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A kind of method for preparing fructo-oligosaccharide and enzyme preparation thereof

A technology of fructo-oligosaccharide 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 reducing manufacturing costs Effect

Active Publication Date: 2019-02-19
森大(天津)生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve and overcome the high cost of enzyme preparation in the above-mentioned fructooligosaccharide production, the production process route is longer, the problems and deficiencies such as many equipments and high cost, the present invention utilizes genetic engineering means to realize the expression of the fructosyltransferase preparation derived from Aspergillus niger, And the protection of fructosyltransferase by hydrophobic combination solvent has successfully realized the industrial production of fructooligosaccharide at high temperature and high concentration sucrose solution

Method used

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  • A kind of method for preparing fructo-oligosaccharide and enzyme preparation thereof
  • A kind of method for preparing fructo-oligosaccharide and enzyme preparation thereof
  • A kind of method for preparing fructo-oligosaccharide and enzyme preparation thereof

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Embodiment 1

[0031] Cloning of embodiment 1 fructosyltransferase coding gene in Bacillus licheniformis

[0032] The total RNA of Aspergillus niger (Aspergillus niger) CBS 513.88 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 a primer to reverse-transcribe the first-strand cDNA, then using the first-strand cDNA as a template, and using primers (sequence SEQ ID NO: 2 and SEQ ID NO: 2 and SEQ ID NO: ID NO:3) Perform PCR amplification on the fructosyltransferase coding gene (sequence SEQ ID NO:1). PCR amplification system and reaction conditions reference HS DNA Polymerase Instruction Sheet (TaKaRa). The PCR product was then cloned into the EcoR I and Sac I sites of the expression vector pHY-WZX (Dandan Niu and Zhengxiang Wang. J Ind Microbiol Biotechnol, 2007, 34:357-362) to obtain the recombinant expression plasmid pHY-fru3.

Embodiment 2

[0033] Example 2 Screening of promoters for secreted expression of fructosyltransferase

[0034] Use NCBI and literature to search for promoters derived from Bacillus (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 SEQ ID NO:4~SEQ ID NO:15), and then through the whole gene synthesis technology (Niu Dandan et al. Journal of Applied and Environmental Biotechnology, 2007,13(4):515-518 ) to obtain the plasmid pUC18-promoter containing these 12 promoters. Construct expression vectors fused with these promoters and fructosyltransferase, and electrotransform into Bacillus licheniformis, and select the recombinant strain with the highest extracellular enzyme activity through the determination of enzyme activity, specifically comprising the following steps:

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

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

Embodiment 3

[0059] Secreted expression of fructosyltransferase in 15L fermenter of embodiment 3

[0060] Further recombinant bacteria CBB3008-P shuttle-09 - fru3 Fermentation experiments were carried out in a 15L fully automatic fermenter (B. Brown, Switzerland).

[0061] The fermentation medium is: yeast extract 3%, peptone 4%, glucose 20%, pH7;

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

[0063] The results are summarized in Table 4, recombinant bacteria CBB3008-P shuttle-09 The level of synthesis and secretion of fructosyltransferase of -fru3 reached 1500U / mL, and the synthesis level of fructosyltransferase of the control strain...

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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 in particular relates to a strain of recombinant genetically engineered bacteria and a method for preparing fructooligosaccharides. Background technique: [0002] Fructooligosaccharides (Fructooligosaccharides), referred to as FOS, also known as oligofructose or saccharotriose oligosaccharides, molecular formula: G-F-Fn, n=1-3 (G is glucose, F is fructose). It is composed of sucrose and 1-3 fructosyls combined with fructosyls in sucrose through β-2-1 glycosidic bonds, such as kestose, kestose, kestose, kestose, kestose and other sugars. mixture. In industry, fructo-oligosaccharides are generally produced from sucrose through the transglycosylation of fructosyltransferase (Fruetosyltransferase, EC 2.4.1.9, FTS). It is low in calories, promotes mineral absorption, does not cause dental caries, lowers blood fat, laxes bowels, is a proliferation factor of bifidobacteria, etc. It is wi...

Claims

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Application Information

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Patent Type & Authority Patents(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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