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Keratinase, and coding gene and application thereof

A technology of keratinase and gene, applied in the field of genetic engineering, to achieve the effect of reducing production cost and great application potential

Active Publication Date: 2013-05-01
GUANGDONG VTR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are no relevant research reports in China, some foreign research results show that under the condition of no sulfide, the hair removal effect of keratinase is very effective

Method used

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  • Keratinase, and coding gene and application thereof
  • Keratinase, and coding gene and application thereof
  • Keratinase, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1, cloning of Aspergillus niger (Aspergillus niger) keratinase VKER gene

[0074] Cultivate Aspergillus niger, its preservation number is: CGMCCNo.4289. Use the RNA extraction kit to extract total RNA from Aspergillus niger, and follow the reverse transcriptase SuperScript TM III Reverse Transcriptase Instructions Synthesis of first-strand cDNA. Using cDNA as a template, design keratinase primers (KER5EcoRI, KER3NotI) for PCR amplification. The PCR product is double-digested by EcoRI and NotI, and then connected to the Pichia pastoris expression vector pPICzaA that has undergone the same digestion, and the ligated product is transformed Escherichia coli Topl0 competent cells were screened by the antibiotic Zeocin, and positive clones were obtained. Plasmids of positive clones were extracted. The samples were sent to Shanghai Yingjun Biological Co., Ltd. for sequencing. The sequencing results showed that the obtained cloned DNA insert contained the complete...

Embodiment 2

[0080] Embodiment 2, the construction of the Pichia pastoris engineering bacterium comprising keratinase gene VKER

[0081] The keratinase gene VKER obtained by PCR amplification was double-digested with EcoRI and NotI, and reconnected to the vector pPICzαA after the same double-digestion to obtain the recombinant expression vector VKER--pPICzαA. Then SacI was used for linearization, and the linearized recombinant vector was electroporated to transform Pichia pastoris X33, and after electrotransformation, it was spread on a YPD (Zeo+) plate for screening to obtain a recombinant strain of Pichia pastoris.

Embodiment 3

[0082] Embodiment 3, high expression of keratinase recombinant strain

[0083] The keratinase recombinant strain VKER-pPICZaA-X33-18 obtained by screening was subjected to high-density fermentation culture.

[0084] Prepare 20L of basic salt medium, sterilize it in a 50L automatic control fermenter, and cool it to room temperature for later use. Use ammonia water and phosphoric acid to adjust the pH value of the fermentation broth to 4.6, control the dissolved oxygen to be greater than 20% by adjusting the rotation speed and air flow rate, and the fermentation temperature is 30°C. The entire fermentation process is divided into three stages: the first stage is the cell culture stage, the recombinant bacteria VKER-pPICZaA-X33-18 is inoculated into the fermenter according to the inoculation amount of 10%, and sterilized 4L of 50% glucose is added. Cultivate for 24-30 hours, marked by the completion of glucose; the second stage is the starvation stage, when the glucose is replen...

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Abstract

The invention relates to the field of gene engineering, and in particular to a keratinase and a coding gene and application thereof. The keratinase has an amino acid sequence shown as a SEQ ID NO.1. Through efficient expression, the recombinant keratinase provided by the invention can reach a fermentation level higher than 20000U / mL. The keratinase provided by the invention can reach high fermentation level through expression compared with the one produced by an existing production technology, and can greatly reduce the production cost in industrial production, so that it shows greater potential in application to industries of feed and unhairing tanning of leather.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a keratinase, its coding gene and application. Background technique [0002] Hundreds of thousands of tons of waste such as feathers and hair are produced in my country every year. The root cause of this phenomenon is that feathers and hair are mainly composed of keratin, which has a very stable structure and is resistant to pepsin and trypsin. Strong resistance, difficult to be degraded, these wastes not only cause waste of resources, but also cause serious environmental pollution. [0003] At present, the commonly used feather treatment method is to use high temperature and high pressure process conditions to soften it, and then process it into animal feed additives, but this process consumes a lot of energy, and essential amino acids such as methionine, lysine and tryptophan are destroyed Poor sex, nutritional instability. The use of keratinase can hydrolyze feather meal th...

Claims

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Application Information

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IPC IPC(8): C12N9/62C12N15/57C12N15/63C12N1/19C12N15/81C12R1/685C12R1/84
Inventor 罗长财李阳源钟开新梁雪霞
Owner GUANGDONG VTR BIO TECH
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