Chitinase-producing desert bacillus and its application

Abstract

The present invention discloses a chitinase-producing desert bacillus and its application, and relates to the field of microbial technology. The present invention discovers a new microbial strain, which is identified as belonging to the genus Desert Bacillus and is named Desert Bacillus sp.MYHB_202305ChI. Experimental verification shows that the strain has the ability to tolerate high salt and secrete chitinase.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a chitinase-producing desert bacillus and application thereof. Background Art

[0002] Chitin is a linear polysaccharide composed of N-acetyl-D-glucosamine (GIcNAc) connected by B-1,4 glycosidic bonds. It is abundant in nature, has high crystallinity, low solubility, stable properties, and is difficult to be effectively utilized. Chitinase is one of the key enzymes for degrading chitin. Chitinase can effectively decompose insoluble chitin and degrade it into low-polymerization chitosan oligosaccharides with higher solubility and physiological activity. Research reports show that bacteria and fungi are considered to be the main chitin decomposers and play a key role in the biogeochemical cycle of carbon and nitrogen. Chitinase can be divided into high-temperature enzymes, low-temperature enzymes, acid-resistant enzymes, and alkali-resistant enzymes according to different enzyme reaction conditions. It is worth noting that most of the reported bacterial chitinases are mesophilic enzymes with an optimal temperature of 40-60°C. Therefore, it is of great significance to explore enzymes that can efficiently degrade chitin in production and life. Summary of the invention

[0003] In order to solve the deficiencies of the prior art, the present invention provides a chitinase-producing desert bacillus and application thereof.

[0004] In order to achieve the purpose of the present invention, the following scheme is proposed:

[0005] The invention separates and cultivates a new microbial strain from the desert Gobi soil in Xinjiang Uygur Autonomous Region, and finds that the strain has the function of high-salt tolerance and chitinase production.

[0006] Genome sequencing and physiological and biochemical experiments confirmed that the bacterium belongs to the genus Desertibacillus and was named Desertibacillus sp. MYHB_202305ChI. The strain has been deposited in the Guangdong Provincial Microbiological Culture Collection Center with the collection number GDMCC No: 64172.

[0007] The beneficial effects of the present invention are: the desert Bacillus MYHB_202305ChI has good salt tolerance, can grow in 2216E agar medium, has the function of producing chitinase, can be directly used as a chitinase production strain, and can be used for industrial production of chitinase in a high-salt environment. At the same time, it may also be used as a new genetic engineering strain to obtain more excellent traits by accepting other excellent genes from outside; in addition, the trait of the improved strain is improved by traditional mutagenesis breeding methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 This is an electron microscope image of Bacillus deserticola MYHB_202305ChI.

[0009] Figure 2 This is the colony morphology of Bacillus deserticola MYHB_202305ChI.

[0010] Figure 3 This is the evolutionary analysis diagram between Bacillus deserticola MYHB_202305ChI and other closely related strains (based on 16SrDNA sequence analysis).

[0011] Figure 4 This is a diagram analyzing the chitinase production ability of Bacillus deserticola MYHB_202305ChI.

[0012] Biological material preservation information: Desertibacillus sp. MYHB_202305ChI, the preservation number is GDMCC No: 64172, the preservation unit is Guangdong Provincial Microbiological Culture Collection Center, and the preservation time is March 6, 2024. DETAILED DESCRIPTION

[0013] Example 1 Obtaining desert Bacillus MYHB_202305ChI

[0014] Soil samples were collected from the desert Gobi in Xinjiang Uygur Autonomous Region. 10 g of soil sample was weighed and placed in a 250 mL conical flask. 90 mL of sterile water and glass beads were added and shaken at 180 r / min and 28 °C for 30 min to prepare a mother solution. After standing for 1 min, 1 mL of the supernatant was taken and placed in 9 mL of sterile water to prepare a 10 dilution. -1 of bacterial suspension.

[0015] PBS buffer was used for 10 -3 , 10 -4 , 10 -5 Dilution, take 200 μL each and apply it on a 2216E medium plate, and culture it in a 28°C incubator until a single colony grows. Pick single colonies with different morphology and color and streak them in solid culture medium, repeat 3-5 times, pick single colonies and inoculate them in the corresponding 2216E liquid culture medium, and culture them in a shaker (28°C, 150r / min) at a temperature of 28°C. The strain of the present invention is obtained.

[0016] The Guangdong Provincial Microbial Culture Collection Center was commissioned to identify the isolated bacteria and confirmed that the bacteria belonged to the genus Bacillus. The 16S rDNA sequence is shown in SEQ ID NO: 1, based on 16S rDNA.

[0017] The culture medium components are as follows:

[0018] 2216E medium: peptone 5.0 g, yeast extract powder 1.0 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 5.9 g, magnesium sulfate 3.24 g, calcium chloride 1.8 g, potassium chloride 0.55 g, sodium bicarbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 34.0 mg, boric acid 22.0 mg, sodium silicate 4.0 mg, sodium fluoride 2.4 mg, ammonium salt 1.6 mg, disodium hydrogen phosphate 8.0 mg, ddH2O 1000 mL, pH 7.6.

[0019] Example 2 Experiment on enzyme production ability of Bacillus deserticola MYHB_202305ChI

[0020] 1. Experimental methods

[0021] The desert Bacillus MYHB_202305ChI strain of the present invention was inoculated into 2216E liquid culture medium and cultured in a shaking incubator at 28°C until OD600 = 1.0. 5 μL of the bacterial solution was inoculated into the identification culture medium respectively. After culture at 28°C and staining, the enzyme production capacity of the strain was analyzed.

[0022] Chitinase identification medium: chitin 10.0g, NaHCO3 1.0g, magnesium sulfate 15.0g, ammonium sulfate 2.5g, magnesium chloride 5.0g, potassium chloride 2.1g, potassium dihydrogen phosphate 1.5g, calcium chloride 0.5g, potassium bromide 0.1g, cesium chloride 0.05g, agar 15g, ddH2O 1000mL pH 7.0.

[0023] 2. Results

[0024] The desert Bacillus obtained by the present invention has the ability to synthesize secretory chitinase and can efficiently degrade chitin. After culturing at 28°C for 24 hours, a huge degradation circle is generated in the chitinase screening culture, and the hydrolysis circle diameter ratio HC value is 2.86, so the strain is named desert Bacillus MYHB_202305ChI.

[0025] The research results show that the desert Bacillus MYHB_202305ChI strain of the present invention has the ability to produce high amounts of chitinase and can be used for the preparation and production of chitinase.

[0026] The desert Bacillus MYHB_202305ChI of the present invention has the following properties:

[0027] 1. Morphological characteristics

[0028] Visible under an electron microscope, they are rod-shaped bacteria, such as Figure 1 shown.

[0029] 2. Characteristics on various culture media:

[0030] On 2216E agar medium, the colonies appear yellow, raised, with a moist surface, neat edges, and no extracellular secretions, e.g. Figure 2 shown.

[0031] 3. Physiological and biochemical characteristics

[0032] The strain of Bacillus deserticola MYHB_202305ChI is a Gram-positive aerobic strain with motility, positive for catalase and oxidase, and negative for urease and arginine dihydrolase.

[0033] 4. Carbon source utilization

[0034] It can use glucose, maltose, mannose, citric acid, etc. as carbon sources.

[0035] 5. Other properties

[0036] The optimum growth temperature is 28°C. The cell wall fatty acids are mainly iso-C15:0, iso-C17:0 and C16:0.

[0037] 6. The culture conditions of Bacillus deserticola MYHB_202305ChI are:

[0038] (1) Culture medium: 2216E agar culture medium was used. The specific formula is: peptone 5g / L, yeast extract 1g / L, ferric citrate 0.1g / L, sodium chloride 19.45g / L, magnesium chloride 5.98g / L, sodium sulfate 3.24g / L, calcium chloride 1.8g / L, potassium chloride 0.55g / L, sodium carbonate 0.16g / L, potassium bromide 0.08g / L, cesium chloride 0.034g / L, boric acid 0.022g / L, sodium silicate 0.004g / L, sodium fluoride 0.0024g / L, sodium nitrate 0.0016g / L, disodium hydrogen phosphate 0.008g / L, agar 15g / L.

[0039] (2) pH 7.4 ± 0.2.

[0040] (3) Culture temperature: 28°C.

[0041] 7. The nucleotide sequence of 16S rDNA of Bacillus deserticola MYHB_202305ChI is shown in SEQ ID NO: 1. The 16S rDNA of Bacillus deserticola MYHB_202305ChI has significant differences compared with the 16S rDNA of other species of the same genus:

[0042] The similarity with Desertibacillus haloalkaliphilus KJ1-10-99 is 98.01%;

[0043] The similarity with Alkalihalobacterium elongatum MEB199 is 97.08%;

[0044] The similarity with Alkalihalobacterium alkalinitrilicum DSM 22532 is 96.27%;

[0045] The phylogenetic tree constructed is as follows Figure 3 shown.

[0046] 8. Comparison of microbiological characteristics with standard strains of the same genus but different closely related species

[0047] Comparative experiments with the standard strain of Desertibacillus haloalkaliphilus KJ1-10-99 showed that the desert Bacillus MYHB_202305ChI discovered in the present invention has significant differences in microbiological characteristics with the standard strain.

[0048] Table 1 Comparison of microbiological characteristics of the desert Bacillus MYHB_202305ChI of the present invention and different closely related strains of the same genus

[0049]

[0050] Note: “+” indicates positive, “-” indicates negative, and “w” indicates weakly positive.

[0051] 9. Enzyme production experiment of Bacillus deserticola MYHB_202305ChI

[0052] Different substrates were used as the sole carbon source to prepare plates for bacterial culture, and the strain was tested for its ability to produce degradation circles. The results showed that the desert Bacillus MYHB_202305ChI of the present invention could grow normally on plates with chitin as the sole substrate, and produce degradation circles on chitinase identification plates, as shown in FIG. Figure 4 .

[0053] The above embodiments are only used to illustrate the technical ideas and features of the present invention, and are not intended to be the only or limit the present invention. It should be understood by those skilled in the art that various changes or equivalent substitutions made to the present invention without departing from the scope of the present invention all fall within the scope of protection of the present invention.

[0054] Sequence Listing

[0055] SEQ ID NO: 1

[0056] TATGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGT

[0057] CGAGCGGACCAACTAAGAGCTTGCTCTTAGTTGGTTAGCGGCGGACGGGTGAGTAACACG

[0058] TGGGCAACCTGCCCTGTAGACTGGGACAACTTCGGGAAACCGAAGCTAATACCAGATAAT

[0059] CAAAAGAAGCTCCTGCTTCTTTTGTAAAAGTTGGGATACTAACACTACAGGATGGGCCCG

[0060] CGGCGCATTAGCTAGTTGGTAAGGTAATGGCTTACCAAGGCGACGATGCGTAGCCGACCT

[0061] GAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCA

[0062] GTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGCGATGAAG

[0063] GCCTTCGGGTCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTATCGTTCGAATAGGGCGAT

[0064] ACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATA

[0065] CGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTCCTTA

[0066] AGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGA

[0067] GTACAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGATATGTGGAGGA

[0068] ACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGG

[0069] GAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTA

[0070] GGGGTTTCGATGCCCTTAGTGCCGAAGTTAACACATTAAGCACTCCGCCTGGGGAGTACG

[0071] ACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGTGGAGCATGTGG

[0072] TTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGA

[0073] GATAGAGCTTTCCCCTTCGGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCG

[0074] TGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGC

[0075] ATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACG

[0076] TCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAG

[0077] GGCAGCGAAACCGCGAGGTTAAGCAAATCCCATAAAGCCATTCTCAGTTCGGATTGCAGG

[0078] CTGCAACTCGCCTGCATGAAGCCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTG

[0079] AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGA

[0080] AGTCGGTGGGGTAACCTTTTGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAA

[0081] GTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTT。

Claims

1. A desert bacillus ( Desertibacillus sp. )MYHB_202305ChI, characterized in that It is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No: 64172.

2. Use of the desert Bacillus MYHB_202305ChI described in claim 1 as a chitinase producing strain.

3. Use of the desert Bacillus MYHB_202305ChI described in claim 1 to construct a chitinase-producing genetically engineered strain.

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