CN110317762A - The polylactic acid degradation bacteria and its application of one plant of production protease - Google Patents
The polylactic acid degradation bacteria and its application of one plant of production protease Download PDFInfo
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- CN110317762A CN110317762A CN201910696442.7A CN201910696442A CN110317762A CN 110317762 A CN110317762 A CN 110317762A CN 201910696442 A CN201910696442 A CN 201910696442A CN 110317762 A CN110317762 A CN 110317762A
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- polylactic acid
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- degradation bacteria
- acid degradation
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- 239000004626 polylactic acid Substances 0.000 title claims abstract description 100
- 229920000747 poly(lactic acid) Polymers 0.000 title claims abstract description 98
- 230000015556 catabolic process Effects 0.000 title claims abstract description 73
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 73
- 241000894006 Bacteria Species 0.000 title claims abstract description 65
- 108091005804 Peptidases Proteins 0.000 title claims abstract description 54
- 239000004365 Protease Substances 0.000 title claims abstract description 49
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims abstract description 49
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 19
- 241000589774 Pseudomonas sp. Species 0.000 claims abstract description 12
- 229920001896 polybutyrate Polymers 0.000 claims abstract description 9
- 239000002689 soil Substances 0.000 claims abstract description 7
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 5
- 238000009629 microbiological culture Methods 0.000 claims abstract description 3
- 235000019419 proteases Nutrition 0.000 claims description 44
- 230000000694 effects Effects 0.000 claims description 13
- 102000035195 Peptidases Human genes 0.000 claims description 5
- 235000019833 protease Nutrition 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 abstract description 13
- 108090000790 Enzymes Proteins 0.000 abstract description 13
- 238000002360 preparation method Methods 0.000 abstract description 6
- 208000034530 PLAA-associated neurodevelopmental disease Diseases 0.000 abstract description 4
- 239000000463 material Substances 0.000 abstract description 4
- 238000005067 remediation Methods 0.000 abstract description 3
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- 150000005690 diesters Chemical class 0.000 abstract 1
- MMHWNKSVQDCUDE-UHFFFAOYSA-N hexanedioic acid;terephthalic acid Chemical compound OC(=O)CCCCC(O)=O.OC(=O)C1=CC=C(C(O)=O)C=C1 MMHWNKSVQDCUDE-UHFFFAOYSA-N 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 description 22
- 239000001963 growth medium Substances 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 8
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- 238000000034 method Methods 0.000 description 6
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- 230000000593 degrading effect Effects 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
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- 238000012360 testing method Methods 0.000 description 4
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
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- 241000193830 Bacillus <bacterium> Species 0.000 description 2
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- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000122971 Stenotrophomonas Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
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- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241001136689 Laceyella Species 0.000 description 1
- 241001503905 Laceyella sacchari Species 0.000 description 1
- 241001661388 Lentzea waywayandensis Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 229920003232 aliphatic polyester Polymers 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 108010079058 casein hydrolysate Proteins 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 238000002856 computational phylogenetic analysis Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
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- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229910052603 melanterite Inorganic materials 0.000 description 1
- VUZPPFZMUPKLLV-UHFFFAOYSA-N methane;hydrate Chemical compound C.O VUZPPFZMUPKLLV-UHFFFAOYSA-N 0.000 description 1
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- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 150000003384 small molecules Chemical group 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 244000000000 soil microbiome Species 0.000 description 1
- 108010079522 solysime Proteins 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229920001169 thermoplastic Polymers 0.000 description 1
- 239000004416 thermosoftening plastic Substances 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
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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
- C12N1/205—Bacterial isolates
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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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
- A62D2101/28—Organic substances containing oxygen, sulfur, selenium or tellurium, i.e. chalcogen
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
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- Chemical Kinetics & Catalysis (AREA)
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Abstract
The present invention provides polylactic acid (PLA) degradation bacterias that one plant produces protease.The present invention is laid with polylactic acid degradation bacteria-pseudomonad that one plant of production protease is separated in polylactic acid/poly terephthalic acid adipic acid fourth diester (PLA/PBAT) mulch soil from Shandong Weifang, and it is named as Pseudomonas sp.strain LXM88, and it is preserved in China General Microbiological culture presevation administrative center, deposit number is (CGMCC 18058).Advantage of the invention is that this bacterium produces the protease of degradation PLA, and the bacterium has good pH tolerance, is provided simultaneously with the ability of good degradation PLA.The pseudomonad and its protease can be used as enzyme preparation, bacteria agent and the biological reinforced dose of environment remediation applied to PLA and PLA base Biodegradable material in environment, have good application value.
Description
Technical field
The present invention relates to polylactic acid degradation techniques, and in particular to the PLA degradation bacteria (Pseudomonas of one plant of production protease
Sp.strain LXM88) and its application.
Background technique
Polylactic acid (PLA) is a kind of Biodegradable material of aliphatic category, due to having both the good thermoplastic of conventional plastic
Property, biocompatibility and service performance, while PLA can by environment microorganism and its enzyme degrade, have it is good can
Biological degradability, so that PLA is widely used in industries such as agricultural, food, packaging and medical treatment.But it has now been found that the drop of PLA
Solution is mainly closely related with the physicochemical properties such as pH, temperature and humidity in environment, and the degradation rate for being embodied in the Northwest PLA is remote
Far below southwest, microbe species and limited amount in the Northwest's environment in addition, it is tight that this receives the application of PLA
The limitation of weight can only be micro- by its furthermore although PLA is aliphatic polyester, but it is difficult by microbial lipase institute catalytic degradation
The protease of bio secretion is degraded.Therefore there is an urgent need to a kind of PLA efficient degrading bacterias of environmental resistance, solve PLA in difference
The low problem of regional degradation rate.
Currently, the research of PLA degradation bacteria is very few, the PLA degradation bacteria reported both at home and abroad mainly has the lake Wei Weida Lenze Salmonella
(Lentzea waywayandensis), bacillus (Bacillus), thermoactinomyces sacchari (Laceyella sacchar),
Stenotrophomonas (Stenotrophomonas) and fungi actinomyces (Actinomadura), but generally existing degradation rate is low asks
Topic, and bacterial strain is limited to the tolerance range of pH.
Researcher has found that the degradation mode of microorganism is mainly two kinds, is on the one hand microorganism secretion protease, Ke Yigong
The ester bond in PLA is hit, macromolecule is made to fragment into small molecule chain；Another aspect microorganism is dropped by metabolism using PLA and PLA
Product lactic acid is solved, carbon dioxide and water are ultimately formed, so that being provided simultaneously with extracellular proteinase and degrading to the PLA of pH tolerance
Bacterial strain becomes research hotspot.
Summary of the invention
The purpose of the present invention is to provide PLA degradation bacterias and its application that one plant produces protease, this bacterium is to the pH in environment
Tolerance is strong, while this bacterium can have good effect to the degradation of PLA with extracellular proteinase.
The present invention is to be achieved through the following technical solutions:
The polylactic acid degradation bacteria of one plant of production protease, polylactic acid degradation bacteria (the Pseudomonas sp.strain
LXM88 deposit number) is CGMCC 18058.
Preferably, the polylactic acid degradation bacteria is isolated from the agricultural land soil that Shandong Weifang is laid with PLA/PBAT.
Preferably, the polylactic acid degradation bacteria has the function of the PLA that degrades in the environment that pH is 5.1~9.1.
Further, the polylactic acid degradation bacteria is higher than 7% to the degradation rate of PLA in the environment that pH is 5.1~9.1.
Preferably, the polylactic acid degradation bacteria has the protease that degradation PLA is generated in the environment that pH is 5.1~9.1
Effect.
Further, the protease for the degradation PLA that the polylactic acid degradation bacteria generates in the environment that pH is 5.1~9.1
Enzyme activity is higher than 25U/mL.
Application of the polylactic acid degradation bacteria of the production protease as bacteria agent in degradation PLA.
The polylactic acid degradation bacteria of the production protease is as the biological reinforced dose of application in degradation PLA.
The polylactic acid degradation bacteria of the production protease is preparing the application in the protease for degrading PLA.
Compared with prior art, the invention has the following beneficial technical effects:
The present invention from Shandong Weifang be laid with PLA/PBAT mulch agricultural land soil in screen isolated one plant lay eggs it is white
The PLA degradation bacteria Pseudomonas sp.strain LXM88 of enzyme, this bacterial strain embody tolerance to pH, pH be 5.1~
In the range of 9.1, there is good white enzymatic property of laying eggs；There is good degradation effect to PLA simultaneously.Drop provided by the invention
Solution bacterium and its protease widen the cognition to PLA degradation bacteria, and compensating for PLA degradation bacteria, pH tolerance range is limited not in the environment
Foot；This bacterium is extensive in distributed in nature, adaptable.
The present invention also provides the application studies that this bacterial strain produces protease and the PLA that degrades in the environment.In the environment for PLA
Biological prosthetic offer technical guarantee, and effectively solve PLA in different regions degradation rate different problems, be the big face of PLA
Product uses offer technical support.
Detailed description of the invention
Fig. 1 colonial morphology of the pseudomonad on LB culture medium thus.
The bio-chemical characteristics characteristic results of the pseudomonad thus of table 1.
The phylogenetic tree of Fig. 2 16S rRNA gene of pseudomonad thus.
Fig. 3 thus test by pseudomonad protease production under condition of different pH.
Fig. 4 degradation experiment of the pseudomonad to PLA thus.
Specific embodiment
Below with reference to specific embodiment, the present invention is described in further detail, it is described be explanation of the invention and
It is not to limit.
The present invention is screened from the agricultural land soil that Shandong Weifang is laid with PLA/PBAT and isolated one plant of production protease
PLA degradation bacteria, and classification naming be Pseudomonas sp.strain LXM88.
The present invention has carried out preservation to the PLA degradation bacteria Pseudomonas sp.strain LXM88 for producing protease:
Preservation day: on July 1st, 2019, depositary institution: China General Microbiological culture presevation administrative center (CGMCC) is protected
Unit address: BeiChen West Road, Chaoyang District, BeiJing City Institute of Microorganism, Academia Sinica is hidden, postcode: 100101, deposit number
CGMCC 18058。
The PLA degradation bacteria Pseudomonas sp.strain LXM88 of the production protease is 5.1~9.1 in pH
It can produce the protease of degradation PLA in environment, enzyme activity is above 25U/mL, reaches as high as 32U/mL.
The PLA degradation bacteria Pseudomonas sp.strain LXM88 of the production protease is 5.1~9.1 in pH
Degradable PLA, degradation rate are above 7% in environment, reach as high as 29%.
The PLA degradation bacteria Pseudomonas sp.strain LXM88 and its enzyme of the described production protease as enzyme preparation,
The environment remediation of PLA and PLA base Biodegradable material in bacteria agent and the biological reinforced dose of environment being applied to.
Its formula of culture medium involved in embodiment is as follows:
Using PLA as sole carbon source minimal medium: the emulsion of PLA (molecular weight is 100,000), 10g；KH2PO4,1g；
Na2HPO4, 1.5g；NH4Cl, 2g；CaCl2·2H2O, 0.1g；KCl, 0.15g；MgSO4·7H2O, 0.2g；FeSO4·7H2O,
0.01g；ZnSO4·7H2O, 0.01g；MnSO4·H2O, 0.001g；Agar, 20g；Distilled water, 1000mL；PH is 7.2~7.4.
LB culture medium: tryptone, 10g；Yeast extract, 5g；NaCl, 10g；Distilled water, 1000mL；PH be 7.2~
7.4。
Skimmed milk power culture medium: skimmed milk power, 40g；Agar, 15g；Distilled water, 1000mL；PH is natural.
The separation of 1 bacterial strain of embodiment
The agricultural land soil that Shandong Weifang is laid with PLA/PBAT is acquired, weighs 10g soil in the 150mL of 90mL sterile water
In triangular flask, configuration 10-1To 10-6The soil bacteria suspension of series of concentrations gradient is inoculated in and trains by sole carbon source inorganic salts of PLA
It supports in base, after cultivating 3 days in 37 DEG C of constant incubator, observes the upgrowth situation of microorganism on culture medium, and picking is single
Bacterium colony, continuous several times are inoculated in using PLA to be isolated and purified in the minimal medium of sole carbon source, until separation obtains one
Then strain bacterium observes it in colonial morphology, as shown in Figure 1, bacterium colony milky is round, the smooth wet, flush edge in surface, no
It is transparent.
The identification of 2 bacterial strain of embodiment
1. the bio-chemical characteristics of bacterial strain
Picking is using PLA as the single bacterium colony of this bacterial strain on the minimal medium of sole carbon source, according to " common bacteria system
System identification handbook " the major physiological biochemical test of this bacterium is tested, as a result as shown in table 1 below, physio-biochemical characteristics are as follows:
V-P experiment, catalase are positive；Gram staining experiment, M.R experiment, Starch Hydrolysis experiment, grease hydrolysis experiment are negative.
1 bio-chemical characteristics result of table
2. the 16S rRNA gene order of bacterial strain is identified
By bacterial strain on LB culture medium, cultivated 3 days on 37 DEG C, the shaking table that revolving speed is 130r/min condition, using boiling
Method extracts the DNA of bacterial strain, and with bacterial universal primers 27F (AGAGTTTGATCCTGGCTCAG) and 1492R
(AAGGAGGTGATCCAAGCC), bacterial strain DNA is expanded, after (the raw work in Shanghai) is sequenced in amplified production, has obtained length
Degree is the 16S rRNA gene order of 1436bp, sees SEQ ID No:1.Test result is uploaded in GENBANK and is logged in
Number it is MK828191, is named as Pseudomonas sp.strain LXM88, and carry out with bacterial strain reported in GENBANK
Sequence analysis, and choose related strain and carry out phylogenetic tree analysis, such as Fig. 2, it is seen that this bacterial strain and Pseudomonas
For mendocina (accession number: BBQC01000018) in the same branch, homology is up to 99%.Therefore present invention screening
The degradation bacteria of acquisition is pseudomonad.
Therefore, this pseudomonad (Pseudomonas sp.strain LXM88) is preserved in Chinese common micro- by inventor
Biological inoculum preservation administrative center (CGMCC), deposit number are CGMCC 18058.
3 bacterial strain of embodiment produces the identification of protease ability
This bacterium is inoculated on skimmed milk power culture medium, 37 DEG C are cultivated 3 days, and observing has apparent transparent hydrolysis under bacterium colony
Circle, it is seen that this bacterium can produce protease.
4 bacterial strain of embodiment produces protease aptitude tests.
The preparation of 1.PLA film
The PLA film that 5g molecular weight is 100,000 is chosen, is dissolved in the chloroform of 95mL, after it fills dissolution, is fallen
Enter mold, volatilizees to chloroform, PLA film is prepared, and be cut into 1*1cm2Fritter, for strains for degrading test.
2. the preparation of culture medium
On the basis of minimal medium, the Co metabolism that other additional carbons such as 2% glucose are added as this bacterium is raw
Long matrix.
3. the preparation of seed liquor
By strain inoculated after purification in the LB culture medium of 100mL, in 37 DEG C, the condition that shaking table speed is 130r/min
Under, 12h is cultivated, obtains the bacterium solution that OD600 is 1.0 as seed liquor.
4. proteinase activity measuring method: being measured using national standard Forint phenol method
Enzyme activity definition: the protease activity (U/mL) of 1 unit is defined as 40 DEG C, under the conditions of pH 7.5 in 1mL supernatant
1min caseinhydrolysate generates albumen enzyme amount required for 1 μ g tyrosine.
5. the production protease of bacterial strain is tested
By this strain inoculated in the 100mL minimal medium of additional 2% glucose, adjust pH to 5.1,6.1,7.1,
8.1 and 9.1, it is inoculated with 1mL seed liquor, under conditions of 37 DEG C, shaking table speed are 130r/min, is cultivated 5 days, using Forint phenol method
Detect the protease content (U/mL) in supernatant.
As a result as shown in figure 3, when pH in culture medium is 5.1,6.1,7.1,8.1 and 9.1, which produces protease
Ability is above 25U/mL, and when pH is 7.1, proteinase activity is up to 26.79U/mL.
In production protease experiment, then adjust the temperature of culture bacterial strain be 22~42 DEG C, the inoculum concentration of bacterial strain be 0.5~
2.5mL considers the influences of the main affecting factors to this strain enzyme-producing such as temperature, inoculum concentration, and optimizes its production using response surface design
Protease condition, it is known that this bacterium produces prolease activity most under conditions of pH is 7.4, inoculum concentration is 1.4% and temperature is 32 DEG C
High reachable 32.15U/mL.It can be seen that this pseudomonad it is excellent can to produce protease ability under conditions of pH is 5.1~9.1
It is good, so the protease of this bacterium secretion degrades PLA with apparent advantage in different environments.
5 strains for degrading PLA of embodiment experiment
By this strain inoculated in the 100mL minimal medium of additional 2% glucose, adjust pH be 5.1,6.1,7.1,
8.1 and 9.1 add 3~5 pieces of PBAT film (every piece of PBAT mass is about 0.0150g), and are inoculated with 1mL seed liquor, 37 DEG C of Yu,
Under conditions of shaking table speed is 130r/min, cultivate 5 days, and measure the mass loss rate (%) of PBAT film.
As a result such as Fig. 4, it is seen that this bacterial strain is good to the degradation capability of bacterial strain in the range of pH is 5.1~9.1, degradation
Rate is above 7%, and better than the level for having reported degradation bacteria at present, under the conditions of meta-alkalescence of the pH 7.1~9.1, this bacterium is to PLA
Degradation capability more preferably, reach as high as 29%.It can be seen that this pseudomonad can secrete the albumen of degradation PLA in the environment
Enzyme is simultaneously good to PLA degradation effect, so this bacterium and its enzyme can be applied to ring as enzyme preparation, bacteria agent and biological reinforced dose
In border in the environment remediation of PLA and PLA base Biodegradable material, there is good use value.
Sequence table
The polylactic acid degradation bacteria and its application of<120>one plants of production protease
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1436
<212> DNA
<213>pseudomonad (Pseudomonas sp. strain LXM88)
<400> 1
ctggggcagc taccatgcag tcgagcggat gagagagctt gctctctgat ttagcggcgg 60
acgggtgagt aatgcctagg aatctgcctg gtagtggggg ataacgttcc gaaaggaacg 120
ctaataccgc gtacgtccta cgggagaaag caggggacct tcgggccttg cgctatcaga 180
tgagcctagg tcggattagc tagttggtga ggtaatggct caccaaggcg acgatccgta 240
actggtctga gaggatgatc agtcacactg gaactgagac acggtccaga ctcctacggg 300
aggcagcagt ggggaatatt srrcawtggr mkwwrgmcwr wtscrssmaw gcygmkyswg 360
yrwrsrrsgt sktsrgawkk ywwmgsaykt waagywskkw rgwrgrgsar krmrktarta 420
myyysywgyt ktkwsrykwm cgacagaata agcaccggcy wacttcgtgc cagcagccgc 480
grkwatacga agggtgcaag cgttaatcgg aattactggg cgtaaagcgc gcgtaggtgg 540
ttcgttaagt tggatgtgaa agccccgggc tcaacctggg aactgcatcc aaaactggcg 600
agctagagta cggtagaggg trgtggaatt tcctgtgtag cggtgaaatg cgtagatata 660
ggaaggaaca ccagtggcga aggcgaccac ctggactgat actgacactg aggtgcgaaa 720
gcgtggggag caaacaggat tagataccct ggtagtccac gccgtaaacg atgtcaacta 780
gccgttggaa tccttgagat tttagtggcg cagctaacgc attaagttga ccgcctgggg 840
agtacggccs caaggttaaa actcaaatga attgacgggg gcccgcacaa gcggtggagc 900
atgtggttta attykaagca acgcgaagaa ccttacctgg ycttgacatg ctgagaactt 960
tccagagatg gattggtgcc ttcgggaamt cagacmcagg tgctgcatgg ctgtygtcag 1020
ctcgtgtcgt ggagatgttg ggttaagtcc cgtaacgagc gcaacccttg tccttagtta 1080
ccagcaacgt tatggtgggc actcctaagg agaactgccg gtgaacaaac cggaagraag 1140
gtggggawkr mgwcragtca tcatggccct tacggccagg gctacacacg tgctacaatg 1200
gtcggtacaa agggttgcca agccgcgagg tggagctaat cccataaaac cgatcgtagt 1260
ccggatcgca gtctgcaact cgactgcgtg aagtcggaat cgctagtaat cgtgaatcag 1320
aatgtcacgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac catgggagtg 1380
ggttgctcca gaagtagcta gtctaacctt cggggggacg gtaccacgag atacga 1436
Claims (9)
1. the polylactic acid degradation bacteria of one plant of production protease, which is characterized in that the polylactic acid degradation bacteria (Pseudomonas
Sp.strain LXM88) on July 1st, 2019 it is stored in China General Microbiological culture presevation administrative center, deposit number
CGMCC 18058。
2. the polylactic acid degradation bacteria according to claim 1 for producing protease, which is characterized in that the polylactic acid degradation bacteria point
From the agricultural land soil for being laid with PLA/PBAT from Shandong Weifang.
3. the polylactic acid degradation bacteria according to claim 1 for producing protease, which is characterized in that the polylactic acid degradation bacteria tool
There is the PLA that degrades in the environment that pH is 5.1~9.1.
4. the polylactic acid degradation bacteria according to claim 3 for producing protease, which is characterized in that the polylactic acid degradation bacteria exists
7% is higher than to the degradation rate of PLA in the environment that pH is 5.1~9.1.
5. the polylactic acid degradation bacteria according to claim 1 for producing protease, which is characterized in that the polylactic acid degradation bacteria tool
Play the role of generating the protease of degradation PLA in the environment that pH is 5.1~9.1.
6. the polylactic acid degradation bacteria according to claim 5 for producing protease, which is characterized in that the polylactic acid degradation bacteria exists
The proteinase activity of the degradation PLA generated in the environment that pH is 5.1~9.1 is higher than 25 U/mL.
7. the polylactic acid degradation bacteria described in any one of claims 1-6 for producing protease is as bacteria agent in degradation PLA
Using.
8. the polylactic acid degradation bacteria described in any one of claims 1-6 for producing protease is as biological reinforced dose in degradation PLA
Application.
9. the polylactic acid degradation bacteria described in any one of claims 1-6 for producing protease is preparing the protease for the PLA that degrades
In application.
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Cited By (2)
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| CN115558624A (en) * | 2022-11-10 | 2023-01-03 | 陕西科技大学 | Low-temperature petroleum degrading bacterium, low-temperature petroleum degrading microbial inoculum and preparation method and application thereof |
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| CN111100815A (en) * | 2020-01-09 | 2020-05-05 | 南京高新工大生物技术研究院有限公司 | Pseudomonas for degrading PBAT (poly (butylene adipate-co-terephthalate)) plastic and application thereof |
| CN111100815B (en) * | 2020-01-09 | 2021-07-23 | 南京高新工大生物技术研究院有限公司 | Pseudomonas for degrading PBAT (poly (butylene adipate-co-terephthalate)) plastic and application thereof |
| CN115558624A (en) * | 2022-11-10 | 2023-01-03 | 陕西科技大学 | Low-temperature petroleum degrading bacterium, low-temperature petroleum degrading microbial inoculum and preparation method and application thereof |
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