Alginate lyase producing strain, alginate lyase and application thereof
An algin lyase and algin-producing technology are applied in the field of algin lyase-producing strains and algin lyase, and can solve the problems of restricting the development of alginate oligosaccharides, single degradation site, low enzymatic activity and the like
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[0034] The preparation method of above-mentioned alginate lyase preferably comprises the following steps:
[0035] (1) Strain activation: Inoculate the above-mentioned mangrove coccus HB182678 on a solid medium, and culture at 28-37°C for 24-48 hours to obtain an activated strain;
[0036] (2) Liquid culture: Pick a ring of bacteria from the purified plate and insert it into the liquid seed medium, shake and culture at 28-37°C, 120-200r / min for 12-15h until the logarithmic growth phase, and produce into seed liquid;
[0037] (3) Fermentation culture: inoculate the seed liquid into the liquid fermentation medium, shake and culture at 28-37°C, 150-200 r / min for 24-60 hours, collect the fermentation liquid, centrifuge at 8000 r / min for 10 minutes, and collect the supernatant to obtain Crude enzyme solution of alginate lyase.
[0038] In the preparation method of the alginate lyase:
[0039] The formula of the solid medium described in step (1) is: sodium alginate 3-12g / L, pept...
Embodiment 1
[0045] Example 1 Isolation and screening of enzyme-producing strain HB182678 GDMCC No.61000
[0046] Brown algae samples were collected in the offshore waters of Qionghai City, Hainan Province. Take a 10g sample, cut it into pieces, dissolve the slurry, and dilute it to 10 -3 ~10 -6 Suspension, draw 0.1mL of series suspension, spread on alginate lyase separation medium, place in 30°C incubator and incubate upside down for 2-5 days. When the colonies grow out, according to the phenotypic characteristics of the colony shape, color, edge state, transparency, surface dry and wet state, etc., pick the colonies that grow well and have different shapes for streaking and purification.
[0047] The activity of alginate lyase was determined for the isolated strains, and the strain HB182678 with strong enzyme activity was screened out.
[0048] The specific process is as follows:
[0049] Pick the strain to be tested and spot it on the detection medium of alginate lyase activity, and...
Embodiment 2
[0054] Example 2 Identification of mangrove coccus HB182678 GDMCC No.61000
[0055] Strain HB182678 GDMCC No.61000 grew well on 2216E agar medium, and clear colonies could be seen after 2 days of culture. The colonies were round, milky white, with neat edges, smooth and raised surfaces, and a diameter of 2-4 mm. Observed under the electron microscope, the cells were ellipsoidal, with a length and width of about 1.3-2.0 μm×1.2-1.5 μm, without flagella. Gram stain was negative.
[0056] A 1425bp 16S rDNA sequence of HB182678 was obtained by PCR amplification, transformation and sequencing, and its nucleotide sequence is shown in SEQ ID No.1. Comparing the sequence with the sequence in the EzBioCloud database, it was found that the strain HB182678 and Mangrovicoccus ximenensis Tlg56 T The homology is the highest (96.3%). Select related strains with high homology, and use the software MEGA7.0 to construct a phylogenetic tree using the Neighbor-joining method ( figure 2 ), it ...
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