CN114657096B - Bacillus safoci PLA1006 strain, screening method and application thereof - Google Patents
Bacillus safoci PLA1006 strain, screening method and application thereof Download PDFInfo
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- CN114657096B CN114657096B CN202210290274.3A CN202210290274A CN114657096B CN 114657096 B CN114657096 B CN 114657096B CN 202210290274 A CN202210290274 A CN 202210290274A CN 114657096 B CN114657096 B CN 114657096B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/62—Plastics recycling; Rubber recycling
Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus subtilis PLA1006 strain, a screening method and application thereof. The strain has the preservation number of: CGMCC No.24188, the preservation date is 2021, 12 months and 24 days, and the method can grow by taking polylactic acid as a carbon source, and the specific degradation process comprises the following steps: (1) Taking a dried polylactic acid film, wiping the surface with 70% ethanol, sterilizing by irradiation of an ultraviolet lamp, and then placing the polylactic acid film into a sterile basic culture medium; (2) The bacillus safoci PLA1006 strain is inoculated into a basic culture medium, and samples are taken regularly to determine the weight loss condition of the film. The strain has the protease production capability and the polylactic acid utilization capability, and the powder, the film and the emulsion of the polylactic acid can be degraded by the bacillus subtilis disclosed by the invention, so that the strain can be applied to the treatment of waste of polylactic acid products.
Description
Technical Field
The invention belongs to the fields of microbial technology and environmental biotechnology, and in particular relates to a protease-producing bacillus safoci Bacillus safensisPLA strain and application thereof in polylactic acid degradation.
Background
The plastic is widely used due to the characteristics of low cost, light weight, corrosion resistance, stable performance, easy processing and forming and the like. However, with the development of the plastic industry and the wide use of disposable plastic products, the environmental pollution problem caused by plastic waste is also increasing. Biodegradable plastics have therefore become a trend in the development of the plastics industry, replacing traditional plastics.
The ideal biodegradable plastic is a polymer material which has excellent usability, can be completely decomposed by environmental microorganisms after being abandoned, and finally becomes an integral part of carbon circulation in the nature by being inorganic. Among biodegradable plastics, polylactic acid (PLA) having high strength and high elastic modulus has been considered as one of the most promising candidates for replacing general-purpose plastics. Polylactic acid may be polymerized from lactide. PLA has good processability and thermoplasticity, can meet the use requirement of common plastics, and can be widely applied to the fields of agricultural films, disposable tableware and the like. In addition, PLA is also used in the fields of drug-eluting materials, orthopedic internal fixation materials, medical sutures, tissue engineering scaffolds, and the like, because of its biocompatibility and non-toxicity. PLA biodegradability is one of the important reasons for its widespread interest.
In general, the biodegradation process of polymers has mainly 3 steps: (1) biodegradation: microorganisms grow on the surface of the polymer, resulting in a change in the physical and chemical properties of the polymer. (2) biological decomposition: microorganisms convert polymers into oligomers and monomers by secreted degrading enzymes; (3) assimilation: microorganisms utilize oligomers and monomers to provide themselves with the necessary carbon, energy, and nutrient sources and convert polymeric carbon into carbon dioxide, water, and biomass. In which the degrading enzymes secreted by the microorganisms play a crucial role.
Microorganisms with PLA-degrading ability secrete corresponding degrading enzymes, in particular proteases, outwards, which tend to accelerate PLA degradation by stimulation with some inducers such as silk fibroin, elastin, gelatin and some peptides and amino acids. Most of these inducers have an L-alanine-like structure, which is similar to the lactic acid units of PLA in the stereochemical position of the chiral carbon. Enzymatic degradation of the degrading enzyme proceeds only through surface erosion and weight loss on the PLA surface, the degrading enzyme selectively degrading such that the enzyme diffuses into the amorphous or less ordered regions of the PLA, and subsequently the crystalline regions of the PLA are also eventually degraded. Degradation by degrading enzymes can break down ester bonds of PLA molecules, resulting in oligomers, dimers, and monomers, which can pass through cell membranes, be absorbed and utilized by microorganisms as a carbon source and energy source, and eventually break down into carbon dioxide and water, etc.
Although PLA has been considered biodegradable, PLA degrading strains and degrading enzymes are relatively few reported and degradation efficiency is low. The reason for this is that PLA is a non-naturally occurring synthetic compound that has emerged in the last decades and microorganisms lack the evolution process for hundreds of millions of years, so that the efficiency of degradation of PLA by microorganisms in nature is relatively low. The PLA degrading enzyme which is separated and identified at present is mostly originally existing in natural environment and does not undergo any evolution process, so that the situation that the binding force with PLA substrate is not strong exists generally. And a method adopting molecular evolution is an effective way for obtaining high-efficiency PLA degrading enzyme.
In conclusion, the method has important theoretical value and practical significance for breeding of PLA degradation strains, research of degradation mechanisms and molecular transformation of degradation enzymes thereof. The researches are favorable for deeply analyzing the PLA degradation process, and are favorable for realizing the effective degradation of biodegradable plastics represented by PLA, thereby laying a foundation for the biodegradable plastics to replace the traditional plastics comprehensively in the early days. Meanwhile, the method provides guidance information for chemical synthesis of new degradable materials and discovery of new microorganisms and enzymes capable of degrading the existing plastics.
Disclosure of Invention
The invention aims to provide a protease-producing bacillus subtilis (Bacillus safensis) PLA1006 strain and a screening method thereof. The strain has protease production capability and polylactic acid utilization capability. In addition, the invention also provides application of the bacillus subtilis (Bacillus safensis) PLA1006 strain in polylactic acid degradation. The polylactic acid powder, film and emulsion can be degraded by bacillus sarefolius.
In order to solve the technical problems, the invention is realized as follows:
bacillus saxifragilis PLA1006 strain, classified and named as Bacillus saxifragilis (Bacillus safensis), was deposited in China general microbiological culture Collection center with a date of deposit of 2021, 12 months and 24 days, address: the Beijing Chaoyang area North Chenxi lu 1, 3, the preservation number is: CGMCC No.24188.
The bacillus safoci (Bacillus safensis) PLA1006 strain was isolated from the smooth municipal domestic waste landfill soil.
The screening method of the bacillus safoci PLA1006 strain is that a soil sample collected from a domestic refuse landfill in Fu city of Liaoning province is placed in a liquid culture medium containing polylactic acid powder, the soil sample is enriched and then is coated on an emulsified flat culture medium, the culture is carried out for 15 to 20 days at the temperature of 30 ℃, the growth of the strain and the formation of a transparent ring are observed, and the strain which can grow on a flat plate and form an obvious transparent hydrolysis ring is separated and screened, namely the bacillus safoci PLA1006 strain.
Further, the liquid culture medium comprises a basal culture medium and polylactic acid powder;
the preparation steps of the basal medium are as follows: KH is carried out 2 PO 4 、Na 2 HPO 4 ·12H 2 O、MgSO 4 ·7H 2 O、 NH 4 Cl、CaCl 2 ·2H 2 O、FeSO4·7H 2 O、MnSO 4 ·H 2 O、ZnSO 4 ·7H 2 O and yeast powder are respectively dissolved in ionized water, the volume is fixed to 1L, and the pH value is regulated to 6.8-7.0;
the preparation steps of the emulsified flat culture medium are as follows: dissolving polylactic acid in dichloromethane, mixing the obtained solution with a basic culture medium containing sodium dodecyl sulfate, performing ultrasonic treatment to obtain polylactic acid emulsion, and adding agar to obtain the emulsified flat culture medium.
Further, the polylactic acid is in a powder, film or emulsion state.
The bacillus safoci PLA1006 strain is applied to the degradation of polylactic acid plastic products.
The application of the bacillus subtilis PLA1006 strain in the aspect of degrading polylactic acid plastic products can be implemented according to the following steps:
(1) Wiping the surface of the dried polylactic acid film with 70% ethanol, irradiating with ultraviolet lamp for sterilization, and placing into sterile basic culture medium;
(2) The bacillus safoci PLA1006 strain is inoculated into a culture medium, and samples are taken regularly to determine the weight loss condition of the film.
Further, the basal medium of the present invention comprises: KH (KH) 2 PO 4 、Na 2 HPO 4 ·12H 2 O、MgSO 4 ·7H 2 O、 NH 4 Cl、CaCl 2 ·2H 2 O、FeSO 4 ·7H 2 O、MnSO 4 ·H 2 O、ZnSO 4 ·7H 2 O and a trace amount of yeast powder.
Further, the culture medium is a mixture of a basic culture medium and a polylactic acid film.
The bacterial cells of the strain PLA1006 of Bacillus safoci (Bacillus safensis) are short bars with length x width of about 1.2-1.4 μm x 0.6-0.7 μm, and the scanning electron microscope photograph of the bacterial cells is shown in figure 1. The strain can form a hydrolysis transparent ring in a culture medium with casein (figure 2) and polylactic acid (figure 3) as the only carbon sources, and the strain has the protease production capability and the polylactic acid utilization capability.
In a second aspect of the present invention, the strain of bacillus sarsasii (Bacillus safensis) PLA1006 is capable of growing on polylactic acid as a carbon source, and the minimal medium includes yeast powder, inorganic salts, and the like in addition to polylactic acid as a carbon source (see specific examples for details).
The invention also provides application of the bacillus subtilis (Bacillus safensis) PLA1006 strain in degrading polylactic acid, wherein the powder, film and emulsion of the polylactic acid can be degraded by the bacillus subtilis.
The bacillus sarefolius disclosed by the invention can secrete corresponding degrading enzymes in the presence of polylactic acid and yeast powder, and the liquid fermentation crude enzyme liquid has obvious hydrolysis effect on corresponding materials, and the main product of enzymolysis is a component monomer or oligomer of the polylactic acid.
The strain provided by the invention has degradation characteristics on polylactic acid, so that the strain can be applied to biodegradation of polylactic acid product waste.
Drawings
The invention is further described below with reference to the drawings and the detailed description. The scope of the present invention is not limited to the following description.
FIG. 1 is an electron micrograph of a strain of Bacillus safoci PLA1006 of the present invention;
FIG. 2 shows the growth of the strain Bacillus safoci PLA1006 on casein medium;
FIG. 3 shows the growth of the strain Bacillus safoci PLA1006 on the polylactic acid emulsion medium;
FIG. 4 is a degradation curve of the polylactic acid film degraded by the Bacillus safoci PLA1006 strain of the present invention;
FIG. 5 is a mass spectrum analysis chart of polylactic acid products degraded by crude enzyme liquid after polylactic acid powder is degraded by the Bacillus safoci PLA1006 strain.
Detailed Description
EXAMPLE 1 screening of degraded Strain
The soil sample collected from the house refuse landfill is put into a liquid culture medium containing polylactic acid powder for enrichment and then is coated on an emulsified flat culture medium, the culture is carried out for 15-20 days at 30 ℃ to observe the growth of the strain and the formation of a transparent ring, and the strain which can grow on a flat plate and form an obvious transparent hydrolysis ring is screened. The components of the screening culture medium and the preparation method are as follows:
the basic culture medium comprises the following components: 4.54g KH 2 PO 4 ，11.94g Na 2 HPO 4 ·12H 2 O，0.5g MgSO 4 ·7H 2 O， 1g NH 4 Cl，0.005g CaCl 2 ·2H 2 O，0.002gFeSO4·7H 2 O、0.001gMnSO 4 ·H 2 O、 0.002gZnSO 4 ·7H 2 O and 0.2g of yeast powder. Deionized water to a constant volume of 1L and a pH of 6.8-7.0.
Polylactic acid liquid medium: 2g of polylactic acid powder was added to the basic medium.
Preparation of an emulsion plate: 0.2g of polylactic acid is dissolved in 10ml of dichloromethane, the obtained solution is mixed with 100ml of basic culture medium containing 0.04g of sodium dodecyl sulfate, the mixture is subjected to ultrasonic treatment to prepare polylactic acid emulsion, the polylactic acid emulsion is heated at 75 ℃ for 90min to remove dichloromethane, the polylactic acid emulsion culture medium is obtained, and 2.0% agar is added to prepare the emulsion plate culture medium.
By the method, the bacillus safoci (Bacillus safensis) PLA1006 strain with the degradation effect on polylactic acid provided by the invention is screened from soil samples from a landfill.
EXAMPLE 2 degradation of polylactic acid film by Bacillus Adefolius (Bacillus safensis) PLA1006 Strain
Cutting polylactic acid film into 3m×3cm, accurately weighing, wiping surface with 70% ethanol, irradiating with ultraviolet lamp for 30min, and placing into sterile basic culture medium. The strain of bacillus safoci (Bacillus safensis) PLA1006 is inoculated into a culture medium, the culture medium is subjected to shaking culture at 30 ℃ and 150rpm, periodic sampling is carried out, the film is dried to constant weight, then the film is weighed, and the weight loss rate is calculated.
In the experiment of degrading the polylactic acid film, after 10 days of culture, the weight loss rate of the polylactic acid film reaches 15% (figure 4), while the weight loss rate of the control film is lower than that of the experiment group although the weight loss rate of the control film is lower than that of the control film, which indicates that the degradation of the polylactic acid film is mainly caused by the action of microorganisms or products thereof, but not by the simple hydrolysis.
EXAMPLE 3 preparation of crude enzyme solution of Bacillus Adefolius (Bacillus safensis) PLA1006 Strain and analysis of polylactic acid degradation products thereof
Bacillus safoci (Bacillus safensis) PLA1006 was cultured in the polylactic acid liquid medium in the same manner as in example 1, and after 7 days of culture, the supernatant was collected by centrifugation at 12000rpm and concentrated by ultrafiltration to obtain a crude enzyme solution. Mixing the crude enzyme solution with PLA emulsion, maintaining the temperature at 40 ℃ for 12 hours, centrifuging at 12000rpm to obtain supernatant reaction solution, and measuring degradation products of the ultrafiltered (with a molecular weight cut-off of 3000 daltons) submembrane solution by using a laser desorption ionization time-of-flight mass spectrometer. The measurement results (fig. 4) show that the polylactic acid enzymatic hydrolysate was found to include lactic acid monomer (mass spectrum peak 89) and lactic acid tetramer (mass spectrum peak 305).
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Shenyang agricultural university
<120> Bacillus safoci PLA1006 strain, screening method and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1458
<212> DNA
<213> Bacillus safoci (Bacillus safensis)
<400> 1
agacctaata catgcagtcg agcggacaga agggagcttg ctccctgatg ttagcggcgg 60
acgggtgagt aacacgtggg taacctgcct gtaagactgg gataactccg ggaaaccgga 120
gctaataccg gatagttcct tgaaccgcat ggttcaagga tgaaagacgg tttcggctgt 180
cacttacaga tggacccgcg gcgcattagc tagttggtgg ggtaatggct caccaaggcg 240
acgatgcgta gccgacctga gagggtgatc ggccacactg ggacggagac accggcaccc 300
agactcctac aggaagcagc agtagggaat cttccgcaat ggacgaaagt ctgaccgagc 360
aacgcagcgt ggagtgatga aggttttcgg atcgtaaagc tctgttgtta gggaagaaca 420
agtgcgagag taactggctc gcaccttgac ggtacctaac ccagaaaggc cacggctaac 480
tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtcccggaa ttattgggcg 540
taaagggctc gcaggcggtt tcttaagtct gatgtgaaag ccccccggct caaccgggga 600
gggtcattgg aaactgggaa acttgagtgc agaagaggag agtggaattc cacgtgtagc 660
ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa 720
ctgacgctga ggagcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg 780
ccgtaaacga tgagtgctaa gtgttagggg gtttccgccc cttagtgctg cagctaacgc 840
attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg 900
gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg 960
tcttgacatc ctctgacaac cctagagata gggctttccc ttcggggaca gagtgacaag 1020
tggtgcatgc ttgtcgtcag ctcgtgtcgt gagatgatgg gttaagtccc gcaacgagcg 1080
ccaactcctt gatcctaggt gccagcattc cagttggcca ctcctaaagt gaactggccc 1140
gaggaaaaac cggaggaaag gtggggatgt acggcaaatc atcatgcccc ttatgacctg 1200
ggctacacac gtgctacaat ggacagaaca aagggctgca agaccgcaag gtttagccaa 1260
tcccataaat ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa 1320
tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc 1380
gcccgtcaca ccacgagagt ttgcaacacc cgaagtcggt gaggtaacct ttatggagcc 1440
agccgccgaa ggggtcag 1458
Claims (5)
1. A bacillus sand foci PLA1006 strain, characterized by the deposit number: CGMCC No.24188.
2. The bacillus safocalis PLA1006 strain according to claim 1, characterized in that: its 16SrDNA sequence has the sequence of SEQ ID NO: 1.
3. The use of the bacillus safoci PLA1006 strain according to claim 1 for degrading polylactic acid plastic products.
4. The use of the strain bacillus safoci PLA1006 for degrading polylactic acid plastic products according to claim 3, characterized by the following steps:
(1) Wiping the surface of the dried polylactic acid film with 70% ethanol, irradiating with ultraviolet lamp for sterilization, and placing into sterile basic culture medium;
(2) Inoculating bacillus safoci PLA1006 strain into culture medium, periodically sampling, and measuring film weightlessness condition; the culture medium is a mixture of a sterile basal culture medium and a polylactic acid film.
5. The use of the bacillus safoci PLA1006 strain according to claim 4 for degrading polylactic acid plastic products, wherein the use is characterized in that: the sterile basal medium comprises: KH (KH) 2 PO 4 、Na 2 HPO 4 ·12H 2 O、MgSO 4 ·7H 2 O、NH 4 Cl、CaCl 2 ·2H 2 O、FeSO 4 ·7H 2 O、MnSO 4 ·H 2 O、ZnSO 4 ·7H 2 O and a trace amount of yeast powder.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007319078A (en) * | 2006-05-31 | 2007-12-13 | National Univ Corp Shizuoka Univ | Method for degrading polylactic acid and microorganism |
| CN113366103A (en) * | 2018-12-21 | 2021-09-07 | 诺维信公司 | Polypeptides having peptidoglycan degrading activity and polynucleotides encoding same |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007319078A (en) * | 2006-05-31 | 2007-12-13 | National Univ Corp Shizuoka Univ | Method for degrading polylactic acid and microorganism |
| CN113366103A (en) * | 2018-12-21 | 2021-09-07 | 诺维信公司 | Polypeptides having peptidoglycan degrading activity and polynucleotides encoding same |
Non-Patent Citations (2)
| Title |
|---|
| Biodegradation of polylactic acid by a mesophilic bacteria Bacillus safensis;Yujun Wang等;Chemosphere;318:137991 * |
| 解淀粉芽孢杆菌在环境保护和农业生产中的应用;胡忠亮;郑催云;田兴一;李警保;樊奔;韩正敏;;农药(04);第241-245页 * |
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