CN113151091A - Pseudomonas rouxii PR415 and application thereof - Google Patents
Pseudomonas rouxii PR415 and application thereof Download PDFInfo
- Publication number
- CN113151091A CN113151091A CN202110449445.8A CN202110449445A CN113151091A CN 113151091 A CN113151091 A CN 113151091A CN 202110449445 A CN202110449445 A CN 202110449445A CN 113151091 A CN113151091 A CN 113151091A
- Authority
- CN
- China
- Prior art keywords
- amylase
- parts
- pseudomonas
- pseudomonas rhodesiae
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- 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
-
- 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)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of microorganisms, and particularly discloses pseudomonas rhodesiae PR415 and application thereof. The strain preservation number of the pseudomonas rhodesiae PR415 is as follows: CCTCC NO: M2021331. The Pseudomonas rouxii PR415 can be used for preparing amylase. The pseudomonas rhodesiae PR415 breaks through the limitation that the amylase meeting the industrial application requirements is mainly derived from bacteria bacillus and fungi aspergillus, the high-activity amylase liquid meeting the industrial application can be obtained through fermentation, the obtained amylase liquid has a wide pH and temperature adaptation range, and high enzyme activity can be kept under the conditions that the pH is 4-11 and the temperature is 30-80 ℃. And the pseudomonas rhodesiae PR415 also has the excellent characteristics of high growth and propagation speed, high salt resistance and short amylase production time, and has great application value in the fields of industry, agriculture and medicine.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to pseudomonas rhodesiae PR415 and application thereof.
Background
Amylases are a generic term for enzymes that hydrolyze starch and glycogen, and are widely used in the industries of food, textile, paper, detergents, agricultural waste treatment, and the like. With the discovery and use of more novel amylases, the application range of amylases has now been expanded from the conventional industrial fields to the fields of clinic, medicine, agriculture and the like. Amylases are also available from a wide variety of sources and can be isolated from animals, plants and microorganisms. The amylase from the microorganism has the characteristics of rich sources, various performances and easy industrial production, can meet various industrial application requirements, and is most widely applied to industry. In the modern industrial starchy treatment process, the traditional chemical hydrolysis method is completely replaced by a microbial amylase hydrolysis method.
Although there are a variety of microorganisms that produce amylase enzymes, including filamentous fungi, yeast, bacteria, actinomycetes, and the like. However, the amylase which can meet the industrial application requirements at present mainly comes from Bacillus (Bacillus) and Aspergillus (Aspergillus), the enzymolysis pH condition range of the existing amylase is narrow, most of the existing amylases have enzymolysis activity only under the conditions of neutrality, acidity and a narrow temperature range, and the amylase is extremely easy to denature and inactivate under the conditions of alkaline conditions or large temperature range change, and the application range of the amylase is severely limited.
Disclosure of Invention
Aiming at the problems of the existing amylase production and amylase, the invention provides the pseudomonas rhodesiae PR415 and the application thereof, the pseudomonas rhodesiae PR415 can rapidly produce high-activity amylase in large quantity, and the amylase has high activity under the conditions of acidity, alkalinity and wider temperature range.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
the pseudomonas rhodesiae PR415 has the strain preservation number: m2021331, wherein the strain belongs to Pseudomonas rhodesiae (Pseudomonas rhodesiae), and is preserved in China center for type culture Collection (Ghan university, Wuhan City, Hubei province, China) at 04.06.2021.
Compared with the prior art, the pseudomonas rhodesiae PR415 breaks through the limitation that the amylase meeting the industrial application requirements is mainly derived from bacteria bacillus and fungi aspergillus, can be fermented to obtain high-activity amylase liquid meeting the industrial application, has a wide pH and temperature adaptation range, and can keep high enzyme activity under the conditions that the pH is 4-11 and the temperature is 30-80 ℃. Meanwhile, the pseudomonas rhodesiae PR415 also has the excellent characteristics of high growth and propagation speed, high salt resistance and short amylase production time, and has great application value in the fields of industry, agriculture and medicine.
The invention also provides application of the pseudomonas rhodesiae PR415 in preparation of amylase.
The pseudomonas rhodesiae PR415 can be used for quickly obtaining amylase with high activity, acid resistance, alkali resistance and wider enzymolysis temperature range, and the obtained amylase is suitable for enzymolysis reaction under more complex enzymolysis environment conditions and has higher application value in the industrial field.
The invention also provides a fermentation medium of the pseudomonas rhodesiae PR415, which comprises the following components in parts by weight:
8-12 parts of tryptone, 4-6 parts of yeast extract powder, 8-12 parts of sodium chloride and 1050 parts of sterile water 950-.
Preferably, the fermentation medium of the pseudomonas rhodesiae PR415 comprises the following components in parts by weight:
10 parts of tryptone, 5 parts of yeast extract powder, 10 parts of sodium chloride and 1000 parts of sterile water.
The invention also provides a culture condition of the pseudomonas rhodesiae PR415, which comprises the following steps: the inoculation concentration is 1.0X 105Per mL-3.0X 106The seed/mL, the inoculation volume is 1-3%, the culture temperature is 27-37 ℃, and the culture time is 24-48 h.
Preferably, the culture temperature is 30 ℃, and the culture time is 32 h.
Preferably, the culture conditions of the pseudomonas rhodesiae PR415 further comprise shaking at the condition of 150rpm-200 rpm.
The selection of the preferable fermentation medium and the preferable culture conditions can further improve the propagation speed and the amylase production efficiency of the pseudomonas rhodesiae PR 415.
The invention also provides a method for preparing the amylase by using the pseudomonas rhodesiae PR415, which comprises the following steps:
s1, inoculating the pseudomonas rhodesiae PR415 into a culture medium for culture to obtain a fermentation culture solution;
s2, centrifuging the fermentation culture solution to remove the precipitate, and obtaining the supernatant which is the amylase liquid containing the amylase.
Drawings
FIG. 1 is a colony morphology of a monoclonal strain isolated in example 1 of the present invention;
FIG. 2 is a gel electrophoresis diagram of the PCR product in example 1 of the present invention;
FIG. 3 is a phylogenetic tree diagram constructed in embodiment 1 of the present invention;
FIG. 4 is a graph showing the enzyme activity at different temperatures of amylase produced by Pseudomonas rosenbergii PR415 detected in example 5 of the present invention;
FIG. 5 is a graph showing the enzymatic activities at different pH values of amylase produced by Pseudomonas rosenbergii PR415 detected in example 5 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
1. Isolation and purification of Pseudomonas roglucida PR415
Weighing 10g of a soil sample (taken from mangrove soil in national wetland park of mangrove in Hailang island, Guangdong province) in a super-clean workbench, adding 90ml of sterile water into the soil sample, and placing the soil sample on an oscillator for oscillation for 60min to uniformly disperse the soil sample in a diluent to form a soil suspension; after the soil is dispersed, 100ul of soil suspension is absorbed into 900ul of sterile water to obtain 10 times of diluent, and then the 10 times of diluent is sequentially diluted to obtain 100 times, 1000 times, 10000 times, 100000 times and 1000000 times of diluent, and the whole process is carried out in a super clean bench.
And (3) coating 100ul of diluent with different dilution times on three LB solid culture plates respectively, and then placing the culture plates in an incubator at 37 ℃ for constant-temperature culture for 2-3 days until bacterial plaques grow on the culture plates.
According to the growth condition of the bacterial colony, single bacterial plaques with different shapes, colors, sizes and the like are selected from different dilution gradient plates to carry out plate streaking, and finally, the single bacterial strain is separated, and the form of the separated single bacterial strain on an LB solid culture plate is shown in figure 1.
The above-mentioned monoclonal strains which had been purified and cultured were picked from the LB plate and inoculated into a 500ml Erlenmeyer flask containing 200ml of LB liquid medium, and cultured on a shaker at 30 ℃ and 180rpm for 24 hours to obtain a seed solution.
Wherein the LB solid medium formula is as follows: 10 parts of tryptone, 5 parts of yeast extract powder, 10 parts of sodium chloride, 15 parts of agar powder and 1000 parts of sterile water; subpackaging the culture medium into conical flasks, sterilizing with high pressure steam at 121 deg.C and 101KPa in a sterilizing pot for 20min, cooling to below 70 deg.C, recovering pressure to 0KPa, taking out, pouring into culture dishes on a clean bench, pouring 10ml of culture medium into each dish, cooling, solidifying, and storing at 4 deg.C.
The LB liquid culture medium comprises the following components in percentage by weight: 10 parts of tryptone, 5 parts of yeast extract powder, 10 parts of sodium chloride and 1000 parts of sterile water; the manufacturing method comprises the following steps: accurately adding the components, diluting to desired volume, packaging into conical flask, sterilizing with high pressure steam at 121 deg.C and 101KPa for 20min in a sterilizing pan, cooling to below 70 deg.C, recovering pressure to 0KPa, and storing.
2. PCR amplification of the 16SrDNA sequence of the monoclonal strain and sequencing
2.1, taking genome DNA: extracting the genome DNA of the monoclonal strain by adopting an Omega Bacterial DNA Kit (D3350-01) Kit, wherein the extraction steps are completely the same as the instruction, and the extracted genome DNA is stored in a refrigerator at the temperature of-20 ℃ for later use;
2.2, PCR amplification ITS sequence: PCR amplification was performed using the genomic DNA obtained in step S1 as a template and Eubac27F and Eubac1492R as primers, and the PCR reaction system (50. mu.l) was as follows:
the sequence of the upstream primer Eubac27F is: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1);
the sequence of the downstream primer Eubac1492R is: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2).
The conditions for PCR amplification were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 30s, renaturation at 55 deg.C for 30s, and extension at 72 deg.C for 1min, and 30 times of denaturation, renaturation and extension cycle; extending for 10min at 72 ℃ to obtain a PCR amplification product, and storing at 4 ℃.
2.3 nucleic acid electrophoresis of PCR products
5 μ L of the PCR product obtained in step 2.2 was spotted and subjected to nucleic acid electrophoresis at 120V for 25 min. As shown in FIG. 2, the amplified 16SrRNA fragment using the isolated genomic DNA of the monoclonal strain as a template has a single band and high brightness, and the length of the amplified 16SrRNA sequence is about 1500 bp.
2.4 sequencing of 16SrRNA sequences
After the PCR product obtained in 2.2 is purified and recovered, 30ul of the purified product is sent to Guangzhou Ongke Biotechnology Limited for sequence two-way sequencing determination, and the sequencing result shows that the sequence length of the 16S rRNA is 1453bp, and the specific sequence is shown as SEQIDNO: 3.
2.5, constructing a phylogenetic tree
Inputting the 16S rRNA sequence into NCBI, performing BLAST comparison, wherein the 16S rRNA sequence has 99% of sequence similarity with the sequence of Pseudomonas rhodesiae DZXOTU1(MT998277.1), is highly similar to most of the sequences in the 16S rRNA barcode database of the genus Pseudomonas rhodesiae, does not have complete superposition, and does not have unique adjacent species sequences; meanwhile, an NJ phylogenetic tree is constructed based on the barcode 16SrRNA segment sequence, as shown in FIG. 3, as can be seen from BLAST comparison and phylogenetic tree construction, the strain obtained by the separation belongs to Pseudomonas rhodesiae in the genus of Pseudomonas rhodesiae in classification, is a new species, is named as Pseudomonas rhodesiae PR415(Pseudomonas rhodesiae. PR415), and is preserved in China center for type culture Collection in 04/06/2021, and the preservation address is located at Wuhan university, Wuhan, Hubei province, China.
Example 2
Culture of Pseudomonas rhodesiae PR415
The seed liquid of Pseudomonas rosenbergii PR415 obtained in example 1 was mixed at a ratio of 1.0X 106Inoculating the strain with the inoculation concentration of one strain/mL and the inoculation volume of 1 percent into a 1000mL conical flask containing 300mL of LB liquid culture medium, and culturing for 48 hours in a constant temperature shaking table with the temperature of 30 ℃ and the rpm of 180 to prepare the pseudomonas rhodesiae PR415 bacterial liquid. Detection shows that the obtained bacterial liquid has higher amylase activity.
The formulation and preparation method of LB liquid medium are the same as in example 1.
Example 3
Production of Amylase Using Pseudomonas Rohdea PR415 isolated in example 1
Inoculation: the seed liquid obtained in example 1 was mixed at a ratio of 1.0X 106The inoculation concentration of one seed/mL and the inoculation volume of 1 percent are inoculated in a 1000mL conical flask containing 300mL of LB liquid culture medium;
culturing: culturing the culture medium inoculated with the seed solution in a constant temperature shaking table at 30 ℃ and 180rpm for 24h to prepare the pseudomonas rhodesiae PR415 bacterial solution.
Centrifugal separation: after the culture is finished, the thalli sediment in the bacteria liquid is removed by centrifugation, and the supernatant is left.
The supernatant obtained in the above way is the crude enzyme solution containing amylase.
Wherein, the formula and the preparation method of the LB liquid culture medium are the same as the example 1.
Example 4
Optimal enzyme-producing fermentation culture condition for screening pseudomonas rhodesiae PR415
1. Optimal enzyme production fermentation culture time
The seed liquid of Pseudomonas rolfsii PR415 obtained in example 1 was mixed at a ratio of 1.0X 106Inoculating the strain/mL of the strain with the inoculation concentration of 1 percent into a 1000mL conical flask containing 300mL of LB liquid culture medium, carrying out constant-temperature shaking culture at 30 ℃ and 180rpm on a shaking table, sucking 10mL of bacterial liquid for 0h, 8h, 16h, 24h, 32h, 40h and 48h respectively, and storing in a refrigerator at 4 ℃;
and taking out the bacterial liquid in each time period from a refrigerator at 4 ℃, centrifuging at 12000rpm for 2min to remove thalli precipitates to obtain a crude enzyme liquid, and detecting the amylase activity of the crude enzyme liquid obtained by different inoculation amounts by using starch as a substrate by adopting a DNS method. The DNS method comprises the following specific steps:
taking 1ml of crude enzyme solution, inactivating in boiling water bath for 5min, centrifuging at 10000r/min for 2min, and taking supernatant as blank control group. Adding 50 μ l of crude enzyme solution and 50 μ l of inactivated crude enzyme solution into 450 μ l of starch solution, respectively, mixing, maintaining in 40 deg.C water bath for 30min, and placing in boiling water bath for 5min to terminate the reaction.
After 500. mu.l of the reaction solution was added to 1.5ml of DNS coloring reagent, the mixture was subjected to a color development reaction in a boiling water bath for 5min, and then cooled in an ice bath to terminate the reaction. Measuring the absorbance at the wavelength of 570nm, and calculating the activity of the amylase.
The amylase activity calculation formula is as follows: u ═ W × N × V × 1000/T/V'
U: amylase activity U/mL; w: (iv) calculated glucose concentration mg/mL; n: dilution factor (N ═ 1); v: reaction volume mL; 1000: the rate of progression from mg to ug; t: reaction time min; v': volume of enzyme solution is mL.
1 enzyme activity unit (U): defined as the amount of enzyme required to catalyze the production of 1. mu.g of reducing sugars in starch per minute at a reaction temperature of 40 ℃.
The detection result is shown in table 1, the pseudomonas rhodesiae PR415 has obvious amylase producing capacity, when the culture time is 32 hours, the enzyme activity reaches the peak value of 647.399U/mL, the positive control amylase standard with the concentration of 0.5mg/mL is taken as a reference, the amylase producing capacity is higher than that of the standard enzyme in 8 hours, and the maximum enzyme activity in 32 hours can reach 3 times of that of the standard enzyme liquid with the concentration. Therefore, the culture time of 32 hours is the optimum culture time for the amylase-producing ability of Pseudomonas rouxii.
TABLE 1 analysis of the influence of fermentation time on the amylase-producing activity of the strain (mean. + -. SD; n ═ 3)
2. Optimum concentration of inorganic salt for enzyme-producing fermentation culture
The seed liquid of Pseudomonas rolfsii PR415 obtained in example 1 was mixed at a ratio of 1.0X 106Inoculating the strain/mL of the strain with the inoculation concentration of 1 percent into a 1000mL conical flask containing 300mL of LB liquid culture medium with different salt concentrations (NaCl), carrying out constant-temperature shaking culture for 32h under the conditions of 30 ℃ and 180rpm shaking table, sucking 10mL of bacterial liquid, and storing in a refrigerator at 4 ℃;
taking out bacterial liquid cultured for 24h at different salt concentrations from a refrigerator at 4 ℃, centrifuging at 12000rpm for 2min to obtain crude enzyme liquid, and detecting the amylase enzyme activity of the crude enzyme liquid obtained at different salt concentrations by adopting the DNS method and taking starch as a substrate. The LB liquid culture medium with different salt concentrations: under the condition that other culture medium components are not changed, the NaCl content is adjusted, and 0 part, 5 parts, 20 parts, 10 parts and 30 parts of sodium chloride are respectively added to form salt concentrations with the NaCl mass content of five different concentrations, namely 0%, 0.5%, 1%, 2% and 3%.
The detection result is shown in table 2, sodium chloride with different concentrations is added into the culture medium, and when the salt concentration in the culture medium is 1%, the enzyme activity of the amylase of the pseudomonas rhodesiae PR415 is the highest and reaches 685.129U/mL; when the salt concentration in the culture medium is 3%, the enzyme activity of the amylase produced by the pseudomonas rhodesiae PR415 can still reach 587.216 +/-2.899, which indicates that the amylase produced by the strain has stronger salt tolerance.
TABLE 2 analysis of the influence of different salt concentrations on the amylase-producing activity of the strains (mean. + -. SD; n ═ 3)
Example 5
Optimum reaction conditions of amylase produced by pseudomonas rhodesiae PR415
1. Optimum temperature
The seed liquid obtained in example 1 was mixed at 1.0X 106Inoculating the strain/mL of the strain with the inoculation concentration of 1% into 300mL of LB liquid medium (same as example 1) with the salt concentration of 1%, carrying out constant-temperature shaking culture at 30 ℃ and 180rpm in a shaking table for 32 hours, sucking 10mL of bacterial liquid, and centrifuging at 12000rpm for 2min to obtain a crude enzyme liquid;
the obtained crude enzyme solution is respectively placed in enzymolysis environments of 4 ℃, 16 ℃, 25 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ and 90 ℃, and the enzymatic activity of the amylase produced by the pseudomonas rhodesiae PR415 under different temperature conditions is detected by using starch as a substrate by using a DNS method in embodiment 4.
When the temperature is 60 ℃, the enzyme activity is the highest and is 759U/mL, the enzyme activity at 60 ℃ is set as 100%, and the enzyme activity measured at the rest temperature is converted into percentage.
As shown in FIG. 4, the detection result shows that the amylase produced by Pseudomonas rosenbergii PR415 has an optimum temperature of about 60 ℃, and the enzyme activity is reduced after the temperature is higher than 60 ℃. When the temperature is 60 ℃, the enzyme activity is set as 100 percent, and the enzyme activity measured at the rest temperature is converted into percentage. As can be seen from FIG. 4, the enzyme activity reaches more than 50% at 30 ℃, the enzyme activity increases most obviously at 30-40 ℃, the enzyme activity is maintained at more than 80% at 40-60 ℃, the enzyme activity is maintained at 60-80 ℃, and the enzyme activity is still maintained at more than 50% even though the enzyme activity is reduced. In conclusion, the amylase has a wide temperature suitable range, belongs to a heat-suitable amylase, has the optimal enzymolysis temperature of 60 ℃, has high enzyme activity within the range of 30-80 ℃, and is suitable for high-temperature environments and environments with complicated and variable temperatures in industrial and agricultural production.
2. Optimum pH value
The seed liquid obtained in example 1 was mixed at 1.0X 106Inoculum concentration of 1% of the inoculum volume/mL in 300mL LB liquid with 1% salt concentrationCulturing the culture medium in a shaking table at 30 ℃ and 180rpm for 32h under constant temperature shaking, sucking 10ml of bacterial liquid, and centrifuging at 12000rpm for 2min to obtain a crude enzyme liquid;
the obtained crude enzyme solution is respectively placed in enzymolysis solutions with pH values of 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, and enzyme activity is measured after 30min of warm bath at 60 ℃. The enzyme activity of the amylase produced by the pseudomonas rhodesiae PR415 under different pH conditions is detected by using the DNS method in example 4 and starch as a substrate.
The results are shown in FIG. 5. When the pH value is 5, the activity of the amylase is the highest and reaches 762U/mL, the enzyme activity under the pH condition is set to be 100%, and the enzyme activities corresponding to the rest pH values are converted into percentages.
As can be seen from FIG. 5, the amylase activity was highest in the crude enzyme solution at pH 5. In addition, the amylase has a wide pH value adaptation range, when the pH value is 10, the enzyme activity can still maintain 80% of the highest activity, and the enzyme activity can still maintain 50% of the highest activity in the range of pH 4-11, so that the amylase can maintain the catalytic activity under the acidic condition and can normally work in the alkaline environment, and the application range of the amylase is further widened.
Example 6
The seed liquid of Pseudomonas rosenbergii PR415 obtained in example 1 was mixed at a ratio of 1.0X 105Inoculating the strain with the inoculation concentration of one strain/mL and the inoculation volume of 2 percent into a 1000mL conical flask containing 300mL of LB liquid culture medium, and culturing for 24h in a constant temperature shaking table with the temperature of 27 ℃ and the rpm of 150 to prepare the pseudomonas rhodesiae PR415 bacterial liquid. Using the DNS method in example 4, the amylase activity in the resulting bacterial suspension was determined to be 518.488U/mL using starch as a substrate.
The formula of the LB liquid culture medium is 8 parts of tryptone, 4 parts of yeast extract powder, 8 parts of sodium chloride and 950 parts of sterile water, and the preparation method is the same as that of the embodiment 1.
The enzyme activity and change of the obtained amylase under different temperature and pH conditions are basically equivalent to those of example 5.
Example 7
The seed liquid of Pseudomonas rosenbergii PR415 obtained in example 1 was mixed at a ratio of 3.0X 106Inoculating the strain with the inoculation concentration of one strain/mL and the inoculation volume of 3 percent into a 1000mL conical flask containing 300mL of LB liquid culture medium, and culturing for 48h in a constant temperature shaking table at 37 ℃ and 200rpm to prepare the pseudomonas rhodesiae PR415 bacterial liquid. Using the DNS method in example 4, using starch as a substrate, the amylase activity in the resulting bacterial suspension was 527.143U/mL.
The formula of the LB liquid culture medium is tryptone 12 parts, yeast extract powder 6 parts, sodium chloride 12 parts and sterile water 1050 parts, and the preparation method is the same as that of example 1.
The enzyme activity and change of the obtained amylase under different temperature and pH conditions are basically equivalent to those of example 5.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
SEQUENCE LISTING
<110> Zhongzhen Green Environment group Limited
<120> Pseudomonas rhodesis PR415 and application thereof
<130> 2021
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Eubac27F
<400> 1
<210> 2
<211> 19
<212> DNA
<213> Eubac1492R
<400> 2
ggttaccttg ttacgactt 19
<210> 3
<211> 1453
<212> DNA
<213> 16S rRNA 1453bp
<400> 3
ggcgtcctcc ccccgggggg gcgcctacca tgcaagtcga gcggtagaga gaagcttgct 60
tctcttgaga gcggcggacg ggtgagtaat gcctaggaat ctgcctggta gtgggggata 120
acgttcggaa acgaacgcta ataccgcata cgtcctacgg gagaaagcag gggaccttcg 180
ggccttgcgc tatcagatga gcctaggtcg gattagctag ttggtggggt aatggctcac 240
caaggcgacg atccgtaact ggtctgagag gatgatcagt cacactggaa ctgagacacg 300
gtccagactc ctacgggagg cagcagtggg gaatattgga caatgggcga aagcctgatc 360
cagccatgcc gcgtgtgtga agaaggtctt cggattgtaa agcactttaa gttgggagga 420
agggccatta cctaatacgt gatggttttg acgttaccga cagaataagc accggctaac 480
tctgtgccag cagccgcggt aatacagagg gtgcaagcgt taatcggaat tactgggcgt 540
aaagcgcgcg taggtggttt gttaagttgg atgtgaaatc cccgggctca acctgggaac 600
tgcattcaaa actgactgac tagagtatgg tagagggtgg tggaatttcc tgtgtagcgg 660
tgaaatgcgt agatatagga aggaacacca gtggcgaagg cgaccacctg gactgatact 720
gacactgagg tgcgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc 780
gtaaacgatg tcaactagcc gttgggagcc ttgagctctt agtggcgcag ctaacgcatt 840
aagttgaccg cctggggagt acggccgcaa ggttaaaact caaatgaatt gacgggggcc 900
cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggcct 960
tgacatccaa tgaactttct agagatagat tggtgccttc gggagcattg agacaggtgc 1020
tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgta acgagcgcaa 1080
cccttgtcct tagttaccag cacgttatgg tgggcactct aaggagactg ccggtgacaa 1140
accggaggaa ggtggggatg acgtcaagtc atcatggccc ttacggcctg ggctacacac 1200
gtgctacaat ggtcggtaca gagggttgcc aagccgcgag gtggagctaa tcccataaaa 1260
ccgatcgtag tccggatcgc agtctgcaac tcgactgcgt gaagtcggaa tcgctagtaa 1320
tcgcgaatca gaatgtcgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 1380
ccatgggagt gggttgcacc agaagtagct agtctaacct tcgggaggac ggtacccacg 1440
gttgatcaga cgc 1453
Claims (8)
1. Pseudomonas rhodesiae PR415, which is characterized in that: the strain preservation number is as follows: CCTCC NO: M2021331.
2. Use of pseudomonas rhodesiae PR415 of claim 1 in the preparation of an amylase.
3. The fermentation medium of pseudomonas rhodesiae PR415 of claim 1, wherein: the paint comprises the following components in parts by weight:
8-12 parts of tryptone, 4-6 parts of yeast extract powder, 8-12 parts of sodium chloride and 1050 parts of sterile water 950-.
4. The fermentation medium of pseudomonas rhodesiae PR415 of claim 3, wherein: the paint comprises the following components in parts by weight:
10 parts of tryptone, 5 parts of yeast extract powder, 10 parts of sodium chloride and 1000 parts of sterile water.
5. The culture conditions for Pseudomonas rhodesiae PR415 as claimed in claim 1, characterized in that: the inoculation concentration is 1.0X 105Per mL-3.0X 106The seed/mL, the inoculation volume is 1-3%, the culture temperature is 27-37 ℃, and the culture time is 24-48 h.
6. The culture conditions for Pseudomonas rhodesiae PR415 according to claim 5, characterized in that: the culture temperature is 30 ℃, and the culture time is 32 h.
7. The culture conditions for Pseudomonas rhodesiae PR415 according to claim 5 or 6, characterized in that: further comprises shaking at 150rpm-200 rpm.
8. A process for producing amylase by using pseudomonas rhodesiae PR415 according to claim 1, wherein: the method comprises the following steps:
s1, inoculating the pseudomonas rhodesiae strain into a culture medium for culture to obtain a fermentation culture solution;
s2, centrifuging the fermentation culture solution to remove the precipitate, and obtaining the supernatant which is the amylase liquid containing the amylase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110449445.8A CN113151091B (en) | 2021-04-25 | 2021-04-25 | Pseudomonas rouxii PR415 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110449445.8A CN113151091B (en) | 2021-04-25 | 2021-04-25 | Pseudomonas rouxii PR415 and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113151091A true CN113151091A (en) | 2021-07-23 |
| CN113151091B CN113151091B (en) | 2021-11-30 |
Family
ID=76870402
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110449445.8A Active CN113151091B (en) | 2021-04-25 | 2021-04-25 | Pseudomonas rouxii PR415 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113151091B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115553170A (en) * | 2022-09-15 | 2023-01-03 | 深圳中绿环境集团有限公司 | Method for constructing hardened seawall vegetation |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008056653A1 (en) * | 2006-11-08 | 2008-05-15 | Nippon Soda Co., Ltd. | Microorganism capable of controlling plant diseases and plant disease-controlling agent using the microorganism |
| JP2011184370A (en) * | 2010-03-09 | 2011-09-22 | Nippon Soda Co Ltd | Microbial agrochemical composition and method for producing field crop with little staining |
| CN109486712A (en) * | 2018-12-05 | 2019-03-19 | 浙江大学 | The Roche pseudomonad PYQ4 and its polyoses producing method of a kind of high-yield extracellular polysaccharide and application |
| CN111763639A (en) * | 2020-06-20 | 2020-10-13 | 山东潍坊烟草有限公司 | Pseudomonas rouxii and application thereof in tobacco |
| CN111876358A (en) * | 2020-08-04 | 2020-11-03 | 陕西均良土壤环境技术有限责任公司 | Livestock and poultry manure deodorization microbial agent and preparation method and application thereof |
| CN112007947A (en) * | 2020-08-19 | 2020-12-01 | 中国科学院城市环境研究所 | Application of pseudomonas rhodesiae in repairing heavy metal polluted environment and heavy metal tolerance |
| CN112080452A (en) * | 2020-08-17 | 2020-12-15 | 天津科技大学 | High-yield phenyllactic acid bacillus licheniformis genetic engineering bacteria, method for producing phenyllactic acid and application |
| CN112592858A (en) * | 2020-12-19 | 2021-04-02 | 深圳市长隆科技有限公司 | Low-temperature bacterium IL-2 and application thereof |
-
2021
- 2021-04-25 CN CN202110449445.8A patent/CN113151091B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008056653A1 (en) * | 2006-11-08 | 2008-05-15 | Nippon Soda Co., Ltd. | Microorganism capable of controlling plant diseases and plant disease-controlling agent using the microorganism |
| JP2011184370A (en) * | 2010-03-09 | 2011-09-22 | Nippon Soda Co Ltd | Microbial agrochemical composition and method for producing field crop with little staining |
| CN109486712A (en) * | 2018-12-05 | 2019-03-19 | 浙江大学 | The Roche pseudomonad PYQ4 and its polyoses producing method of a kind of high-yield extracellular polysaccharide and application |
| CN111763639A (en) * | 2020-06-20 | 2020-10-13 | 山东潍坊烟草有限公司 | Pseudomonas rouxii and application thereof in tobacco |
| CN111876358A (en) * | 2020-08-04 | 2020-11-03 | 陕西均良土壤环境技术有限责任公司 | Livestock and poultry manure deodorization microbial agent and preparation method and application thereof |
| CN112080452A (en) * | 2020-08-17 | 2020-12-15 | 天津科技大学 | High-yield phenyllactic acid bacillus licheniformis genetic engineering bacteria, method for producing phenyllactic acid and application |
| CN112007947A (en) * | 2020-08-19 | 2020-12-01 | 中国科学院城市环境研究所 | Application of pseudomonas rhodesiae in repairing heavy metal polluted environment and heavy metal tolerance |
| CN112592858A (en) * | 2020-12-19 | 2021-04-02 | 深圳市长隆科技有限公司 | Low-temperature bacterium IL-2 and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| HEE-YOUNG ET AL.: "Isolation and Characterization of Yam-Putrefactive Psychrotrophic Bacteria from Rotted Yam.Ryu", 《KOR. J. MIROBIOL. BIOTECHNOL.》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115553170A (en) * | 2022-09-15 | 2023-01-03 | 深圳中绿环境集团有限公司 | Method for constructing hardened seawall vegetation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113151091B (en) | 2021-11-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103695341B (en) | A kind of algin catenase secreted by marine bacteria and preparation method thereof | |
| CN111100827B (en) | Bacillus capable of producing high-activity alginate lyase and application thereof | |
| CN109456921B (en) | Paenibacillus polymyxa, application thereof, microbial agent, powder and granules | |
| CN112175880A (en) | Saline-alkali-resistant phosphate solubilizing bacterium and application thereof | |
| CN113416657B (en) | Paecilomyces lilacinus M-1 and application thereof | |
| CN109251914B (en) | Bacillus cereus and application thereof in production of cellulase | |
| CN113106044A (en) | Streptomyces modified bacterium and application thereof in feather degradation | |
| CN113151091B (en) | Pseudomonas rouxii PR415 and application thereof | |
| CN113214999B (en) | Geotrichum TN42 and application thereof in sewage treatment | |
| CN112322535B (en) | Pseudomonas phosphate solubilizing bacteria and application thereof | |
| CN112409100A (en) | Microbial organic functional fertilizer for preventing and treating ginger soil-borne diseases and preparation method thereof | |
| CN106754486A (en) | One plant height produces pseudomonad and its enzymatic production method of trehalose synthase | |
| CN112175876B (en) | Bacillus decolorationis and application thereof in antagonism of fruit and vegetable fungal diseases | |
| CN113151090B (en) | Copper pollution treating agent and application thereof | |
| CN108728370B (en) | Salmonidae nephrobacter strain QD-01 capable of efficiently producing chitosanase and fermentation method and application thereof | |
| CN115305222A (en) | Bacillus strain and application thereof | |
| CN114933974A (en) | Trichoderma pseudokoningii C5-9 and application thereof in antagonism of botrytis cinerea | |
| CN111778165A (en) | Aspergillus niger DFY1 and application thereof | |
| CN110257266B (en) | Near-rose color-locked yeast strain and application thereof in production of lactase | |
| CN112725220B (en) | Lysobacter xylosus JZ3-4-7 and application thereof | |
| CN116121099B (en) | Mangrove bacillus and application thereof and straw cellulose degradation method | |
| CN114591856B (en) | Bacillus amyloliquefaciens for producing alkaline phosphatase and application thereof | |
| CN109207407B (en) | Haicheng station bacterium K4-1 and application thereof | |
| CN117660267B (en) | Burkholderia for preventing and controlling tea tree pathogenic fungi and application thereof | |
| CN116286557B (en) | Salt-tolerant bacillus beijerinckii for producing cellulase and culture method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |