CN117126773A - Sugar-degrading sugar-phagocytosis bacterium FZY0027 and application thereof - Google Patents
Sugar-degrading sugar-phagocytosis bacterium FZY0027 and application thereof Download PDFInfo
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- CN117126773A CN117126773A CN202310983048.8A CN202310983048A CN117126773A CN 117126773 A CN117126773 A CN 117126773A CN 202310983048 A CN202310983048 A CN 202310983048A CN 117126773 A CN117126773 A CN 117126773A
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Abstract
The invention discloses a sugar-degrading saccharophaga bacterium FZY0027 and application thereof, wherein the strain shows higher agar degradation activity in an agar culture medium and has the potential of degrading various biological polysaccharides. The genomic sequence of strain FZY0027 was sequenced and saved in the NCBI database. And "super degradation agent" Saccharophagus degradans2-40 T In contrast, strain FZY0027 contains a greater number of genes for the glycoside hydrolase (Glycoside Hydrolases, GHs) domain. Bacterial strain FZY0027 degradation in extracellular polysaccharide degradation systemThe concentration of reducing sugar generated by polysaccharide has stronger degradation capability to starch, xylan and manna, and can show higher polysaccharide degradation activity at the normal temperature of 32-37 ℃. Therefore, the strain Saccharophagus degradans FZY0027 can provide a novel research material for the degradation development and utilization of polysaccharide.
Description
Technical Field
The invention relates to the technical field of degradation and development of polysaccharide, in particular to a bacterium strain for degrading sugar and phagocytic bacteria (Saccharophagus degradans) FZY0027 from surface seawater of an intertidal zone and application thereof in degradation of polysaccharide.
Background
The saccharophilic bacteria (Saccharophagus degradans) for degrading the polysaccharide is one of the most diverse marine microorganisms with polysaccharide degrading functions reported at present, and is known as a super degradation person. In recent years, with the continuous and intensive research on ecology and application value thereof, the saccharophiles have wide research and application value in the aspects of environment, food, medicine, energy and the like. From the classification status, the saccharophiles belong to the phylum gamma-proteobacteria, and are closely related to the strains of marine sediments alike such as marine decomposers (Marinobacter) and Pasteurella (Pseudomonas). However, there are also some differences between them at the level of degradation of polysaccharides. The saccharophilic bacteria have high-efficiency polysaccharide degrading function, and various polysaccharides such as agar, alginate, carrageenan, carboxymethyl cellulose, chitin, beta-glucan, laminarin, pectin, amylopectin, starch and xylan and the like are likely to play an important role in ocean carbon circulation. This makes it an important bacterial species for marine material decomposition and organic matter degradation. The saccharophilic bacteria can quickly degrade various polysaccharides, and simultaneously optimize the growth conditions so as to facilitate the generation of biomass, and has great potential in the aspects of production of biofuels, pharmaceutical preparations, biological materials and the like. From the application value, the polysaccharide degrading ability of the saccharophiles is widely used in various fields. In the aspect of environmental management, the method can decompose pollutants such as seaweed, organic waste in sewage and the like, and effectively reduce the influence of the pollutants on the environment. In the food industry, polysaccharide degrading enzymes that degrade saccharophilia have been widely used in production processes, for example, in the development and production of foods such as bread, dairy products, cheese, pudding, candy, and jelly, which not only contribute to improving the quality and taste of the product, but also can reduce the processing cost of the food. In addition, the saccharophila is also used for preparing various biological materials, such as important intermediates of cellulose derivatives, lignin derivatives and mannan derivatives, fireproof materials, adsorbing materials, biological coatings and the like.
The published patent 25 on degrading sugar and phagoshage Saccharophagus degradans was retrieved from the "Chinese well-known net" (https:// www.cnki.net), and the related strains are mostly Saccharophagus degradans2-40 T The strain is an excellent model of microbial degradation of insoluble complex polysaccharides, and can degrade complex polysaccharides from a variety of sources including algae, terrestrial plants, crustaceans, bacteria and fungi: agar, alginate, chitin, cellulose, laminarin, pectin, amylopectin, starch, xylan, and the like. Thus, a variety of plant, animal and microbial complex polysaccharides can be recovered and are believed to play an important role in the recovery of carbon and nitrogen in marine environments. One of the most effective degrading agents of complex polysaccharides identified so far is degrading sugar-phagocytes Saccharophagus degradans2-40 T In relation to Saccharophagus degradans2-40 T The agarase, chitinase and phycoprotein systems have been widely demonstrated and have been studied in a number of fields such as: analysis of polysaccharide degrading activity of alpha-neoagarobiose hydrolase (alpha-NABH) and obtaining monosaccharides by disrupting polymers on plant cell walls and degrading cellulose to monosaccharides by cellulolytic enzymes.
The discovery of the novel saccharophila degrading bacteria has a huge application prospect.
Disclosure of Invention
The invention discloses a sugar-degrading saccharophaga bacterium FZY0027, which is classified and named as Saccharophagus degradans FZY0027, and has the preservation time of: 2023, 3, 23; preservation address: the institute of microbiology, national academy of sciences, north chen xi lu 1, 3, the region of the morning sun in beijing; preservation unit: the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.26885.
The sugar-degrading sugar-phagocytosis bacterium FZY0027 is collected from a surface sea water sample (sample number L6) of an intertidal zone (33 degrees 6 '59' N,120 degrees 51 '9' E) of a salt city Dafeng farm of yellow sea in China, and the sample L6 forms larger degradation pits on a R2A (supplemented with 2.5.5% sea salt) (the R2A culture medium used in the invention is added with 2.5% sea salt, hereinafter referred to as R2A) flat plate, so that the agar degradation activity is strong. Single colonies were picked for pure culture after 5 days of culture at 28℃in R2A medium using the dilution plating method. The strain Saccharophagus degradans FZY0027 has a certain hydrolysis capability on polysaccharide and has good hydrolysis activity on starch, sodium alginate, xylan and mannan; the hydrolysis effect on starch is more obvious when the culture temperature is 37 ℃; the hydrolysis effect on sodium alginate and mannan is remarkable at 32 ℃ compared with 37 ℃ when the culture is carried out. Wherein, R2A medium was purchased from BD company, america, and was configured according to the product instructions, cat# 218263.
Morphological characteristics of the strain Saccharophagus degradans FZY0027 with polysaccharide degrading ability of the invention:
strain Saccharophagus degradans FZY0027 is gram negative bacteria, streaked onto R2A solid plates and incubated at 28℃for 72 hours, the colonies are viscous, round, smooth in surface, red in color and approximately 1.0-1.5mm in size. After streaking onto 2216E solid plates and incubation at 28℃for 72h, the colonies were viscous, round, smooth, black in surface, and showed significant agar degradation. Under transmission electron microscope, cells of strain FZY0027 are rod-shaped and have a size of 2.5-3.5X10.8-1.2 μm with terminal flagella. Wherein 2216E medium was purchased from Beijing Soy Bao technology Co., ltd and was configured according to the product specifications, cat# LA0341.
Culture characteristics of the strain Saccharophagus degradans FZY0027 of the invention:
aerobic bacteria grow at 4-42 deg.c and optimal growth temperature of 24-32 deg.c. Can grow in the environment with the NaCl concentration of 1-5 percent, and the NaCl concentration of 2-3 percent is the optimal concentration.
Genomic characteristics of strain Saccharophagus degradans FZY0027 of the invention:
the genome of strain Saccharophagus degradans FZY0027 was sequenced in real time single molecule (third generation sequencing) by wubi scientific company using Illumina Novaseq 6000 sequencer and Oxford Nanopore PromethION sequencer, and the genome sequencing results were assembled using Unicycler (v 1.0). The resulting genome contained only 1 circular chromosome with a total length of 5.2Mb, with a G+C content of 45.5%. The genome was uploaded to NCBI, resulting in GenBank accession No. CP123764, bioProject accession No. PRJNA958120, biosample accession No. SAMN34277932.
The 16S rRNA sequence fragment size of the strain FZY0027 is 1335bp, and according to the result of NCBI alignment, the strain with the highest 16S rRNA sequence similarity of the strain Saccharophagus degradans FZY0027 comprises the following steps: "Saccharophagus sp." AG21 (99.9%), saccharophagus degradans2-40 T (99.9%), "Saccharomyces phagus sp." Myt-1 (99.9%), "Gamma proteobacterium" R001 (99.9%), "Saccharophagus degradans" HME8281 (99.9%), "Saccharomyces phagus sp." HK-S109 (99.9%) and "Saccharomyces phagussp." MM1-2b (98.8%).
Wherein, the agar-decomposing bacterium Saccharophagus sp.AG21 (https:// doi.org/10.4014/jmb.1209.09009) isolated from red seaweed (Gelidium amansii) collected from south coast of Jizhou island in Korea, 16S rRNA gene GenBank accession No. JQ743648. A novel beta-agarase gene (beta-agarase II) was found in the strain Saccharophagus sp.AG21, designated agy1, which was published in NCBI database under GenBank accession No. AFR90184, but without complete genomic data. Agy1 the GH16 (glycosyl hy)drolase family 16) family, and Saccharophagus degradans2-40 T Agal6B of (A) was 93.7% similar to the agarase gene (beta-agarase) of Saccharophagus degradans FZY0027 (carbohydrate binding modules family in CBM6 family) was 93.4%. Agy1 agarose is degraded mainly neoagaroses and neoagaroses, showing the best activity at a relatively high temperature of 55 ℃, and a weakly basic ph=7.5. Saccharophagus degradans2-40 T (formerly Microbulbifer degradans 2-40) is a saprophytic marine bacterium isolated from decayed brine grass spartina alterniflora (Spartina alterniflora). Is the first marine bacterium demonstrated to degrade cellulosic algae and higher plant material, 16S rDNA analysis showed strain 2-40 T Is a member of the phylum gamma-Proteus, the GenBank accession number of the 16S rRNA gene sequence is CP000282, and the GenBank accession number of the genome is ASM1366v1. In previous studies, saccharomyces sp.Myt-1 (https:// doi.org/10.4014/jmb.1301.01075), a basophilic alginate lyase (alginate lyase) with a conserved amino acid sequence (RTELREM, QIH, YFKAGVYNQ), designated AlgMytC, belonging to the PL7 (polysaccharide lyase family 7) family member, was isolated from the sediment of Fushan Bay in Japan as a novel degrading sugar-phagosaccharide species, 16S rRNA gene GenBank accession No. AB778303, and a specific alginate lyase gene GenBank accession No. BAM76722, but no genomic information. BLAST homology analysis shows that AlgMytC is as high as Saccharophagus degradans2-40 T The amino acid sequence similarity of alg7A is 95.9%, and the amino acid sequence similarity of algL with Saccharophagus degradans FZY0027 is also 95.9%, but alginate lyase needs to show stronger enzyme activity in alkaline environment (pH is 8.5-10.0). Cellulolytic bacteria isolated from deep sea using nanocellulose plates: gamma proteobacterium R001 (https:// doi.org/10.1016/j.isci.2022.104732), genBank accession numbers of the 16S rRNA gene sequences are JF317346, and Saccharophagus degradans2-40 T Shows 99% homology and no genome information. Saccharophagus degradans HME8281 was isolated from a seawater sample, genBank accession No. JQ762407, without genomic information. Saccharophagus sp.HK-S109 was isolated from the atlantic ocean under GenBank accession number KJ123696 without genomic information. Saccharophaguss MM1-2b was isolated from the island of Korea under GenBank accession No. HQ882703, without genomic information.
Using Burkholderia cepacia ATCT 25416 T (Access number: AF 097530) as an outer branch, a phylogenetic tree constructed by adopting a contiguous method based on the 16S rRNA gene, saccharophagus degradans FZY0027 and "Saccharomyces cerevisiae sp." Myt-1,Saccharophagus degradans2-40 T "Saccharophagus sp." AG21, "Gamma proteobacterium" R001, "Saccharophagus degradans" HME8281 and "Saccharophagus sp." HK-S109 are clustered together to form a defined monocase clade, and the bootstrap value of this clade is 100%. Thus, strain FZY0027 is a degrading sugar-phagosaccharide. In addition, bootstrap values (percentage of 1000 replicates) exceeding 70% are shown on the branching nodes; in brackets are GenBank accession numbers of the corresponding 16S rRNA gene sequences of the strains. Burkholderia cepacia ATCT 25416 T The exosomes were 0.05 as genetic variation. Clade includes strains: eionea nigra 17X/A02/237 T (AY576771)，Pseudoteredinibacter isoporae SW-11 T (FJ347760)，Aestuariicella hydrocarbonica SM-6 T (KF982858)，Maricurvus nonylphenolicus KU41E T (AB626730)，Pseudomaricurvus alkylphenolicus KU41G T (AB809161)，Pseudomaricurvus alcaniphilus MEBiC06469 T (JQ672628)，Agarilytica rhodophyticola 017 T (CP020038)，Thalassocella blandensis ISS155 T (MH732325)，Teredinibacter franksiae Bsc2 T (MT416121)，Simiduia litorea KMM 9504 T (AB894237)，Simiduia aestuariiviva J-MY2 T (KJ742851)，Marinibactrum halimedae Q-192 T (AB900126)，Teredinibacter turnerae T7902 T (ARAH01000054)，Teredinibacter haidensis Bs08 T (MT416119)，Teredinibacter franksiae Bsc2 T (MT 416121) and Teredinibacter purpureus Bs12 T (MT416120B)。
To further determine the phylogenetic tree of strain FZY0027, 9 genomes in Cellvibrionaceae and were selectedOuter group Haliea salexigens X/A02/235 T Genome constructs bac120 evolutionary tree. Strains FZY0027 and Saccharophagus degradans2-40 T And Saccharophagus degradans E M17 T In recent relatedness, a defined single line clade was formed, with a bootstrap value of 100%, which was similar to the 16S rRNA developmental tree. In addition, by aligning genomic sequences with protein sequences, strains FZY0027 and Saccharophagus degradans2-40 T And Saccharophagus degradans E M17 T The ANI values of 96.5% and 97.8% (above the threshold for species division: 95-96%), AAI values of 96.7% and 97.5% (above the threshold for species division: 95-96%), dDDH values of 70.0% and 79.2% (above the threshold for species division: 70%), respectively, indicate that strain FZY0027 is a degrading sugar-phagostimulant.
Analyses were carried out using GGDC (http:// GGDC. Dsmz. De/GGDC. Php#), average Nucleotide Identity calculator and Average Amino acid Identity calculator (http:// end-omics. Ce. Gatech. Edu) on-line tools, strains FZY0027 and Saccharophagus degradans2-40 T The dDDH, ANI and AAI values were 69.6%,96.5% and 96.7%, respectively, with the degrading sugar-phagosaccharide bacteria being different subspecies of the same bacteria. Comparing KEGG (Kyoto Encyclopedia of Genes and Genomes) with CAzymes database (Carbohydrate-Active Enzymes Database) to analyze two strains, and KEGG annotation result shows that the difference of the gene function enrichment results of the two strains is smaller, and only a part of metabolic pathways have relatively larger difference; the CAZymes online annotation result showed a large difference in the number of genes containing the glycoside hydrolase (Glycoside Hydrolases, GHs) domain alone, strain Saccharophagus degradans FZY0027 containing GHs domain compared to strain Saccharophagus degradans2-40 T And 7 more.
The strain Saccharophagus degradans FZY0027 of the invention has the characteristic of degradation capability of agar polysaccharide:
strain Saccharophagus degradans FZY0027 was grown in 2216E solid medium at 28 ℃ for 3 days with large colonies and more pronounced dishing on the plate surface. After being dyed by Lugol's iodine solution, the strain Saccharophagus degradans FZY0027 forms a large transparent ring around the colony, and the reason for the transparent ring is that Lugol's iodine solution can dye agar polysaccharide into dark brown and can not dye agar oligosaccharide generated after the hydrolysis of the agar. By observing the size of a hydrolysis ring of a colony and the staining identification result of Lugol's iodine solution, the strain Saccharophagus degradans FZY0027 is found to have stronger agar degradation capability. The DNS colorimetric sugar determination method is adopted, an ultraviolet-visible spectrophotometer is used for measuring the absorbance at the wavelength of 540nm, the amount of reducing sugar generated by the reaction is determined according to a galactose standard curve, the agar enzyme activity of the reaction strain Saccharophagus degradans FZY0027 is higher, and the more the yield of the reducing sugar is, the higher the agar enzyme activity is. The reducing sugar concentration is in an ascending trend in the logarithmic growth phase and the stationary phase of 1-4d, the number of living cells of the strain is stable in the stationary phase and the primary metabolic accumulation reaches the highest peak, and reaches the highest value in the fourth day.
Polysaccharide degrading ability characteristics of the strain Saccharophagus degradans FZY 0027:
the invention verifies that the strain FZY0027 is used for preparing starch, sodium alginate, xylan, mannans, chitosan, chitin, microcrystalline cellulose, carrageenan, xanthan gum, pectin and chondroitin sulfate by a DNS method. In the extracellular polysaccharide degradation system, the strain FZY0027 has the strongest degradation capability on starch, and the concentration of the generated reducing sugar is 2.28mg/mL, and then xylan (1.83 mg/mL), mannan (1.15 mg/mL), sodium alginate (0.41 mg/mL), pectin (0.22 mg/mL), chondroitin sulfate (0.15 mg/mL), microcrystalline cellulose (0.12 mg/mL), chitin (0.12 mg/mL), carrageenan (0.12 mg/mL), xanthan gum (0.11 mg/mL) and chitosan (0.11 mg/mL). The strain FZY0027 has strong polysaccharide degradation capability on starch, xylan, mannan and sodium alginate, but can not degrade pectin, microcrystalline cellulose, chitin, carrageenan, xanthan gum and chitosan. In addition, with strains 2-40 T In comparison, strain FZY0027 has a greater capacity to degrade starch, xylan and mannan in the concentration of reducing sugars produced by the degradation of polysaccharides in an extracellular polysaccharide degradation system. Strain 2-40 according to LA Enson (https:// doi.org/10.1038/sj.jim.2900696) T During degradation of starch and xylan, the resulting concentration of reducing sugars was 1.60mg/mL and 1.02mg/mL, respectively.
Strain Saccharophagus degradans FZY0027 and strains Saccharophagus degradans2-40 of the invention T Genomic profile comparison analysis of (c):
strains Saccharophagus degradans FZY0027 and Saccharophagus degradans2-40 T All have the activity of degrading various complex polysaccharides, and are analyzed from comparative genomics of two strains: strains Saccharophagus degradans FZY0027 and Saccharophagus degradans2-40 T Has extremely high similarity in genome sequence. Strain Saccharophagus degradans FZY0027 is annotated with 4080 protein sequences, of which 249 are predicted to produce domains associated with six broad classes of carbohydrate active enzymes, and part of the protein sequences are predicted to be able to produce two and more domains associated with CAZymes simultaneously.
The invention analyzes agarose-degrading agarose domain of strain Saccharophagus degradans FZY0027 and finds out strain Saccharophagus degradans FZY0027 and strain Saccharophagus degradans2-40 T Enzymes and corresponding protein sequences that perform similar functions. Thus, from strains Saccharophagus degradans FZY0027 and Saccharophagus degradans2-40 T Protein sequences capable of producing enzymes with agar (oligo) saccharides as substrates were extracted for further analysis, strain Saccharophagus degradans FZY0027 contained in the same manner as strain Saccharophagus degradans2-40 T Six protein sequences with nearly identical results were annotated by CAZymes. Wherein, protein ID of strain Saccharophagus degradans FZY0027 the Protein sequence is at accession number of the NCBIProtein database; strain Saccharophagus degradans FZY0027 locus_tag was obtained when annotated by PGAP; ec# is the number of enzymes; HMMER columns represent the domain of the enzyme predicted by the tool HMMER dbCAN; the Substrate is a predicted Substrate for the enzyme; signal Peptide is a prediction of the Signal Peptide of the protein sequence, Y: yes, N: no. In addition to the six genes QFX18_15130 to QFX18_07375 in Table 1, the genes of strain Saccharophagus degradans FZY0027 whose locus_tag is QFX18_01135 and QFX18_06020 can also find homologous genes in strain 2-40, the predicted products β of both genesThe galactosidase is essential for the degradation of agarose, mainly involved in the hydrolysis of the beta-galactoside bond, and the monomer G of agarose is linked to LA by means of the beta-1, 4-glycosidic bond. Further shown are the CAZymes domain predicted results corresponding to wp_ 011467657.1-wp_ 011469134.1 and qqx18_15130-qqx18_ 07375, respectively, in pairs.
Gene corresponding to protein sequences QFX18_15130-QFX18_ 07375 of strain Saccharophagus degradans FZY0027 and strain Saccharophagus degradans2-40 were found by combining domain prediction results of CAzymes T The genes encoding WP_011467657.1 to WP_011469134.1 of (A) should be homologous genes. Of the predictions, attention was drawn to the Protein ID wp_011469134.1 and its homologous gene qfx18_07375, both of which are predicted to produce two domains of GH117, and the two domains of GH117 have overlapping parts at the beginning and end of the two domains, further analyzing both in the tool HMMER: as a result of detailed annotation under bCAN, strains Saccharophagus degradans FZY0027 and Saccharophagus degradans2-40 were found T Both predicted lower E Value for the first GH117 domain than the second and higher Coverage. Smaller E Value indicates more reliable prediction results, and higher Coverage indicates more Coverage of the identified peptide fragment on the protein sequence. Thus, the prediction of the first GH117 domain is more accurate. In addition, according to the previous study, strain Saccharophagus degradans2-40 T The mechanism for degrading agar uses 5 β -agarases, named Aga50A, aga16B, aga86C, aga D and Aga86E, respectively, and a putative new agarobiose agarase Aga117F. It appears that the six protein sequences which can act as agar (oligo) saccharides with substrates are predicted under the HMMER dbCAN-sub tool to correspond to the encoded enzymes which correspond to the five beta-agarases and the assumed enzyme Aga117, and Aga117F is an intracellular enzyme, and the corresponding Signal Peptide prediction result of QFX18_07375 is also No, which indicates that the enzyme is not an extracellular enzyme. The comparative genomic analysis results described above verify the strain Saccharophagus degradans FZY0027 and model strain Saccharophagus degradans2-40 from the genetic level T Also has the ability to degrade polysaccharides.
The strain Saccharophagus degradans FZY0027, mutants, cultures of variants, cell contents, bacterial solutions of bacterial strains, fermentation broths and filtrates of broths, various metabolites and derivatives can be used for degrading polysaccharides. Can be used for degrading polysaccharide such as agar, starch, xylan, mannan and sodium alginate to produce various metabolites and derivatives.
Saccharophagus degradans FZY0027 genes involved in the coding of agarase: the QFX18_15130, QFX18_15125, QFX18_07410, QFX18_07385, QFX18_07440 and QFX18_07375 genes are useful in the preparation of various biological agents involved in the expression of agarose enzyme products and in the inclusion of these protein products.
The beneficial effects achieved by the invention are as follows: the strain Saccharophagus degradans FZY0027 of the invention shows higher agar degradation activity in an agar culture medium and has the potential of degrading various biological polysaccharides. In addition, most degrading glycophages have no complete genomic sequence, but the strain FZY0027 genomic sequence has been sequenced and saved in the NCBI database under GenBank accession No. CP123764, bioprject accession No. PRJNA958120, biosmple accession No. SAMN34277932. And "super degradation agent" Saccharophagus degradans2-40 T In contrast, strain FZY0027 contains a greater number of genes for the glycoside hydrolase (Glycoside Hydrolases, GHs) domain. The concentration of reducing sugar produced by degrading polysaccharide in extracellular polysaccharide degradation system of strain Saccharophagus degradans FZY0027 is measured by DNS method, and strain FZY0027 has stronger capability to starch, xylan and mannan, and can show higher polysaccharide degradation activity at normal temperature of 32-37 ℃. Therefore, the strain Saccharophagus degradans FZY0027 can provide a novel research material for the degradation development and utilization of polysaccharide.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 shows the morphology of strain Saccharophagus degradans FZY0027 by transmission electron microscopy in 2216E solid medium at 28℃for 3 days. Bar,1.0 μm;
FIG. 2 is a phylogenetic tree of the adjacent method constructed by strain Saccharophagus degradans FZY0027 based on the 16S rRNA gene sequence;
FIG. 3 is a maximum likelihood phylogenetic tree of strain Saccharophagus degradans FZY0027 constructed based on 120 highly conserved genes;
FIG. 4 shows strain Saccharophagus degradans FZY0027 and strain Saccharophagus degradans-40 T Is annotated with a result comparison graph by the CAZymes database;
fig. 5 shows the results of lugol's iodine staining of strain Saccharophagus degradans FZY 0027;
FIG. 6 is a graph showing the degradation ability verification of the agarose of strain Saccharophagus degradans FZY 0027;
FIG. 7 is a diagram showing the polysaccharide degrading ability of strain Saccharophagus degradans FZY 0027;
FIG. 8 shows strain Saccharophagus degradans FZY0027 and strain Saccharophagus degradans-40 T Genome comparison circle of (a);
FIG. 9 shows strains Saccharophagus degradans FZY0027 and Saccharophagus degradans2-40 of Table 1 T A protein sequence predicted structural map of (2);
FIG. 10 shows D-galactose standard curve.
Detailed Description
The preferred embodiments of the present invention will be described below with reference to the accompanying drawings, it being understood that the preferred embodiments described herein are for illustration and explanation of the present invention only, and are not intended to limit the present invention.
Examples
1. The strain Saccharophagus degradans FZY0027 isolation method comprises the following steps:
co-cultures of flora L6-3 were isolated from the water sample of the intertidal zone of the Minchen Dafeng farm (sample number L6) and formed significant agar degradation pits on the R2A agar medium. Subjecting the stable sub-population culture (L6-3) to 10-fold gradient dilution -6 Concentration gradient dilutions were then run from 10 -6 0.1mL of the concentration gradient suspension was taken and applied with a sterile applicator rodIs coated on R2A medium added with 2.5% sea salt. After being smeared evenly, the mixture is placed into an incubator for inversion culture at 28 ℃; after 5 days of culture, strains with different colony morphologies were picked and streaked on R2A plates for purification culture. Colonies showing the pits were picked and re-streaked, and pure colonies were selected by repeating streaking under the same conditions. After the degrading sugar and saccharophaga strain is obtained, the strain is placed at the temperature of 4 ℃ for standby, a proper amount of bacterial colony is scraped and evenly stirred in 1mL of 0.5% dimethyl sulfoxide, and the strain is placed at the temperature of-80 ℃ for storage.
2. The identification method of the strain Saccharophagus degradans FZY0027 with good polysaccharide degradation capability comprises the following steps:
(1) Extraction of genomic DNA from strains
Bacterial genomic DNA was extracted according to the instructions of the Ezup column type bacterial genomic DNA extraction kit (Shanghai Co., ltd., product No. B518255).
(2) 16S rRNA Gene amplification
PCR amplification was performed using the proposed DNA as a template. The PCR reaction system is as follows:
the primer sequence is (5 '-3'): 27F, AGAGTTTGATCCTGGCTCAG;
1492R，GGTTACCTTGTTACGACTT。
the PCR amplification procedure was: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 54℃for 30s, elongation at 72℃for 100s, 34 cycles total; extending at 72℃for 5min. mu.L of PCR product was taken, electrophoresed on 0.8% agarose gel containing the SuperRed/GeLRed nucleic acid dye for 20min at 160V, and photographed under observation under a gel imager.
(3) 16S rRNA sequence determination and phylogenetic analysis thereof
16s rRNA gene sequencing was performed by biological engineering (Shanghai) Inc., and the results were aligned with the reference sequences obtained on the NCBI database using BioEid software and phylogenetic analysis was performed using MEGA X software. And (3) calculating genetic distances by adopting a Kimura double-parameter model, and constructing a phylogenetic tree by adopting a neighbor-joining (NJ) method.
The strain was inoculated into 2216E liquid medium, and after culturing at 28℃and 200r/min for 72 hours, the cells were collected. The extraction and sequencing of genomic DNA was accomplished by using Illumina Novaseq 6000 sequencer for the second generation and Oxford Nanopore PromethION sequencer for the third generation, by martial arts, ltd. The result of corporate delivery is clean data after quality control, genome assembly in Ubuntu system using SPADes program (http:// cab. Spbu. Ru/software/spots /) in UGENE software package, deleting sequence with Coverage less than 5 or length less than 500bp, submitting genome data to GenBank and annotating genome using NCBI's PGAP online tool (https:// www.ncbi.nlm.nih.gov/genome/section_prok /). 120 highly conserved housekeeping genes were selected using the EasyCGTree software package @https://github.com/zdf1987/EasyCGTree) Constructing bac120 phylogenetic tree of strain FZY0027 and using an iTOL online toolhttps://itol.embl.de/) And visually displaying the result of the phylogenetic tree. The above analysis, unless specifically stated, is performed under default parameters of the software or tool.
On the basis of a phylogenetic tree of the bac120 core gene set, an online tool of Kostas Lab is used for cleaning the whole planthttp:// enve-omics.ce.gatech.edu/g) The strain FZY0027 was analyzed for average nucleotide identity (average nucleotide identity, ANI) to average amino acid identity (average amino acid identity, AAI) to its closely related strain. In addition, utilizing Genome-to-Genome Distance Caculator (GGDC) on-line Server version 3.0https://ggdc.dsmz.de) The evolution status of strain FZY0027 was further determined by calculating the value of digital DNA-DNA molecular hybridization (digital DNA-DNA hybridization, dDDH) between strains.
(4) Determination of optimal growth conditions for strains
Strains were streaked onto R2A, 2216E, LB and TSA common solid media, respectively, and 2216E was determined to be the optimal medium. The strain was streaked onto 2216E solid medium, and cultured in the environment of 4deg.C, 20deg.C, 24deg.C, 28deg.C, 37deg.C, 42deg.C and 45deg.C for 7 days, respectively, and the growth of the strain under different temperature conditions was observed every 24 h. 2216E solid media were prepared at sea salt concentrations of 0%, 0.5%, 1%, 2%, 3%, 4%, 5%, 7%, 10% and 12%, respectively. The strains were streaked and inoculated into 2216E solid culture media with different salt concentrations, placed into an incubator at 28 ℃ for 7 days, and the growth conditions of the strains under different salt concentrations were observed every 24 hours.
3. The method for identifying the degradation agarose of the strain Saccharophagus degradans FZY0027 with better degradation capability of polysaccharide comprises the following steps:
lugol's iodine solution dyeing
Identification principle: lugol's iodine solution is a staining reagent that can make agarose black, but cannot stain agarose oligosaccharides, and thus can be used to detect the agarose degradation ability of bacteria. After staining with lugol's iodine solution, it was judged whether the bacteria had agarose degrading ability by observing whether a transparent ring formed around the colony, the size of the transparent ring reflecting the ability of the colony to utilize agarose.
Lugol's iodine solution (Lugol's iodine solution formulation: 5g iodine (I) 2 ) And 10g of potassium iodide (KI) in 85mL of distilled water) were added to 2216E solid plates of active growth of strain FZY0027, the plates were gently shaken side to side, left to stand for several minutes, after full staining the staining results were observed again, and photographed for recording.
(II) DNS colorimetric sugar determination method
Experimental principle: by adopting a DNS colorimetric sugar determination method, because the agarase can decompose agarose to generate agarose containing a reducing end, the DNS can also perform oxidation-reduction reaction with the agarose under an alkaline condition to oxidize a reducing group in an oligosaccharide structure into carboxyl, and the DNS is reduced into 3-amino-5-nitro salicylic acid, and the substance is reddish brown after being heated in a boiling water bath. The reacted solution was measured for absorbance (OD) at a wavelength of 540nm, and then the content of agarose was calculated by using a D-galactose standard curve, thereby realizing rapid quantification of agarose.
3, 5-dinitrosalicylic acid (DNS) reagent: 6.3g of 3, 5-dinitrosalicylic acid and 262mL of 2mol/L sodium hydroxide are added into 500mL of hot water solution containing 182g of potassium sodium tartrate, 5g of heavy phenol and 5g of sodium sulfite are added, stirred and dissolved, cooled, added with water to 1000mL of constant volume, thus preparing the DNS reagent, and the DNS reagent is stored in a brown bottle for standby after a week.
D-galactose standard curve preparation:
(1) Preparing a D-galactose standard solution: accurately weighing 100mg of D-galactose which is dried to constant weight at 105 ℃ in advance, dissolving in a small amount of distilled water, transferring to a volumetric flask of 100mL, and then fixing the volume to a scale mark, wherein the final concentration is 1mg/mL.
(2) 6 centrifuge tubes of 15mL are taken, the reagent is added according to the table 4.3, the mixture is heated for 5min in a boiling water bath, after the mixture is cooled to room temperature, distilled water is used for supplementing the mixture to 15mL, and the OD value at the wavelength of 540nm is measured by an ultraviolet-visible spectrophotometer.
(3) Plotted on the abscissa with D-galactose content (mg/mL) and on OD 540 D-galactose standard curve with values on the ordinate:
wherein: the units of the D-galactose standard solution, distilled water and DNS reagent are all mL.
Sample preparation:
the present invention prepares a sample by co-culturing the strain FZY0027 with agarose, which is herein abbreviated as co-culture, and explores the agarose degrading activity of the strain FZY0027 for producing agarase.
(1) FZY0027 colonies actively growing on 2216E agar plates were picked and inoculated into 100mL 2216E liquid medium containing 0.1% (w/v) agarose, shake flask cultured at 25℃for 150 r/min;
note that: the unvaccinated strain FZY0027 was set as a blank control group and the experimental groups were 3-fold-aligned.
(2) After every 24 hours, uniformly sucking 20mL of liquid from each liquid culture medium in a sterile environment, and centrifuging for 10min at a relative centrifugal force of 12000 Xg;
(3) Taking 1mL of supernatant after centrifugation in a new centrifuge tube, immediately adding 2mL of DNS reagent, and heating in a boiling water bath for 5min;
(4) After cooling to room temperature, distilled water is filled up to 15mL, and OD value at 540nm wavelength is measured;
(5) Samples were tested as described above for 5 consecutive days.
Analysis of the agar Lipase Activity:
according to the method for preparing the D-galactose standard curve, the D-galactose content (mg/mL) and the corresponding OD 540 The data are shown in the table. D-galactose standard curves in the following figures were plotted according to the data in the tables. The linear regression equation is obtained from the graph as Y= 1.21843X-0.03738, R 2 ＝0.99174。
The co-cultivation method requires to detect the OD value for 5 days altogether, record experimental data and calculate the corresponding reducing sugar content through a D-galactose standard curve. The 5-day reduced sugar data was plotted using the software Origin 2023 to give the bar graph of FIG. 6.
4. The method for identifying the polysaccharide degradation capability of the strain Saccharophagus degradans FZY0027 with good polysaccharide degradation capability comprises the following steps:
the degrading saccharophilic bacteria can degrade various biomass polysaccharides to produce oligosaccharides with reducing ends. 3, 5-dinitrosalicylic acid (DNS) can perform oxidation-reduction reaction with the agar oligosaccharide under alkaline condition, so that the reducing group in the oligosaccharide structure is oxidized into carboxyl. The DNS is reduced into 3-amino-5-nitro salicylic acid, the substance is reddish brown after being heated in boiling water bath, and the color depth and the oligosaccharide content are in a direct proportion, so that the DNS can be detected by a colorimetric method, and the simple and rapid quantification of the seaweed oligosaccharide is realized.
(1) Preparation of glucose standard curve
Glucose standard solutions (1 mg/mL) 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL are respectively taken and put in 15mL test tubes, distilled water is used for supplementing to 1.0mL, DNS reagent 2mL is accurately added respectively, boiling water bath heating is carried out for 2min, running water cooling is carried out, and water is used for supplementing to 15mL scale. Absorbance was measured at 540nm wavelength. Drawn at 540The absorbance at nm wavelength is a glucose standard curve with the glucose solution concentration on the abscissa, as shown in fig. 10. Linear regression equation: y=0.0737x+0.00388, r 2 ＝0.95815。
Selecting bacterial strains with obvious pits or hydrolysis rings around bacterial colonies on a flat plate, adding the bacterial strains into sterile water containing 2.5% sea salt to prepare bacterial suspension with turbidity of 0.5, fully and uniformly mixing, adding 100 mu L of bacterial suspension into 10mL of prepared liquid culture medium with polysaccharide (xanthan gum, pectin, chondroitin sulfate, chitosan, starch, sodium alginate, xylan, mannan, microcrystalline cellulose, chitin and carrageenan) as a unique carbon source, setting 1 group of control groups without bacterial liquid and 3 groups of parallel groups for each polysaccharide, fully and uniformly mixing, and carrying out shake culture for 7 days at the temperature of 32 ℃ at 150 r/min.
Centrifuging 3mL of culture medium in a centrifuge tube with 3mL to 15mL of culture medium taking polysaccharide as a unique carbon source for 10min at 12000r/min, centrifuging 1mL of supernatant in a test tube with 15mL, adding 2mL of DNS reagent, heating with boiling water for 5min to enable reducing sugar to fully react with the DNS reagent, supplementing the reducing sugar with 15mL with distilled water after cooling, measuring absorbance at 540nm wavelength, and measuring the liquid to be measured in each test tube for 3 times to reduce errors. The concentration corresponding to glucose was found from the standard curve, the reducing sugar content in the sample was calculated, and a histogram was drawn using the software Origin 2023 to give fig. 7.
4. The strain Saccharophagus degradans FZY0027 and the strain Saccharophagus degradans-40 with better polysaccharide degradation capability T Is characterized by comprising the following steps:
in software BRIG (v 0.95)https://sourceforge.net/projects/brig/) In the above, blast-2.9.0+ was set as an alignment of genome sequencesSoftware (E-value set to 1E-5, consistency threshold 50%), based on the results of the bac120 phylogenetic tree, selected to use strain Saccharophagus degradans FZY0027 as the central reference sequence, the genomic sequence of strain Saccharophagus degradans-40 as the comparative reference sequence, and uploaded the genome annotation files annotated by both strains by PGAP to construct strain Saccharophagus degradans FZY0027 and strain Saccharophagus degradans2-40 T Genome circle map.
KEGG database resources for understanding high level functions and utilizations of biological systems such as cells, organisms and ecosystems from molecular level information, especially genome sequencing and other high throughput generated large scale molecular datasets. Wherein KEGG PATHWAY is a set of manually drawn pathway graphs representing an understanding of molecular interactions, responses and relationship networks from seven broad categories, metanolism (Metabolism), genetic Information Processing (genetic information processing), environmental Information Processing (environmental information processing), cellular Processes (cellular processes), organismal Systems (organism systems), human Diseases and Drug Development. Based on phylogenetic tree results of strain Saccharophagus degradans FZY0027, standard strain Saccharophagus degradans-40 was selected for this study as being a saccharophaga T (GCA_ 000013665.1) as a reference. The protein amino acid sequences of the two strains are respectively submitted to an EggNOG online website, default parameters are selected for functional annotation, different KEGG metabolic pathway types of the two strains and the number of genes corresponding to the metabolic pathway are counted, then the KEGG functional annotation statistical results of the strain Saccharophagus degradans FZY0027 and the reference strain 2-40 are compared and analyzed, and a transverse cluster bar graph is drawn by using Origin 2023.
CAZymes database [ ]http://www.cazy.org/) Is a database resource for enzymes capable of synthesizing or decomposing complex carbohydrates and sugar complexes. The database mainly divides the carbohydrate-active enzymes into six major classes, including glycoside hydrolases (Glycoside Hydrolases, GHs), glycoside transferases (GlycosylTransferases, GTs), polysaccharide lyases (Polysaccharide Lyases, PL)s), carbohydrate esterases (Carbohydrate Esterases, CEs), accessory module enzymes (Auxiliary Activities, AAs) and Carbohydrate-Binding Modules (CBMs). On-line annotation of CAzymes database mainly through dbCAN #https://bcb.unl.edu/dbCAN2/index.php) The online server and database are based mainly on searches of conserved domain databases and selection of documents, defining a tag domain for each CAZyme family explicitly, and building a hidden Markov model for this domain (Hidden Markov models). The dbCAN data is mainly derived from the CAzymes database and updated with the database update. Selection of Strain Saccharophagus degradans-40 T As reference strains, protein sequences of the two strains are respectively submitted to a dbCAN website for online annotation, and HMMER is selected to be dbCAN (E-value<1e-15,coverage>0.35 dbCAN-sub (E-value)<1e-15,coverage>0.35 As a tool, annotation results were obtained, the total number of genes containing six major CAZymes domains was counted for each of the two strains, and the differences between the two were compared by plotting a bar graph using software Origin 2023. The annotation result is based on the predicted structural domain of HMMER-dbCAN, and the HMMER-dbCAN-sub is mainly used for predicting the action substrate corresponding to the enzyme.
4. Analysis of results
The morphological structure of the strain Saccharophagus degradans FZY0027 under a transmission electron microscope is shown in figure 1, and cells are ellipsoidal and have flagellum.
The adjacent method system development tree constructed by the strain Saccharophagus degradans FZY0027 based on the 16S rRNA gene sequence is shown in FIG. 2. The strain FZY0027 and the strain degrading glycophagosaccharide Saccharophagus degradans are aggregated together and belong to the same genus.
Strain Saccharophagus degradans FZY0027 a maximum likelihood phylogenetic tree constructed based on 120 highly conserved genes is shown in figure 3. Strain FZY0027 and strain Saccharophagus degradans2-40 T And strain Saccharophagus degradans E M17 T Together, strain FZY0027 belongs to the taxonomic group of glycophagostimulants (Saccharophagus degradans).
Strain Saccharophagus degradans FZY0027 and strains Saccharophagus degradans2-40 T The result pairs of the CAzymes database annotation result are shown in figure 4, the difference of the gene function enrichment result is small, and the difference is relatively large only in part of metabolic pathways; the CAZymes online annotation result showed a large difference in the number of genes containing the glycoside hydrolase (Glycoside Hydrolases, GHs) domain alone, strain Saccharophagus degradans FZY0027 containing GHs domain compared to strain Saccharophagus degradans2-40 T And 7 more.
The results of lugol's iodine staining of strain Saccharophagus degradans FZY0027 are shown in fig. 5, with strain Saccharophagus degradans FZY0027 forming a large transparent ring around the colonies after staining with lugol's iodine.
And verifying the degradation capacity of the strain Saccharophagus degradans FZY0027 agarose, measuring the absorbance at 540nm wavelength by adopting a DNS colorimetric sugar determination method and using an ultraviolet-visible spectrophotometer, determining the amount of reducing sugar generated by the reaction according to a galactose standard curve, and reacting the agarase activity of the strain Saccharophagus degradans FZY0027, wherein the more the yield of the reducing sugar is, the higher the agarase activity is. As shown in FIG. 6, the concentration of reducing sugar was in an ascending trend in the logarithmic growth phase and stationary phase 1-4d, the number of living cells of the strain was stable in the stationary phase and the primary metabolite accumulated at the highest peak, and at the fourth day.
The ability of the strain Saccharophagus degradans FZY0027 to degrade polysaccharide was verified, as shown in fig. 7, the strain FZY0027 showed strong polysaccharide degrading ability to starch, xylan, mannan and sodium alginate, but could not degrade pectin, microcrystalline cellulose, chitin, carrageenan, xanthan gum and chitosan.
Strain Saccharophagus degradans FZY0027 and strain Saccharophagus degradans2-40 T As shown in FIG. 8, the genome sequences of the two strains have extremely high similarity. From inside to outside in fig. 8: circle 1, gc content; circle 2, gc offset; circle 3, forward coding gene; circle 4, reverse coding gene; circle 5, rna encoding gene; rings 6,Saccharophagus degradans2-40 T Homologous sequences, color tableShown is similarity; circle 7, cazymes database annotates results with red-GHs, orange-GTs, yellow-PLs, green-CEs, blue-AAs, purple-CBMs.
Comparison of Strain FZY0027 with Strain 2-40T
Strains Saccharophagus degradans FZY0027 and Saccharophagus degradans-40 T A comparison of the predicted structure of the protein sequence of (C) is shown in FIG. 9.
As shown in FIG. 9, the predicted domains of protein sequences corresponding to each other are shown in Table 1, which are arranged in order from top to bottom. Red represents GHs domain and blue represents CBMs domain.
From strains Saccharophagus degradans FZY0027 and Saccharophagus degradans2-40 T Protein sequences capable of producing an enzyme with agar (oligo) saccharides as a substrate were extracted for further analysis, and strain Saccharophagus degradans FZY0027 contained the same as strain Saccharophagus degradans-40 as shown in the above table T Six protein sequences with nearly identical results were annotated by CAZymes.
The domain prediction results for wp_011469134.1 and qfx18_07375 are shown in the following table.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. The degrading sugar phagocytes FZY0027, which is classified and named Saccharophagus degradans, are preserved in the North Chen Silu No. 1 and 3 of the Korean area of Beijing in 2023 and 23 days, and the preservation number is CGMCC No.26885.
2. Use of a glycophagosaccharide degrading bacterium FZY0027 according to claim 1 for degradation of a polysaccharide.
3. The use of the culture, cell content, bacterial liquid, fermentation broth, and filtrate of the broth of the sugar-degrading sugar-phagosaccharide bacterium FZY0027 of claim 1 for degrading polysaccharides.
4. Use of the mutant or variant of the sugar degrading glycophagostimulant FZY0027 according to claim 1 for degrading polysaccharides.
5. The use of the mutants, cultures of variants, cell contents, bacterial solutions, fermentation broths, and filtrate of broths of the degrading sugar-forming bacterium FZY0027 according to claim 1 for degrading polysaccharides.
6. The use according to any one of claims 2 to 5, wherein the polysaccharide is any one or more of agar, starch, xylan, mannan and sodium alginate.
7. Use of a gene encoding an agarase in a sugar degrading sugar-phages FZY0027, said gene encoding an agarase being qfx18_15130, qfx18_15125, qfx18_07410, qfx18_07385, qfx18_07440 and qfx18_07375, for the preparation of agarase products and various biological agents comprising these protein products.
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