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Xylose isomerase, and encoding gene and use thereof

A technology of xylose isomerase and encoding, applied in the field of enzyme genetic engineering, can solve the problem of insufficient specific enzyme activity

Inactive Publication Date: 2008-12-17
HENAN TIANGUAN GRP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the previous work, we cloned the xylose isomerase gene xylA of S. celluiosum, which can show activity in Saccharomyces cerevisiae, but the specific enzyme activity is not high enough, so further modification is necessary

Method used

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  • Xylose isomerase, and encoding gene and use thereof
  • Xylose isomerase, and encoding gene and use thereof
  • Xylose isomerase, and encoding gene and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0068] The xylose isomerase gene mxylA provided by the present invention is obtained by cloning the xylose isomerase gene xylA (Genebank EU643621) from Sorangium cellulosum 157-2, and then combined with DNA shuffling by error-prone PCR In vitro molecular directed evolution technology introduces mutation points and obtains them through screening. The specific steps are as follows:

[0069] (1) Total DNA was extracted from Sorangium cellulosum 157-2:

[0070] Collect 0.1 g of cells, add 0.75 mL of GTE buffer (25% sucrose, 50 mmol / L Tris-Cl, 100 mmol / L LEDTA, pH 8.0) and appropriate amount of glass beads, vortex, and mix the cells thoroughly. Transfer the bacteria to a 5mL centrifuge tube, add 0.5mL GTE, mix with a vortex shaker, add 150μL of 10% SDS, 15μL of 20mg / mL proteinase K, mix well, and incubate at 37°C for 1h. Invert and mix at regular intervals during this period. Add 0.25mL 5mol / L NaCl, mix well and let stand for 10min, then add 0.25mL CTAB / NaCl (10%CTAB / 0.7mol / L NaCl)...

Embodiment 2

[0087] Enzyme activity assay of evolution enzyme S1:

[0088] (1) Using the total DNA of Sorangium cellulosum 157-2 as a template, use primer 1 and primer 2 to amplify the wild-type xylose isomerase gene xylA, digest with BglII and SacI, and adopt a directional ligation strategy Directly ligated with the plasmid vector pYPX251 (GenBank AY178046) treated with the same enzyme digestion to obtain the recombinant plasmid pYPX251-SXI.

[0089] (2) Escherichia coli HB101 was transformed with the recombinant plasmid in step (1) to obtain recombinant strain HR101-SXI.

[0090] (3) According to the enzyme activity assay method described in step (11) in Example 1, measure different bacterial strains at different temperatures

[0091] The specific activity of xylose isomerase contained in the source crude enzyme solution is shown in Table 1.

[0092] Table 1 Specific activity of xylose isomerase in different strains at different temperatures

[0093]

[0094] (4) According to the e...

Embodiment 3

[0096] Example 3 Active Expression of Mutant Xylose Isomerase Gene in Saccharomyces cerevisiae

[0097] (1) Design primers according to the upstream-375bp--1bp sequence of the HXT7 gene in the Saccharomyces cerevisiae genome database:

[0098] Primer 3: 5'-GATC CCCGGG CGGGCCCCTGCGTG-3'

[0099] Primer 4: 5'-TTGC GGTACC TTTTTGATTAAAATTAAAAAAAAC-3'

[0100] The HXT7 promoter (HXT7p) fragment was amplified by PCR using the Saccharomyces cerevisiae chromosome as a template, and the PCR product was double-digested with SmaI and KpnI.

[0101] (2) Design primers according to the nucleotide sequence shown in SEQ NO.1:

[0102] Primer 5: 5'-AGCT GGTACC ATGACCGTCGTGATTGGAAAC-3'

[0103] Primer 6: 5'-TTGC AGATCT TTACCGGATCCACCGATTGAC-3'

[0104] Using pYPX251-S1 as a template, mxylA was amplified by PCR, and the PCR product was double-digested with KpnI and BglII.

[0105] (3) Design primers according to the nucleotide sequence shown in SEQ NO.1:

[0106] Primer 5: 5'-AGCT ...

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Abstract

The invention relates to a xylose isomerase and the coded protein and the application thereof, pertaining to the technical field of enzyme genetic engineering. A coded xylose isomerase DNA sequence is obtained from mutation at partial positions of a xylose isomerase gene xylA(Genebank EU643621) in Sorangium cellulosum 157-2. The invention also relates to enzyme coded by the DNA sequence and the application of the coded enzyme in the fields of chemical industry, food and medicine. In addition, compared with the traditional xylose isomerase, the activity of xylose isomerase of the invention in saccharomyces cerevisiae is increased by 2.8 times.

Description

technical field [0001] The invention relates to a xylose isomerase and its encoding gene and application, belonging to the technical field of enzyme gene engineering. Background technique [0002] Xylose isomerase (D-Xylose Isomerase, XI) catalyzes the conversion of the five-carbon sugar D-xylose into D-xylulose in vivo, and catalyzes the isomerization reaction of the six-carbon sugar D-glucose into D-fructose in vitro , also known as glucose isomerase (GlucoseIsomerase, GI), widely exists in bacteria, a small part of fungi. [0003] Lignocellulose (lignocelluiosic) is the most abundant renewable resource on the earth, and it mainly exists in agricultural by-products and woody waste in forestry industry. Its main components are: cellulose, hemicellulose and lignin. Cellulose is an unbranched crystalline polymer composed of glucose residues connected by β-1,4 glycosidic bonds; hemicellulose is a variety of five-carbon sugars including xylose, arabinose, glucose, galactose, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12N9/90C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21
Inventor 鲍晓明沈煜
Owner HENAN TIANGUAN GRP
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