阅读说明：本技术 一种芽孢杆菌来源碱性蛋白酶的新型抑制肽 (Novel inhibitory peptide of bacillus-derived alkaline protease ) 是由 路福平 王洪彬 李雪 李奕鸣 于 2021-02-20 设计创作，主要内容包括：本发明涉及芽孢杆菌来源碱性蛋白酶的竞争性抑制剂,发明获得了一种新型抑制肽,该抑制肽及其衍生物对芽孢杆菌来源碱性蛋白酶具有明显的抑制作用,在芽孢杆菌来源碱性蛋白酶的液体酶制剂中添加该抑制肽,蛋白酶活力保存的稳定性明显增强。该抑制剂在环保和安全性上具有明显的优势,有望应用于液体加酶洗涤剂领域,替代传统的硼砂类抑制剂。(The invention relates to a competitive inhibitor of bacillus-derived alkaline protease, and obtains a novel inhibitory peptide, wherein the inhibitory peptide and derivatives thereof have obvious inhibitory action on the bacillus-derived alkaline protease, and the stability of protease activity preservation is obviously enhanced by adding the inhibitory peptide into a liquid enzyme preparation of the bacillus-derived alkaline protease. The inhibitor has obvious advantages in environmental protection and safety, is expected to be applied to the field of liquid enzyme-added detergents, and replaces the traditional borax inhibitor.)

1. An inhibitory peptide of alkaline protease derived from Bacillus, characterized by the sequence R₁-A-A-P-F-R₂Wherein A is alanine, P is proline, F is phenylalanine, R₁Represents N-terminal having no or a protecting group, R₂Representing no or containing group modification at the C-terminal.

2. The inhibitory peptide of claim 1, preferably the N-terminal modification is an acylated protecting group.

3. The inhibitory peptide of claim 1, preferably, the C-terminal carboxyl group is modified by hydroformylation.

4. A liquid enzyme composition preparation comprising an alkaline protease derived from Bacillus, characterized by comprising the inhibitory peptide according to claim 1.

Technical Field

The invention belongs to the field of enzyme engineering, and particularly relates to inhibitory peptide of alkaline protease derived from bacillus.

Background

The alkaline protease from the spore bacteria has the highest ratio in industrial protease preparations due to the advantages of strong hydrolysis capacity, high production efficiency and the like, and is widely applied to the industries of washing, food, textile and the like. Protease formulations are divided into solid and liquid formulations, with liquid formulations being increasingly used, especially in the liquid enzyme-containing detergent industry. With the continuous development of the industry of Chinese washing products, the industrial structure of the Chinese washing products is greatly changed, and the market proportion of the liquid detergent is increased year by year. The bacillus-derived microbial alkaline protease is a main enzyme for adding a liquid detergent, has high dirt-removing capacity under alkaline conditions, and has the best effect on protein dirt such as blood stains, sweat stains, oil stains and the like. Since proteases hydrolyze proteins and hydrolyze enzymes in liquid systems, alkaline proteases derived from spores need to be added to a stable system product of liquid formulations or liquid detergents to inhibit their activity so as to maintain their stability during storage. The protease inhibitor widely used in the industry of the liquid enzyme-containing detergent at present is borax, is not friendly to human health and environment, and has potential reproductive toxicity. With the environmental protection concept being deepened gradually, the development of the alkaline protease inhibitor which is green, environment-friendly and high in safety has very important significance. The polypeptide inhibitor has higher advantages in environmental protection and safety, and is expected to become the best substitute of the existing inhibitor in the fields of liquid enzyme and liquid enzyme-added detergent.

Disclosure of Invention

The object of the present invention is to find novel polypeptide inhibitors for bacillus-derived alkaline proteases. The invention researches and discovers that the sequence is characterized by R₁-A-A-P-F-R₂The short peptide has obvious inhibition and stabilization effects on bacillus-derived alkaline protease, wherein A is alanine; p is proline; f is phenylalanine; r₁Represents N-terminal having no or a protecting group, R₂Representing no or containing group modification at the C-terminal. Preferably, the N-terminal protecting group is an acyl protecting group such as succinyl (Suc), acetyl (Ac) and the like, and the C-terminal carboxyl group is modified by hydroformylation (-H), so that the effects of inhibiting and stabilizing the enzyme activity are better. The stability of the preservation of the activity of the protease in the liquid enzyme composition preparation containing the bacillus-derived alkaline protease is significantly enhanced by adding the inhibitory peptide of the present invention.

The invention proves that the technical scheme of the novel polypeptide inhibitor for inhibiting and stabilizing bacillus alkaline protease is as follows:

1. determining and comparing the inhibition rate of different inhibitors on enzyme activity:

(1) preparing enzyme solution of bacillus alkaline protease.

(2) On the basis of enzyme solution, different additions are respectively carried out, and the experiment is divided into a blank group, a control group and an experiment group. Buffer solution is added to a blank group, enzyme solution of 4-formylphenylboronic acid (4-FPBA) is added to a control group, and polypeptides AAPF, Suc-AAPF and AAPF-H, Suc-AAPF-H are respectively added to an experimental group. 4-FPBA is a competitive inhibitor for alkaline protease from spore bacteria at present.

(3) The activity of the alkaline protease is determined according to the national standard GB/T23527-2009, the inhibition rate of the enzyme activity is calculated, and the inhibition performances of different inhibitors are compared.

2. Determination and comparison of the stability of different inhibitors towards enzyme activity:

(1) preparing stabilizer (CaCl as main component)₂Glycerol, propylene glycol, etc., and does not contain an enzyme activity inhibitor).

(2) On the basis of enzyme solution containing a stabilizing agent, different additions are respectively carried out, and experiments are divided into a blank group, a control group and an experiment group. Buffer solution is added to a blank group, 4-formylphenylboronic acid (4-FPBA) is added to a control group, and AAPF, Suc-AAPF and AAPF-H, Suc-AAPF-H are respectively added to an experimental group. All solutions were sealed and incubated in an incubator at 37 ℃ and samples were taken at different time points to determine the residual enzyme activity.

(3) Calculating the enzyme activity retention rates at different time points, and comparing the stability performance of different inhibitors on the alkaline protease.

The invention has the beneficial effects that:

(1) the inventive inhibitory peptide has certain inhibitory and stabilizing effects on alkaline protease from spore, and can be used for compounding liquid enzyme preparation and prolonging the stability of liquid enzyme.

(2) The inhibitory peptide can replace borax inhibitors in liquid enzyme-containing detergents and has higher safety on environment and human health.

Drawings

FIG. 1, inhibitory Effect of different inhibitors on subtilisin

FIG. 2, stabilization of subtilisin by different inhibitors in a stabilizer matrix

Detailed Description

The technical content of the present invention is further illustrated by the following examples, but the present invention is not limited to these examples, and the following examples should not be construed as limiting the scope of the present invention.

Example 1: inhibitory Effect of inhibitory peptides on Bacillus alkaline protease

1. A mother liquor of 100mM 4-FPBA, Suc-AAPF or Suc-AAPF-H was prepared using a boric acid buffer solution (pH 10.5).

2. An enzyme solution of alkaline protease derived from Bacillus clausii was diluted with a boric acid buffer solution (pH 10.5), and then 2mM of 4-FPBA, inhibitory peptides AAPF and Suc-AAPF-H were added, respectively, and no inhibitor was added to the blank group. The enzyme activity of the alkaline protease is determined according to the national standard GBT23527-2009, and then the enzyme activity inhibition rate is calculated.

3. The result of enzyme activity inhibition is shown in FIG. 1, under the condition of 2mM of the addition concentration, the enzyme activity inhibition rate of Suc-AAPF to alkaline protease is 42%, while the inhibition rate of Suc-AAPF-H is 46%, which reaches 84% of the positive control 4-FPBA inhibition rate (55%). Suc-AAPF-H, Suc-AAPF all has inhibitory activity to bacillus derived alkaline protease, and the inhibitory activity is also enhanced after hydroformylation modification.

Example 2: influence of inhibitory peptides on enzyme activity retention rate of bacillus alkaline protease

1. A boric acid buffer (pH 10.5) was used to prepare 100mM stock solutions of 4-FPBA, Suc-AAPF and Suc-AAPF-H.

2. Preparation of a stabilizer (5mM CaCl)₂3% glycerol, 7% propylene glycol) was added to the bacillus alkaline protease solution to a final concentration of 2mM, 4-FPBA, Suc-AAPF, and Suc-AAPF-H, respectively. Buffer was added to the blank. All solutions were sealed and incubated in an incubator at 37 ℃ and samples were taken at different time points to determine the residual enzyme activity.

3. The trend of the residual enzyme activity with time is shown in FIG. 2. As can be seen from the figure, the enzyme activity of the blank group without any inhibitor is reduced fastest, the enzyme activity of the 4-FPBA group is reduced slowest, the effect of the Suc-AAPF-H group is obviously better than that of the blank group and is also better than that of the Suc-AAPF group, and the aldehyde modification can enhance the stabilizing effect of the inhibitory peptide on the enzyme activity. Although the enzyme activity retention effect of Suc-AAPF-H is not as good as that of 4-FPBA under the condition of 2mM addition, the enzyme activity of the alkaline protease has obvious effect on stabilizing the enzyme activity of the alkaline protease, and the stabilizing effect can be enhanced by increasing the use concentration in practical application.