CN108060170B

CN108060170B - Novel metal ion-tolerant keratinase and application thereof

Info

Publication number: CN108060170B
Application number: CN201711468398.1A
Authority: CN
Inventors: 史劲松; 龚劲松; 陶丽妍; 许正宏; 李恒; 苏畅
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2020-05-15
Anticipated expiration: 2037-12-29
Also published as: CN108060170A

Abstract

The invention provides a novel metal ion tolerant keratinase and application thereof, provides a keratinase excavated based on metagenome technology and an application method thereof, and belongs to the technical field of industrial biology. The whole length of the keratin enzyme gene is 1,149bp, and 382 amino acids are coded; bacillus subtilis WB600 was taken as an example to successfully achieve heterologous expression of the keratinase gene. The acquisition method of the keratinase gene is convenient and feasible, the gene is subjected to recombinant expression, the constructed recombinant strain can realize the secretory expression of the recombinant keratinase, and the recombinant keratinase has better tolerance to metal ions and surfactants and better enzyme stability. The recombinant strain has short fermentation period and is suitable for industrial production. In addition, the enzyme has good reducing capability, can be applied to the research of preparing nano silver particles by a biological method, and the prepared nano silver particles have good morphology and good bacteriostatic activity.

Description

Novel metal ion-tolerant keratinase and application thereof

Technical Field

The invention provides gene excavation, expression and property research of novel metal ion tolerant keratinase and an application method of the novel metal ion tolerant keratinase in synthesizing nano-silver by a biological method, and belongs to the technical field of industrial biology.

Background

Keratinases are special proteases capable of specifically degrading keratin-rich feather, wool, hair, nails, etc. The characteristic that the insoluble keratin can be degraded and is different from general protease enables the keratinase to be widely applied to the animal husbandry industry, the feed industry, the tanning industry and the medicine industry. The early research on the microbial keratinase in China mainly focuses on the breeding of keratinase producing bacteria, the separation and purification and property research of the keratinase and the application of the keratinase. In 2003, bin et al, from Jilin university, succeeded for the first time in isolating the keratinase gene from Bacillus licheniformis L-25 and in achieving exogenous expression in Bacillus subtilis, and from this point on, studies on molecular biology and genetic engineering, such as the isolation and expression of the keratinase gene, have been receiving increasing attention from researchers in more and more countries. In recent years, keratinase from different sources has been successfully expressed in recombinant keratinase in bacillus, escherichia coli and pichia pastoris expression systems, but the expression level and the degradation activity of the recombinant keratinase are not ideal, so that the keratinase is difficult to be used in industrial fermentation in large quantities, and the industrial application of the keratinase is limited. In recent years, researchers turned their efforts to the application of keratinase in the fields of cleaning of pipeline hair waste, skin and nail related medicines, personal care products, and the like. Two keratinases purified from Bacillus halodurans PPKS-2 wild strain by a research team in India were found to have high disulfide bond reduction activity in one of them (Applied Microbiology and Biotechnology,2010,87,2: 625-); indicating that the keratinase has good reducing ability.

In recent years, with the continuous development of nano-silver preparation technology, the application of nano-silver is more and more extensive. The silver nano material has special physicochemical properties and is widely applied to important fields of industry, medicine and the like. In industrial production, the nano silver has better catalytic activity compared with silver, and the optical, thermal and magnetic properties of the product can be effectively improved after the nano silver is added. In the field of medicine, nano-silver is often used as a drug for prevention and treatment of diseases. The preparation method of the nano silver particles comprises a physical method, a chemical method and a biological method. The physical method comprises a vacuum deposition method, a ball milling method, an evaporation condensation method, a plasma method and the like, is suitable for large-scale preparation, but has higher requirements on equipment, high reaction energy consumption and low yield. The chemical method reduces silver ions in a solution into simple substance silver through a chemical reduction reagent, then the simple substance silver is aggregated to be changed into nano-grade silver particles, and other additives such as a dispersing agent, a stabilizing agent, a protective agent and the like are often required to be introduced in the reaction. The additives are free of organic solvents with high toxicity and serious pollution, so that the additives are not beneficial to the wide application of the prepared nano silver, and particularly limit the application of the nano silver in the field of medicine (CN 106180753A). The biological method for synthesizing the nano silver mainly reduces simple substance silver through enzymes, proteins and other substances in a biological sample, and can be used as a stabilizer to prepare nano silver particles.

Disclosure of Invention

The invention aims to provide gene excavation, expression and property research of novel high metal ion tolerance keratinase and an application method of the novel high metal ion tolerance keratinase in synthesizing nano-silver by a biological method, and belongs to the technical field of industrial biology. Provides a plasmid containing the gene of the invention, a host cell containing the expression plasmid, a method for producing keratinase by utilizing the expression of the recombinant strain, and an application method for synthesizing nano-silver by utilizing the keratinase biological method.

The technical scheme of the invention is as follows:

based on the metagenome technology, the soil metagenome DNA of leather factory piled fur is taken as a template, the conserved sequence of keratinase is used for carrying out degenerate primer design and PCR amplification, the target keratinase gene is screened, and then the primer is further designed according to the sequencing result to obtain the complete gene coding sequence through PCR technology amplification. The entire coding sequence obtained is constructed into expression vectors, such as the Bacillus subtilis-large intestine shuttle plasmid pMA5, pET series vectors, pPIC series vectors, and the like. The constructed recombinant plasmid is transformed into host bacteria bacillus subtilis competent cells, but not limited to bacillus subtilis, and also comprises escherichia coli, filamentous fungi, corynebacterium glutamicum, corynebacterium crenatum, pichia pastoris, saccharomyces cerevisiae and the like, and heterologous expression of keratinase is realized after fermentation culture. The keratinase is subjected to enzymatic property analysis, the characteristics of high metal ion tolerance and high reducing capability of the keratinase are utilized to be applied to the preparation of the nano silver synthesized by a biological method, and the prepared nano silver is subjected to multi-aspect characterization and bacteriostatic activity verification.

(1) Screening of keratinase gene by metagenome technology

Collecting soil samples from places where furs are piled in leather factories, and extracting total DNA of the samples by using a soil genome extraction kit. Designing degenerate primer for PCR amplification reaction based on the conserved sequence of keratinase, sequencing the amplified fragment, comparing with NCBI database, further designing primer based on the result, and amplifying to obtain complete keratinase gene coding sequence. The method is based on the metagenome technology, combines the conserved sequences of keratinase in a database to carry out direct amplification screening, and has the advantages of simple and easy operation method, short screening period and the like compared with the construction screening method based on the metagenome library.

(2) Establishment of keratinase bacillus subtilis expression system

Designing a primer with an enzyme cutting site according to a keratinase gene sequence, and obtaining a keratinase coding gene sequence through PCR amplification:

an upstream primer: 5' -CGGGATCCATGAGAGGCAAAAAGGTATGG-3’

A downstream primer: 5' -CGACGCGTTTACTGAGCTGCCGCCTGTA-3’

The PCR amplification reaction was carried out in a 40. mu.L system to which 20. mu.L of PrimeSTAR HS, 17. mu.L of LddH were added₂O, 1. mu.L of template DNA, 1. mu.L of each of the upstream and downstream primers. The reaction conditions were such that cycling was initiated after 5min of pre-denaturation at 95 ℃: denaturation at 95 ℃ for 30s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; final extension at 72 ℃ for 10 min.

And performing nucleic acid electrophoresis identification on the PCR product, tapping, recovering and purifying, cloning the recovered product to a vector to construct recombinant plasmids, transforming the plasmids to a cloning host, selecting a plurality of positive transformants to an LB liquid culture medium with corresponding antibiotics, culturing at 37 ℃ and 220rpm for 10-12 h, extracting the plasmids, and performing PCR, double enzyme digestion and sequencing verification on the plasmids. And (3) transforming the correctly verified recombinant plasmid into a bacillus subtilis expression host bacterium, coating a solid culture medium with a corresponding antibiotic, and further screening a positive transformant by using a PCR verification method. Successfully constructed keratinase producing bacteria are streaked on a solid culture medium to separate a single colony, then the single colony is picked into a 10mL liquid LB culture medium for overnight culture, and the single colony is inoculated into a 30mL TB fermentation culture medium by the inoculation amount of 2% the next day for keratinase enzyme production research.

(3) Recombinant keratinase enzyme activity detection method

Preparation of 1% keratin substrate: first, a solution of 0.1M Tris-HCl (pH 9.0) was prepared, then 5% soluble keratin stock solution was added, and deionized water was added to dilute the Tris-HCl solution to 0.05M and the keratin solution to 1%. Enzyme reaction: taking 100 mu L of enzyme solution which is diluted moderately, adding 0.1mL of substrate solution, placing in a water bath at 50 ℃ for accurate reaction for 15min, and immediately adding 200 mu L of 5% TCA to terminate the reaction after the reaction is finished. In the control group, 200. mu.L of TCA was added first, and 0.1mL of substrate solution was added after the completion of the enzyme reaction. After the above reaction, the reaction mixture was centrifuged at 12000rpm for 5min, 200. mu.L of the supernatant was pipetted into a new 1.5mL EP tube, and 1mL of 0.4M Na was added₂CO₃Then 200 mul of forskolin phenol is added, the mixture is placed in a water bath at 40 ℃ for color reaction for 20min, and then the absorbance value is detected at 680 nm. Definition of enzyme activity: under the above reaction conditions, the difference in absorbance at 680nm of the substrate hydrolyzed by the enzyme solution was defined as one unit of enzyme activity U. The enzyme activity calculation formula is as follows: u ═ O (OD)_{680 experimental group}-OD_{680 control group}) X 100 x dilution factor

(4) Enzymatic Properties of recombinant keratinases

Recombinant keratinase was subjected to enzymatic property studies, focusing on the property of resistance to chemical agents and metal ions:

A. effect of Metal ions on recombinant keratinase Activity

Adding different metal ions (Mg) into the enzyme activity determination reaction system²⁺、Ca²⁺、Zn²⁺、Mn²⁺、Co²⁺、Ni²⁺、Fe²⁺、Cd²⁺、Ba²⁺、Li⁺、Fe³⁺、Al³⁺、Pb²⁺、Ag⁺) The final concentrations were adjusted to 1mM and 5mM, respectively, and the enzyme activities were measured, taking the reaction tube to which no metal ion was added as 100%.

B. Effect of chemical Agents on recombinant keratinase enzyme Activity

PMSF, EDTA, DTT, β -mercaptoethanol and different surfactants SDS, Tween20, Tween80, TritonX-100, TritonX-114 and H are respectively added into an enzyme activity determination reaction system₂O₂DMSO was adjusted to final concentrations of 1mM, 5mM and 1% respectively, and the enzyme activities were measured according to the enzyme activity measurement method, taking the reaction without addition of chemical reagents as 100%.

C. Analysis of the reducing power of keratinase

To 300. mu.L of the sample keratinase enzyme solution diluted appropriately, 300. mu.L of 0.2M PBS (pH 6.6) and 300. mu.L of 1% (w/v) potassium ferricyanide solution were added, and the mixture was reacted in a water bath at 50 ℃ for 20 min. Then 300. mu.L of 10% (w/v) TCA was added and centrifuged at 4000rpm for 10 min. Finally, 200. mu.L of the supernatant was pipetted into 800. mu.L of a 0.01% (w/v) ferric trichloride solution. The reaction was carried out at 30 ℃ for 10min, and the absorbance at 700nm was measured. The higher the absorbance value, the higher the reducing power. The control was a sample solution without keratinase.

(5) Keratinase biological method for synthesizing nano silver

Adding 2mL of the filtered and sterilized keratinase solution into 50mL of 1mM silver nitrate solution, adding no reducing agent or stabilizing agent, and oscillating at 37 ℃ for reaction to obtain the nano-silver solution, wherein the reaction solution gradually changes from colorless to darker orange yellow. The nano silver prepared by the method is subjected to characterization in various aspects such as ultraviolet-visible light spectrum analysis, dynamic light scattering analysis, transmission electron microscope analysis, Fourier infrared analysis and the like. And finally, carrying out bacteriostatic activity detection on the prepared nano silver.

The invention has the advantages and beneficial effects that:

the invention provides gene excavation, expression and property research of novel high metal ion tolerance keratinase and an application method of synthesizing nano silver by a biological method. It has the nucleotide sequence shown in SEQ ID NO. 1,1,149 nucleotides in total length and 382 amino acids in code. Taking bacillus subtilis as an example, the heterologous expression of the keratinase gene is successfully realized. The keratinase exhibits good tolerance to surfactants. The keratinase has good tolerance to metal ions and high reduction capacity, and can be used for synthesizing nano silver particles by a biological method. The method for preparing the nano silver particles by the keratinase biological method is simple to operate, does not need to add a reducing agent and a stabilizing agent, is green and environment-friendly, and has mild and controllable reaction conditions. The nano silver particles prepared by the method have a maximum absorption peak at 430nm, which is a plasma resonance energy band of the silver nanoparticles, the nanoparticle solution has better dispersibility, a transmission electron microscope shows that the nano silver particles have an average particle size of 6.54 +/-2.62 nm and have a good spherical shape, and finally, infrared spectroscopic analysis 3100-^-1And 1650cm^-1The absorption peak proves that the reduction of the keratinase causes the generation of the nano-silver, and the bacteriostatic analysis shows that compared with the nano-silver prepared by a chemical method, the nano-silver synthesized by the keratinase biological method has better bacteriostatic activity.

Drawings

FIG. 1 phylogenetic tree analysis of the keratinase gene

FIG. 2 is a graph showing the effect of metal ions on keratinase activity

FIG. 3 is a keratinase reducing power test

1: a control sample; 2: keratinase samples.

FIG. 4 shows the plasma resonance energy band of keratinase biological method for synthesizing nano silver particles

FIG. 5 is a transmission electron microscope image of keratinase biological method for synthesizing nano silver particles

FIG. 6 is the analysis of the particle size of the nano silver particles synthesized by keratinase biological method

FIG. 7 is Fourier infrared spectrum analysis of keratinase biological method for synthesizing nano silver particles

FIG. 8 shows the bacteriostatic activity analysis of the keratinase biological method for synthesizing nano silver particles

1: blank control; 2: preparing nano silver antibacterial activity by a chemical method; 3: the bacteriostatic activity of the nano silver is synthesized by a keratinase biological method.

Detailed description of the preferred embodiments

The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to these examples.

Example 1

This example illustrates a method for screening a keratinase gene based on metagenomic technology.

Collecting soil samples from places where furs are piled in leather factories, and extracting total DNA of the samples by using a soil genome extraction kit. Designing degenerate primer for PCR amplification reaction based on the conserved sequence of keratinase, sequencing the amplified fragment, comparing with NCBI database, further designing primer based on the result, and amplifying to obtain complete keratinase gene coding sequence. The obtained keratinase sequence was analyzed by phylogenetic tree and found to be most similar to the sequence of B.velezensis derived keratinase (FIG. 1). The method is based on the metagenome technology, combines the conserved sequences of keratinase in a database to carry out direct amplification screening, and has the advantages of simple and easy operation method, short screening period and the like compared with the construction screening method based on the metagenome library.

Example 2

This example illustrates the construction of the keratinase Bacillus subtilis expression system.

an upstream primer: 5' -CGGGATCCATGAGAGGCAAAAAGGTATGG-3’

A downstream primer: 5' -CGACGCGTTTACTGAGCTGCCGCCTGTA-3’

The PCR amplification reaction was carried out in a 40. mu.L systemTo this line 20. mu.L PrimeSTAR HS, 17. mu.L ddH were added₂O, 1. mu.L of template DNA, 1. mu.L of each of the upstream and downstream primers. The reaction conditions were such that cycling was initiated after 5min of pre-denaturation at 95 ℃: denaturation at 95 ℃ for 30s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; final extension at 72 ℃ for 10 min.

Example 3

This example illustrates a method for detecting recombinant keratinase enzyme activity.

Example 4

This example illustrates the catalytic characteristics of keratinase.

(1) Respectively adding different metal ions into a reaction system for enzyme activity determination to make the final concentration reach 1mM, and comparing with a blank control group without any metal ions, 6 metal ions (Mg) are provided²⁺、Ca²⁺、Zn²⁺、Ni²⁺、Cd²⁺、Fe³⁺) Has effect in promoting enzyme activity, wherein Cd is²⁺The promotion effect of ions on recombinant keratinase is most obvious; and Mn²⁺The ions obviously inhibit the enzyme activity. When the concentration of metal ions reaches 5mM, except Mg²⁺And Li⁺The ions have a weak promoting effect on the enzyme activity, and the remaining metal ions all show a more or less inhibitory effect (fig. 2). In conclusion, the keratinase has high tolerance to various metal ions, can keep more than 80% of relative activity under the silver ion concentration conditions of 1mM and 5mM, and has great potential advantages when being applied to the synthesis research of nano silver particles by a biological method.

(2) Different chemical reagents are respectively added into an enzyme activity determination reaction system, and then the enzyme activity is respectively determined. As shown in Table 1, PMSF showed significant inhibition of the enzyme at a concentration of 1mM and was able to completely inhibit the activity of the enzyme at a concentration of 5mM, indicating that the recombinant keratinase belongs to the class of serine proteases; surfactants such as Tween and Triton have a promoting effect on recombinant keratinase, and the enzyme has good stability in the surfactant, so that the enzyme has good application prospect in the field of detergents.

TABLE 1 Effect of chemical reagents on recombinant keratinase enzyme Activity

(3) Compared with a control sample, the keratinase shows obvious high reducing power, and the unique property of the keratinase can be applied to the preparation research of nano silver by a biological method (figure 3).

Example 5

This example illustrates the use of keratinase for nanosilver biosynthesis.

Adding 2mL of the recombinant keratinase solution subjected to filtration sterilization into 50mL of 1mM silver nitrate solution, and oscillating for reaction at 37 ℃ to find that the reaction solution is changed into darker orange yellow from colorless, namely the prepared nano silver solution. The ultraviolet-visible light spectrum analysis of the nano silver solution prepared by the biological method shows that the maximum absorption peak is at 430nm, which is the SPR energy band of the nano silver, and further confirms the synthesis of the nano silver (figure 4). Dynamic light scattering analysis shows that the nano silver solution has better dispersion characteristics. The appearance of the nano silver particles can be clearly observed by transmission electron microscopy analysis (fig. 5), and the average particle diameter of 6.54 ± 2.62nm is obtained (fig. 6). Characterization by fourier infrared analysis showed that nanosilver synthesis was protein dependent (fig. 7), also confirming the effect of keratinase. And finally, carrying out bacteriostatic activity detection on the prepared nano silver particles. The result shows that the nano silver synthesized by the keratinase biological method has better bacteriostatic activity (figure 8).

The present invention is not limited to the embodiments disclosed, and it is intended to cover various modifications and equivalent changes within the scope of the claims and described technical solution, and any modifications or improvements which can be easily made by those skilled in the art without departing from the technical solution of the present invention are within the scope of the claims.

Sequence listing

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Claims

1. The nucleotide sequence of the gene for coding the keratinase is shown as SEQ ID NO. 1.

2. The amino acid sequence of keratinase is shown in SEQ ID NO 2.

3. A vector for carrying a gene encoding keratinase, characterized in that: comprising the nucleotide sequence of claim 1, said vector being selected from the group consisting of the pMA5 plasmid, the pET series, the pPICZ series, the pDXW series.

4. A method for preparing a recombinant strain for producing keratinase, characterized in that: the vector of claim 3 introduced into an exogenous host selected from the group consisting of Escherichia coli, Bacillus, Corynebacterium, Yeast Yeasts, and filamentous fungus flamentous fungi.

5. A method for preparing nano-silver by applying keratinase to a biological method is characterized in that: placing keratinase produced by the recombinant strain of claim 4 and silver nitrate solution into a reactor, reacting for 48-72h at 25-40 ℃ in the dark without adding any reducing agent and stabilizing agent, reducing silver ions by the keratinase to replace silver simple substances, and aggregating the silver simple substances into clusters to become nano silver particles, namely the nano silver prepared by a biological method.