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Pichia pastoris strain, keratinase produced by fermentation of pichia pastoris strain and application of keratinase

A technology of Pichia pastoris and keratinase, which is applied in the application field of keratinase and keratinase, can solve the problems of animal absorption and utilization rate, low degradation rate and gene mutation that affect the nutritional value of feed, so as to facilitate animal digestion and absorption and preparation Simple method and mild degradation conditions

Inactive Publication Date: 2020-01-03
HUAZHONG AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of the prior art are: gene mutations are prone to occur in the process of using genetic engineering technology to construct protease-producing engineered bacteria, and genetically engineered bacteria may not be able to correctly translate foreign protease genes, etc.
Due to the above existing problems, the existing genetically engineered bacteria expressing keratinase has a low degradation rate for animal feathers, resulting in low utilization efficiency of feather meal in feed, affecting the nutritional value of feed and the absorption and utilization rate of animals.

Method used

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  • Pichia pastoris strain, keratinase produced by fermentation of pichia pastoris strain and application of keratinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Breeding method of Pichia pastoris.

[0025] A: The target gene Fpk encoding keratinase (as shown in SEQ ID NO: 1) was cloned from Aspergillus fumigatus; the expressed protein is shown in SEQ ID NO: 2.

[0026] B: Design primers according to the target gene, and perform PCR (including 10 times buffer and Taq enzyme, dNTP and other In-Fusion kits) to obtain the target gene sequence Fpk (as shown in SEQ ID NO: 1);

[0027] Primer F: 5'-ctcctgccaagctgaagc-3',

[0028] Primer R: 5'-gatcatggaacggattc-3',

[0029] Amplification program: 95°C for 2min; 95°C for 30s, 56°C for 30s, 72°C for 1min, 45 cycles; 72°C for 5min; store at 4°C.

[0030] C: Insert the obtained target genes into the expression vector pPIC9k respectively, and then transfer them into E. coli DH5α competent cells, use the LB plate containing kanamycin to screen the positive clones, verify the colony PCR and enzyme digestion, the fragment size is correct and then Sequencing identification was carried out to...

Embodiment 2

[0034] Induced expression and detection of Pichia pastoris AN-PK-9K-GS115

[0035] (1) Transfer the obtained Pichia pastoris AN-PK-9K-GS115 to the YPD plate, then select a single colony and transfer it to the fermentation medium, and culture it for 20-24 hours at 30°C and 180rpm as a seed solution.

[0036] Fermentation medium: 85% phosphoric acid 26.7mL / L, CaSO 4 0.93g / L,K 2 SO 4 18.2g / L, MgSO 4 ·7H 2 O14.9g / L, KOH 4.13g / L, glycerin 40g / L.

[0037] (2) The mass fraction of the seed liquid is 5% inserted in a 50L fermenter containing 35L BSM inorganic salt medium (Shanghai Yubo Biotechnology Co., Ltd.), the pH value is controlled at 5, the temperature is 30 ° C, and the stirring speed is adjusted. Be 600rpm, dissolved oxygen DO is controlled at 10%, along with the carrying out of fermentation, dissolved oxygen rises to 30% and when continuing 30-60min, regulating stirring speed is 800rpm, and replenishes methyl alcohol once (replenishment is 1.5% of total volume ), an...

Embodiment 3

[0041] Feather meal keratinase enzymolysis feather meal

[0042] The application method of feather powder keratinase of the present invention in the enzymatic hydrolysis of chicken feather powder is to dry the feather powder keratinase crude enzyme liquid fermented for 60h in Example 4, and then use the dried feather powder keratinase crude enzyme enzymolysis Chicken feather powder, specifically:

[0043] (1) Mix chicken feather powder with water, prepare chicken feather powder and water according to the mass ratio of 1:5, 1:9, and 1:10 respectively, adjust the pH value to 6, and add dry feather powder keratinase crude oil at 25°C Enzyme, the amount added is 4500U per gram of chicken feather powder, enzymatic hydrolysis for 35h, and the decomposition rate of chicken feather powder is determined.

[0044] According to the above enzymatic hydrolysis, it was found that chicken feather powder and water were prepared at a mass ratio of 1:5, the feather decomposition rate was 60.5%...

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Abstract

The invention discloses a pichia pastoris strain (Classification and Nomenclature, pichia pastoris strain AN-PK-9K-GS115; Preservation Number, CCTCC NO: M 2019593; Preservation Date, July 30, 2019; Preservation Unit, China Center for Type Culture Collection; and Preservation Address, Wuhan University, Wuhan, China). Meanwhile, the invention also discloses a breeding selection method of the pichiapastoris strain, a feather meal keratinase produced by fermentation of the pichia pastoris strain and an application of the feather meal keratinase. A recombinant pichia pastoris strain capable of efficiently producing the feather meal keratinase is obtained through breeding selection, and the decomposition of feather of different species of animals under mild conditions is realized, so that proteins in the feather meal are decomposed into micromolecular peptide or amino acid, thus facilitating the efficient absorption and utilization of feather protein feed.

Description

technical field [0001] The invention belongs to the technical field of fermentation engineering, and in particular relates to a Pichia pastoris and its fermented keratinase and the application of the keratinase. Background technique [0002] 75% of industrial enzyme preparations are proteolytic enzymes, and protease is the enzyme with the largest proportion of industrial enzymes, accounting for about 60% of the world's total annual sales. Among them, feather powder keratinase is the most popular research in the feed industry. Protein is the main nutrient component for animal development and growth. The protein content in feathers is higher than 85% (keratin). Effective use of protein in animal feathers will have great potential value. The breeding methods of protease strains include physical and chemical factors single or compound mutagenesis, protoplast technology, genetic engineering technology and so on. The disadvantages of the prior art are: genetic mutations are pron...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N9/62A23K10/26A23K20/189C12R1/84
CPCA23K10/26A23K20/189C12N9/62C12N15/815
Inventor 彭楠常章兵田建平梁运祥
Owner HUAZHONG AGRI UNIV
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