Composite neutral protease stabilizer and its application

Abstract

The present invention relates to a composite protease stabilizer and its application, and in particular to a composite neutral protease stabilizer and its application. The composite neutral protease stabilizer is prepared by using an effective amount of Na2SO4, peptone and Aprotinin. The present invention solves the problems of low enzyme activity preservation rate of neutral protease JW-3 existing in the prior art. The present invention has the advantages of raw material source and simple and easy preparation, and the enzyme activity preservation rate after being applied to neutral protease JW-3 is significantly better than that of existing products and literature reports.

Description

Technical Field

[0001] The present invention relates to a composite protease stabilizer and application thereof, in particular to a composite neutral protease stabilizer and application thereof. Background Art

[0002] Neutral protease is a kind of endonuclease composed of endopeptidases. It is a general term for enzymes that can catalyze protein hydrolysis to produce free amino acids and polypeptide fragments in a neutral or weakly acidic or weakly alkaline environment. Its optimal pH value is 6.0-7.5. Neutral protease has mild action conditions and high catalytic efficiency. It is widely used in food, medicine, leather, feed, chemical industry and waste treatment.

[0003] At present, commercial neutral protease preparations are mainly in the form of solid powder. Although the powder form is convenient to store and transport, the neutral protease is prone to autolysis, and even if it is freeze-dried at low temperature, a large amount of enzyme activity will be lost. Due to the limitations of external conditions (high temperature and high humidity, etc.), liquid protease has great problems in storage stability during transportation, which are mainly manifested in flatulence during long-distance transportation, a sharp drop in enzyme activity under high temperature conditions, a drop in enzyme activity of the enzyme preparation due to long storage time, and turbidity.

[0004] There are very few types of liquid neutral proteases on sale in China, all of which are imported products and are expensive. Bacillus subtilis, whose Latin name is Bacillus subtilis and whose preservation number is CGMCC No.14837, is an engineered bacterium for depilation constructed by the applicant through genetic recombination, and the final product produced by its fermentation is neutral protease JW-3.

[0005] It is well known to those skilled in the art that borax can better slow down the loss of enzyme activity under storage conditions of 25°C to 40°C. The applicant's experiment confirmed that the enzyme activity preservation rate of neutral protease JW-3 after adding borax stabilizer and storing at a temperature of 40°C for 2 days was about 60%; the enzyme activity preservation rate of neutral protease JW-3 without adding any protective agent was about 70% after storing at a temperature of 40°C for 2 days. The patent document with publication number CN108048441A discloses a composite stabilizer for stabilizing protease in laundry detergent, the stabilizer comprises calcium ammonium citrate and alkyl glycoside surfactant, and the enzyme activity preservation rate reaches 84.3% and 71.5% after storing at 37°C for 3 weeks under the condition of adding composite stabilizer. However, the stability of neutral protease JW-3 in calcium ammonium citrate and alkyl glycoside surfactant is poor, and the enzyme activity preservation rate is about 60% after storing at a temperature of 37°C for 3 weeks. Patent document with publication number CN108977428A discloses a bromelain stabilizer, a preparation method and an application thereof. The stabilizer is composed of sodium D-isoascorbate, L-cysteine, disodium ethylenediaminetetraacetic acid, sodium acetate, potassium sorbate and vitamin B9, but the enzyme activity preservation rate of neutral protease JW-3 under this component is about 60%. Summary of the invention

[0006] The object of the present invention is to provide a composite neutral protease stabilizer and application thereof, which can effectively solve the problem of low enzyme activity preservation rate of neutral protease JW-3.

[0007] The overall technical concept of the present invention is:

[0008] Composite neutral protease stabilizer, including raw materials per unit mass:

[0009] Na2SO4 10~20; Peptone 2~10; Aprotinin 60~200U / ml; pH=6.0-7.0.

[0010] Application of composite neutral protease stabilizer in the preparation of neutral protease preparations.

[0011] The specific technical features of the present invention also include:

[0012] The preferred technical solution is that Aprotinin is produced by Beijing Solebow Technology Co., Ltd., and its CAS number is 90087-70-1 and the product number is A8260.

[0013] The preferred technical solution is that the pH is adjusted using NaOH or HCl.

[0014] The application of the composite neutral protease stabilizer in the preparation of a neutral protease preparation comprises the following steps:

[0015] A. Select neutral protease JW-3 prepared by fermentation of Bacillus subtilis, whose Latin name is Bacillus subtilis and whose deposit number is CGMCC No.14837, centrifuge at high speed to obtain the supernatant, filter it, and dilute it to prepare a protease aqueous solution with an enzyme activity of 2000U / mL;

[0016] B. Add Na2SO4 at a ratio of 10% to 20% of the mass of the protease aqueous solution, add peptone at a ratio of 2% to 10% of the mass of the protease aqueous solution, add Aprotinin at a ratio of 60 to 200 U / mL, and adjust the pH of the mixture to 6.0-7.0.

[0017] In the step A, the speed of high-speed centrifugation is 7000 rpm and the time is 15 minutes.

[0018] In the step A, No. 10 diatomaceous earth is used for filtration.

[0019] In order to further determine the functional indicators of the composite neutral protease stabilizer obtained by the method of the present invention and conduct a more comprehensive and scientific evaluation of the product, the applicant conducted the following tests:

[0020] 1. Accelerated temperature test

[0021] The inactivation rate of proteases at different temperatures is different. As the temperature rises, the inactivation rate accelerates. In order to shorten the test time, the present invention adopts a temperature acceleration test, that is, by examining the inactivation of the enzyme at 40°C to infer the stability of the enzyme solution at 25°C. To this end, the enzyme activity and relative enzyme activity of the enzyme after being stored at 25°C and 40°C for 48 hours were measured respectively.

[0022] Temperature / ℃ <![CDATA[Enzyme activity / U.mL -1 > Enzyme activity preservation rate / % 40 496.63±13.61 34.85±0.96 25 1330.76±24.03 93.38±1.69

[0023] Note: Initial enzyme activity is 1425.14±40.04U.mL -1

[0024] 1. Determination of inactivation rate constant Kd

[0025] When no substrate is added to the enzyme solution, the inactivation model assumes that the active enzyme molecule [Ea] becomes an inactive protein [Ei] through irreversible conformational or chemical changes. The inactivation reaction is a first-order reaction, that is, the enzyme inactivation rate is proportional to the active enzyme concentration [Ea]:

[0026] d Ea / d t =-Kd[Ea] (1)

[0027] From (1), we can get: K d =-1 / t*ln(E a,t / Ea,0 ) (2)

[0028] Where: K d ——Inactivation rate constant (1 / h)

[0029] E a,0 、E a,t ——Enzyme activity at 0 and t hours respectively (U / mL)

[0030] t——time of inactivation process (h)

[0031] From formula (2) and the table data, we can get:

[0032] K at 25℃ d,298 =1.42×10 -3 / h40℃K d,313 =2.20×10 -2 / h

[0033] (298 and 313 are the absolute temperatures at 25°C and 40°C respectively)

[0034] 2. Determination of activation energy ΔE

[0035] From the Arrhenius equation: K d =Ae -ΔE / RT (3)

[0036] We can get: ln(K d,T2 / K d,T )=ΔE / R×(1 / T1-1 / T2) (4)

[0037] Where: ΔE——deactivation activation energy (KJ / mol)

[0038] R——gas constant (8.31 J / K·mol)

[0039] T——absolute temperature (K)

[0040] A——Frequency factor (1 / h)

[0041] Substitute K d,298 , K d,313 The value of inactivation activation energy ΔE (25-40°C) of the enzyme was calculated to be 141.61 KJ / mol.

[0042] From (3), we can get the frequency factor A = 9.707 × 10 -21 / h

[0043] (3) Inactivation time t 40 From formula (2), we can know that the time required to achieve the same enzyme inactivation rate at 40℃ and 25℃ has the following relationship: t 40 =t25 ×(K d,298 / K d,313 ) (5)

[0044] According to the light industry standard "Industrial Protease Preparation" (QB1805.3-1993), liquid protease is qualified if the enzyme activity retention rate is ≥ 80% when stored at 25°C for 3 months, i.e. 90 days. The present invention adopts a 40°C temperature rise test, so the corresponding storage time t 40 =139.42h≈139h, that is, the result obtained by storing at 40℃ for 139 hours is approximately equivalent to the enzyme activity retention rate stored at 25℃ for 3 months. In addition, since the activation energy of enzyme inactivation ΔE increases after adding stabilizer, the ln(K d,298 / K d,313 ) value will be larger than when no stabilizer is added, that is, the storage time at 40℃ after adding stabilizer is less than h. However, in order to ensure that the selected formula still meets the national standard requirements when stored at 25℃ for 3 months, in the subsequent longer-term storage test, the enzyme activity retention rate under accelerated experimental conditions (40℃, insulation for 139h) is still used as the criterion for whether the stability of the standard liquid enzyme meets the standard.

[0045] 2. Detection of enzyme activity preservation rate

[0046] The method for determining the activity of protease is based on the method in Appendix B of GB / T 23527-2009 Protease Preparations. The calculation formula for the enzyme activity preservation rate is: w(%) = A i / A0×100(w: enzyme activity preservation rate; A i : Enzyme activity measured after storage at 40°C for 139 h; A0: initial enzyme activity).

[0047] The substantial features and significant technical advances achieved by the present invention are:

[0048] The raw material composition and preparation method of the neutral protease stabilizer prepared by the present invention are simple and easy to implement. After being applied to the neutral protease JW-3, the enzyme activity preservation rate is significantly better than the existing products or literature reports known to the applicant. DETAILED DESCRIPTION

[0049] The present invention is further described below in conjunction with embodiments, but it should not be construed as limiting the present invention. The protection scope of the present invention shall be based on the contents recorded in the claims, and any replacement of equivalent technical means made according to the description shall not depart from the protection scope of the present invention.

[0050] Example 1

[0051] The neutral protease JW-3 prepared by fermentation of Bacillus subtilis with the Latin name of Bacillus subtilis and the preservation number of CGMCC No.14837 was selected, the supernatant was taken after centrifugation at 7000 rpm for 15 minutes, filtered through No. 10 diatomaceous earth, diluted to an enzyme activity of 2000 U / mL, Na2SO4 was added at a ratio of 10% by mass of the protease aqueous solution, peptone was added at a mass of 2% by mass of the protease aqueous solution, Aprotinin was added at an addition amount of 60 U / mL, and the pH of the mixed solution was adjusted to 6.0-7.0 with NaOH or HCl.

[0052] Example 2

[0053] The difference between this embodiment and embodiment 1 is that:

[0054] Na2SO4 was added at a ratio of 20% by mass of the protease aqueous solution, peptone was added at a ratio of 10% by mass of the protease aqueous solution, Aprotinin was added at a ratio of 200 U / mL, and the pH of the mixture was adjusted to 6.0-7.0 with NaOH or HCl.

[0055] The rest of the contents are the same as those in Example 1.

[0056] Example 3

[0057] The difference between this embodiment and embodiment 1 is that:

[0058] Na2SO4 was added at a ratio of 15% by mass of the protease aqueous solution, peptone was added at a ratio of 6% by mass of the protease aqueous solution, Aprotinin was added at a ratio of 130 U / mL, and the pH of the mixture was adjusted to 6.0-7.0 with NaOH or HCl.

[0059] The rest of the contents are the same as those in Example 1.

[0060] Example 4

[0061] The difference between this embodiment and embodiment 1 is that:

[0062] Na2SO4 was added at a ratio of 15% by mass of the protease aqueous solution, peptone was added at a ratio of 4% by mass of the protease aqueous solution, Aprotinin was added at a ratio of 90 U / mL, and the pH of the mixture was adjusted to 6.0-7.0 with NaOH or HCl.

[0063] The rest of the contents are the same as those in Example 1.

[0064] Example 5

[0065] The difference between this embodiment and embodiment 1 is that:

[0066] Na2SO4 was added at a ratio of 16% by mass of the protease aqueous solution, peptone was added at a ratio of 8% by mass of the protease aqueous solution, Aprotinin was added at a ratio of 150 U / mL, and the pH of the mixture was adjusted to 6.0-7.0 with NaOH or HCl.

[0067] The rest of the contents are the same as those in Example 1.

[0068] The applicant tested the products involved in Examples 1 to 5 of the present application according to the determination method of enzyme activity preservation rate recorded in this specification, as follows:

[0069] Testing location: The Third Research Laboratory of Hebei Institute of Microbiology Co., Ltd.

[0070] Tester: Wu Fangtong

[0071] The test results are as follows:

[0072] The neutral protease JW-3 liquid preparations obtained in Examples 1 to 5 were stored at 40°C for 139 hours, and the enzyme activity preservation rates were measured and shown in Table 1. Enzyme preparations are easily affected by stability, pH, etc., resulting in a sharp drop in enzyme activity. Adding a protective agent can significantly improve the stability of the neutral protease JW-3.

[0073] Table 1 Protease activity preservation rate

[0074] Test batch Enzyme activity preservation rate (%) Neutral protease JW-3 without adding protective agent 23.41±0.20％ Example 1 93.02±0.24％ Example 2 82.27±2.01％ Example 3 84.30±1.32％ Example 4 93.33±1.64％ Example 5 85.76±0.88％

Claims

1. A composite neutral protease stabilizer suitable for neutral protease JW-3, characterized in that Including raw materials per unit mass: Na2SO4 10~20; Peptone 2~10; Aprotinin 60~200U / ml; pH=6.0-7.0; The neutral protease JW-3 is prepared from Bacillus subtilis (CGMCC No. 14837). Bacillus subtilis ) Fermentation preparation.

2. The composite neutral protease stabilizer suitable for neutral protease JW-3 according to claim 1, characterized in that Aprotinin is produced by Beijing Solebow Technology Co., Ltd., with CAS number 90087-70-1 and product number A8260.

3. The composite neutral protease stabilizer suitable for neutral protease JW-3 according to claim 1, characterized in that The pH was adjusted with NaOH or HCl.

4. Use of the composite neutral protease stabilizer suitable for neutral protease JW-3 according to any one of claims 1 to 3 in the preparation of neutral protease JW-3, wherein the neutral protease JW-3 is fermented and prepared by Bacillus subtilis with a deposit number of CGMCC No. 14837.

5. The use according to claim 4, characterized in that The steps include: A. Choose the Latin name Bacillus subtilis, The neutral protease JW-3 prepared by fermentation of Bacillus subtilis with a deposit number of CGMCC No. 14837 was centrifuged at high speed to obtain the supernatant, filtered, and diluted to prepare a protease aqueous solution with an enzyme activity of 2000 U / mL; B. Add Na2SO4 at a ratio of 10% to 20% of the mass of the protease aqueous solution, add peptone at a ratio of 2% to 10% of the mass of the protease aqueous solution, add Aprotinin at a ratio of 60 to 200 U / mL, and adjust the pH of the mixture to 6.0-7.

0.

6. The use according to claim 5, characterized in that In the step A, the speed of high-speed centrifugation is 7000 rpm and the time is 15 minutes.

7. The use according to claim 5, characterized in that In the step A, No. 10 diatomaceous earth is used for filtration.