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Penicillium source chitosanase gene and preparation method thereof

A technology of chitosanase and gene, which is applied in the field of genetic engineering and microorganisms, can solve the problems of less research on cloning and expression, and achieve the effect of effective and reliable method, saving time and high activity

Inactive Publication Date: 2011-11-23
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, only a few fungal strains have been found to produce chitosanase, and there are few studies on its cloning and expression

Method used

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  • Penicillium source chitosanase gene and preparation method thereof
  • Penicillium source chitosanase gene and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The acquisition of embodiment 1 penicillium

[0024] Using chitosan as a single carbon source medium (NaNO 3 0.2%, K 2 HPO 4 0.1%, MgSO 4 0.05%, KCl 0.05%, FeSO 4 0.001%, colloidal chitosan 0.5%, pH 5.0) cultured and screened to obtain the common Penicillium sp) that can express and produce chitosanase.

Embodiment 2

[0025] Cloning of embodiment 2 chitosanase gene

[0026] After the Penicillium thallus DNA of Example 1 was extracted, the chitosanase gene was obtained by inverse PCR (I-PCR). Firstly, the primers DFP (5'-GCGGATCCAAYATGGAYATHGAYTGYGA-3') and DRP (5'-GCGAAGCTTRTCDCCCCADATNCCRTA-3') were designed through the chitosanase highly conserved sequence of the fungus, and the genomic DNA of Penicillium was used as a template for PCR. The program was 94 Pre-denaturation at ℃ for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 1 min, constant temperature at 72 °C for 2 min, 30 cycles, and finally extension at 72 °C for 10 min. The obtained fragments were sequenced, and then nested primers FP1 (5-CCA GAG CAC GTT GGC ATC AA-3), FP2 (5'-GGT CTG CAA CAA CAA GCT CAT C-3') and RP1 were further designed according to the obtained sequences (5-ACC ATA GTC GGA CTT GAC CT-3), RP2 (5'-GAG TCG ATG CCG TCT TGA TC-3'). Then use Pst I, BamHI, EcoRI and HindIII to digest genomic DNA respe...

Embodiment 3

[0027]According to the chitosanase full-length coding sequence (SEQ ID NO.1) obtained by gene annotation in the examples, primers capable of amplifying the complete coding reading frame were designed to construct an expression vector. Penicillium DNA was used as a template to amplify the full-length chitosanase gene. Recombined into the expression vector pET28a under the premise of ensuring the correct reading frame, and then transformed the recombinant vector into Escherichia coli DH5α competent cells (the transformation method was CaCl 2 method or electrotransformation method), and antibiotic marker method was used to screen (in this case, kanamycin resistance) positive recombinant bacteria pET28a-csn / DH5α. Pick a single colony of engineered bacteria pET28a-csn / DH5α, shake and culture in 5ml LB medium containing 30μg / ml kanamycin overnight at 37°C, extract the recombinant plasmid and transform it into Escherichia coli BL21(DE3) competent cells , Screening (in this case, kan...

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Abstract

The invention discloses a method for cloning and expressing a penicillium chitosanase gene. A special culture medium with a single carbon source is used for screening to obtain penicillium for manufacturing chitosanase; in the method provided by the invention, a unique degenerate primer is utilized to amplify a segment of the penicillium chitosanase gene, and then a whole-length gene is obtained through a reversed PCR (Polymerase Chain Reaction) method; and the novel enzyme gene is proved to contain no introns. The gene is cloned to a suitable expression carrier and can realize heterologous expression in colon bacillus; and an enzymological research represents that the obtained chitosanase gene has higher activity. The method provided by the invention is effective and reliable, and a set of effective method for cloning the penicillium chitosanase gene is established, so that a large amount of inducible expression is finally realized and the method provides chitosanase starting materials with low cost for subsequent industrial production.

Description

technical field [0001] The invention belongs to the field of genetic engineering and microorganism technology, and in particular relates to a penicillium-derived chitosanase gene and a cloning and expression method thereof. Background technique [0002] Cellulose, chitin, and chitosan are the most abundant polysaccharides in nature, and they contain huge sugar resources. They are all connected by glucopyranose through β-1, 4 glycosidic bonds. The difference lies in the carbon 2 The functional group attached to it. Among them, chitosan is composed of D-glucosamine residues, which can be obtained by partial or complete deacetylation of chitin. In nature, it is abundant in fungi and insects. The scientific community pays great attention to chitosan and its hydrolyzate, because they have many important and potential biological functions, such as sterilization, anti-cancer, etc., and some functions have been practically used in agriculture, food and pharmaceutical industries. ...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/21C12N1/19C12N5/10C12N15/10C12R1/19C12R1/80
Inventor 朱旭芬吴敏张文武
Owner ZHEJIANG UNIV
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