CN112760313B

CN112760313B - Complex enzyme stabilizing system and application thereof in cleaning of medical instruments

Info

Publication number: CN112760313B
Application number: CN202011638219.6A
Authority: CN
Inventors: 张小玉; 赵彰; 吴冬梅
Original assignee: Shanghai Dai Di Biotechnology Co ltd; Zaozhuang Maternal And Child Health Hospital
Current assignee: Shanghai Dai Di Biotechnology Co ltd; Zaozhuang Maternal And Child Health Hospital
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2022-10-28
Anticipated expiration: 2040-12-31
Also published as: CN112760313A; CN115109772A

Abstract

The invention provides a complex enzyme stabilizing system and application thereof in cleaning of medical instruments, and particularly relates to a short peptide stabilizer capable of improving the stability of protease and lipase, which is obtained through a large number of tests after high-throughput screening is carried out through computer simulation according to the three-dimensional structure information of enzyme, so that the complex enzyme stabilizing system and a complex enzyme cleaning agent are developed.

Description

Complex enzyme stabilizing system and application thereof in cleaning of medical instruments

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a complex enzyme stabilizing system and application thereof in cleaning of medical instruments.

Background

For medical instruments which are repeatedly used, strict cleaning and sterilization work is required. In the using process, blood, secretion and the like of a patient can cause serious pollution to medical instruments, and if the cleaning and the disinfection are not thorough, serious secondary pollution and even medical accidents can be caused when the medical instruments are used again. For example, the equipment used in obstetrics in hospitals is not only large in number but also complex in structure. These obstetric instruments must be rigorously cleaned and sterilized after use before they can be reused. In the cleaning and disinfection process, the early cleaning work is particularly important. The early-stage cleaning of the obstetrical apparatus is not thorough, a biological film is easily formed, the biological film can seriously influence the sterilization effect, and the occurrence of cross infection is caused.

The cleaning agent used in the cleaning of the medical apparatus at present comprises a chemical cleaning agent and a compound enzyme cleaning agent. The compound enzyme cleaning agent has mild action, can be used for automatic cleaning equipment and hand washing operation, is made of completely biodegradable materials, has no residue after washing, and has no corrosion and aging effect on various precise instruments and medical appliances. Therefore, complex enzyme cleaning agents are increasingly used in medical systems.

The general compound enzyme cleaning agent contains protease, lipase, cellulase, amylase and the like, and can effectively decompose and remove pollutants adhered to medical instruments. Wherein, the protease and the lipase are main enzyme species in the compound enzyme cleaning agent. Because the protease is characterized by decomposing protein, the protease and other enzymes can degrade other enzymes when compounded together, and even the protease has certain self-degradation, so that the application effect of the compound enzyme cleaning agent is unstable, and even the compound enzyme cleaning agent is completely ineffective. Therefore, it is very important to ensure the stable existence of various enzymes in the complex enzyme cleaning agent.

In addition, a certain content of surfactant is required to be prepared in the compound enzyme cleaning agent so as to improve the cleaning effect of dirt. However, these surfactants, especially anionic surfactants (such as sodium linear alkylbenzene sulphonate) can significantly affect the activity of the enzyme. The anionic surfactant has strong ionic strength and divalent metal ion chelating capacity, so that the surface charge and the space structure of the biological enzyme molecule are changed violently, and the enzyme loses catalytic action due to the change of tertiary conformation. Therefore, the problem of compatibility between the enzyme and the surfactant also needs to be considered in the complex enzyme cleaning agent.

Therefore, technical personnel in the field are dedicated to developing complex enzyme cleaning agent products with stable enzyme activity, convenient use and storage, long quality guarantee period and high cleaning efficiency.

Disclosure of Invention

The invention aims to provide a complex enzyme stabilizing system and application thereof in cleaning of medical instruments.

In a first aspect of the present invention, there is provided a use of a short peptide as a stabilizer for a complex enzyme, the short peptide being one or more short peptides selected from the group consisting of: short peptide VST, short peptide PST, and short peptide FPR.

In another preferred embodiment, the short peptide is one or more short peptides selected from the group consisting of: short peptide PST, and short peptide FPR.

In another preferred embodiment, the complex enzyme comprises protease and lipase.

In another preferred embodiment, the compound enzyme also comprises cellulase.

In another preferred embodiment, the compound enzyme also comprises amylase.

In another preferred embodiment, the protease is a protease prepared by fermentation of strain Aspergillus niger ATCC 16404.

In a second aspect of the invention, there is provided a complex enzyme stabilizing system comprising one or more short peptides selected from the group consisting of: short peptide VST, short peptide PST, and short peptide FPR.

In another preferred embodiment, the complex enzyme stabilizing system comprises short peptide PST and/or short peptide FPR.

In another preferred embodiment, the complex enzyme stabilizing system further comprises: sodium sulfate, calcium chloride, and glycerol.

In a third aspect of the invention, a complex enzyme is provided, wherein the complex enzyme comprises protease, lipase and one or more short peptides selected from the following group: short peptide VST, short peptide PST, and short peptide FPR.

In another preferred embodiment, the complex enzyme comprises short peptide PST and/or short peptide FPR.

In another preferred embodiment, the compound enzyme also comprises cellulase.

In another preferred embodiment, the compound enzyme also comprises amylase.

In another preferred example, the enzyme activity of the protease in the compound enzyme is 5000-100000U/ml.

In another preferred embodiment, the enzyme activity of the lipase in the compound enzyme is 1000-50000U/ml.

In another preferred embodiment, the mass fraction of the short peptide in the compound enzyme is 0.01-0.5%.

In another preferred embodiment, the complex enzyme also comprises sodium sulfate, calcium chloride and glycerol.

The fourth aspect of the invention provides a compound enzyme cleaning agent, which comprises the compound enzyme of the third aspect of the invention and a surfactant.

In another preferred embodiment, the surfactant is selected from one or more of the following group: anionic surfactants, cationic surfactants, and nonionic surfactants.

In another preferred embodiment, the surfactant consists of an anionic surfactant Triton H-66, a surfactant Plurafac LF221, and sodium fatty alcohol polyoxyethylene ether sulfate.

The fifth aspect of the invention provides the application of the compound enzyme cleaning agent of the fourth aspect of the invention in cleaning medical instruments.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1: example 2 product appearance after 2 months of storage at 35 ℃ in experimental group 1;

FIG. 2 is a schematic diagram: example 2 product appearance after 2 months of storage at 35 ℃ in experimental group 2;

FIG. 3: example 2 product appearance after 2 months of storage at 35 ℃ in experimental group 3;

FIG. 4: example 2 product appearance after storage of the control group for 2 months at 35 ℃.

Detailed Description

In the existing complex enzyme stabilizing system, a boron-containing stabilizer is generally used as a reversible inhibitor of protease, so that the protease can be stabilized in a liquid complex enzyme preparation. For example, CN201811046808.8 incorporates boron-containing stabilizers for protease stabilization. Boron-containing stabilizers, however, have potential reproductive toxicity and should therefore be avoided in medical device cleaning.

It has been reported (for example CN 201710162091.2) that some short peptides have a certain stabilizing effect on protease. However, in the research, the stabilizing effect of the short peptide stabilizer on different types of protease is found to be greatly different. In addition, document CN201710779678.8 discloses that the use of an active short peptide can improve the stability, enzyme activity, and heat resistance of lipase, but the active short peptide sequence used therein contains 27 amino acid residues, which results in high synthesis cost.

In a preferred embodiment of the invention, the protease used in the invention is prepared by fermentation of Aspergillus niger ATCC 16404 strain, which is commercially available from Biotechnology Ltd, cangzhou Xia Chengmei. Aspergillus niger has food-grade production permission approved by FDA in the United states, and the protease expression amount is considerable, which is not possessed by many other integrated expression systems (such as pichia pastoris expression systems and the like), thereby providing quality guarantee for the application of the protease obtained by Aspergillus niger fermentation in the fields of food and medical treatment.

In a preferred embodiment of the present invention, the lipase used in the present invention is alkaline lipase GFY-3310 manufactured by Xia Chengmei, cangzhou, biotechnology Ltd.

In a preferred embodiment of the invention, the amylase used in the invention is food grade fungal alpha-amylase FDY-2247, produced by Biotechnology, inc., xia Chengmei, cangzhou.

In a preferred embodiment of the present invention, the cellulase used in the present invention is food grade cellulase (neutral) FDY-2236, manufactured by Cangzhou Xia Chengmei Biotechnology Inc.

The enzyme activity detection of protease and amylase is carried out according to GB 1886.174-2016.

Protease activity is defined as: hydrolyzing casein for 1min at 40 deg.C and pH of 7.5 to generate 1 microgram of tyrosine, which is an enzyme activity unit.

Amylase (α -amylase) enzyme activity is defined as: 1g of solid enzyme powder (or 1ml of liquid enzyme) is liquefied at 60 ℃ for 1 hour to obtain 1g of soluble starch, namely an enzyme activity unit.

The detection of the cellulase activity is carried out according to Q/NXM 026-2018. The enzyme activity is defined as: . The amount of enzyme required to hydrolyze sodium carboxymethylcellulose at 50 ℃ and pH 6.0 per minute to produce 1mol of reducing sugar (in terms of glucose) is defined as one unit of enzyme activity.

Detection of lipase activity is carried out according to GB/T23535-2009. Hydrolyzing the substrate for 1min at 40 ℃ and pH of 7.5 to generate 1 micromole of titratable fatty acid, namely an enzyme activity unit.

The main advantages of the present invention include:

(1) The short peptide stabilizer capable of improving the stability of the protease from Aspergillus niger is screened out.

(2) Short peptide stabilizers capable of improving the stability of protease and lipase in the presence of surfactants are screened out.

(3) The invention provides a compound enzyme formula suitable for cleaning medical instruments, and the product is stable and good.

(4) The short peptide stabilizer provided by the invention is tripeptide, has low synthesis cost and is suitable for industrial use.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are examples of experimental procedures not specified under specific conditions, generally according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

EXAMPLE 1 establishment of Complex enzyme stabilization System

The invention designs a large number of short peptide sequences according to the three-dimensional structure information of enzyme, carries out high-throughput screening through computer simulation, and finally selects dozens of potential short peptides for synthesis.

Firstly, by investigating the enzyme activity preservation rates of protease and lipase, short peptide stabilizers with excellent stability to the protease and the lipase are screened out.

Representative short peptides are listed in table 1 herein, wherein amino acid residues are indicated by single letter abbreviations.

In this embodiment, the complex enzyme stabilizing system comprises:

2g of sodium sulfate;

6g of calcium chloride;

10g of glycerol;

0.1g of short peptide stabilizer;

water is added to 100g.

Respectively weighing the above materials, mixing, stirring, and dissolving to obtain clear solution.

Under the stirring condition, 10g of protease enzyme solution (250000U/ml) is respectively added into 90g of the prepared complex enzyme stabilizing system, and after the mixture is stored for 2 months at 35 ℃, the storage rate of the protease enzyme activity is detected. In addition, 10g of lipase enzyme solution (100000U/ml) is added into 90g of the prepared complex enzyme stabilizing system, and after the lipase enzyme solution is stored for 2 months at 35 ℃, the lipase enzyme activity storage rate is detected.

The enzyme activity was measured by Xia Cheng (Shanghai) Biotech limited and reported.

The method for calculating the preservation rate of the enzyme activity comprises the following steps:

X＝E ₁ /E×100％

x is the preservation rate of the enzyme activity of the sample%

E ₁ : the sample actually measured the enzyme activity (U/ml)

E: sample designation (before preservation) enzyme Activity (U/ml)

The results of the experiment are shown in table 1.

TABLE 1

Experimental results show that the short peptide VST, the short peptide PST, the short peptide SNP and the short peptide FPR have good protease stabilizing effect. Although the stability of lipase is obviously superior to that of protease in a conventional system, and the enzyme activity preservation rate of the lipase can reach 76.3%, short peptides VST, PST, KNP and KNY are still observed to promote the stability of the lipase. And selecting short peptide VST, short peptide PST and short peptide FPR for further test.

EXAMPLE 2 preparation of Complex enzyme detergent

In this embodiment, the complex enzyme comprises:

respectively weighing the above materials, mixing, stirring, and dissolving to obtain clear solution. The short peptide stabilizers VST, PST and FPR selected in example 1 are respectively added into the complex enzyme to prepare different complex enzymes so as to verify the effect.

Adding 20g of anionic surfactant Triton H-66 (Dow, USA), 10g of surfactant Plurafac LF221 (Pasteur) and 5g of fatty alcohol-polyoxyethylene ether sodium sulfate (Li Chen) into 65g of water, heating to 45 ℃, and continuously stirring until the surfactant is completely dissolved to obtain a surfactant solution.

And mixing 100g of the prepared complex enzyme preparation and 100g of the surfactant solution, and uniformly stirring to obtain the complex enzyme cleaning agent. After 2 months of preservation at 35 ℃, the preservation rate of the enzyme activity is detected.

TABLE 2

Experimental results show that in the presence of a surfactant, the short peptide PST and the short peptide FPR have good stabilizing effect on protease and lipase, and the lipase stabilizing effect of the short peptide VST is greatly reduced. The cellulase and the amylase have better stability in the system. In the control group, in the absence of the short peptide stabilizer, the degradation of protease and lipase was severe, and the loss of the enzyme activity of cellulase and amylase was also large. Fig. 1, 2, and 3 are the appearances of the products after the experimental group 1, the experimental group 2, and the experimental group 3 were stored at 35 ℃ for 2 months, respectively, and the whole was clear and transparent, indicating that the product stability was good. FIG. 4 is the appearance of the product after storage at 35 ℃ for 2 months in the control group, showing a distinct turbidity.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. The application of the short peptide as a stabilizer of the composite enzyme is characterized in that the short peptide has a PST sequence, the composite enzyme is composed of protease, lipase, cellulase and amylase, and the protease is derived from Aspergillus niger.

2. A complex enzyme stabilizing system is characterized by comprising short peptides, sodium sulfate, calcium chloride, glycerol and a complex enzyme, wherein the sequence of the short peptides is PST;

and the complex enzyme in the complex enzyme stabilizing system consists of protease, lipase, cellulase and amylase, wherein the protease is protease from aspergillus niger.

3. A complex enzyme cleaning agent, characterized in that the complex enzyme cleaning agent comprises the complex enzyme stabilizing system of claim 2 and a surfactant.

4. The complex enzyme cleaning agent as claimed in claim 3, wherein the surfactant is selected from one or more of the following group: anionic surfactants, cationic surfactants, and nonionic surfactants.

5. The complex enzyme cleaning agent as claimed in claim 4, wherein the surfactant is composed of anionic surfactant Triton H-66, surfactant Plurafac LF221, and sodium fatty alcohol polyoxyethylene ether sulfate.

6. The use of the complex enzyme cleaning agent as claimed in claim 3 in cleaning medical instruments.