Acinetobacter juni. X8 and application thereof in preparing algin lyase
A technology of Acinetobacter angnei and alginate lyase, which is applied in the direction of lyase, bacteria, and microorganism-based methods, and can solve the problems of difficult separation, poor storage stability, and low production of alginate lyase
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Embodiment 1
[0048] Example 1: Enrichment, domestication and isolation and screening of strains
[0049] Inoculate the spoiled Hijiki leaves and bacterial suspension into a 500mL Erlenmeyer flask filled with 100mL No. 1 medium, and enrich and culture them at 30°C and 180rpm for 72 hours. If the growth is good, pipette 5mL of the culture medium to inoculate Cultivate in No. 2 medium at 30°C and 180 rpm for the same time, otherwise continue to inoculate into No. 1 medium for cultivation. Repeat this for many times, and then move to the culture medium with sodium alginate as the only carbon source for separation and screening.
[0050] The formula of No. 1 and No. 2 liquids and separation and screening medium is as follows:
[0051] ① Medium 1: 0.5% sodium alginate, 0.4% peptone, 2.5% NaCl, 1.0% MgSO 4 ·7H 2 O, 0.2%K 2 HPO 4 ·3H 2 O, 0.001% FeSO 4 ·7H 2 O, the solvent is water, the pH value is 7.2 to 7.4;
[0052] ② Medium 2: 0.5% sodium alginate, 0.5% (NH 4 ) 2 SO 4 , 2.5% NaCl, ...
Embodiment 2
[0055] Embodiment 2: Preparation of alginate lyase enzyme liquid
[0056] The preparation method of alginate lyase is as follows:
[0057] (1) Inoculate the strain Acinetobacter juni X8 stored in a slant test tube into an isolation medium plate, culture for 48-72 hours, and transfer several times in this way to activate the strain; the isolation medium is prepared according to the following composition: sodium alginate 5g, (NH 4 ) 2 SO 4 5g, NaCl 25g, K 2 HPO 4 ·3H 2 O2g, MgSO 4 ·7H 2 O 1g, 20g agar, make up to 1000mL with water, pH 7.2-7.4, heat to dissolve the agar, cool to get a plate;
[0058] (2) The activated strain is inoculated into a 250mL Erlenmeyer flask containing 50mL liquid seed medium, cultivated at 30°C and 150rpm for 8h to obtain a seed liquid; the liquid seed medium formula is: 0.5% sodium alginate, 0.5 %(NH 4 ) 2 SO 4 , 2.5% NaCl, 0.2% K 2 HPO 4 ·3H 2 O, the solvent is water, pH7.5;
[0059] (3) Inoculate 1.0 mL of seed liquid into a 250 mL E...
Embodiment 3
[0068] Embodiment 3: Preparation of fucoidan oligosaccharides
[0069]The preparation process of alginate oligosaccharides is as follows: add 1mL 0.75% (w / w) alginate substrate solution, 1mL 0.1moL / L pH7.5 sodium phosphate buffer solution, and 1mL enzyme solution prepared in Example 2 to the test tube in sequence , and react at 40.0°C for 5 minutes to obtain unpurified alginate oligosaccharides. As determined by the 3,5-dinitrosalicylic acid method, about 1395 μg of alginate can be obtained per mg of purified alginate lyase Fucoidan oligosaccharides.
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