rpm; the centrifugal temperature is 3-5°C, preferably 4°C; and the centrifugal time is 10-20 min, preferably 15 min.

[0043] In some embodiments, in S1, the concentration of PBS phosphate buffer is 0.01-0.02 mol / L, preferably 0.01 mol / L, and the pH is 7.2-7.4.

[0044] In some embodiments, in S2, the concentration of Tris-HCl buffer is 0.03-0.06 mol / L, pH is 7.9-8.1, preferably 0.05 mol / L, pH is 8.0; the amount of Tris-HCl buffer added is 5-10 times the mass of wet bacteria. As a further optimization, the Tris-HCl buffer may contain 150 mmol / L NaCl and 1 mmol / L PMSF (phenylmethylsulfonyl fluoride). The Tris-HCl buffer can maintain the stability of pH during the lysis process, prevent the negative impact of pH changes on the structure and activity of guanosine deaminase, provide a stable acid-base environment for guanosine deaminase, and ensure that guanosine deaminase maintains its original structure and functional characteristics during the lysis process. NaCl and PMSF can work together with the Tris-HCl buffer to ensure the stability of environmental conditions during the lysis process, ensure the integrity of the structure and functional characteristics of guanosine deaminase, and further improve the reliability of the enzyme activity test results.

[0045] In some embodiments, in S2, the amount of lysozyme added is 0.1-2 mg per gram of wet bacteria, the lysis temperature is 3-5 ° C, preferably 4 ° C, and the lysis time is 20-60 min. Adding lysozyme to the bacterial solution for lysis treatment at a low temperature of 3-5 ° C is beneficial to improve the stability and activity of lysozyme and enhance the lysis effect. Under low temperature conditions, the activity of the bacteria is reduced, which enables lysozyme to act more effectively on the bacterial cell wall and effectively destroy the cell wall structure, thereby achieving a better lysis effect. The improvement of the lysis effect is more conducive to the thorough separation of bacterial cell tissue and guanosine deaminase, thereby minimizing the impurities in the crude enzyme solution, thereby reducing the impurities in the reaction system, making the reaction of guanosine deaminase and guanosine more sufficient, and further improving the accuracy, authenticity and reliability of the enzyme activity test results.

[0046] In some embodiments, in S2, the temperature of ultrasonic crushing is 3-5°C, preferably 4°C, the ultrasonic power of ultrasonic crushing is 250-350W, the ultrasonic time is 8-12s, the interval time is 10-20s, and the total duration is 10-20min. Preferably, the ultrasonic power is 300W, the ultrasonic time is 10s, and the interval time is 15s.

[0047] In some embodiments, the centrifugation speed in S2 is 14000 rpm, the centrifugation temperature is 4° C., and the centrifugation time is 10 min.

[0048] In some embodiments, in S3, the amount of crude enzyme solution added is 0.5-10% of the volume of the reaction system.

[0049] In some embodiments, in S3, the reaction system includes 15-25mmol / L Tris-HCl, 130-160mmol / L NaCl and 5-50mmol / L guanosine, preferably, Tris-HCl is 20mmol / L (pH8.0), and NaCl is 150mmol / L. The reaction temperature is 37±1°C, the reaction time is 30-150min, and after the reaction is completed, 1mol / L NaOH is added to terminate the reaction, and the final concentration of 1mol / L NaOH in the reaction system is 0.1mol / L.

[0050] The HPLC conditions in S4 were as follows: liquid chromatography column (SinoChrom ODS-BP, 5 μm, 4.6 mm×250 mm), mobile phase A was methanol, mobile phase B was 50 mmol / L diammonium phosphate solution, elution program was 0-8 min: 100% B; 9-21 min: 75% B; 22-30 min: 100% B; flow rate was 1.0 mL / min, column temperature was 25°C, injection volume was 10 μL, and detection wavelength was 259 nm.

[0051] In S5, the xanthosine content of the experimental group and the blank control group can be calculated by the external standard method, thereby calculating the amount of enzyme required for guanosine deaminase to convert 1 μmol of substrate guanosine into product xanthosine in 1 minute, and the enzyme activity per gram of wet bacteria can be obtained. By directly detecting the content of the reaction product and calculating the enzyme activity, not only can false positive results be avoided, but also the activity level of guanosine deaminase can be more accurately reflected, and the calculation results are more accurate, true and reliable.

[0052] Specifically, the enzyme activity per gram of wet bacteria is calculated as follows:

[0053]

[0054] C1: Xanthoglobin concentration in the experimental group (μmol / L);

[0055] C2: Xanthoglobin concentration in the blank control group (μmol / L);

[0056] V1: volume of the reaction system (L);

[0057] V2: total volume of crude enzyme solution (mL);

[0058] V3: volume of crude enzyme solution added to the reaction system (mL);

[0059] T: reaction time (min);

[0060] M: wet cell mass (g).

[0061] Example 1

[0062] This embodiment provides a method for detecting guanosine deaminase activity, comprising the following steps:

[0063] S1. The fermentation broth producing guanosine deaminase was centrifuged at a speed of 8000 rpm and a temperature of 4°C for 15 min, and the wet cells of Escherichia coli expressing guanosine deaminase were collected. The supernatant fermentation broth was discarded, and the cells were resuspended with PBS phosphate buffer (0.01 mol / L, pH 7.2-7.4), and centrifuged again to collect the wet cells. The centrifugation speed was 8000 rpm, the temperature was 4°C, and the time was set to 15 min. The mass of the wet cells was recorded as 8 g (M).

[0064] S2. Resuspend the wet cells with Tris-HCl buffer (0.05 mol / L, pH 8.0) containing 150 mmol / L NaCl and 1 mmol / L PMSF. The amount of Tris-HCl buffer added is 5 times the mass of the wet cells. At the same time, add lysozyme at a rate of 0.1 mg per gram of wet cells and lyse at 4°C for 60 min. Then, perform ultrasonic disruption at 4°C with an ultrasonic power of 300 W, an ultrasonic time of 10 s, an interval of 15 s, and a total duration of 10 min. After the ultrasonic treatment, centrifuge at 4°C and 14,000 rpm for 10 min, retain the supernatant (i.e., crude enzyme solution), and record the total volume of the crude enzyme solution as 40 mL (V2).

[0065] S3. Prepare 0.1L (V1) reaction system containing 20mmol / L Tris-HCl (pH 8.0), 150mmol / L NaCl and 5mmol / L guanosine. Add crude enzyme solution to the reaction system as the experimental group. The amount of crude enzyme solution added to the experimental group is 0.5mL (V3). Add Tris-HCl buffer in the same amount as the crude enzyme solution to the reaction system as the blank control group. Both the experimental group and the blank control group are shaken for reaction at 37°C for 30min (T). After the reaction, add 1mol / L NaOH to terminate the reaction.

[0066] S4. Dilute the experimental group and the blank control group respectively, and use high performance liquid chromatography to measure the peak area of ​​the conversion product xanthoglobin in the experimental group and the blank control group respectively; the high performance liquid chromatography conditions are: liquid chromatography column (SinoChrom ODS-BP, 5μm, 4.6mm×250mm), mobile phase A is methanol, mobile phase B is 50mmol / L ammonium dihydrogen phosphate solution, elution program is 0-8min: 100% B; 9-21min: 75% B; 22-30min: 100% B; flow rate is 1.0mL / min, column temperature is 25℃, injection volume is 10μL, detection wavelength is 259nm. The xanthoglobin concentration of the experimental group is calculated by external standard method to be 1250μmol / L (C1), and the xanthoglobin concentration of the blank control group is 0μmol / L (C2).

[0067] S5. The amount of guanosine deaminase required to convert 1 μmol of substrate guanosine into product xanthosine in 1 min is defined as 1 activity unit IU. The enzyme activity per gram of wet bacteria is calculated by the following formula:

[0068]

[0069] C1: Xanthoglobin concentration in the experimental group (μmol / L);

[0070] C2: Xanthoglobin concentration in the blank control group (μmol / L);

[0071] V1: volume of the reaction system (L);

[0072] V2: total volume of crude enzyme solution (mL);

[0073] V3: volume of crude enzyme solution added to the reaction system (mL);

[0074] T: reaction time (min);

[0075] M: wet cell mass (g).

[0076] The enzyme activity per gram of wet bacteria was calculated by the above formula to be 41.67 IU / g.

[0077] Example 2

[0078] This embodiment provides a method for detecting guanosine deaminase activity, comprising the following steps:

[0079] S1. The fermentation broth producing guanosine deaminase was centrifuged at a speed of 8000 rpm and a temperature of 4°C for 15 min, and the wet cells of Escherichia coli expressing guanosine deaminase were collected. The supernatant fermentation broth was discarded, and the cells were resuspended with PBS phosphate buffer (0.01 mol / L, pH 7.2-7.4), and centrifuged again to collect the wet cells. The centrifugation speed was 8000 rpm, the temperature was 4°C, and the time was set to 15 min. The mass of the wet cells was recorded as 8 g (M).

[0080] S2. Resuspend the wet cells with Tris-HCl buffer (0.05 mol / L, pH 8.0) containing 150 mmol / L NaCl and 1 mmol / L PMSF. The amount of Tris-HCl buffer added is 10 times the mass of the wet cells. At the same time, add lysozyme at a rate of 0.5 mg per gram of wet cells and lyse at 4°C for 40 min. Then, perform ultrasonic disruption at 4°C with an ultrasonic power of 300 W, an ultrasonic time of 10 s, an interval of 15 s, and a total duration of 10 min. After the ultrasonic treatment, centrifuge at 4°C and 14,000 rpm for 10 min, retain the supernatant (i.e., crude enzyme solution), and record the total volume of the crude enzyme solution, 80 mL (V2).

[0081] S3. Prepare 0.1L (V1) reaction system containing 20mmol / L Tris-HCl (pH 8.0), 150mmol / L NaCl and 50mmol / L guanosine. Add crude enzyme solution to the reaction system as the experimental group. The amount of crude enzyme solution added to the experimental group is 10mL (V3). Add Tris-HCl buffer equal to the amount of crude enzyme solution to the reaction system as the blank control group. Both the experimental group and the blank control group are shaken for reaction at 37°C for 30min (T). After the reaction, add 1mol / L NaOH to terminate the reaction.

[0082] S4. Dilute the experimental group and the blank control group respectively, and use high performance liquid chromatography to measure the peak area of ​​the conversion product xanthoglobin in the experimental group and the blank control group respectively; the high performance liquid chromatography conditions are: liquid chromatography column (SinoChrom ODS-BP, 5μm, 4.6mm×250mm), mobile phase A is methanol, mobile phase B is 50mmol / L ammonium dihydrogen phosphate solution, elution program is 0-8min: 100% B; 9-21min: 75% B; 22-30min: 100% B; flow rate is 1.0mL / min, column temperature is 25℃, injection volume is 10μL, detection wavelength is 259nm. The xanthoglobin concentration of the experimental group is calculated by external standard method to be 31500μmol / L (C1), and the xanthoglobin concentration of the blank control group is 0μmol / L (C2).

[0083] S5. The amount of guanosine deaminase required to convert 1 μmol of substrate guanosine into product xanthosine in 1 min is defined as 1 activity unit IU. The enzyme activity per gram of wet bacteria is calculated by the following formula:

[0084]

[0085] C1: Xanthoglobin concentration in the experimental group (μmol / L);

[0086] C2: Xanthoglobin concentration in the blank control group (μmol / L);

[0087] V1: volume of the reaction system (L);

[0088] V2: total volume of crude enzyme solution (mL);

[0089] V3: volume of crude enzyme solution added to the reaction system (mL);

[0090] T: reaction time (min);

[0091] M: wet cell mass (g).

[0092] The enzyme activity per gram of wet bacteria was calculated by the above formula to be 52.50 IU / g.

[0093] Example 3

[0094] This embodiment provides a method for detecting guanosine deaminase activity, comprising the following steps:

[0095] S1. The fermentation broth producing guanosine deaminase was centrifuged at a speed of 8000 rpm and a temperature of 4°C for 15 min, and the wet cells of Escherichia coli expressing guanosine deaminase were collected. The supernatant fermentation broth was discarded, and the cells were resuspended with PBS phosphate buffer (0.01 mol / L, pH 7.2-7.4), and centrifuged again to collect the wet cells. The centrifugation speed was 8000 rpm, the temperature was 4°C, and the time was set to 15 min. The mass of the wet cells was recorded as 8 g (M).

[0096] S2. Resuspend the wet cells with Tris-HCl buffer (0.05 mol / L, pH 8.0) containing 150 mmol / L NaCl and 1 mmol / L PMSF. The amount of Tris-HCl buffer added is 10 times the mass of the wet cells. At the same time, add lysozyme at a rate of 1 mg per gram of wet cells and lyse at 4°C for 30 min. Then, perform ultrasonic disruption at 4°C with an ultrasonic power of 300 W, an ultrasonic time of 10 s, an interval of 15 s, and a total duration of 15 min. After the ultrasonic treatment, centrifuge at 4°C and 14,000 rpm for 10 min, retain the supernatant (i.e., crude enzyme solution), and record the total volume of the crude enzyme solution, 80 mL (V2).

[0097] S3. Prepare 0.1L (V1) reaction system containing 20mmol / L Tris-HCl (pH 8.0), 150mmol / L NaCl and 5mmol / L guanosine. Add crude enzyme solution to the reaction system as the experimental group. The amount of crude enzyme solution added to the experimental group is 0.5mL (V3). Add Tris-HCl buffer in the same amount as the crude enzyme solution to the reaction system as the blank control group. Both the experimental group and the blank control group are shaken for reaction at 37°C for 30min (T). After the reaction, add 1mol / L NaOH to terminate the reaction.

[0098] S4. Dilute the experimental group and the blank control group respectively, and use high performance liquid chromatography to measure the peak area of ​​the conversion product xanthoglobin in the experimental group and the blank control group respectively; the high performance liquid chromatography conditions are: liquid chromatography column (SinoChrom ODS-BP, 5μm, 4.6mm×250mm), mobile phase A is methanol, mobile phase B is 50mmol / L diammonium phosphate solution, elution program is 0~8min: 100%B; 9~21min: 75%B; 22~30min: 100%B; flow rate is 1.0mL / min, column temperature is 25℃, injection volume is 10μL, detection wavelength is 259nm. The xanthoglobin concentration of the experimental group is calculated by external standard method to be 29440μmol / L (C1), and the xanthoglobin concentration of the blank control group is 0μmol / L (C2).

[0099] S5. The amount of guanosine deaminase required to convert 1 μmol of substrate guanosine into product xanthosine in 1 min is defined as 1 activity unit IU. The enzyme activity per gram of wet bacteria is calculated by the following formula:

[0100]

[0101] C1: Xanthoglobin concentration in the experimental group (μmol / L);

[0102] C2: Xanthoglobin concentration in the blank control group (μmol / L);

[0103] V1: volume of the reaction system (L);

[0104] V2: total volume of crude enzyme solution (mL);

[0105] V3: volume of crude enzyme solution added to the reaction system (mL);

[0106] T: reaction time (min);

[0107] M: wet cell mass (g).

[0108] The enzyme activity per gram of wet bacteria was calculated by the above formula to be 58.88 IU / g.

[0109] Example 4

[0110] This embodiment provides a method for detecting guanosine deaminase activity, comprising the following steps:

[0111] S1. The fermentation broth producing guanosine deaminase was centrifuged at a speed of 8000 rpm and a temperature of 4°C for 15 min, and the wet cells of Escherichia coli expressing guanosine deaminase were collected. The supernatant fermentation broth was discarded, and the cells were resuspended with PBS phosphate buffer (0.01 mol / L, pH 7.2-7.4), and centrifuged again to collect the wet cells. The centrifugation speed was 8000 rpm, the temperature was 4°C, and the time was set to 15 min. The mass of the wet cells was recorded as 8 g (M).

[0112] S2. Resuspend the wet cells with Tris-HCl buffer (0.05 mol / L, pH 8.0) containing 150 mmol / L NaCl and 1 mmol / L PMSF. The amount of Tris-HCl buffer added is 8 times the mass of the wet cells. At the same time, add lysozyme at a rate of 2 mg per gram of wet cells and lyse at 4°C for 20 min. Then, perform ultrasonic disruption at 4°C with an ultrasonic power of 300 W, an ultrasonic time of 10 s, an interval of 15 s, and a total duration of 20 min. After the ultrasonic treatment, centrifuge at 4°C and 14,000 rpm for 10 min, retain the supernatant (i.e., crude enzyme solution), and record the total volume of the crude enzyme solution as 64 mL (V2).

[0113] S3. Prepare 0.1L (V1) reaction system containing 20mmol / L Tris-HCl (pH 8.0), 150mmol / L NaCl and 5mmol / L guanosine. Add crude enzyme solution to the reaction system as the experimental group. The amount of crude enzyme solution added in the experimental group is 1.0mL (V3). Add Tris-HCl buffer in the same amount as the crude enzyme solution to the reaction system as the blank control group. Both the experimental group and the blank control group are shaken for reaction at 37°C for 30min (T). After the reaction, add 1mol / L NaOH to terminate the reaction.

[0114] S4. Dilute the experimental group and the blank control group respectively, and use high performance liquid chromatography to measure the peak area of ​​the conversion product xanthoglobin in the experimental group and the blank control group respectively; the high performance liquid chromatography conditions are: liquid chromatography column (SinoChrom ODS-BP, 5μm, 4.6mm×250mm), mobile phase A is methanol, mobile phase B is 50mmol / L ammonium dihydrogen phosphate solution, elution program is 0~8min: 100% B; 9~21min: 75% B; 22~30min: 100% B; flow rate is 1.0mL / min, column temperature is 25℃, injection volume is 10μL, detection wavelength is 259nm. The xanthoglobin concentration of the experimental group is calculated by external standard method to be 11475μmol / L (C1), and the xanthoglobin concentration of the blank control group is 0μmol / L (C2).

[0115] S5. The amount of guanosine deaminase required to convert 1 μmol of substrate guanosine into product xanthosine in 1 min is defined as 1 activity unit IU. The enzyme activity per gram of wet bacteria is calculated by the following formula:

[0116]

[0117] C1: Xanthoglobin concentration in the experimental group (μmol / L);

[0118] C2: Xanthoglobin concentration in the blank control group (μmol / L);

[0119] V1: volume of the reaction system (L);

[0120] V2: total volume of crude enzyme solution (mL);

[0121] V3: volume of crude enzyme solution added to the reaction system (mL);

[0122] T: reaction time (min);

[0123] M: wet cell mass (g).

[0124] The enzyme activity per gram of wet bacteria was calculated by the above formula to be 61.20 IU / g.

[0125] The above contents are merely examples and illustrations of the structure of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and these obvious replacement forms all belong to the protection scope of the present invention.

Claims

1. A method for detecting guanosine deaminase activity, characterized in that: The following steps are involved: S1. Centrifuge the fermentation broth producing guanosine deaminase, collect the wet cells, discard the supernatant, resuspend with PBS phosphate buffer, centrifuge again, collect the wet cells, and record the mass of the wet cells; S2, resuspend the wet cells in Tris-HCl buffer, add lysozyme to lyse, then ultrasonically disrupt under low temperature, centrifuge, retain the supernatant to obtain a crude enzyme solution, and record the total volume of the crude enzyme solution; S3, preparing a reaction system, wherein the reaction system includes Tris-HCl, NaCl and guanosine, adding a crude enzyme solution to the reaction system as an experimental group, adding a Tris-HCl buffer solution equal to the crude enzyme solution to the reaction system as a blank control group, both the experimental group and the blank control group are subjected to oscillation reaction, and after the reaction is completed, NaOH is added to both the experimental group and the blank control group to terminate the reaction, and the reaction time is recorded; S4, diluting the reaction solution of the experimental group and the blank control group respectively, measuring the peak area of ​​the conversion product xanthoglobin in the experimental group and the blank control group respectively by high performance liquid chromatography, and calculating the xanthoglobin content in the experimental group and the blank control group by external standard method; S5. The amount of guanosine deaminase required to convert 1 μmol of substrate guanosine into product xanthosine per minute is defined as 1 activity unit (IU), and the enzyme activity per gram of wet bacteria is calculated.

2. The method for detecting guanosine deaminase activity according to claim 1, characterized in that: The wet bacteria are Escherichia coli that recombinantly express guanosine deaminase.

3. The method for detecting guanosine deaminase activity according to claim 1, characterized in that: In the S1, the centrifugal speed is 7000-8000 rpm, the centrifugal temperature is 3-5°C, and the centrifugal time is 10-20 min.

4. The method for detecting guanosine deaminase activity according to claim 1, characterized in that: In the S1, the concentration of PBS phosphate buffer is 0.01-0.02 mol / L, and the pH is 7.2-7.

4.

5. The method for detecting guanosine deaminase activity according to claim 1, characterized in that: In the S2, the concentration of the Tris-HCl buffer is 0.03-0.06 mol / L, the pH is 7.9-8.1, and the amount of the Tris-HCl buffer added is 5-10 times the mass of the wet bacteria.

6. The method for detecting guanosine deaminase activity according to claim 1, characterized in that: In the S2, the amount of lysozyme added is 0.1-2 mg per gram of wet bacteria, the lysis temperature is 3-5° C., and the lysis time is 20-60 min.

7. The method for detecting guanosine deaminase activity according to claim 1, characterized in that: In the S2, the temperature of ultrasonic crushing is 3-5°C, the ultrasonic power of ultrasonic crushing is 250-350W, the ultrasonic time is 8-12s, the interval time is 10-20s, and the total time is 10-20min.

8. The method for detecting guanosine deaminase activity according to claim 1, characterized in that: In the step S3, the amount of crude enzyme solution added is 0.5-10% of the volume of the reaction system.

9. The method for detecting guanosine deaminase activity according to claim 1, characterized in that: In S3, the reaction system includes 15-25 mmol / L Tris-HCl, 130-160 mmol / L NaCl and 5-50 mmol / L guanosine, the reaction temperature is 37±1° C., and the reaction time is 30-150 min.

10. The method for detecting guanosine deaminase activity according to claim 1, characterized in that: The enzyme activity calculation formula per gram of wet bacteria is: Among them, C1 is the xanthoglobin concentration of the experimental group (μmol / L); C2 is the xanthoglobin concentration of the blank control group (μmol / L); V1 is the volume of the reaction system (L); V2 is the total volume of crude enzyme solution (mL); V3 is the volume of crude enzyme solution added to the reaction system (mL); T is the reaction time (min); M is the wet cell mass (g).

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