Pseudomonas aeruginosa 24F6 and application thereof

Abstract

The invention discloses pseudomonas aeruginosa 24F6 and application thereof, and belongs to the technical field of microorganisms. The invention discloses a pseudomonas aeruginosa strain 24F6 with the preservation number of CGMCC No.35032. The chicken feather degradation rate is 86%, and the wool degradation rate is 40%. The specific activity of the strain cultured for 24 hours under the conditions that the pH value is 7.0 and the temperature is 40 DEG C is 119.67 U / mg. The invention also relates to application of the keratin degrading agent in preparation of keratin degrading products. The pseudomonas aeruginosa 24F6 has the following advantages that a plurality of bacteria are separated from the excrement of the larva of the costustomoth, strains with different keratin degradation capacities are screened out through a keratin selective culture medium, and the pseudomonas aeruginosa 24F6 is a strain which is reported for the first time and has unique keratin degradation characteristics; the specific activity of the strain under the optimal fermentation condition is obviously higher than that of reported similar strains, the degradation rate of natural chicken feathers is as high as 86%, and the strain shows good industrial application potential.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a Pseudomonas aeruginosa 24F6 and application thereof. BACKGROUND

[0002] Natural resource shortage and environmental pollution have become key problems restricting human sustainable development, and protein-rich biomass waste (such as poultry feathers, crop processing by-products, etc.) generated in industrial and agricultural production is an important renewable resource. How to efficiently and high-value convert such waste, especially the recalcitrant biomass rich in keratin, has become a research hotspot in the field of biotechnology.

[0003] Protein biomass resources are widely sourced from multiple fields such as agriculture, animal husbandry, food industry, etc. For example, soybean dregs and bran generated in crop processing, fur and feathers in animal husbandry by-products, meat residues in food processing waste, and shells generated in aquaculture, etc. are all rich in protein. According to statistics, more than 4.7 million tons of chicken feathers were generated in global poultry processing in 2019. Such resources have stable carbon skeletons and are rich in nitrogen, and can be converted into high-value-added products (such as bioactive peptides, feed additives, etc.) through reasonable utilization, which can not only reduce resource waste, but also reduce environmental burden.

[0004] However, there are significant challenges in the utilization of keratin waste: first, the resources are distributed in a scattered manner, and the collection and transportation costs are high; second, keratin is difficult to degrade due to its compact structure caused by a large number of hydrogen bonds, disulfide bonds and hydrophobic interactions. Traditional physical (high temperature and high pressure) and chemical (strong acid and strong base) treatment methods not only have high energy consumption and strict equipment requirements, but also cause degradation of heat-sensitive amino acids and produce toxic products, which limits the large-scale application. Therefore, biological conversion technology centered on microorganisms and their secreted enzymes has become a research focus due to its high efficiency and environmental protection.

[0005] Keratin is a complex structural fibrous protein belonging to the intermediate filament protein superfamily, and is divided into α-keratin and β-keratin. α-keratin mainly exists in tissues such as hair and hoof, and is mainly in the form of spiral curl structure; β-keratin is mainly found in feathers, claws, etc., and is characterized by β-sheet structure. Both of them form rigid structures through disulfide bonds and hydrogen bonds, especially the cysteine content of hard keratin (such as feathers) is as high as 10% to 22%, and the disulfide bonds are dense, which become the main barrier to degradation.

[0006] The annual production of keratin waste is more than 10 million tons globally (1182 million tons in 2020), of which feathers account for about 85%. If not properly treated, such waste will cause serious pollution; if effectively degraded, it can be converted into nutrients such as amino acids and polypeptides. However, the compact structure of keratin makes it difficult to be hydrolyzed by ordinary proteases, and the breaking of disulfide bonds becomes the key rate-limiting step of degradation, which also makes the efficient treatment of keratin waste a difficult point for environmental protection and resource utilization.

[0007] Keratinase is a kind of protease that can specifically degrade keratin and is considered an ideal catalyst for keratin conversion. Its unique feature is that it can bind to insoluble keratin substrates (such as feathers and hair) and achieve degradation by breaking disulfide bonds and hydrolyzing peptide bonds. According to the catalytic mechanism, keratinases belong to multiple families (such as S8, M4, etc.), among which S8 family serine proteases and M4 family metalloproteases are the most widely studied.

[0008] Microbial sources of keratinase are diverse, including Bacillus, Streptomyces, fungi, etc. For example, Bacillus is an important keratinase-producing bacterium due to its strong secretion ability and high safety (GRAS certification). However, the keratinases reported so far have low catalytic efficiency and long reaction time, making it difficult to meet industrial needs. In addition, the complete degradation of keratin requires the cooperation of multiple enzymes: disulfide bond-reducing enzymes (such as γ-glutamyltransferase) break disulfide bonds, endopeptidases (such as S8 serine proteases) cut the internal polypeptide chain, exopeptidases (such as S13 carboxypeptidases) hydrolyze from the end, and oligopeptidases further decompose into amino acids.

[0009] With the development of omics technology, degradation omics (studying the system of all proteases and their substrates, inhibitors of an organism) provides a new tool for analyzing microbial degradation mechanisms. Genomics can predict the protease-encoding genes of a strain, proteomics can analyze the dynamic expression of extracellular enzymes, and integrating multiple omics technologies can systematically elucidate the mechanism of enzyme system cooperation. For example, through genome mining, the degradation group of a strain (such as the distribution of protease families in Bacillus) can be identified, and combined with proteomics, key functional enzymes (such as S8 serine proteases and M4 metalloproteases) can be located, providing a basis for the screening and modification of high-efficiency enzyme systems.

[0010] At present, efficient degradation of keratin-rich waste still faces challenges: one is the lack of high-efficiency degradation strains and enzyme systems, especially specific enzymes for keratin; two is that the synergistic mechanism of microbial degradation system is not clear. Although the microbial degradation method has the above advantages, the degradation effect is not ideal. The reason is that the degradation ability of many strains isolated at present is limited in degradation, and in fact, there are very few strains that actually have the activity of degrading keratin, that is, not all Bacillus or Brevibacterium bacteria have this activity, only specific strains have the activity. Therefore, more new strains that can degrade keratin need to be isolated, and these strains may carry unique degradation genes or metabolic pathways, which can not only degrade keratin, but also synchronously produce high-value-added products (such as amino acids, polypeptides, bioactive substances), so as to expand their application in the fields of feed, medicine or cosmetics.

[0011] Tinea pellionella Tineolabisselliella ) belongs to Lepidoptera Tineidae Tinea, and is a globally distributed insect. The larval stage has significant destructive power on natural materials rich in keratin. The larva is small in size and white in color, and the body length changes dynamically with the growth stage: only 1-2 mm at the initial hatching, and up to 5-10 mm after maturation. The larvae usually hide in the silk cocoon clothes bags woven by themselves and move as a protective barrier. The larvae have a highly specialized diet, mainly feeding on keratin-rich materials such as animal hair and hoof, and especially causing serious damage to wool products, natural fiber fabrics and clothes, and are therefore recognized as typical warehouse pests.

[0012] In view of the physiological characteristics of Tinea pellionella larvae relying on keratin for survival, there is a possibility that there is a special mechanism in the digestive system of the larvae to adapt to the degradation of keratin. It is speculated that in the intestinal tract and feces of the larvae, there may be a microbial community involved in the process of keratin decomposition. These microorganisms can secrete keratinase, disulfide bond reductase and other functional enzymes to help the host break down the dense structure of keratin (such as breaking the disulfide bond and hydrolyzing the peptide chain), and then realize efficient utilization of the recalcitrant substrate. This characteristic provides a unique research model for exploring the mechanism of keratin biodegradation and screening new functional microorganisms and enzyme resources. SUMMARY

[0013] The first object of the present application is to disclose a Pseudomonas aeruginosa 24F6.

[0014] The second object of the present application is to disclose the application of the above-mentioned Pseudomonas aeruginosa 24F6.

[0015] The object of the present application is realized by the following technical scheme: A Pseudomonas aeruginosa 24F6 ( Pseudomonas aeruginosa ), CGMCC No. 35032.

[0016] The Pseudomonas aeruginosa 24F6 has a degradation rate of 86% for chicken feather and a degradation rate of 40% for sheep wool.

[0017] The Pseudomonas aeruginosa 24F6 has a specific activity of 119.67 U / mg after being cultured for 24 hours under the condition of pH 7.0 and temperature 40 DEG C.

[0018] The Pseudomonas aeruginosa 24F6 has an enzyme activity of 35.9 U / ml after being fermented for 24 hours under the condition of pH 7.0 and temperature 40 DEG C.

[0019] The Pseudomonas aeruginosa 24F6 is a gram-negative bacterium.

[0020] The Pseudomonas aeruginosa 24F6 is used for preparing a keratin degradation product.

[0021] The application has the insoluble keratin as the keratin.

[0022] The application has the insoluble keratin as the chicken feather.

[0023] The application has the following beneficial effects: The application has the following beneficial effects: The application has the following beneficial effects:

[0024] Strain preservation information: 1. The Pseudomonas aeruginosa 24F6 is classified and named as Pseudomonas aeruginosa. Pseudomonas aeruginosa It has been preserved in the China General Microbiological Culture Collection Center, the address of which is No. 1, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC NO. 35032, and the preservation date is June 27, 2025. DETAILED DESCRIPTION

[0025] Figure 1 It is an optical microscope graph of the 24F6 strain. Figure 2 It is an electron microscope graph of the 24F6 strain. Figure 3Figure 1 is a solid plate map of 24F6 strain; Figure 4 Figure 2 is a phylogenetic tree of 24F6 strain; Figure 5 Figure 3 is the result of response surface interaction of enzyme activity of 24F6 strain with fermentation time and pH; Figure 6 Figure 4 is the result of response surface interaction of enzyme activity of 24F6 strain with fermentation time and temperature; Figure 7 Figure 5 is the result of response surface interaction of enzyme activity of 24F6 strain with temperature and pH; Figure 8 Figure 6 is the degradation result of chicken feather and sheep wool by 24F6 strain; wherein Figure 8 A is before chicken feather degradation, Figure 8 B is after chicken feather degradation (3d); Figure 8 C is before sheep wool degradation, Figure 8 D is after sheep wool degradation (3d); Figure 9 Figure 7 is a dynamic change gel electrophoresis map of 24F6 extracellular protease. DETAILED DESCRIPTION

[0026] In order to facilitate the understanding of the technical solutions of the present application, a strain of Pseudomonas aeruginosa 24F6 and its application will be further described in combination with specific examples.

[0027] Note: The chicken feather powder in the examples is ground into powder after being dried at 80℃; the chicken feather is pure chicken feather treated by high-temperature sterilization.

[0028] The medium formula required in the examples of the present application is shown in Table 1, Table 2 and Table 3:

[0029] Table 2 Deficient medium

[0030] Table 3 Liquid medium

[0031] Example 1: Isolation and morphological identification of Pseudomonas aeruginosa 24F6: 1. Method: Isolation and purification of strain and morphological identification: Rearing of Tinea clostera larvae: The Tinea clostera larvae were stored in a light-proof environment with a temperature of 20℃~25℃ and a relative humidity of 70%~80%. The only food source was small pieces of sheep wool, which were divided into multiple rearing boxes. The larvae feces were collected regularly and immediately stored in a-80℃ ultra-low temperature refrigerator to maximize the activity and stability of the metabolites in the feces.

[0032] Homogenization and preparation of suspension: 0.1-0.5 g of fecal sample was weighed, 5-10 mL of sterile normal saline or PBS buffer was added, and the sample was vortexed for 15 minutes to fully disperse, and the supernatant was taken as the stock solution (10 0 ).

[0033] Enrichment culture: In a sterile operating table, 1 mL of the original bacterial solution was inoculated into a triangular flask containing 100 mL of selective medium, and was placed in a 37°C constant temperature shaker, and was cultured at 220 rpm for 48 h, so that the target bacterial flora was enriched.

[0034] Gradient dilution and separation: 1 mL of the enriched bacterial solution was added to 9 mL of sterile normal saline to prepare a 10 -1 dilution solution. According to the 10-fold gradient dilution method, serial dilutions of 10 -1 to 10 -7 were prepared. 100 μL of 10 -3 to 10 -7 concentration dilutions were taken and uniformly coated on the separation medium plate, with 3 repeats for each concentration. The plate was inverted and cultured in a 37°C constant temperature incubator for 2-5 days.

[0035] First purification (continuous streaking method): Under sterile conditions, single colonies with obvious morphological characteristics and no bacterial contamination were picked up, and continuous streaking was performed on fresh medium plates with an inoculation loop. When streaking, the inoculation loop was gently contacted with the plate surface at an angle of 30°-40° to avoid breaking the medium. The inoculation loop was sterilized by flame burning after each streaking, and the cooling was continued for the next zone streaking, and the operation was repeated until the whole plate was covered. The plate was inverted and cultured at 37°C for 1-2 days.

[0036] Second purification (zoning streaking method): Single colonies were picked from the first purification plate and further purified by four-zone streaking method. The specific operation is as follows: the plate is divided into A, B, C, and D four regions, first streaking inoculation in A region, then burning the inoculation loop after cooling and taking bacteria from A region to B region, and so on to complete C and D region streaking. The inoculation loop needs to be burned after each streaking to ensure that the bacterial amount gradually decreases to obtain single colonies. Invert the plate and culture at 37°C for 1-2 days.

[0037] Multiple purification verification: Repeat the above zoning streaking operation at least 9 times, and each time select single colonies for transfer until pure culture with consistent morphology and no bacterial contamination is obtained.

[0038] 2. Results: Isolation and purification of strains and morphological identification results: Ten strains of bacteria were isolated from the feces of *Chicken Feather Moth* larvae. Analysis of their substrate utilization capabilities showed that more strains could decompose and utilize natural chicken feathers (insoluble keratin) than those that could decompose and utilize soluble keratin. This phenomenon may be related to the host's feeding characteristics—*Chicken Feather Moth* larvae feed directly on natural keratin (such as feathers and hair), and the microorganisms in their intestines and feces have long been adapted to such substrates, making them more likely to evolve degradation mechanisms targeting insoluble keratin.

[0039] Among all isolated strains, strain 24F6 significantly outperformed other strains in key indicators such as enzyme activity and chicken feather degradation rate, exhibiting the strongest keratin degradation efficiency. Therefore, strain 24F6 was selected as the research subject. The colony morphology characteristics of strain 24F6 are as follows: Figure 1 As shown in Table 4, strain 24F6 appears as a rod-shaped structure under an optical microscope (objective lens 40×0.65, eyepiece 10×22). Figure 2 As shown; its morphology under an electron microscope is rod-shaped (e.g.) Figure 3 As shown), the electron microscope's equipment model is SU8600, accelerating voltage is 5.00kV, working distance is 8.8mm, magnification is ×22.0k, imaging mode is UD(+LD), and scale bar is 2.00μm; Table 4. Description of 24F6 colony morphology

[0040] Example 2: Gram staining and 16S rRNA identification of strain 24F6: 1. Methods: Gram staining was performed on the selected strain 24F6 to observe its morphological characteristics. Simultaneously, pure cultures were sent to Xinjiang Youkang Biotechnology Co., Ltd. for 16S rRNA gene sequencing. The sequenced sequences were compared with publicly available 16S rDNA sequences in GenBank using the BLAST tool in the NCBI database for nucleotide homology analysis. A phylogenetic tree was constructed using MEGA 11.0 software to complete the molecular biological identification of the strain.

[0041] 2. Results: Gram staining and 16S rRNA identification of strain 24F6 (1) The Gram staining result of strain 24F6 was negative. Based on the cell wall structure characteristics of Gram-negative bacteria, it is speculated that the peptidoglycan layer in its cell wall is relatively thin (usually 2~3nm) and wrapped with a lipopolysaccharide layer on the outside. The overall structure is loose and multi-layered.

[0042] Table 5 Gram staining results

[0043] Note: Positive is +, negative is -.

[0044] (2) 16S rRNA identification: the nucleotide sequence of 24F6's 16S rRNA is PP930722.1 (GenBank), which is identified by Figure 4 phylogenetic tree analysis Pseudomonas aeruginosa 24F6.

[0045] Example 3: Enzyme activity determination of strains under different physiological and biochemical conditions: 1. Method: (1) Strain culture and preparation of crude enzyme solution: Gradient culture: first, scrape the high-efficiency enzyme-producing strain from the slope medium, inoculate into 5 mL liquid LB medium, and shake culture for 24 h; then inoculate 4% into 50 mL liquid LB medium, and shake culture for 24 h to expand the strain number; finally, inoculate 4% into 100 mL fermentation medium, and culture for 0 h, 12 h, 24 h, 48 h, 72 h, and 84 h at 37°C and 220 r / min.

[0046] Preparation of crude enzyme solution: centrifuge the fermentation broth with different culture times at 4°C and 10,000 r / min for 10 min, and take the supernatant as the crude enzyme solution, which is stored at 4°C for standby.

[0047] (2) Keratinase enzyme activity determination: Method and system: according to Gradisar (2000) method, take 500 μL of crude enzyme solution supernatant, add 1 mL of 0.05 mol / L Tris / HCl buffer (pH 5-9 gradient setting) and 5 mg of chicken feather powder substrate to construct the reaction system.

[0048] Reaction and termination: incubate in a constant temperature water bath at 20°C-50°C gradient temperature for 2 h, then add 1.0 mL of 10% TCA to terminate the reaction, and centrifuge at 4°C and 10,000 rpm for 15 min.

[0049] Determination and calculation: measure the absorbance value at 280 nm (add TCA to the control group in advance), and define the enzyme activity unit as 1 U per 0.01 increase in A280 value, and take the average value of three repeated experiments.

[0050] 2. Results: The response surface interaction results of 24F6 strain enzyme activity with fermentation time and pH are shown in Figure 5 , the response surface interaction results of 24F6 strain enzyme activity with fermentation time and temperature are shown in Figure 6 , the response surface interaction results of 24F6 strain enzyme activity with temperature and pH are shown in Figure 7 , and the comprehensive results are shown in Figure 5 , Figure 6 and Figure 7As a result, the enzyme activity of strain 24F6 was 35.9 U / ml when it was fermented for 24 h at pH 7.0 and 40℃.

[0051] Example 4: Determination of protein content by Coomassie brilliant blue method (Bradford method): 1. Method: 2 ml of the prepared enzyme fermentation broth was taken, then centrifuged at 10000 r / min for 5 min, the precipitate was discarded, and the supernatant was transferred into a 25 ml volumetric flask, which was diluted with distilled water to the mark, and the sample extract was obtained.

[0052] The standard curve was drawn with the standard protein concentration as the abscissa and the absorbance value as the ordinate.

[0053] 2. Results: After determining the protein content by Coomassie brilliant blue method, the absorbance value of the measured enzyme activity was substituted into the formula y = 1.9721x + 0.116 according to the standard curve, and the enzyme activity of strain 24F6 under the optimal enzyme production conditions was calculated.

[0054] Table 6: Keratinase activity determination results of strain 24F6

[0055] Example 5: Specific activity and degradation rate determination experiment: 1. Method: Specific activity determination of Malacosoma neustria larva feces strain 24F6: The maximum enzyme activity of the strain under the optimal pH and temperature conditions was determined by the foregoing experiment, and the protein content data was determined by Coomassie brilliant blue method (Bradford method). The specific activity of the strain was calculated according to the formula "specific activity = enzyme activity / (protein content x solution volume)".

[0056] Degradation rate determination of chicken feather and sheep wool by keratin-degrading bacteria (Pseudomonas aeruginosa 24F6): 0.02 g of chicken feather and 0.02 g of sheep wool were accurately weighed and placed in different conical flasks. According to the proportion of adding a certain amount of selective medium corresponding to 0.02 g of solid substrate, the corresponding degradation solution was prepared. Then, the strain 24F6 obtained by screening was inoculated into each prepared degradation solution at an inoculation amount of 8%. After the inoculation operation was completed, the conical flasks were sealed with sealing film and placed in a constant temperature incubator at 37℃ for culture. The degradation rate was calculated according to the formula "degradation rate = (degradation before mass - degradation after mass) / degradation before mass x 100%".

[0057] 2. Results: Specific activity and degradation rate determination experiment results: (1) The specific activity of strain 24F6 is 119.67 (U / mg).

[0058] Table 7 Calculation results of strain specific activity

[0059] (2) The degradation results of chicken feather and wool by strain 24F6: The degradation results of chicken feather and wool by strain 24F6 are shown in Table 7. Figure 8 Before degradation, chicken feather and wool are complete solid substrates (A and C, respectively). Figure 8 After 3 days of degradation, the solid substrate in the chicken feather group is significantly reduced, and the bottle is mainly yellow-green liquid (B), indicating that the chicken feather substrate is effectively degraded by P. aeruginosa, the solid structure is decomposed, and the degradation products are dissolved into the liquid, showing typical characteristics of the post-degradation system. Figure 8 Comparing the degradation of chicken feather and wool, the degradation of wool is more slight, reflecting the difference in the degradation of different keratin substrates by the strain. Figure 8 Figure 8 The degradation rate is shown in Table 8.

[0060] The degradation rate of chicken feather by strain 24F6 can reach 86%, and the degradation rate of wool is 40%.

[0061] Table 8 Calculation results of degradation rate

[0062] Example 6: Analysis of the degradation potential of extracellular protease of strain 24F6: 1. Method: First, centrifuge the degradation fermentation broth at different time periods at 10000 r / min for 10 min at 4°C to obtain the supernatant, mix it with denaturing loading buffer containing β-mercaptoethanol at a ratio of 4:1, heat in a boiling water bath for 8 min, and then cool it for standby use. For electrophoresis, prepare 12% separation gel and 5% concentration gel according to the molecular weight of extracellular protease, load the denatured sample (8 μL) and 10-190 kDa protein marker (2 μL), and perform the entire electrophoresis process on ice. After electrophoresis, the gel is stained with Coomassie Brilliant Blue R-250 for 20-30 min, and then decolorized with decolorizing solution until the bands are clear.

[0063] 2. Results: Analysis of the degradation potential of extracellular protease: The secretion of most microbial proteases is strictly regulated by the type of substrate. Generally, keratinases can degrade keratin into absorbable nutrients under nutritional stress. The dynamic change of extracellular protease of 24F6 is shown in​Figure 9 As shown in the figure, the gel results show that when P. aeruginosa 24F6 grows with chicken feather as substrate, the extracellular proteases are secreted from 1d, and the types and concentrations of the secreted proteases increase with the extension of fermentation time. In the late fermentation stage, the types of the extracellular proteases tend to be stable, and the abundance of the individual proteases slightly increases with the extension of fermentation time.

[0064] The above is only the preferred embodiment of the present application, and does not limit the present application in any form and substance. Any equivalent changes, modifications and evolutions made by those skilled in the art within the scope of the technical solutions of the present application, using the technical content disclosed above, are equivalent embodiments of the present application. Meanwhile, any equivalent changes, modifications and evolutions made according to the essential technology of the above embodiments are still within the scope of the technical solutions of the present application.

Claims

1. A Pseudomonas aeruginosa 24F6 (P. aeruginosa 24F6) Pseudomonas aeruginosa ), CGMCC No. 35032.

2. The P. aeruginosa 24F6 of claim 1, wherein: The degradation rate of the strain to chicken feather is 86%, and to wool is 40%.

3. The P. aeruginosa 24F6 of claim 1, wherein: The specific activity of the strain cultured at pH 7.0 and 40 DEG C for 24 hours is 119.67 U / mg.

4. The P. aeruginosa 24F6 of claim 1, wherein: The enzyme activity of the strain fermented at pH 7.0 and 40 DEG C for 24 hours is 35.9 U / ml.

5. The P. aeruginosa 24F6 of claim 1, wherein: The strain is gram-negative bacteria.

6. Use of Pseudomonas aeruginosa 24F6 according to any one of claims 1 to 5 for the preparation of a keratin-degrading product.

7. Use according to claim 6, characterized in that: The keratin is insoluble keratin.

8. Use according to claim 7, characterized in that: The insoluble keratin is chicken feather.

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