A kind of aspergillus oryzae keratinase gene and its expression vector and application

A keratinase and keratin technology, applied in the field of genetic engineering, can solve the problems of difficult large-scale application, low enzyme activity, etc., and achieve the effect of rich sources

Active Publication Date: 2022-04-15
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are few keratinases reported so far, and the enzyme activity is low, so it is difficult to apply on a large scale

Method used

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  • A kind of aspergillus oryzae keratinase gene and its expression vector and application
  • A kind of aspergillus oryzae keratinase gene and its expression vector and application
  • A kind of aspergillus oryzae keratinase gene and its expression vector and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment one: Obtain the bacterial strain that can express keratinase

[0021] Aspergillus oryzae strain CGMCC 3.13905 was selected and purchased from the China General Microorganisms Culture Preservation and Management Center. After culturing in PDA medium at 28°C for 3 days, the mycelium was collected and total mRNA was extracted. cDNA was obtained by reverse transcription using mRNA as a template. Then, using the cDNA as a template, the coding sequence of the AoKerase11 gene was obtained by cloning by PCR technology, as shown in SEQ ID No:1. The coding sequence DNA fragment was double-digested with restriction endonucleases EcoR I and Not I to obtain the coding sequence DNA fragment after digestion. The constitutive expression plasmid pGAPZA and the methanol-inducible plasmid pPIC9K were digested simultaneously with restriction endonucleases EcoR I and Not I to obtain two plasmid fragments. Among them, plasmid pGAPZA and plasmid pPIC9K were purchased from Thermo...

Embodiment 2

[0023] Example 2: Demonstration of enzymolysis performance of keratinase

[0024] 1. Inoculate the expression strain of Pichia pastoris carrying pGAPZA-AoKerase11 obtained in Example 1 into BMMY medium, culture at 30° C. for 1 day, and centrifuge to obtain bacterial cells. The promoter of plasmid pGAPZA is GAP promoter, and carbon sources such as glycerol can induce the expression of downstream genes. Therefore, the bacterial cells obtained by centrifugation were reinoculated into fresh BMMY medium, 1% glycerol (v / v) was added to the medium, cultured for 5-7 days, and then the bacterial cells were collected by centrifugation.

[0025] Ultrasonic disrupted the bacteria, centrifuged to remove the precipitate, took 1.0 mL of the crushed solution (pH=6.5), added 10 mg of feather meal as a substrate, and reacted for 3 minutes at 30° C. and 150 rpm. The results showed that some feather powder particles disappeared after being hydrolyzed, and the absorbance value at 280nm measured b...

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Abstract

The invention belongs to the technical field of genetic engineering, and in particular relates to an aspergillus oryzae keratinase gene and its expression vector and application. The specific technical scheme is: a keratinase expression gene, the DNA sequence of which is shown in SEQ ID No:1. The gene expresses a keratinase, the amino acid sequence of which is shown in SEQ ID No:2. The invention provides a new gene resource capable of expressing keratinase, combines the gene with a plasmid, and transfers the gene into a bacterial strain to produce keratinase, thereby enriching the source of keratinase.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an aspergillus oryzae keratinase gene and its expression vector and application. Background technique [0002] my country's livestock and poultry resources are extremely rich, with an annual output of about 2 million tons of feathers, most of which have not been rationally utilized. If the feathers are directly discharged into the environment, it will not only be a huge waste, but also cause pollution. [0003] The feather meal formed by crushing the feather is rich in nutrition, and the crude protein content in the feather meal reaches more than 80%. But at the same time, feather meal is rich in keratin, which is difficult to be directly digested and utilized, thereby reducing the feeding value of feather meal. Keratinase (Keratinase) is a protease that can degrade keratin. It can effectively hydrolyze raw materials rich in keratin, such as animal hair ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/57C12N9/62C12N15/81C12P21/06
CPCC12N9/62C12N15/815C12P21/06
Inventor 李牧杜芸
Owner HUAZHONG AGRI UNIV
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