High efficiency transglyco beta galactoside gene

Abstract

The invention discloses a high efficiency turning glycosyl beta-galactosidase gene. The genes are gained by enterobacter cloacae B5 CGMCC No.1401 gene set DNA under PCR amplification. The whole length of gene nucleotide sequence is 3090 basic groups, and coding 1029 amino acid. The recombinase features are that enzyme is quart-subunit albumen; polymer molecular weight are 391kD; mono subunit are 97.8kD; the enzyme to lactose Km value is 0.32mmol / L, Vmax is 210.70umol / (L.min); and it to onitrophenol-beta-D-galactoside Km value is close to 0; its optimum reaction temperature is 35 centigrade degree; the pH value is from 6.5-9.5; it can be stored at least two months at room temperature; oligomerization galactose yield is 55%; receptor substrate specificity of the enzyme turning glycosyl is very wild; it can used to compose multi galactoside compounds. The gained enzyme gene can be used to modify gene to gain new beta-galactoside synthetase, and improve its combined efficiency fundamentally.

Description

field of invention

[0001] The invention relates to a β-galactosidase gene, in particular to a high-efficiency transglycosyl β-galactosidase gene and the application of the high-efficiency transglycosyl β-galactosidase gene. Background of the invention

[0002] Oligosaccharides are one of the elements of glycoproteins and glycolipids on the surface of mammalian cells 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 structural analysis of oligosaccharides and the development of oligosaccharide synthesis technology have attracted great attention. Although oligosaccharides are recognized by scientists as having considerable potential as a therapy, their important role has not been fully realized so far. One of the reasons for its slow development is that it is very difficult to synthesize oligosaccharides in sufficient quant...

Examples

Embodiment 1

[0025] Example 1 Extraction of Enterobacter cloacae B5 Genomic DNA

[0026]Inoculate Enterobacter cloacae B5 CGMCC No.1401 in 5 mL of LB medium, culture on a shaker at 37°C for 16 hours, take 1.5 mL of the culture, centrifuge at 12,000 rpm for 2 minutes, and suspend the obtained pellet in 565 μL of TE buffer solution; add 30 μL 10% (mass volume ratio) sodium dodecyl sulfate (SDS) and 5 μL 20 mg / mL proteinase K, mix well, and incubate at 37°C for 1 hour; add 100 μL 5mol / L NaCl and mix well, then Add 80 μL CTAB / NaCl solution, mix well, and incubate at 65°C for 20 minutes; then ice-bath for 30 minutes; add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1, v / v / v), mix Centrifuge at 12,000 rpm for 5 minutes, transfer the supernatant to a new tube, add an equal volume of phenol / chloroform / isoamyl alcohol, 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 to the tube, mix gently until ...

Embodiment 2

[0030] Example 2 PCR amplification of E.cloacae B5β-galactosidase gene

[0031] Design primers to introduce the BamHI restriction site that can be inserted into the pET-15b plasmid (Novagen) (the pET-15b plasmid vector has an N-terminal 6His tag, which is convenient for the purification of expressed proteins), and the sequence is as follows:

[0032] F-B5: 5'-GATCGGATCCGATGCCCAACACTCTATCG-3'

[0033] R-B5: 5'-GAAGGGATCCTTAAGGGTTCTGCTGCCA-3'

[0034] Using the extracted Enterobacter cloacae genomic DNA as a template, the above primers were used for PCR amplification. 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 LA 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°C for 3 minutes); extensio...

Embodiment 3

[0035] Cloning and DNA sequence determination of embodiment 3 PCR products

[0036] The PCR recovery product of embodiment 2 is connected on the pMD18-T plasmid carrier (TA clone), transforms Escherichia coli Top10 competent cell, spreads ampicillin plate, screens recombinant bacteria with colony PCR method, then sequenced, sequenced by Shanghai Bo Subbiotech Co., Ltd. completed the analysis of the measured sequence, showing that there is an open reading frame consisting of 3090 bases, encoding 1029 amino acids, which is the complete gene sequence of E.cloacae B5β-galactosidase, such as SEQ ID Shown in No.1.

field of invention

[0001] The invention relates to a β-galactosidase gene, in particular to a high-efficiency transglycosyl β-galactosidase gene and the application of the high-efficiency transglycosyl β-galactosidase gene. Background of the invention

[0002] Oligosaccharides are one of the elements of glycoproteins and glycolipids on the surface of mammalian cells 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 structural analysis of oligosaccharides and the development of oligosaccharide synthesis technology have attracted great attention. Although oligosaccharides are recognized by scientists as having considerable potential as a therapy, their important role has not been fully realized so far. One of the reasons for its slow development is that it is very difficult to synthesize oligosaccharides in sufficient quant...

Examples

Embodiment 1

[0025] Example 1 Extraction of Enterobacter cloacae B5 Genomic DNA

[0026]Inoculate Enterobacter cloacae B5 CGMCC No.1401 in 5 mL of LB medium, culture on a shaker at 37°C for 16 hours, take 1.5 mL of the culture, centrifuge at 12,000 rpm for 2 minutes, and suspend the obtained pellet in 565 μL of TE buffer solution; add 30 μL 10% (mass volume ratio) sodium dodecyl sulfate (SDS) and 5 μL 20 mg / mL proteinase K, mix well, and incubate at 37°C for 1 hour; add 100 μL 5mol / L NaCl and mix well, then Add 80 μL CTAB / NaCl solution, mix well, and incubate at 65°C for 20 minutes; then ice-bath for 30 minutes; add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1, v / v / v), mix Centrifuge at 12,000 rpm for 5 minutes, transfer the supernatant to a new tube, add an equal volume of phenol / chloroform / isoamyl alcohol, 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 to the tube, mix gently until ...

Embodiment 2

[0030] Example 2 PCR amplification of E.cloacae B5β-galactosidase gene

[0031] Design primers to introduce the BamHI restriction site that can be inserted into the pET-15b plasmid (Novagen) (the pET-15b plasmid vector has an N-terminal 6His tag, which is convenient for the purification of expressed proteins), and the sequence is as follows:

[0032] F-B5: 5'-GATCGGATCCGATGCCCAACACTCTATCG-3'

[0033] R-B5: 5'-GAAGGGATCCTTAAGGGTTCTGCTGCCA-3'

[0034] Using the extracted Enterobacter cloacae genomic DNA as a template, the above primers were used for PCR amplification. 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 LA 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°C for 3 minutes); extensio...

Embodiment 3

[0035] Cloning and DNA sequence determination of embodiment 3 PCR products

[0036] The PCR recovery product of embodiment 2 is connected on the pMD18-T plasmid carrier (TA clone), transforms Escherichia coli Top10 competent cell, spreads ampicillin plate, screens recombinant bacteria with colony PCR method, then sequenced, sequenced by Shanghai Bo Subbiotech Co., Ltd. completed the analysis of the measured sequence, showing that there is an open reading frame consisting of 3090 bases, encoding 1029 amino acids, which is the complete gene sequence of E.cloacae B5β-galactosidase, such as SEQ ID Shown in No.1.

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