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Transglycosyl alpha-galactoglucosidezyme gene

A technology of galactosidase and transglycosylation, which can be used in genetic engineering, plant gene improvement, enzymes, etc., and can solve the problem of lack of Bifidobacterium breve transglycosylation α-galactosidase gene.

Inactive Publication Date: 2006-05-31
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] After searching, there is currently no report on the transglycosyl α-galactosidase gene of Bifidobacterium breve in the world

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1 Extraction of Bifidobacterium breve (Bifidobacterium breve) ATCC 15700 Chromosomal DNA

[0025] Inoculate Bifidobacterium breve (Bifidobacterium breve) ATCC 15700 in 5mL MRS medium, culture anaerobically at 37°C for 24 hours, take 1.5ml of the culture, centrifuge at 12,000 rpm for 2 minutes, and suspend the obtained pellet in 565μL of TE buffer Add lysozyme to a final concentration of 10 mg / ml, incubate at 37°C for 30 minutes; add 30 μL of 10% (mass volume ratio) sodium dodecyl sulfate (SDS) and 5 μL of 20 mg / mL proteinase K, mix well, and Incubate at 37°C for 1 hour; add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1, v / v / v), mix well; centrifuge at 12,000 rpm for 5 minutes, and transfer the supernatant to a fresh Add an equal volume of phenol / chloroform / isoamyl alcohol to the tube, and mix well; centrifuge at 12,000 rpm for 5 minutes, transfer the supernatant to a new tube, add 2 times the volume of absolute ethanol, and mix gently until the D...

Embodiment 2B

[0028] Example 2B.breve ATCC 15700α-galactosidase gene highly conserved DNA fragment cloning

[0029] The DNA sequences of the α-galactosidase genes of different strains were retrieved from GenBank, the sequence homology was compared with the online software Clustal W, and a pair of degenerate PCR primers were designed using the primer design software Primer5.0:

[0030] Wtz-F: 5'-(TC)TI AA(TC)A(AC)(N)TGG GA(AG)G-3'

[0031] Wtz-R: 5'-(GA)TC CCA(CT)TT(N)A(CT)(GA)TA(GA)TC-3'

[0032] Using the extracted B. breve ATCC 15700 chromosomal DNA as a template, PCR was carried out with the above primers. The total volume of the PCR amplification system is 50 μL, containing 35 μL of ultrapure water, 5 μL of 10×PCR buffer, dNTP 2.4 mmol / L, primers 1 μmol / L, MgCl 2 1.5mmol / L, template DNA 100ng, TaKaRa Taq 2.5U. PCR amplification conditions: pre-denaturation at 95°C for 5 minutes; reaction for 28 cycles (denaturation at 95°C for 1 minute, annealing at 52°C for 1 minute, extension at 72...

Embodiment 3B

[0043] Example 3B.breve ATCC 15700α-galactosidase whole gene cloning

[0044] (1) Construction of partial gene library of B.breve ATCC 15700

[0045] After B.breve ATCC 15700 chromosomal DNA is extracted, six sets of complete enzyme digestion are carried out at the same time. The six sets of enzymes are BamH I-HindIII, BamH I-Nde I, BamH I-Xba I, HindIII-Nde I, HindIII-Xba I, BamH I (TaKaRa). After electrophoresis, the membrane was transferred, and the cloned 457bp DNA fragment was used as a probe for Southern hybridization. The results showed that the chromosomal DNA of B.breve ATCC 15700 was digested with BamH I and Nde I, and the fragment that could hybridize with the probe was about 6kb, which could be used to construct a partial gene library. It was estimated by experiments that about 81.3% of the recombinant bacteria in some gene libraries had foreign fragments, and the library was successfully constructed.

[0046] (2) Screening, verification and sequence determinati...

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Abstract

The invention presents a transfer glycation alpha-D-galactosides enzyme gene, this gene is gained with the PCR amplified of Bifidobacteriumbreve gene group DNA to get the high conservative DNA fragments and crossbreeds with Southern. The full length of its nucleotide sequence is 2226 base, and codes 741 amino acids. The attribute of the recombination enzyme ,which is expressed by this gene, is that the enzyme is double-subunit albumen, the molecular weight of polymer is about 160kD, the molecular weight of sigle-subunit is about 80kD, the Km of this enzyme to nitrophenol alpha-D-galactosides is 0.17mmol / L, Vmax is 4.43 mol / (Líñmin); the best reaction temperature is 37 deg.C, and the best PH is 5.5-6.0, the enzyme has transfer glycation activity in the reaction system of hydrolysis melibinose, or raffinose, this reaction can create transfer glycation production; the different of the enzymeí»s glycosides substrate can be used to compound many glycoconjugate of a-galactosides oligosaccharide and alpha-D-galactosides. The gains of the enzyme can be used to get alpha-galactosides compound enyzme by gene alteration, and improve the compound efficiency of alpha-galactosides oligosaccharide with glycoconjugate radically.

Description

field of invention [0001] The invention relates to an α-galactosidase gene, in particular to a transglycosyl α-galactosidase gene and the application of the transglycosyl α-galactosidase gene. Background of the invention [0002] Oligosaccharides are one of the elements of mammalian cell surface glycoproteins and glycolipids and physiologically active substances derived from microorganisms, and have powerful functions such as biological information transmission. Therefore, the use of oligosaccharides in drug modification, the analysis of oligosaccharide structure and the development of oligosaccharide synthesis technology have attracted great attention. Although oligosaccharides as a therapy have been recognized by scientists as having considerable potential, their important role has not been fully realized so far. One of the reasons for its slow development is the difficulty of synthesizing oligosaccharides in sufficient quantities for clinical use. Multiple reactive hydr...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/38C12N15/70C12P19/14
Inventor 肖敏赵晗王勤鹏王鹏
Owner SHANDONG UNIV
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