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Gene capable of increasing expression level of aspergillus niger xylanase, recombinant plasmid and host cell thereof

A technique for recombining plasmids and Aspergillus niger, applied in the fields of molecular biology and genetic engineering

Inactive Publication Date: 2012-04-25
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there has been no research report on the overexpression of Aspergillus niger xylanase gene in Pichia pastoris through directional modification

Method used

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  • Gene capable of increasing expression level of aspergillus niger xylanase, recombinant plasmid and host cell thereof
  • Gene capable of increasing expression level of aspergillus niger xylanase, recombinant plasmid and host cell thereof
  • Gene capable of increasing expression level of aspergillus niger xylanase, recombinant plasmid and host cell thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: Cloning and expression of Aspergillus niger xylanase gene

[0027] 1.1 Extraction of Aspergillus niger Genomic DNA (Refined Molecular Biology Experiment Guide (Fourth Edition) Beijing: Science Press, 2005)

[0028] (1) Inoculate the activated Aspergillus niger strain on the liquid medium, culture at 28-30° C., 150 rpm, for 2 days.

[0029] (2) Collect the mycelium by filtration, wash with sterile water 3-4 times until the culture medium is completely washed, drain it, wrap it in tin foil, and freeze it in liquid nitrogen.

[0030] (3) Put 2 g of mycelium into a pre-cooled mortar, add an appropriate amount of liquid nitrogen and grind until the Aspergillus niger mycelium is ground into powder.

[0031] (4) Put the mycelia powder in several EP tubes, add CTAB extract solution preheated to 65° C. (adding mercaptoethanol accounting for 2%wt of the extract solution). Shake to make it fully mixed, incubate at 65°C for 45-60min, and mix from time to time.

[0...

Embodiment 2

[0058] Embodiment 2: The xynB sequence of xylanase gene xynB of Aspergillus niger carries out directional transformation

[0059] By optimizing the codon sequence of the gene, it can adapt to the abundance of modified nucleotides at the swing position of the antisense codon of the tRNA isoacceptor and the host, and is also conducive to the formation of the secondary structure of translation. Genes that are highly expressed in yeast tend to use codons that are preferred by yeast itself, and studies have also shown that 25 of the 61 codons are preferred by yeast. If the exogenous gene contains more rare codons expressed by Pichia pastoris, a bottleneck effect will be generated in the translation process and the expression will be affected. There is a significant difference in codon usage bias between Aspergillus niger and Pichia pastoris, and most of the codons in the xynB gene xynB of Aspergillus niger are less frequently used in Pichia pastoris. According to the DNA sequence ...

Embodiment 3

[0064] Example 3: Construction of recombinant plasmid pPICZα-xynBT and preparation of recombinant xylanase

[0065] 3.1 Construction of recombinant plasmid pPICZα-xynBT and restriction restriction linearization of expression vector

[0066] The obtained transformed genes xynBT and pPICZα-A were digested step by step with endonucleases EcoR I and Xba I, respectively, recovered by tapping the rubber, concentrated and ligated overnight at 16°C. Competent cells of Top10F' were used as host bacteria, spread on LLB plate (containing 25 μg / mL Zeocin), inverted the plate overnight, picked a single colony into 5 mL liquid LLB (containing 25 μg / mL Zeocin), and cultured at 37°C, 200rpm for 8 hours. The recombinant plasmid was extracted for sequencing verification, and the recombinant plasmid pPICZα-xynBT containing the optimized gene xynBT was obtained (Figure-1). Correctly identified positive transformants were cultured in large quantities, and the recombinant plasmid pPICZα-xynBT was ...

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Abstract

A gene capable of increasing an expression level of aspergillus niger xylanase, a recombinant plasmid and a host cell thereof relate to optimal codon optimization, GC content adjusting and optimization of cis-acting element and sequential-repetitive sequence on a xylanase gene from aspergillus niger. According to design of a site-directed mutagenesis primer, artificial reconstruction on an original aspergillus niger xylanase gene is carried out to obtain an xylanase gene xynBT over-expressed in pichia pastoris. The reconstructed aspergillus niger xylanase gene xynBT has an expression level in pichia pastoris increased by 2.6 times, compared with that of an original gene xynB; during high density fermentation in a 3 L fermentation cylinder, the xylanase gene xynBT has an expression level in pichia pastoris reaching 20424.2 U / mL. The invention can be applied to molecule reconstruction on the xylanase gene and production of a recombinant xylanase, and can substantially increase the expression level of the xylanase.

Description

technical field [0001] The invention belongs to the fields of molecular biology, genetic engineering and the like, and in particular relates to an improved aspergillus niger xylanase gene, its recombinant plasmid and Pichia cells after the recombinant plasmid. Background technique [0002] Xylan is one of the main components of the cell walls of broad-leaved plants and grasses, and widely exists in fruits, grains and straws. The full name of xylanase is β-1,4-endoxylanase or 1,4-β-xylan xylohydrolase (EC 3.2.1.8), and its function is to hydrolyze the interior of xylan backbone chain The 1,4 glycosidic bonds of Xylooligosaccharides or oligosaccharides with side branches (Curr Opin Biotechnol, 1996, 7(3):337-342). [0003] With the development of biotechnology, people gradually realize that xylanase has very broad application prospects in paper industry, biochemical industry, and food and feed industries. In the papermaking process, xylan deposited on the fiber is the main o...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/63C12N15/81C12N15/66C12N1/19C12R1/84
Inventor 赵林果李飞
Owner NANJING FORESTRY UNIV
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