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Organic solvent-tolerant glycosidase Fru6, its mutant and its applications

A technology resistant to organic solvents and glycosidase, applied in the directions of application, hydrolase, botanical equipment and methods, etc., can solve the problems of complex, inability to meet high-efficiency synthesis and large-scale production of oligosaccharides, and achieve the effect of excellent tolerance

Active Publication Date: 2013-12-18
NANJING TECH UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In recent years, in order to solve the constraints of multiple protective group operations and intermediate separation in the chemical synthesis of oligosaccharides, sugar chemists have developed techniques such as one-pot synthesis and solid-phase synthesis. Phase synthesis), tag-assisted synthesis and other new strategies and methods, these strategies and methods have promoted the development of oligosaccharide chemical synthesis technology to a certain extent, but they are still very complicated and cannot meet the needs of efficient synthesis and the need for large-scale production of oligosaccharides

Method used

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  • Organic solvent-tolerant glycosidase Fru6, its mutant and its applications
  • Organic solvent-tolerant glycosidase Fru6, its mutant and its applications
  • Organic solvent-tolerant glycosidase Fru6, its mutant and its applications

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Effect test

Embodiment 1

[0060] This experiment illustrates the screening procedure for natural strains producing the organic solvent-tolerant glycosidase Fru6.

[0061] Using 10%-20% DMSO (v / v) organic solvent as the screening pressure, the extremophiles resistant to organic solvents were screened from the soil samples of chemical plant sewage outlets. The primary screening medium used is: corn steep liquor (1.5-2.5) mL / L, sucrose (10-20) g / L, NH 4 Cl or urea 3.0g / L, KH 2 PO 4 3.0g / L, MgSO 4 2g / L (adjust the pH to 7.0-7.5). Inoculate the screened organic solvent-resistant microorganisms in the primary screening culture based on 30°C, 180rpm culture for two days, then transfer, and inoculate into the sterilized medium according to the inoculation amount of 0.5%-1% (v / v); add 10% -20%DMSO (v / v) organic solvent DMSO, need to transfer twice. Gradiently dilute the culture medium obtained from the enrichment and transfer twice to an appropriate multiple, and spread it on the LB plate. LB plate mediu...

Embodiment 2

[0064] This experiment illustrates the purification procedure of the organic solvent-resistant glycosidase Fru6.

[0065] First, Arthrobacter arilaitensis NJEM01 strain was cultured in the plate medium for 24 hours, then transferred twice, cultured for 24 hours respectively, then connected to the seed solution, cultivated for 12 hours, then connected to the fermentation medium, cultured again for 12 hours, 10,000rpm at 4°C Centrifuge for 15 min, and take the supernatant as the crude enzyme solution. Put the crude enzyme solution in an ice bath, add solid powdered ammonium sulfate to 50%, let stand overnight at 4°C and then centrifuge (10000rpm×30min, 4°C), dissolve the precipitate with 20mmol / L Tris-HCl pH7 buffer, Then add solid powdered ammonium sulfate to the solution again to 90%, let stand overnight at 4°C and then centrifuge (10000rpm×30min, 4°C), the precipitate is still dissolved in Tris-HCl pH7 buffer. The dialyzed precipitate lysate was subjected to DEAE Sepharose ...

Embodiment 3

[0068] This experiment illustrates the determination method of the enzymatic properties of the organic solvent-resistant glycosidase Fru6.

[0069] Determination of the optimal reaction pH value and pH stability of organic solvent-resistant glycosidase Fru6:

[0070] In order to investigate the effect of different pH on the activity of glycosidase Fru6 and determine the optimum pH of the reaction, buffers with pH 3.0, 4.0, 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 10.0 were prepared respectively, and purified The final glycosidase Fru6 was reacted with different pH value substrate reaction solutions, and the activity of glycosidase Fru6 was detected respectively. The enzyme activity was as follows: figure 2 shown. From figure 2 It can be seen that the enzyme activity is seriously lost when the pH is lower than 5.0 or higher than 9.0, and the enzyme activity is higher (over 85%) at pH 5.0-6.5, and the optimum pH of the enzyme activity is 5.0. In order to investigate the pH s...

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Abstract

The invention relates to an organic solvent-tolerant glycosidase producing strain, an organic solvent-tolerant glycosidase Fru6 generated by the strain, an autoploid and a mutant of the organic solvent-tolerant glycosidase Fru6, and applications of the organic solvent-tolerant glycosidase Fru6 in glycosylation modification of flavonoid substances in a water or non-water phase, and the synthesis of high-quality oligosaccharides. The organic solvent-tolerant glycosidase Fru6 can tolerate a certain concentration of a plurality of hydrophilic organic solvents. According to the invention, an organic solvent-tolerant glycosidase Fru6 coding gene is separated and cloned, and has a nucleotide sequence represented by SEQ ID NO:1, and an amino acid sequence represented by SEQ ID NO:2. The organic solvent-tolerant glycosidase Fru6 / Fru6 mutant has a strong transglycosylation synthesis effect, so the organic solvent-tolerant glycosidase Fru6 / Fru6 mutant has industrial application values in the glycosylation modification of the flavonoid substances in a water or non-water phase, and the synthesis of the oligosaccharides.

Description

technical field [0001] The present invention relates to a kind of organic solvent resistant glycosidase Fru6 and its mutant and application, specifically, relate to a kind of organic solvent resistant glycosidase producing bacteria and the organic solvent resistant glycosidase Fru6 and its mutant that it produces, and its in water The application of catalyzing fructosylation reaction to generate -O-glucoside in phase or non-aqueous phase, has the ability of high-efficiency glycosyl synthesis, and can efficiently modify flavonoids and synthesize oligosaccharides in aqueous phase or non-aqueous phase. It belongs to microorganisms science and enzymology. Background technique [0002] Glycoside compounds are widely used in the fields of food, pharmacy and feed. Oligosaccharides (4-8 carbon sugars, oligosaccharides), as physiologically active substances, have the functions of adjusting the structure of intestinal flora and enhancing immunity; in 2002, the global output of oligos...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/24C12N15/56C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12P19/14C12P19/44C12R1/06
Inventor 何冰芳王瑞吴薛明周晨吴斌
Owner NANJING TECH UNIV
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