New strain of Zabelella producing amylase and its application

Abstract

The present invention discloses a new amylase-producing Zobellella species and its application, and relates to the field of microbial technology. The present invention discovers a new microbial species, which is identified as belonging to the genus Zobellella and is named Zobellella sp. MYHB_202302AmY. Experimental verification shows that the bacteria has the ability to tolerate high salt and secrete amylase.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a new strain of amylase-producing Zabel's bacterium and application thereof. Background Art

[0002] Starch is a high-energy polymer composed of multiple glucose molecules, and its structure includes straight-chain starch and amylopectin. The main component of straight-chain starch is glucose molecules connected by α-1,4 glycosidic bonds. The glycosidic bonds at the branch points are different from the glycosidic bonds of the main chain and are connected by α-1,6 glycosidic bonds. Amylopectin is different. It has more branches based on a main chain. Its main chain and branches are connected by α-1,4 glycosidic bonds, and the branch points are connected by α-1,6 glycosidic bonds. The proportion of branches contained in amylopectin is relatively high, accounting for about 70-85% in potatoes and cereals, and reaching 100% in glutinous crops.

[0003] There are three types of starch hydrolysis: acid hydrolysis, enzymatic hydrolysis and composite method. Among them, the enzymatic hydrolysis method has the characteristics of mild conditions. The first step of hydrolysis is carried out by amylase to obtain oligosaccharides and dextrins, and the second step of hydrolysis is carried out by saccharifying enzymes, and the final product is glucose. Amylase can directly catalyze the hydrolysis of starch into oligosaccharides such as glucose and maltose, and is most widely used in the enzyme preparation industry. Amylase is mainly derived from microorganisms, including bacteria such as Bacillus and Actinomycetes and fungi such as Aspergillus oryzae and Monascus. At present, the amylase production strains are relatively single, so it is necessary to seek different types of amylase production strains. Summary of the invention

[0004] In order to solve the deficiencies of the prior art, the present invention provides a new strain of amylase-producing Zymoxella and its application.

[0005] In order to achieve the purpose of the present invention, the following scheme is proposed:

[0006] The invention separates and cultivates a new microbial strain from the soil of Xinjiang desert Gobi, and finds that the strain has the function of producing amylase in a high-salt-resistant manner.

[0007] Genome sequencing and physiological and biochemical experiments confirmed that the bacterium belongs to the genus Zobellella and was named Zobellella sp. MYHB_202302AmY. The strain has been deposited in the Guangdong Provincial Microbiological Culture Collection Center with the collection number GDMCC No: 64175.

[0008] The beneficial effects of the present invention are: the MYHB_202302AmY strain has good salt tolerance, can grow in 2216E agar medium, has the function of producing amylase, can be directly used as a starch production strain, and can be used for industrial production of amylase 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 strain with improved traits can be obtained by traditional mutagenesis breeding methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a transmission electron microscope image of Zymoxema MYHB_202302AmY.

[0010] Figure 2 This is the colony morphology of Zymoxema MYHB_202302AmY.

[0011] Figure 3 This is the evolutionary analysis diagram between Zymox zona MYHB_202302AmY and other closely related strains (based on 16SrDNA sequence analysis).

[0012] Figure 4 This is an analysis chart of the amylase production ability of Zymox zona MYHB_202302AmY.

[0013] Biological material preservation information: Zobellella sp. MYHB_202302AmY, the preservation number is GDMCC No: 64175, the preservation unit is Guangdong Provincial Microbiological Culture Collection Center, and the preservation time is March 6, 2024. DETAILED DESCRIPTION

[0014] Example 1 Obtaining Zygella MYHB_202302AmY

[0015] Soil samples were collected from the Gobi Desert in Xinjiang. 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.

[0016] PBS buffer was used for 10 -3 , 10 -4 , 10 -5Dilution, 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.

[0017] The Guangdong Provincial Microbial Culture Collection Center was commissioned to identify the isolated bacteria and confirmed that the bacteria belonged to the genus Chabelella. The 16S rDNA sequence is shown in SEQ ID NO: 1, based on 16S rDNA.

[0018] The culture medium components are as follows:

[0019] 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.

[0020] Example 2 Experiment on the enzyme production ability of Zymela MYHB_202302AmY

[0021] 1. Experimental methods

[0022] The MYHB_202302AmY 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.

[0023] Amylase identification medium: soluble starch 10.0 g, peptone 10.0 g, yeast powder 5 g, sodium chloride 10 g, agar 15 g, ddH2O 1000 mL, pH 7.0.

[0024] 2. Results

[0025] The strain obtained by the present invention has the ability to synthesize secretory amylase and can efficiently degrade starch. After culturing at 28°C for 12 hours, a huge degradation circle is generated in the amylase screening culture, and the hydrolysis circle diameter ratio HC value is 2.65, so the strain is named as the strain of ...

[0026] The research results show that the strain Zymox MYHB_202302AmY of the present invention has the ability of high-yield amylase and can be used for the preparation and production of amylase.

[0027] The Zhuobeier's bacteria MYHB_202302AmY of the present invention has the following properties:

[0028] 1. Morphological characteristics

[0029] Visible under an electron microscope, they are rod-shaped bacteria, such as Figure 1 shown.

[0030] 2. Characteristics on various culture media:

[0031] On 2216E agar medium, the colonies appear white, raised, with a moist surface, neat edges, and no extracellular secretions, e.g. Figure 2 shown.

[0032] 3. Physiological and biochemical characteristics

[0033] The strain of Zymex MYHB_202302AmY is a Gram-negative aerobic strain with motility, positive for catalase, oxidase, and urease, and negative for arginine dihydrolase, lysine decarboxylase, and ornithine decarboxylase.

[0034] 4. Carbon source utilization

[0035] It can use glucose, maltose, mannose and sucrose as carbon sources and can weakly utilize citric acid.

[0036] 5. Other properties

[0037] The optimum growth temperature is 28°C. The cell wall fatty acids contain C18:1, C16:1 and iso-C16:0.

[0038] 6. The culture conditions of Zolpidem MYHB_202302AmY are:

[0039] (1) The culture medium used was 2216E agar medium. The specific formula was: 5 g / L peptone, 1 g / L yeast extract, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L cesium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L sodium nitrate, 0.008 g / L sodium hydrogen phosphate, and 15 g / L agar.

[0040] (2) pH 7.4 ± 0.2.

[0041] (3) Culture temperature: 28℃.

[0042] 7. The nucleotide sequence of 16S rDNA of Z. chrysogenum MYHB_202302AmY is shown in SEQ ID NO: 1. Compared with the 16S rDNA of other species of the same genus, the 16S rDNA of Z. chrysogenum MYHB_202302AmY has significant differences:

[0043] The similarity with Zobellella endophytica 59N8 is 97.89%;

[0044] The similarity with Zobellella iuensis CPY4 is 97.80%;

[0045] The similarity with Zobellella taiwanensis ZT1 is 97.20%;

[0046] The similarity with Zobellella denitrificans ZD1 is 96.86%;

[0047] The similarity with Oceanimonas doudoroffii MBIC1298 is 97.27%;

[0048] The similarity with Oceanimonas marisflavi 102-Na3 is 97.2%, etc.

[0049] The phylogenetic tree constructed is as follows Figure 3 shown.

[0050] 8. Comparison of microbiological characteristics with standard strains of the same genus but different closely related species

[0051] Comparative experiments with the standard strain of Zobellella endophytica 59N8T showed that the microbiological characteristics of Zobellella MYHB_202302AmY discovered in the present invention were significantly different from those of the standard strain.

[0052] Table 1 Comparison of microbiological characteristics of the present invention's Zygella MYHB_202302AmY and different closely related strains of the same genus

[0053]

[0054] Note: “+” indicates positive, “-” indicates negative, and “w” indicates weakly positive.

[0055] 9. Enzyme production experiment of Zygella MYHB_202302AmY

[0056] 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 present invention's Zybelella MYHB_202302AmY could grow normally on plates with starch as the sole substrate, and produce degradation circles on amylase identification plates, as shown in FIG. Figure 4 .

[0057] 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.

[0058] Sequence Listing

[0059] SEQ ID NO: 1

[0060] TTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTC

[0061] GAGCGGCAGCGGGAGAGTAGCTTGCTACTTTTGCCGGCGAGCGGCGGACGGGTGAGTAAG

[0062] GCTTGGGTATCTGCCCGGTCGAGGGGGATAACCGTTGGAAACGACGGCTAATACCGCATA

[0063] CGCCCTACGGGGGAAAGCAGGGGATCTTCGGACCTTGCGCGATTGGATGAGCCCAAGTGA

[0064] GATTAGCTGGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCTCTAGCTGGTCTGAGAG

[0065] GATGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGG

[0066] GAATATTGCACAATGGGGGCAACCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTT

[0067] CGGGTTGTAAAGCACTTTCATGGGGAGGAAAGGTACACCGTTAATACCGGTGTGCTGTG

[0068] ACGTTACCCACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGG

[0069] GTGCAAGCGTTAATCGGAATGACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGCCAG

[0070] ATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATTTGGAACTGGCAAGCTAGAGTCTTG

[0071] GAGAGGGGGGTAGAATTTCCGGTGTAGCGGTGAAATGCGTAGAGATCGGAAGGAATACCA

[0072] GTGGCGAAGGCGGCCCCCTGGCCAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAA

[0073] ACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCAACTTGGAGTCTGTGCCA

[0074] TTTGAGCGCGGGTTCCGGAGCTAACGCGTTAAGTTGACCGCCTGGGGAGTACGGCCGCAA

[0075] GGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATT

[0076] CGATGCAACGCGAAGAACCTTACCTACCCTTGACATACAGCGAAGTTTTCAGAGATGAAT

[0077] TCGTGCCTTCGGGAACGCTGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAG

[0078] ATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGATTCGGTC

[0079] GGGAACTCAAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCA

[0080] TCATGGCCCTTACGGGTAGGGCTACACACGTGCTACAATGGCGCGTACAGAGGGCGGCGA

[0081] ACCAGCGATGGTGAGCGAATCCCAAAAAGCGCGTCGTAGTCCGGATCGGAGTCTGCAACT

[0082] CGACTCCGTGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTT

[0083] CCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGATA

[0084] GCTTAACCTTCGGGAGGGCGTTTACCACGGAGTGATTCATGACTGGGGTGAAGTCGTAAC

[0085] AAGGTAACCGTAGGGGAACCTGCGGTTGGATCACCTCCTT

Claims

1. A strain of Zygella MYHB_202302AmY, characterized in that The taxonomic name is Zobellella sp., and it is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No: 64175.

2. Use of the strain Zymox MYHB_202302AmY described in claim 1 as an amylase producing strain.

Images