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
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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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