Auxiliary agent for improving nuclease stability and preparation method and application thereof

Abstract

The present invention belongs to the technical field of enzyme preparations, and specifically provides an auxiliary agent for improving the stability of nucleases, including a carrier and a nuclease fermentation broth; the addition amount of the nuclease fermentation broth is 0.01-0.1% of the mass of the carrier. The carrier includes at least one of wheat starch, corn starch, and cassava starch. The auxiliary agent for improving the stability of nucleases provided by the present invention is mixed with the nuclease fermentation broth to obtain a stable nuclease preparation, which has a retention rate of more than 90% when stored at 4°C for one year, and has other excellent enzymatic properties at the same time, a retention rate of more than 95% when treated at 75°C for 5 minutes, is not easily inactivated at high temperature, is convenient to produce and transport, is suitable for high-temperature production processes, and effectively improves the application prospects of nucleases.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme preparations, and in particular relates to an auxiliary agent for improving the stability of nucleases, a preparation method and an application thereof. Background Art

[0002] Nuclease is a biological catalyst that can accelerate the hydrolysis and shearing of nucleic acid molecules. It plays an important role in life activities and participates in many gene expression and metabolic processes. Nuclease plays an important role in biological research. It can be used to change the structure and function of DNA and RNA, detect specific DNA and RNA sequences, and can also be used for gene therapy, anti-tumor therapy, vaccine research, protein purification, etc. Nuclease is also widely used in the food industry and agriculture. It is used to produce mononucleotide products and remove nucleic acid impurities in biological products such as proteins and sugars. It has great development potential. Most nucleases are room-temperature nucleases, which are easily inactivated at high temperatures, which brings inconvenience to production and transportation, especially not suitable for high-temperature production processes. In addition, the product also has the problem of poor stability during actual storage. Summary of the invention

[0003] The purpose of the present invention is to overcome the problems of poor temperature resistance and storage stability of nuclease in the prior art.

[0004] To this end, the present invention provides an auxiliary agent for improving the stability of nuclease, comprising a carrier and a nuclease fermentation liquid; the addition amount of the nuclease fermentation liquid is 0.01-0.1% of the mass of the carrier.

[0005] Specifically, the nuclease fermentation broth is the fermentation broth obtained by producing nuclease with Pichia pastoris.

[0006] Specifically, the carrier includes at least one of wheat starch, corn starch and tapioca starch.

[0007] The present invention also provides a method for preparing the above-mentioned auxiliary agent for improving the stability of nuclease, comprising the following steps: weighing a carrier, adding nuclease fermentation liquid thereto, and then adding water to degrade the carrier to obtain the auxiliary agent.

[0008] Specifically, the degradation temperature is 25-40°C, and the degradation time is 16-24h.

[0009] Specifically, the moisture content of the mixture after adding water is 25-60%.

[0010] The present invention also provides a stable nuclease preparation, comprising nuclease and the above-mentioned auxiliary agent for improving the stability of nuclease.

[0011] The present invention also provides a method for preparing the above-mentioned stable nuclease preparation, comprising the following steps: mixing the nuclease fermentation liquid with the auxiliary agent for improving the stability of the nuclease, adjusting the pH of the mixed liquid, and drying the mixed liquid to obtain the stable nuclease preparation.

[0012] Specifically, the mass ratio of the nuclease fermentation broth to the auxiliary agent for improving the stability of the nuclease is (1-5):1.

[0013] Specifically, the pH of the mixed solution is adjusted to 4.0-5.2.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0015] The auxiliary agent for improving the stability of nuclease provided by the present invention has low cost and is easy to prepare. The stable nuclease preparation obtained after being mixed with the nuclease fermentation liquid can reach a retention rate of more than 90% when stored at 4°C for one year, and at the same time has other excellent enzymatic properties. The retention rate reaches more than 95% after being treated at 75°C for 5 minutes, is not easily inactivated at high temperature, is easy to produce and transport, is suitable for high-temperature production processes, and effectively improves the application prospects of nuclease. DETAILED DESCRIPTION

[0016] The technical scheme in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be limited by the attached claims and their equivalents.

[0017] The invention provides an auxiliary agent for improving the stability of nuclease, comprising a carrier and a nuclease fermentation liquid; the addition amount of the nuclease fermentation liquid is 0.01-0.1% of the mass of the carrier. The nuclease fermentation liquid is a fermentation liquid obtained by producing nuclease by Pichia pastoris. The carrier comprises at least one of wheat starch, corn starch and cassava starch.

[0018] The nuclease fermentation broth can be obtained by conventional fermentation methods in the art, preferably by the following steps:

[0019] Take a Pichia glycerol tube, culture it on a slant, pick a single colony and place it in a liquid culture medium, and culture it on a shaker to form a seed solution. Inoculate the seed solution into a fermentation tank, culture temperature 35°C, pH 6.5, tank pressure 0.035-0.05MPa, ventilation ratio 1.0m 3 / (m 3·min), stirring speed 100 rpm, culture period 96 h. Foam was controlled by adding silicone oil Silicone 1520 or other feasible methods.

[0020] The present invention also provides a method for preparing the above-mentioned auxiliary agent for improving the stability of nuclease, comprising the following steps: weighing a carrier, adding nuclease fermentation liquid thereto and then adding water to make the moisture content of the mixture 25-60%, degrading at a constant temperature of 25-40°C for 16-24h to obtain the auxiliary agent, and drying or directly using it on a wet basis.

[0021] The present invention also provides a stable nuclease preparation, comprising nuclease and the above-mentioned auxiliary agent for improving the stability of nuclease.

[0022] The present invention also provides a method for preparing the above-mentioned stable nuclease preparation, comprising the following steps:

[0023] The nuclease fermentation liquid is mixed with the auxiliary agent for improving the stability of the nuclease in a mass ratio of (1-5): 1, preferably 1000 mL of the auxiliary agent wet base or 200 g of the drying auxiliary agent is added to 1000 mL of the fermentation liquid. Citric acid or sodium citrate is used to adjust the pH of the mixed solution to 4.0-5.2, preferably 4.5-5.2, more preferably 5.0±0.2. The mixed solution is dried to obtain the stable nuclease preparation.

[0024] The auxiliary agent for improving the stability of nuclease of the present invention, its preparation method and the effect of its application are studied through specific examples below.

[0025] The nuclease fermentation broth used in the embodiment of the present invention is obtained by the following steps: take a Pichia glycerol tube, streak it on a YPD slant medium, culture it at 35°C for 36 hours, pick a single colony in a YPD liquid medium, and culture it on a shaker at 35°C and 180rpm for 10 hours to form a seed solution. 100mL of seed solution is inoculated into a 30-L tank (loading volume 15L YPD medium), the culture temperature is 35°C, pH 6.5, the tank pressure is 0.035-0.05MPa, and the ventilation ratio is 1.0m 3 / (m 3 The foam was controlled by adding silicone oil 1520.

[0026] Embodiment 1:

[0027] The present embodiment provides an auxiliary agent for improving the stability of nuclease, which is prepared by the following steps: weighing wheat starch, adding 0.01% nuclease fermentation liquid to it based on the mass of wheat starch, and then adding water to make the moisture content of the mixture 50%, degrading it at a constant temperature of 30°C for 20 hours, and drying to obtain the auxiliary agent.

[0028] Based on the above-mentioned auxiliary agents, this embodiment also provides three stable nuclease preparations, which are prepared by the following steps: 1000 ml of nuclease fermentation broth is mixed with 200 g of the above-mentioned auxiliary agents and divided into three parts, and citric acid is used to adjust the pH of the mixed solution to 4.0 (sample 1), 4.5 (sample 2) and 5.0 (sample 3), respectively, and the mixed solution is dried to obtain three stable nuclease preparations.

[0029] Embodiment 2:

[0030] The present embodiment provides an auxiliary agent for improving the stability of nuclease, which is prepared by the following steps: weigh corn starch, add 0.01% nuclease fermentation liquid to it based on the mass of corn starch, add water to make the moisture content of the mixture 50%, degrade it at a constant temperature of 30°C for 20 hours, and dry it to obtain the auxiliary agent.

[0031] Based on the above-mentioned auxiliary agents, this embodiment also provides three stable nuclease preparations, which are prepared by the following steps: 1000 ml of nuclease fermentation broth is mixed with 200 g of the above-mentioned auxiliary agents and divided into three parts, and citric acid is used to adjust the pH of the mixed solution to 4.0 (sample 4), 4.5 (sample 5) and 5.0 (sample 6), respectively, and the mixed solution is dried to obtain three stable nuclease preparations.

[0032] Embodiment 3:

[0033] The present embodiment provides an auxiliary agent for improving the stability of nuclease, which is prepared by the following steps: weighing cassava starch, adding 0.01% nuclease fermentation liquid to it based on the mass of cassava starch, and then adding water to make the moisture content of the mixture 50%, degrading it at a constant temperature of 30°C for 20 hours, and drying to obtain the auxiliary agent.

[0034] Based on the above-mentioned auxiliary agents, this embodiment also provides three stable nuclease preparations, which are prepared by the following steps: 1000 ml of nuclease fermentation broth is mixed with 200 g of the above-mentioned auxiliary agents and divided into three parts, and citric acid is used to adjust the pH of the mixed solution to 4.0 (sample 7), 4.5 (sample 8) and 5.0 (sample 9), respectively, and the mixed solution is dried to obtain three stable nuclease preparations.

[0035] Embodiment 4:

[0036] In this example, the wet heat resistance evaluation method was used to verify the temperature resistance of the stable nuclease preparation, and the long-term storage stability of the stable nuclease preparation was evaluated by storage at 4°C, as follows.

[0037] 1. Detection of initial nuclease activity

[0038] The initial enzyme activity was detected according to the method specified in GB / T 34222-2017 and recorded as U.

[0039] 2. Moist heat treatment

[0040] Weigh 2.0g of the enzyme sample to be tested and spread it in a plate (9cm in diameter) in three parallel portions, then place it in a hollow test tube basket, place the test tube basket in a stable constant temperature and humidity chamber (75°C, relative humidity 95%), accurately time 5min, take it out and cool it for use. Transfer all the samples in the plate to a conical flask (record the mass), extract with 100mL buffer for 30min, let it stand for 10min, and the supernatant is the enzyme solution to be tested. Subsequently, the enzyme activity test was performed according to the method specified in GB / T 34222-2017, recorded as U1.

[0041] Wet heat box temperature resistance retention rate K1 = (U1 / U) × 100%

[0042] Where: K1: thermotolerant enzyme activity retention rate; U: original enzyme activity; U1: thermotolerant enzyme activity

[0043] 3. Long-term storage stability

[0044] The nuclease powder was packaged in aluminum foil bags of 200 g each, and three parallel samples were sealed and stored in a refrigerator at 4°C for one year. The enzyme activity U2 was measured and the long-term storage retention rate was calculated.

[0045] Retention rate: K2 = (U2 / U) × 100%

[0046] Where: K2: long-term storage retention rate; U: original enzyme activity; U2: enzyme activity after one year of storage

[0047] The test results are shown in Table 1, where the groups of tested samples are:

[0048] Control group 1-3: 1000 ml of nuclease fermentation liquid was mixed with 200 g of wheat starch and divided into three parts. The pH of the three mixed solutions was adjusted to 4.0 (control group 1), 4.5 (control group 2) and 5.0 (control group 3) with citric acid, and the mixed solutions were dried to obtain samples to be tested.

[0049] Control groups 4-6: 1000 ml of nuclease fermentation liquid was mixed with 200 g of corn starch and divided into three portions. The pH of the three mixed solutions was adjusted to 4.0 (control group 4), 4.5 (control group 5) and 5.0 (control group 6) with citric acid, and the mixed solutions were dried to obtain samples to be tested.

[0050] Control groups 7-9: 1000 ml of nuclease fermentation liquid was mixed with 200 g of cassava starch and divided into three portions. The pH of the three mixed solutions was adjusted to 4.0 (control group 7), 4.5 (control group 8) and 5.0 (control group 9) using citric acid, and the mixed solutions were dried to obtain samples to be tested.

[0051] Blank control group 1: Use citric acid to adjust the pH of the nuclease fermentation liquid to 4.0, and dry the mixed liquid to obtain the sample to be tested.

[0052] Blank control group 2: Use citric acid to adjust the pH of the nuclease fermentation liquid to 4.5, and dry the mixed liquid to obtain the sample to be tested.

[0053] Blank control group 3: Use citric acid to adjust the pH of the nuclease fermentation liquid to 5.0, and dry the mixed liquid to obtain the sample to be tested.

[0054] Experimental group 1: Sample 1 prepared in Example 1.

[0055] Experimental group 2: Sample 2 prepared in Example 1.

[0056] Experimental group 3: Sample 3 prepared in Example 1.

[0057] Experimental group 4: Sample 4 prepared in Example 2.

[0058] Experimental group 5: Sample 5 prepared in Example 2.

[0059] Experimental group 6: sample 6 prepared in Example 2.

[0060] Experimental group 7: Sample 7 prepared in Example 3.

[0061] Experimental group 8: sample 8 prepared in Example 3.

[0062] Experimental group 9: sample 9 prepared in Example 3.

[0063] Table 1 Nuclease temperature resistance and storage retention rate - undigested carrier

[0064]

[0065]

[0066] As can be seen from Table 1, the 75°C heat-resistant retention rate and long-term storage retention rate are different under different carriers and different pH conditions. With the increase of pH value, both the heat-resistant retention rate and storage stability are improved. The product with added carrier is significantly better than the product without addition and spray-dried directly, and the effect of the carrier after enzymatic hydrolysis is better than the product without enzymatic hydrolysis. The stable nuclease preparation obtained by mixing the auxiliary agent for improving the stability of nuclease provided by the present invention with the nuclease fermentation broth can have a retention rate of more than 90% when stored at 4°C for one year, and at the same time has other excellent enzymatic properties. The retention rate after treatment at 75°C for 5 minutes is more than 95%. It is not easy to be inactivated at high temperature, and it is convenient to produce and transport. It is suitable for high-temperature production processes, which effectively improves the application prospects of nucleases.

[0067] The above examples are merely illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. All designs that are the same or similar to the present invention fall within the protection scope of the present invention.

Claims

1. An auxiliary agent for improving the stability of nuclease, characterized in that The invention comprises a carrier and a nuclease fermentation liquid; the addition amount of the nuclease fermentation liquid is 0.01-0.1% of the mass of the carrier; the nuclease fermentation liquid is a fermentation liquid obtained by producing nuclease by Pichia pastoris; the carrier comprises at least one of wheat starch, corn starch and cassava starch; the auxiliary agent is prepared by the following steps: weighing the carrier, adding the nuclease fermentation liquid thereto and then adding water to degrade the carrier to obtain the auxiliary agent.

2. The auxiliary agent for improving the stability of nuclease according to claim 1, characterized in that: The degradation temperature is 25-40℃ and the degradation time is 16-24h.

3. The auxiliary agent for improving the stability of nuclease according to claim 1, characterized in that: After adding water, the moisture content of the mixture is 25-60%.

4. A stable nuclease preparation, characterized in that: The invention comprises a nuclease and the auxiliary agent for improving the stability of the nuclease as claimed in claim 1.

5. The method for preparing a stable nuclease preparation according to claim 4, characterized in that: The following steps are involved: The nuclease fermentation liquid is mixed with the auxiliary agent for improving the stability of the nuclease, the pH of the mixed liquid is adjusted, and the mixed liquid is dried to obtain the stable nuclease preparation.

6. The method for preparing a stable nuclease preparation according to claim 5, characterized in that: The mass ratio of the nuclease fermentation broth to the auxiliary agent for improving the stability of the nuclease is (1-5):

1.

7. The method for preparing a stable nuclease preparation according to claim 5, characterized in that: Adjust the pH of the mixture to 4.0-5.2.

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