CN108929859B - Bacillus-like strain HB172198 and application thereof - Google Patents
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Abstract
The invention discloses a bacillus like strain HB172198, which is classified and named as bacillus like (Paenibacillus sp.) with the preservation number as follows: CGMCC No.15412, the preservation date is: 03 and 05 days 2018, the preservation unit is: the China general microbiological culture Collection center has the following preservation addresses: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing. The invention also discloses an alginate lyase and a preparation method thereof, and application of the bacillus-like strain HB172198 and the alginate lyase in degradation of brown algae.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus-like strain HB172198 and application thereof.
Background
Algin, also known as sodium alginate, is a linear macromolecule formed by linking and randomly arranging two uronic acid monomers, 1, 4-beta-D-mannuronic acid (1, 4-beta-D-mannuronic acid, M) and 1, 4-alpha-L-guluronic acid (1, 4-alpha-L-guluronic acid, G), through alpha/beta-1, 4 glycosidic bonds, and is widely present in cells of brown algae such as kelp, gulfweed, kelp, carrageen, and the like. The algin, as a polysaccharide substance with great application value, can be degraded into oligosaccharide fragments polymerized by 2-20 monosaccharides, thereby showing various biological activities and being widely applied in the industries of food, medical treatment, industrial production and the like. The degradation method of algin is divided into chemical degradation method, physical degradation method and enzyme degradation method. The enzymolysis method is characterized in that macromolecule algin is degraded into micromolecule oligosaccharide fragments by algin lyase, the reaction condition is mild, the efficiency is high, the substrate specificity is strong, byproducts harmful to human bodies and the environment are not generated, and the method gradually becomes a main means for producing algin oligosaccharide by industrially degrading algin.
The source of the alginate lyase in nature is wide, and the alginate lyase can be separated from various marine animals, oceans and terrestrial microorganisms, wherein the microorganisms are a main group for producing the alginate lyase. The microorganisms are also highly capable of producing enzymes by bacteria and are widely researched, such as Vibrio (Vibrio), Pseudomonas (Pseudomonas), Halomonas (Halomonas), Klebsiella (Klebsiella), Flavobacterium (Flavobacterium) and Bacillus (Bacillus), and the majority of the microorganisms are gram-negative bacteria, while the reports of gram-positive bacteria are few. The bacillus has the characteristics of strong adaptability, high growth speed, long survival time and the like, and fewer bacilli producing the alginate lyase are reported at present. The alginate lyase produced by the bacillus can act on a specific glycosidic bond to form a fixed product, and has wide application prospect.
Disclosure of Invention
The invention aims to provide a bacillus like strain HB172198 which can effectively degrade algin, can also be used as a novel alginate lyase producing strain, and can also be directly used as a microbial resource for alginate biodegradation.
The invention also aims to provide the alginate lyase and the preparation method thereof, the alginate lyase can effectively degrade the algin, the method has mature process, and a large amount of the alginate lyase can be obtained.
The third purpose of the invention is to provide the bacillus like strain HB172198 and the application of the algin lyase in degrading algin.
The first object of the present invention is achieved by the following technical solutions: a bacillus like strain HB172198 is classified and named as bacillus like (Paenibacillus sp.) with the deposit number as follows: CGMCC No.15412, the preservation date is: 03 and 05 days 2018, the preservation unit is: china general microbiological culture Collection center (CGMCC), the preservation address is as follows: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.
The bacillus like HB172198 of the invention belongs to the bacterial domain, the phylum firmicutes, the class of bacilli, the order of bacillales, the family of bacillaceae and the genus of bacilli.
The bacillus like HB172198 is obtained by separating and screening epiphytic bacteria of brown algae.
The second object of the present invention is achieved by the following technical solutions: an alginate lyase is prepared by fermenting the bacillus-like strain HB 172198.
The above method for preparing alginate lyase preferably comprises the following steps:
(1) strain activation: inoculating the bacillus like HB172198 into a solid culture medium, and culturing at 28-37 ℃ for 24-48 h to obtain an activated strain;
(2) liquid culture: inoculating the activated strain into a liquid seed culture medium, and performing shaking culture at 28-37 ℃ and 120-200 r/min for 12-24 h to prepare a seed solution;
(3) fermentation culture: inoculating the seed solution into a liquid fermentation culture medium according to the volume percentage of 2-10%, carrying out shaking culture at the temperature of 28-37 ℃ and at the speed of 150-200 r/min for 24-60 h, collecting fermentation liquor, centrifuging, and collecting supernatant to obtain crude enzyme liquid of the alginate lyase.
The preparation method of the alginate lyase comprises the following steps:
the solid medium in the step (1) is preferably sodium alginate 2216E solid medium.
The sodium alginate 2216E solid medium comprises the following components: 2-15 g/L of sodium alginate, 3-10 g/L of peptone, 0.5-5 g/L of yeast powder, 5-35 g/L of sodium chloride, 18-20 g/L of agar powder, and 6.5-7.8 of pH, and is prepared by using distilled water.
Sodium alginate is added into the culture medium, so that the bacteria can be promoted to produce alginate lyase. The liquid seed culture medium in the step (2) and the liquid fermentation culture medium in the step (3) are preferably sodium alginate 2216E liquid culture medium.
The sodium alginate 2216E liquid medium comprises the following components: 2-15 g/L of sodium alginate, 3-10 g/L of peptone, 0.5-5 g/L of yeast powder, 5-35 g/L of sodium chloride and pH of 6.5-7.8, and is prepared by distilled water.
The third object of the present invention is achieved by the following technical solutions: the bacillus like strain HB172198 and the application of the alginate lyase in degrading brown algae are provided.
The brown algae preferably include brown algae such as Sargassum and herba Zosterae Marinae.
The invention has the following advantages:
(1) the bacillus-like strain HB172198 can effectively degrade algin, can be used as a novel alginate lyase producing strain, and can also be directly used as a microbial resource for alginate biodegradation.
(2) The alginate lyase can effectively degrade algin, and the preparation method and the process of the alginate lyase are mature, and a large amount of the alginate lyase can be obtained.
(3) The bacillus-like strain HB172198 and the algin lyase can be used for degrading algin, gulfweed, kelp and other brown algae.
Drawings
FIG. 1 is an electron micrograph of strain HB172198 obtained in example 1;
FIG. 2 is a 16S rDNA-based phylogenetic tree of strain HB172198 in example 2;
FIG. 3 is a thin-layer chromatography of oligosaccharide formed by degradation of sodium alginate by crude enzyme solution of strain HB172198 in example 5, wherein 1 is standard (monosaccharide, trisaccharide and pentasaccharide) and 2-8 are enzymolysis samples of 2 nd, 6 th, 10 th, 14 th, 20 th, 36 th and 48 th hours, respectively.
The invention will be further illustrated with reference to specific examples:
Detailed Description
Example 1 isolation and screening of Bacillus bacteroides HB172198 CGMCC No.15412
Brown algae samples were collected offshore from the water bay of Wenchang city, Hainan province. Collecting 10g sample, cutting under sterile condition, dissolving, placing in 250mL conical flask containing 90mL sterile water and glass beads, shaking in constant temperature shaking table at 28 deg.C and 200r/min for 30min, standing for 5min, collecting supernatant, diluting to 10 times of gradient, and diluting to 10-1～10-3A series of suspensions. And (3) killing non-spore bacteria by adopting a water bath treatment method at 80 ℃ for 20min, sucking 0.1mL of serial suspensions, coating the serial suspensions on an alginate lyase separation culture medium, and placing the algin lyase separation culture medium in an incubator at 30 ℃ for inverted culture for 2-5 days.
When the bacterial colony grows out, the bacterial colony with good growth and different forms is selected for streak purification according to the phenotypic characteristics of the shape, the color, the edge state, the transparency, the surface dry and wet state and the like of the bacterial colony.
And (3) carrying out alginate lyase activity determination on the bacillus obtained by separation, and screening out bacillus HB172198 with strong enzyme activity.
The specific process is as follows:
selecting the strain to be detected with a sterilized toothpick, inoculating to the alginate lyase activity detection culture medium, and culturing at 30 deg.C for 2 d. After a distinct colony has grown on the plate, the colony diameter (d) is measured. Adding 1mol/L CaCl into a plate2Standing the solution for 30-60 min. And after the hydrolysis ring is displayed on the flat plate, measuring the diameter (D) of the hydrolysis ring, and taking the ratio (D/D) of the diameter of the hydrolysis ring to the diameter of the bacterial colony as a primary screening index.
Wherein the D/D value of the bacillus HB172198 is 10.5, and the activity is obvious.
The formula of the culture medium used is as follows:
alginate lyase separation culture medium: 5g of sodium alginate, 1g of yeast powder, 5g of peptone, 0.01g of high-iron phosphate and 18g of agar, and the volume is adjusted to 1L by seawater and the pH value is 7.6.
Alginate lyase activity detection culture medium: sodium alginate 5g, (NH)4)2SO4 5g，K2HPO4 2g，NaCl 20g，MgSO4·7H2O 1g，FeSO4·7H20.01g of O and 18g of agar, and the volume is adjusted to 1L by seawater, and the pH value is 7.6.
Example 2 species identification of Bacillus subtilis HB172198 CGMCC No.15412
After the strain HB172198 CGMCC No.15412 is subjected to streak culture for 3d on a 2216E agar culture medium, the bacterial colony is circular, has regular edges, and has a matt, flat, light yellow and opaque surface; the thallus is rod-shaped, produces spores, and has flagella at both ends (figure 1). The gram stain is variable, nitrate is not reduced, gelatin is not hydrolyzed, and indole is not generated.
The 16S rDNA sequence 1477bp of HB172198 is obtained by PCR amplification and sequencing, and the nucleotide sequence is shown in SEQ ID No. 1. The sequence is compared with the sequence in an EzBioCloud database, and the strains HB172198 and Paenibacillus lemnae L7-75 are foundTThe homology is highest (97.6%), and the homology with other strains is below 97.0%. The related strains with high homology were selected, and a phylogenetic tree (FIG. 2) was constructed by the Neighbor-join method using the software MEGA5.0, in which the strains HB172198 and Paenibacillus lemnae L7-75 were foundTOn the same branch, the two are closer in relationship. The genomic ANI values for both were compared to only 75.3%, less than the 95% species limit as defined by the International Commission on bacterial systems taxonomy.
The morphological, physiological, biochemical and molecular characteristics of the two strains are compared, and the strain HB172198(CGMCC No.15412) in the invention is identified as a new species of the bacillus like, which is tentatively named as Paenibacillus sp.HB172198.
The strain is preserved in China general microbiological culture Collection center (CGMCC) at 3 and 05 months in 2018, and the preservation registration number is CGMCC No.15412, and the survival is proved. The preservation address is the microbiological research institute of China academy of sciences, No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.
The strain has stronger activity for producing the alginate lyase, can effectively degrade the algin bodies such as gulfweed, kelp and the like, can be used for preparing the alginate lyase by fermentation, and can also be developed into a novel microbial agent applied to the biodegradation of the algin.
Example 3 alginate lyase and preparation method thereof
The algin lyase is prepared by fermenting the bacillus licheniformis HB172198 with the preservation number of CGMCC No.15412 in the embodiment 1, and the specific method comprises the following steps:
(1) strain activation: inoculating the bacillus like HB172198 in example 1 into a solid culture medium, and culturing at 30 ℃ for 48h to obtain an activated strain;
(2) liquid culture: inoculating the activated strain into a liquid seed culture medium, and performing shaking culture at 30 ℃ and 180r/min for 12-24 h to prepare a seed solution;
(3) fermentation culture: inoculating the seed solution into a liquid fermentation culture medium according to the volume percentage of 3%, carrying out shaking culture at 30 ℃ and 180r/min for 36h, collecting fermentation liquor, centrifuging, and collecting supernatant to obtain crude enzyme solution of the alginate lyase.
The solid culture medium in the step (1) is sodium alginate 2216E solid culture medium.
The sodium alginate 2216E solid medium comprises the following components: 2-15 g/L of sodium alginate, 3-10 g/L of peptone, 0.5-5 g/L of yeast powder, 5-35 g/L of sodium chloride, 18-20 g/L of agar powder, and 6.5-7.8 of pH, and is prepared by using distilled water.
The liquid seed culture medium in the step (2) is sodium alginate 2216E liquid culture medium.
The liquid fermentation medium in the step (3) is sodium alginate 2216E liquid medium.
The sodium alginate 2216E liquid medium consists of: 2-15 g/L of sodium alginate, 3-10 g/L of peptone, 0.5-5 g/L of yeast powder, 5-35 g/L of sodium chloride and pH of 6.5-7.8, and is prepared by distilled water.
Example 4 determination of enzyme Activity of alginate lyase
And (3) adopting an ultraviolet absorption method to carry out the determination of the activity of the alginate lyase. The process is as follows:
the fermentation broth was collected and centrifuged, and the supernatant was used as a crude enzyme solution (prepared in example 3).
Preheating 1.8mL of substrate (3.0g sodium alginate dissolved in 1L of 50mM phosphate buffer solution pH 7.0) at 40 deg.C for 5min, adding 0.2mL of crude enzyme solution to be tested, warm-bathing at 40 deg.C for 10min, determining the OD of reaction system with inactivated enzyme solution system as blank control235Ultraviolet absorption value of (1). Define OD in the above enzyme activity measuring method235The amount of enzyme whose UV absorbance increased by 0.1 per minute was one unit of activity (1U) of the enzyme.
The result shows that the alginate lyase is the largest when the strain HB172198 is fermented for 36 hours, and the enzyme activity is 14.6U/mL.
Example 5 degradation of sodium alginate by alginate lyase
The method for determining the effect of degrading sodium alginate by using the alginate lyase comprises the following steps: collecting fermentation liquor, centrifuging, taking the supernatant as a crude enzyme solution (namely alginate lyase), preparing a sodium alginate solution with the mass percentage of 2%, adding the crude enzyme solution with the volume ratio of 20%, reacting at the temperature of 30 ℃, taking 5mL of the solution respectively when reacting for 2, 6, 10, 14, 20, 32 and 48 hours, adding absolute ethyl alcohol until the final concentration is 70%, centrifuging, taking supernatant, freeze-drying, adding a small amount of water to dissolve, and performing Thin Layer Chromatography (TLC). The developing agent is n-butanol, formic acid and water at ratio of 4: 5: 1, 5% ethanol sulfate is used as developer, and the developer is kept at 110 deg.C for 10 min. The polymerization degree of the main degradation products is 1-7, a thin-layer chromatography spectrum is shown in figure 3, in the figure, a sample 1 is a standard product (monosaccharide, trisaccharide and pentasaccharide), and samples 2-8 are enzymolysis samples of 2 nd, 6 th, 10 th, 14 th, 20 th, 36 th and 48 th hours respectively, and the result shows that the alginate lyase generated by the strain HB172198 can effectively degrade sodium alginate.
Example 6 degradation of Sargassum by alginate lyase
Inoculating the alginate lyase prepared in example 3 into a sterilized sargassum powder solid matrix according to an inoculation amount (mass percentage, preferably 3%) of 2-10%, wherein the matrix comprises the following components: 30-50% of sargassum powder, 10-30% of corn flour, 10-30% of bean pulp, 10-30% of bran and 30-60% of water content (preferably 30% of sargassum powder, 30% of corn flour, 20% of bean pulp, 20% of bran and 45% of water content). Standing and culturing in an incubator at 28-37 deg.C (preferably 30 deg.C), shaking to disperse the solid culture medium several times per day, and culturing for 10-20d (preferably 12 d).
Weighing an appropriate amount of solid fermentation sample, placing in a centrifuge tube, adding an appropriate amount of pure water, mixing, centrifuging, collecting supernatant, and performing Thin Layer Chromatography (TLC). The developing agent is n-butanol, formic acid and water at ratio of 4: 5: 1, 5% ethanol sulfate is used as developer, and the developer is kept at 110 deg.C for 10min for developing, and the main degradation product is oligosaccharide with polymerization degree of 2-7.
Example 7 degradation of kelp by Strain HB172198
Soaking dried kelp for 1h, cleaning, cutting into pieces (1cm multiplied by 1cm), horizontally placing in a culture dish, adding a proper amount of inorganic salt culture solution, and adding the following components: (NH)4)2SO4 5g，K2HPO4 2g，NaCl 20g，MgSO4·7H2O 1g，FeSO4·7H2O0.01 g, and distilled water was added thereto to make 1L, pH 7.5.
The fresh bacterial solution of HB172198 prepared in example 3 at the late logarithmic growth stage was inoculated, the inoculum size was 2%, mixed, cultured at 30 ℃ for 7 days, and the turbidity of the medium and the shape of the sea tangle block at different time points were observed.
Observing and finding that the kelp blocks are gradually dissolved along with the prolonging of the culture time, the color of the colorless culture solution is gradually deepened to brown, and the kelp blocks are completely degraded to be in an unfixed state by the 7 th day, so that the kelp blocks are completely degraded. This indicates that strain HB172198 and its fermentation liquor can effectively degrade kelp.
The present invention has been described above by referring to a part of specific embodiments, and it should be noted that the above specific embodiments are only used for further description of the present invention and do not represent a limitation to the protection scope of the present invention. Other insubstantial modifications and adaptations of the present invention can be made without departing from the scope of the present invention.
Sequence listing
<110> research institute of tropical biotechnology of Chinese tropical academy of agricultural sciences
<120> bacillus sp
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1477
<212> DNA
<213> Bacillus sp
<400> 1
gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggagttaatg gagtgcttgc 60
actcctgatg cttagcggcg gacgggtgag taacacgtag gcaacctgcc ctcaagactg 120
ggataactac cggaaacggt agctaatacc ggatagttga tttcgctgca tagtgagatc 180
tggaaaggcg gagcaatctg tcacttgagg atgggcctgc ggcgcattag ctagttggtg 240
gggtaacggc ctaccaaggc gacgatgcgt agccgacctg agagggtgaa cggccacact 300
gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg 360
acgaaagtct gacggagcaa cgccgcgtga gtgatgaagg ttttcggatc gtaaagctct 420
gttgccaggg aagaacgtct tctagagtaa ctgctagaag agtgacggta cctgagaaga 480
aagccccggc taactacgtg ccagcagccg cggtaatacg tagggggcaa gcgttgtccg 540
gaattattgg gcgtaaagcg cgcgcaggcg gtttgttaag tctggtgttt aaacctgggg 600
ctcaacctca ggtcgcactg gaaactggga aacttgagtg cagaagagga gagtggaatt 660
ccacgtgtag cggtgaaatg cgtagatatg tggaggaaca ccagtggcga aggcgactct 720
ctgggctgta actgacgctg aggcgcgaaa gcgtggggag caaacaggat tagataccct 780
ggtagtccac gccgtaaacg atgaatgcta ggtgttaggg gtttcgatac ccttggtgcc 840
gaagttaaca cattaagcat tccgcctggg gagtacggtc gcaagactga aactcaaagg 900
aattgacggg gacccgcaca agcagtggag tatgtggttt aattcgaagc aacgcgaaga 960
accttaccag gtcttgacat ccctctgacc ggtatagaga tatacctttc cttcgggaca 1020
gaggagacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080
cgcaacgagc gcaacccttg atcttagttg ccagcaggtt atgctgggca ctctaaggtg 1140
actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga 1200
cctgggctac acacgtacta caatggctgg tacaacggga agcgaaggag cgatctggag 1260
cgaatcctaa aaagccagtc tcagttcgga ttgcaggctg caactcgcct gcatgaagtc 1320
ggaattgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gtcttgtaca 1380
caccgcccgt cacaccacga gagtttacaa cacccgaagt cggtggggta acccgcaagg 1440
gagccagccg ccgaaggtgg ggtagatgat tggggtg 1477
Claims (3)
1. Paenibacillus strain（Paenibacillussp.）HB172198, characterized by: the preservation number is: CGMCC No.15412, the preservation date is: 03 and 05 days 2018, the preservation unit is: the China general microbiological culture Collection center has the following preservation addresses: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.
2. A preparation method of alginate lyase is characterized by comprising the following steps:
(1) strain activation: inoculating the bacillus like HB172198 of claim 1 into a solid culture medium, and culturing at 28-37 ℃ for 24-48 h to obtain an activated strain;
(2) liquid culture: inoculating the activated strain into a liquid seed culture medium, and performing shaking culture at 28-37 ℃ and 120-200 r/min for 12-24 h to prepare a seed solution;
(3) fermentation culture: inoculating the seed solution into a liquid fermentation culture medium according to the volume percentage of 2-10%, carrying out shaking culture at the temperature of 28-37 ℃ and at the speed of 150-200 r/min for 24-60 h, collecting fermentation liquor, centrifuging, and collecting supernatant to obtain crude enzyme liquid of the alginate lyase.
3. A use of the Bacillus pumilus strain HB172198 according to claim 1 in degrading brown algae.
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