Expression of high temperature resistant xylanase gene in Kluyveromyces lactis

A technology of Kluyveromyces and xylanase, which is applied in the field of secretion and expression in Kluyveromyces lactis, can solve the problems of increased cost of separation and purification, achieve the effect of reducing the cost of separation and purification and improving expression efficiency

Inactive Publication Date: 2009-02-25
CHINA AGRI UNIV
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there have been reports of expressing mxynB, a xylanase gene derived from Thermotoga maritima MSB8, in Escherichia coli. Although the expression level of the target gene is high, the product is mainly concentrated in the cell, which increases the cost of subsequent separation and purification.

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  • Expression of high temperature resistant xylanase gene in Kluyveromyces lactis
  • Expression of high temperature resistant xylanase gene in Kluyveromyces lactis
  • Expression of high temperature resistant xylanase gene in Kluyveromyces lactis

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Embodiment 1

[0024] Example 1, Kluyveromyces lactis expression plasmid pKLAC1-mxynB (64) build

[0025] 1.1 Primer design

[0026] mxynB according to GenBank (64) Based on the sequence of the gene (GenBank accession number is AY340789) and the characteristics of the multiple cloning site of Kluyveromyces lactis expression vector pKLAC1, the following pair of primers were designed and synthesized:

[0027] PX1: FW: 5'-CCC ctcgag AAAAGAATGAAAATATTACCTTC-3'

[0028] PX2: REV: 5'-TTT ggtacc TCATTTTCTTTCTTCTATCTTTTTCT-3'

[0029] The two ends of PX1 and PX2 are designed with XhoI and KpnI restriction sites respectively (see the lowercase and underlined part in the above sequence)

[0030] 1.2 Thermostable xylanase gene mxynB (64) PCR amplification

[0031] Using PX1 and PX2 primers, extract the genomic DNA of Thermotoga maritima, MSB8 (Thermotoga maritima, MSB8) as a template, (Thermotoga maritima, MSB8 genome registration number in GenBank: NC 000853, MSB8 bacterial strain can be fou...

Embodiment 2

[0041] Embodiment 2 thermostable xylanase gene (mxynB (64) ) secretory expression in Kluyveromyces lactis GG799

[0042] 2.1 Preparation of electroporation competent cells of Kluyveromyces lactis GG799 [purchased from New England BioLabs Inc. (NEB)] and electroporation transformation

[0043] (1) Pick a fresh single colony in 5ml YPD liquid medium (yeast powder 1%, peptone 2%, glucose 2%), and culture overnight at 30°C and 250rpm.

[0044] (2) Inoculate 100ml of freshly prepared YPD liquid at room temperature with an inoculation volume of 0.1% by volume

[0045] culture medium at 30°C, 250rpm to OD 600 0.4 to 0.5.

[0046] (3) Centrifuge at 3000 rpm for 5 minutes at 4°C to collect the cells;

[0047] (4) Wash the cells once with 250ml ice-cold sterile water;

[0048] (5) Wash the cells once with 50 ml of ice-cold sterile electroporation buffer EB (g / L): 92.4 sucrose, 2.03 magnesium chloride, 1 L of 10 mmol / L pH7.5 MES buffer;

[0049] (6) Resuspend the cells in 50ml ice-...

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Abstract

The invention provides a high-temperature-resistant xylanase secrete expression vector and a method for high-efficiency expression of the high-temperature-resistant xylanase by a lactic acid Kluyveromyces lactis. By adopting the method, most of the xylanase expressed is secreted outside cells, which lowers the cost of separation and purification and greatly improves the expression rate; wherein, under the condition of shaking culture, the amount of the xylanase secreted outside the cells can be up to 70mg / L when galactose is used for induction culture; and when RBB-xylan is taken as a substrate, the activity of the xylanase is up to 6,100U / L.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the secretion and expression of a thermostable xylanase gene from eubacteria in Kluyveromyces lactis. Background technique [0002] Xylanase is mainly produced by saprophytic fungi and bacteria, it can degrade xylan components in plant cell walls, and has been proved to have important application value in many fields of agriculture and industry. Xylan is heterogeneous, so the complete degradation of xylan requires the joint action of various enzymes, among which β-1,4-D-xylanase is one of the key enzymes, which can break the D on the main chain. - β-1,4 glycosidic bonds between xylose residues, thereby degrading the polysaccharide backbone into short-chain xylooligosaccharides. Therefore, β-1,4-D-xylanase has a very wide application in food, feed and paper industry. Among them, the thermostable xylanase XynB belongs to a kind of β-1,4-D-xylanase, and the coding gene of thermostable...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N1/19C12N9/24
Inventor 李颖印铁关国华王珍芳李季伦
Owner CHINA AGRI UNIV
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