A strain producing alginate lyase and its application
An algin lyase and strain technology are applied to the preparation of alginate oligosaccharides, the field of Hecobetella and the fermentation field for preparing algin lyase, and can solve the problem of low yield of algin lyase, limited popularization and application, Problems such as increasing the production cost of enzymatic hydrolysis
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Embodiment 1
[0037] Example 1: Screening of strains producing alginate lyase
[0038] (1), 50mL enrichment medium was divided into 250mL Erlenmeyer flasks, a small amount of sea mud was shaken with sterile water for 30min, and then the oscillation liquid and the collected seawater were respectively inoculated into the enrichment medium. Both were 5 mL, cultured at 30°C for 48h.
[0039] (2), the primary screening medium is poured on the plate, and the seawater and sea mud bacteria liquid of the enrichment culture are respectively carried out for 10 -1 ~10 -7 Serial dilution, for 10 -3 ~10 -7 The diluted bacterial solution was applied and incubated at 30°C for 48h. Observe the size of the transparent circle around the colony, select the strain with good growth condition and a large transparent circle as the starting strain, and carry out plate streak separation and purification.
[0040] (3), put 50mL liquid seed medium into a 250mL Erlenmeyer flask, inoculate the strains separated by ...
Embodiment 2
[0047] Example 2: Identification of strains producing alginate lyase
[0048] The bacterial strain screened out in Example 1 was extracted using a bacterial genomic DNA extraction kit to extract the whole genome of the cell, and then the universal 27F / 1492R primer (forward primer 5'-AGAGTTTGATCCTGGCTCAG-3', reverse primer 5'-GGTTACCTTGTTACGACTT- 3') Perform PCR amplification of 16S rDNA, the PCR amplification conditions are: 95°C for 5 minutes; 95°C for 30s, 55°C for 30s, 72°C for 60s, 35 cycles; 72°C for 10 minutes. After PCR amplification, the Guangzhou Branch of Beijing Liuhe Huada Gene Technology Co., Ltd. was entrusted with sequencing.
[0049] The deoxynucleic acid sequence of the 16S rDNA of the strain is shown in the sequence table. The above 16S rDNA deoxynucleic acid sequence was compared with the 16S rDNA sequence in the gene bank of the National Center for Biotechnology Information USA (NCBI), and it was 99% homologous to Cobetia marina strain JCM 21022, and then ...
Embodiment 3
[0050] Example 3: Optimization of fermentation conditions for the production of alginate lyase by Cobetia marina HQZ08
[0051] (1) Effect of medium pH on enzyme production of Cobetia marina HQZ08
[0052] Six experimental groups were set up, and the influence of the initial pH (6.0, 6.5, 7.0, 7.5, 8.0, 8.5) of the enzyme-producing medium on the enzyme production of the strain was investigated. ~5g / L, yeast powder 1~2g / L, sodium chloride 25~30g / L, pH 7~7.5 liquid seed culture medium after 12~24h, inoculate with 2% inoculation amount in the different pH , in a liquid enzyme-producing medium composed of sodium alginate 5-7g / L, peptone 3-5g / L, sodium chloride 25-30g, dipotassium hydrogen phosphate trihydrate 1-2g / L, cultured at 30°C 24h. The initial pH of the medium can directly affect the permeability, stability and activity of metabolite enzymes of the bacterial cell membrane. The bacterium can grow normally in the range of 6.0 to 8.5, and the enzyme production of the strain...
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