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Method for efficiently preparing fructo-oligosaccharide and enzymic preparation thereof

A technology of fructooligosaccharide and fructosyltransferase, which is applied in the field of applied enzyme engineering, can solve the problems of long production process route, high cost of enzyme preparation, and many equipments, and achieve the goal of reducing equipment investment, improving protection, and efficient secretion and expression Effect

Active Publication Date: 2016-03-30
森大(天津)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve and overcome the above-mentioned problems and deficiencies such as high cost of enzyme preparations in the production of fructooligosaccharides, long production process routes, many equipments, and high costs, the present invention utilizes genetic engineering means to realize the high-efficiency expression of fructosyltransferase preparations derived from Aspergillus niger , and using the protection of fructosyltransferase by hydrophobic combined solvent, the industrial production of fructooligosaccharides at high temperature and high concentration of sucrose was successfully realized

Method used

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  • Method for efficiently preparing fructo-oligosaccharide and enzymic preparation thereof
  • Method for efficiently preparing fructo-oligosaccharide and enzymic preparation thereof
  • Method for efficiently preparing fructo-oligosaccharide and enzymic preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Cloning of fructosyltransferase encoding gene in Bacillus licheniformis

[0032] The total RNA of Aspergillus niger CBS513.88 was extracted with TRNzol total RNA extraction reagent. Using total RNA as a template, referring to the RT-PCR kit instructions, using oligo (dT) as primers to reverse transcribe first-strand cDNA, and then using the first-strand cDNA as a template, using primers (sequences of SEQ ID NO: 2 and SEQ ID NO: 3). ) The fructosyl transferase encoding gene (sequence SEQ ID NO: 1) was subjected to PCR amplification. PCR amplification system and reaction conditions reference HSDNAPolymerase instructions (TaKaRa). Then the PCR product was cloned into the EcoRI and SacI sites of the expression vector pHY-WZX (Dandan Niu and Zhengxiang Wang.JInd Microbiol Biotechnol, 2007, 34: 357-362) to obtain the recombinant expression plasmid pHY-fru3.

Embodiment 2

[0033] Example 2 Screening of promoters for efficient secretion and expression of fructosyl transferase

[0034] Use NCBI and literature to search for Bacillus-derived promoters (including P 43 , P shuttle-09 , P grac , P xylA , P sacB , P mtlA , P glv , P aprE , P aprN , P manP , P HapII , P ituD ) Gene sequence (sequence SEQIDNO: 4 ~ SEQIDNO: 15), and then through total gene synthesis technology (Niu Dandan et al. Journal of Applied and Environmental Biotechnology, 2007, 13(4): 515-518) Plasmid pUC18-promoter with 12 kinds of promoters. Constructing expression vectors in which these promoters are fused with fructosyltransferase, and electrotransformed into Bacillus licheniformis, and selecting recombinant strains with the highest extracellular enzyme activity by measuring the enzyme activity, specifically including the following steps:

[0035] 1. Construction of plasmid pHY-Promoter-SP-fru3

[0036] Using the plasmid pUC18-promoter containing the above 12 different prom...

Embodiment 315

[0059] Example Efficient secretory expression of fructosyltransferase in 315L fermenter

[0060] The recombinant strain CBB3008-P shuttle-09 -fru3 was tested in a 15L fully automatic fermentation tank (B.Brown, Switzerland).

[0061] The fermentation medium is: yeast extract 3%, peptone 4%, glucose 20%, pH 7;

[0062] The working fermentation volume is 10L; the fermentation temperature is 42±1℃; the dissolved oxygen is maintained above 20% during the fermentation process; after 12 hours of fermentation, 50% glucose solution is added to maintain the glucose concentration at 8g / L; sulfuric acid or ammonia is used during the fermentation process Control the pH to 7; the fermentation time is 150h;

[0063] The results are summarized in Table 4. Recombinant strain CBB3008-P shuttle-09 -The level of fructosyltransferase synthesis and secretion of fru3 reached 1500U / mL, which was greatly improved compared with the level of fructosyltransferase of the control strain by 329%.

[0064] Table 415...

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Abstract

The invention belongs to the field of enzyme application engineering and particularly relates to a recombinant genetic engineering bacterium and a method for preparing fructo-oligosaccharide through the bacterium. The recombinant bacterium is obtained by efficiently expressing fructosyl transferase encoding gene fru3 genes with the aspergillus niger source in bacillus host cells. The recombinant bacterium further comprises a promoter Pshuttle-09 capable of allowing the fructosyl transferase encoding gene fru3 genes to be efficiently expressed in bacillus licheniformis. In addition, through protectiveness of a hydrophobic combined solvent on fructosyl transferase, by means of the mode of adding the hydrophobic solvent in the production process of fructo-oligosaccharide, the heat resistance of enzymes is improved, the concentration of a substrate saccharose solution is increased accordingly, and no later decoloring, refining and concentration technologies or other technologies are needed for the prepared fructo-oligosaccharide. Through the technology, the production process route of fructo-oligosaccharide is greatly shortened, equipment investment is reduced, and production cost is reduced.

Description

Technical field: [0001] The invention belongs to the field of applied enzyme engineering, and specifically relates to a recombinant genetic engineering strain and a method for preparing oligofructose. Background technique: [0002] Fructooligosaccharides (Fructooligosaccharides), referred to as FOS, is also known as oligofructose or fructotriose oligosaccharides, the molecular formula is: G-F-Fn, n=1-3 (G is glucose, F is fructose). It is composed of sucrose and 1-3 fructose groups combined with fructose groups in sucrose through β-2-1 glycosidic bonds, sucrose triose, sucrose tetraose, sucrose pentaose, sucrose hexaose and its mixture. In industry, fructosyltransferase (EC2.4.1.9, FTS) transglycosylation of sucrose is generally used to produce fructo-oligosaccharides. Fructo-oligosaccharides are a health food with many beneficial functions to the human body, such as It is low in calories, promotes the absorption of minerals, does not cause dental caries, lowers blood fat, mois...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/18C12P19/12C12P19/04
CPCC12P19/04C12P19/12C12P19/18
Inventor 王正祥路福平
Owner 森大(天津)生物科技有限公司
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