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Bacillus sp capable of producing alginate lyase and application thereof

A technology of alginate lyase and Bacillus, applied in the direction of lyase, bacteria, and microorganism-based methods, can solve the problems of unsafe strains, poor enzyme stability, narrow enzyme substrate spectrum, etc., achieve high activity and simple preparation , nutritional requirements simple effect

Active Publication Date: 2014-12-10
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the product development of alginate lyase still faces problems such as unsafe strains, low enzyme activity, poor enzyme stability, and narrow substrate spectrum of the enzyme.

Method used

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  • Bacillus sp capable of producing alginate lyase and application thereof
  • Bacillus sp capable of producing alginate lyase and application thereof
  • Bacillus sp capable of producing alginate lyase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Screening identification and preservation of Bacillus sp. Alg07

[0026] (1) Screening of strains

[0027] The rotten kelp samples were obtained from Weihai Qingyutan Sea Treasure Farm, and 5g samples were put into a 250mL Erlenmeyer flask filled with 50mL enrichment medium, and fermented by shaking the flask at 30°C and 180r / min. After culturing for 48 h, transfer 5 mL of the culture solution to another 250 mL Erlenmeyer flask filled with 50 mL of fresh enrichment medium, and pass 5 times continuously. The culture medium of continuous acclimatization for 5 generations was mixed with sterile saline according to 10 -3 to 10 -7 Carry out gradient dilution, take 0.1mL of the diluted solution and spread it on the primary screening solid plate, and after 48 hours of constant temperature incubation at 30°C, use the plate streaking method to determine the strains of single colonies that can grow on the primary screening medium and produce transparent circles. sep...

Embodiment 2

[0038] Example 2, Bacillus sp. (Bacillus sp.) Alg07 CGMCC No.9391 production medium composition optimization of alginate lyase

[0039] Put 50ml of fermentation medium into a 250mL conical flask, sterilize with high-temperature steam at 121°C for 30 minutes, and aseptically inoculate the seed liquid cultivated to the late logarithmic phase into the conical flask with an inoculum volume of 2%, at 30°C and 180r / min Under culture for 48h, the enzyme activity was measured, and all experiments were designed in parallel three times. The results of the previous optimization step are used in subsequent experiments.

[0040] (1) Effect of carbon source on enzyme production of Bacillus sp. Alg07 CGMCC No.9391

[0041] Using 0.5% glucose, lactose, mannose, D-mannitol, maltose, starch and sodium alginate as the sole carbon source to prepare the fermentation medium, the results showed that the strain Alg07 could only produce enzymes when sodium alginate was the sole carbon source , indic...

Embodiment 3

[0045] Example 3, Optimization of fermentation conditions for the production of alginate lyase by Bacillus sp. Alg07 CGMCC No.9391

[0046] The optimized fermentation medium formula was used to prepare the fermentation medium, and the optimal conditions for the production of alginate lyase by strain Alg07 were optimized through single factor experiments. All experiments were designed in triplicate. The results of the previous optimization step are used in subsequent experiments.

[0047] (1) Effect of culture temperature on enzyme production of Bacillus sp. Alg07 CGMCC No.9391

[0048] Temperature is an important environmental factor affecting the growth and metabolism of the strain. Under the optimized medium conditions, the strain was fermented and cultured at 15°C, 20°C, 25°C, 30°C, 35°C, and 40°C for 48 hours, and the alginate lyase in the fermentation broth was detected. Activity, the results showed that when the temperature was 20°C to 30°C, the enzyme activity was high...

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Abstract

The invention provides Bacillus sp Alg07 capable of producing an alginate lyase. The preservation number of the Bacillus sp Alg07 is CGMCC No.9391. The Bacillus sp Alg07 provided by the invention has the advantages that requirement on nutrition is low and fermentation time is short; a crude enzyme is easily prepared, and the crude enzyme can be obtained through centrifugation; the alginate lyase produced by the Bacillus sp Alg07 has high enzyme activity, the specific enzyme activity of an unpurified crude enzyme can reach 20000-40000 U / mg (563U / mL); the alginate lyase produced by the Bacillus sp Alg07 has good stability, is not obviously changed in enzyme activity after being preserved at 40 DEG C for 24 hours and is high and stable in enzyme activity at pH of 5.5-8.5; and the produced alginate lyase is capable of degrading sodium alginate to generate alginate-derived oligosaccharide with biological activity.

Description

technical field [0001] The invention belongs to the technical field of functional microorganism screening, and in particular relates to a bacillus strain producing alginate lyase and application thereof. Background technique [0002] Alginate is composed of β-D-mannuronic acid (β-D-1,4-mannuronic acid, referred to as M) and its 5-position epimer α-L-guluronic acid (α-L- 1,4-guluronic acid, referred to as G) is a linear molecule formed by non-homopolymerization of C-1,4 glycosidic bonds. Studies have found that the degradation product of alginate, alginate oligosaccharides, has many physiological activities such as anti-tumor, anti-coagulation, enhancing plant stress resistance, and promoting the growth of bifidobacteria in the intestinal tract. Its functional evaluation and development research have been continuously deepened. Value research has become a new hot spot. There are many ways to prepare fucoidan oligosaccharides, such as acid degradation, oxidative degradation,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/88C12R1/07
Inventor 孙媛霞朱玥明陈朋门燕马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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