Method for producing oligo-galactose by cyclic utilization of recombinant Saccharomyces cerevisiae

A technology for recombining Saccharomyces cerevisiae and galactooligosaccharides, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, botany equipment and methods, etc., can solve the problems of enzyme loss, lower use efficiency, etc., and achieve low cost and solution Inhibition, the effect of broadening the prospects for industrialization

Active Publication Date: 2009-07-08
SHANDONG UNIV
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  • Description
  • Claims
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Problems solved by technology

However, there are problems such as the inhibition of by-products (glucose) and the continuous loss of enzymes during the immobilized enzyme reaction process, which reduces its use efficiency.

Method used

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  • Method for producing oligo-galactose by cyclic utilization of recombinant Saccharomyces cerevisiae
  • Method for producing oligo-galactose by cyclic utilization of recombinant Saccharomyces cerevisiae
  • Method for producing oligo-galactose by cyclic utilization of recombinant Saccharomyces cerevisiae

Examples

Experimental program
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Embodiment 1

[0023] Example 1: Construction of Saccharomyces cerevisiae surface display vector for β-galactosidase and its expression on yeast surface

[0024] Primers F and R were designed according to Penicillium expansica CGMCC No.2352β-galactosidase gene sequence (GenBank Accession No.EU543998), respectively introduced into the insertable plasmid pYD1 (purchased from Invitrogen, the plasmid vector has α-lectin receptor Partial sequence of the gene, the EcoR I and Not I restriction endonuclease sites (shown underlined), the recombinant protein can be anchored on the yeast cell surface), the sequence is as follows:

[0025] F: 5'-CAT GAATTC GGAGTTGCTTCAAAAATATGTGACTTGGG-3'

[0026] R: 5'-GCAT GCGGCCGC GTACGCCCCCTTTCGAGAC-3'

[0027] Using the extracted total RNA of Penicillium expansica as a template, the first strand of cDNA was synthesized by reverse transcription using the above-mentioned R primers. Using the first strand of cDNA as a template, carry out PCR amplification with F...

Embodiment 2

[0032] Example 2: Recycling of cell surface recombinant yeast to ferment lactose to produce galacto-oligosaccharides

[0033] The recombinant yeast preserved in Example 1 was inoculated in YNB-CAA-glucose medium, and cultured at 30°C until OD 600After reaching 2.0-5.0, centrifuge at 3000 rpm for 5 minutes to collect the cells, discard the supernatant, resuspend the cells with YNB-CAA-galactose culture medium, and adjust the OD 600 0.5-1.0, galactose induction at 20°C for 40 hours, then centrifuged at 3000 rpm for 5 minutes, collected recombinant Saccharomyces cerevisiae EBY-100 displaying β-galactosidase on the surface, resuspended in YNB-CAA-lactose culture medium, Tune OD 600 8.0 to 10.0, cultured at 25°C for 4 to 6 days, centrifuged at 3000 rpm for 5 minutes, and the supernatant obtained was analyzed by high performance liquid chromatography for the content of galactooligosaccharides, and the obtained yeast cells were resuspended in new YNB-CAA-lactose The culture medium ...

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Abstract

The present invention relates to a method of using recombinant Saccharomyces cerevisiae to produce galactooligosaccharides, specifically to a recombinant Saccharomyces cerevisiae with surface display of beta-galactosidase and method for making same and a method of recycling the recombinant Saccharomyces cerevisiae fermentation lactose to produce galactooligosaccharides. The method includes: first of all, building a beta-galactosidase yeast surface display vector, displaying the beta-galactosidase on cell surface of the Saccharomyces cerevisiae, and then recycling the recombinant yeast fermentation lactose to produce galactooligosaccharides. The inventive recombinant Saccharomyces cerevisiae of the surface display beta-galactosidase can be recycled to produce galactooligosaccharides, and has high yields, low production cost, simple process and broad industrialization application prospects.

Description

technical field [0001] The invention relates to a method for producing galacto-oligosaccharides by using recombinant Saccharomyces cerevisiae, in particular to a recombinant Saccharomyces cerevisiae surface-displayed β-galactosidase and its preparation method, and recycling of the recombinant Saccharomyces cerevisiae to ferment lactose to produce galacto-oligosaccharides Lactose method. technical background [0002] Galacto-oligosaccharides (Galacto-oligosaccharides, GOS) is a kind of non-digestible oligosaccharides with natural properties. It is the main component in human breast milk. It can promote the proliferation of bifidobacteria; reduce the incidence of dental caries; Absorption of minerals such as zinc; improving lipid metabolism, lowering blood fat and cholesterol and other functions. This type of oligosaccharide can be synthesized by β-galactosidase produced by microorganisms using lactose as a substrate to transglycosylate. There are two preparation methods, one...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/56C12P19/00C12R1/865
Inventor 肖敏李玉梅卢丽丽
Owner SHANDONG UNIV
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