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Alginate lyase secreted from marine bacterium and preparation method thereof

A technology of alginate lyase and bacterial strain, applied in the field of alginate lyase and its preparation

Active Publication Date: 2014-04-02
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

patent published Pseudomonas The molecular weight of alginate lyase produced by sp.HZJ216 strain is 60.25 KDa, Vibrio The molecular weight of the alginate lyase produced by sp. JG07-007 is 34.6 KDa, etc., but there is no such thing after searching. Cobetia Bacteria of the genus produce the same alginate lyase

Method used

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  • Alginate lyase secreted from marine bacterium and preparation method thereof
  • Alginate lyase secreted from marine bacterium and preparation method thereof
  • Alginate lyase secreted from marine bacterium and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: bacterial strains Cobetia Isolation of sp. WG-007

[0024] The present invention obtains soil samples and waste samples from domestic chemical plants producing sodium alginate. Sodium alginate was used as the sole carbon source to screen high-yielding alginate lyases. Weigh 1.0-1.5 g of soil samples in the media of different concentrations of sodium alginate, ferment and culture at 25 °C for 40 h, take 200 μl of the diluted solution and spread it on the screening solid medium, and culture at 25 °C for 16-18 h , Pick a single colony that can grow on a sodium alginate plate and inoculate it into a fresh self-made liquid medium for 20 h, then inoculate the cell liquid culture at a 1% (w / v) inoculum in a 50 mL fermentation medium In a 250 mL Erlenmeyer flask with a base, shake culture at 25 °C at a speed of 150 r / min, collect the fermentation broth after 40 h, and centrifuge at 8000-12000 r / min for 20-30 min to remove the bacteria, collect the supernatant, and...

Embodiment 2

[0025] Example 2: Strains Cobetia Molecular biological characterization of sp. WG-007

[0026] Strain WG-007 was inoculated in the growth medium, and after culturing for 30 h, part of the fermentation liquid was taken in a 1.5 mL sterile centrifuge tube, and the genome of the bacteria was extracted using a bacterial genome rapid extraction kit, and the genome was used as a template Amplifies the 16S rRNA gene sequence. The PCR reaction conditions were: pre-denaturation at 94 °C for 5 min; denaturation at 95 °C for 45 s, annealing at 55 °C for 60 s, and extension at 72 °C for 60 s; after 30 cycles, post-extension at 72 °C for 10 min.

[0027] The obtained PCR product was purified by tapping recovery kit (Axygen) and ligated with TA cloning vector. After the ligation solution was transformed into competent cells, positive clones were picked and sequenced by Sangon Bioengineering (Shanghai) Co., Ltd. The 16S rDNA sequence of strain WG-007 is 1508 bp in length. The sequence inf...

Embodiment 3

[0028] Example 3: Morphology and Physiological Characteristic Identification of Bacterial Strain WG-007

[0029] The strain is short rod-shaped, round-ended and arranged individually. Gram staining is red, and after being cultured on the plate for 24 hours, the colony is round, with a diameter of about 2-3mm, smooth surface, moist, neat edges, the same color on both sides, slightly raised, and beige in color. No flagella, no sporulation. Biochemical characteristics: Gram staining is negative. The strain can grow under the condition of salt concentration of 30%, and the growth temperature is 15-40°C, but the optimum growth temperature is 25°C, and the optimum NaCl concentration is 6.0%. Catalase is positive, oxidase is negative, M.R is positive, V.P reaction is negative, glucose, lactose, mannitol, etc. can be used; starch and gelatin cannot be hydrolyzed; it has physiological and biochemical characteristics such as nitrate reduction.

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Abstract

The invention belongs to the field of biotechnologies, and particularly relates to a marine bacterium Cobetia sp. WG-007 screened from a waste material sample and an alginate lyase secreted from the marine bacterium and a preparation method thereof. By combining morphology and physiological and biochemical characters, the strain belongs to a Cobetia category, and is preserved in China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC No.7964. The strain secretes the alginate lyase, and the alginate lyase is purified by ultrafiltration and concentration, Q-Sepharose strong anion exchange chromatography, Phenyl Sepharose 6 Fast Flow hydrophobic chromatography and Superdex-G100 gel filtration technologies so as to obtain the alginate lyase with electrophoresis purity; SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) determines that the molecular weight of the alginate lyase generated by the strain is 35.0KDa.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically involving a marine bacterium Cobetia Alginate lyase secreted by sp. WG-007 and its preparation method. Background technique [0002] Alginate, also known as alginic acid, is a linear polysaccharide composed of β-D-mannuronic acid (mannuronate, M) and α-L-guluronic acid (guluronate, G). Alginate is mainly derived from seaweed, kelp, sargassum and macroalgae, etc. It has a complex structure and various physical and chemical properties, and is widely used in medicine, food-grade cosmetics and other industries. Alginate lyase is a kind of enzyme that breaks alginate through β-elimination reaction. Because of its specificity, high efficiency and mild reaction, it is regarded as a powerful tool for modifying alginate. In addition, fucoidan oligosaccharides not only have good antitumor and anticoagulant activities, but also can be used as plant growth promoters. The preparation methods of f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/88C12R1/01
Inventor 史劲松刘旭梅许正宏李恒龚劲松蒋敏
Owner JIANGNAN UNIV
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