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Vibrio natriegens for producing agarase and application of vibrio natriegens

A technology requiring sodium vibrio and agarase, applied in the production of bacteria, hydrolase, bulk chemicals, etc., can solve problems such as poor product uniformity, complex process, ecological environment pollution, etc., and achieve mild reaction conditions and simple preparation methods. , The effect of separation and purification is simple

Active Publication Date: 2015-11-25
HUAQIAO UNIVERSITY
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AI Technical Summary

Problems solved by technology

The preparation of agar oligosaccharides by acid degradation has poor uniformity and poor repeatability. In addition, the acidic substances used in the acid degradation method will cause serious pollution to the surrounding ecological environment; the derivatization degradation method is mainly used for the component structure analysis of polysaccharides, and the process is complicated when used for the synthesis of oligosaccharides; the redox degradation method The hydroxyl free radicals generated in the reaction are used to break the sugar chain non-specifically. This method has poor specificity and cannot be repeated, which is not conducive to the preparation of agar oligosaccharides; the preparation of agar oligosaccharides by enzymatic hydrolysis has high catalytic efficiency. , product specificity, mild reaction conditions, easy product control, no pollution and many other advantages. However, due to the lack of strains with high enzyme activity, the yield of agarase is low, which increases the production cost of enzymatic hydrolysis and limits the method. Promoted application of

Method used

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  • Vibrio natriegens for producing agarase and application of vibrio natriegens
  • Vibrio natriegens for producing agarase and application of vibrio natriegens
  • Vibrio natriegens for producing agarase and application of vibrio natriegens

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1: the screening of producing agarase strain

[0037] (1) Dispense 50 mL of 2216E medium containing 1‰ agar into 250 mL Erlenmeyer flasks, take a small amount of rooted laver and rootless laver (from a laver breeding area in Zhangzhou City, Fujian Province) Shake for 30 minutes, then inoculate the shaking solution mixed with seawater into the enrichment medium, and enrich and culture for 2 to 3 generations, so that the number of the target strain increases.

[0038] (2) Pour the 2216E medium containing 15‰ agar onto the plate, and carry out 10 -1 ~10 -8 Serially diluted, the 10 -7 、10 -8 Two dilutions of bacterial solutions were spread on the plate, and stained with Lugol's iodine solution for observation after 48 hours of cultivation (the bacterial strain capable of producing agarase can degrade agar, and after the agar is degraded, it cannot be stained with Lugol's iodine solution, so will produce an obvious transparent circle), and the strains that can...

Embodiment 2

[0048] Embodiment 2: the identification of producing agarase strain

[0049] The 16SrDNA sequence analysis of the sodium-demanding Vibrio HJPHYXJ-1 screened in Example 1: use the SK1201 column bacterial genomic DNA extraction kit to extract the whole genome of the cell, and then use the universal 27F / 1492R primer (forward primer 5'- AGAGTTTGATCCTGGCTCAG-3' (shown in SEQIDNO01); reverse primer 5'-GGTTACCTTGTTACGACTT-3' (shown in SEQIDNO02)) for PCR amplification of 16SrDNA, PCR amplification conditions: 98°C for 5min; 94°C for 35s, 55°C 35s, 72°C 90s, 35 cycles; 72°C 8min. After the PCR amplification products were purified with SK1131 column DNA gel kit, they were sequenced by Beijing Sanbo Polygala Biotechnology Co., Ltd.

[0050] The partial deoxynucleic acid sequence of the 16S rDNA of the Narophilicus HJPHYXJ-1 is shown in SEQ ID NO03, specifically as follows:

[0051] AGCGGAACGAGTTAACTGAACCTTCGGGGGACGTTAACGGCGTCGAGCGGCGGACGGGTGAGTAATGCCTAGGAAATTGCCCTGATGTGGGGGATAACCATTGG...

Embodiment 3

[0053] Embodiment 3: Fermentation preparation of agarase by sodium vibrio

[0054] (1) Cultivate the Vibrio natrigenus (Vibrionatriegens) HJPHYXJ-1 strain on the slant medium at 30-35° C. for 24 to 48 hours to obtain the activated Vibrio natrigenus strain; the slant medium The formula is: agar 15-20g / L, yeast extract 1.0-2.0g / L, peptone 3.0-5.0g / L, beef extract 1.0-2.0g / L, iron phosphate 0.01g / L, solvent is old sea water or synthetic Seawater, pH=7.4~7.6.

[0055] (2) inoculate the Vibrionatriegens HJPHYXJ-1 strain after the activation culture in step (1) into the seed liquid culture medium, shake and cultivate at 30-35°C and 150-200r / min for 8-24h, Obtain seed liquid; the formula of described seed liquid culture medium is: yeast extract 1.0~2.0g / L, peptone 3.0~5.0g / L, beef extract 1.0~2.0g / L, iron phosphate 0.01g / L, solvent It is aged seawater or synthetic seawater, pH=7.4~7.6.

[0056] (3) Transplant the seed solution of step (2) into the liquid medium with the inoculatio...

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Abstract

The invention discloses vibrio natriegens HJPH YXJ-1 for producing agarase and application of the vibrio natriegens; the strain is preserved in China Center for Type Culture Collection with preservation number of CCTCC M 2015244 on April 23, 2015, wherein the China Center for Type Culture Collection is located in Wuhan University, and post code is 430072. The agarase generated from the strain is an extracellular enzyme, which is simple in separation and purification and is free from cell breakage; enzyme-producing activity is relatively high, and under optimized conditions, the activity of unpurified crude enzyme reaches 103U / mL; to degrade agar, the agarase produced from the strain is mild in reaction condition, good in specificity and free from environmental pollution, and the is suitable for the large-scale preparation of neoagaro-oligosaccharides.

Description

technical field [0001] The invention belongs to the technical field of microorganism application, and in particular relates to an agarase-producing natrivibrio and application thereof. Background technique [0002] Agar (agar) is a polysaccharide extracted from marine red algae. It has a long history of application in food, medicine, health and other industries. Certain restrictions. Agar oligosaccharides are oligosaccharides with a degree of polymerization of 2 to 10 obtained from the degradation of agar, also known as agar oligosaccharides, which are mainly composed of repeating units of agarose. Due to their good water solubility, they are easily absorbed by the human body. , so it has high application value in the field of medicine. Studies have shown that agar oligosaccharides can inhibit the rise of blood sugar, improve human immunity, anti-tumor, reduce oxidative damage of cells, etc., and are a kind of oligosaccharides with great development potential. [0003] Th...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/24C12P19/14C12R1/63
CPCY02P20/52
Inventor 肖美添黄雅燕叶静韩军萍
Owner HUAQIAO UNIVERSITY
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