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Aspergillus oryzae phospholipase C as well as encoding gene and application thereof

A phospholipase and encoding technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of poor stability and low yield of phospholipase C

Active Publication Date: 2021-03-19
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been some research reports on the heterologous expression of phospholipase C gene, the yield of phospholipase C is still low, the substrate specificity is highly concentrated, and the stability is poor (Yu Zhenzhen, Chang Ming, Liu Ruijie, etc. Phospholipase C Research progress in enzymatic degumming. China Oils, 2013,38(7):19-22)

Method used

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  • Aspergillus oryzae phospholipase C as well as encoding gene and application thereof
  • Aspergillus oryzae phospholipase C as well as encoding gene and application thereof
  • Aspergillus oryzae phospholipase C as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Embodiment 1, cloning and expression of Aspergillus oryzae phospholipase C gene AoPC

[0099] 1. Amplification of gene AoPC

[0100] 1. Extract the RNA of Aspergillus oryzae, reverse transcribe it into cDNA, use the cDNA as a template, and carry out PCR amplification with the following primers:

[0101] Upstream primer: 5'-AAA TACGTA AGCCCTGTCACGTCCGAG-3' (the underline indicates the SnaBI restriction site)

[0102] Downstream primer: 5'-AAA GCGGCCGC TTATACCGAAAAGGTATGGGGAGTCTTG-3' (the underline indicates the NotI restriction site).

[0103] 2. The PCR conditions in step 1 are pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 90 seconds, 30 cycles; extension at 72°C for 5 minutes.

[0104] The amplified PCR product has the nucleotide sequence shown in Sequence Table 1 in the Sequence Listing, and the gene indicated by the nucleotide is named Gene AoPC, wherein the 58th-1395th posit...

Embodiment 2

[0147] Example 2, Purification and enzymatic property determination of recombinant phospholipase C AoPC

[0148] 1. Purification of recombinant phospholipase C AoPC

[0149] 1. Dialyze the fermentation supernatant of recombinant bacteria GS115 / pPIC9K-AoPC-PDI with 5L of 20mM Tris-HCl pH 7.5 buffer solution (buffer solution A) for 12h, and incubate at 4° C. , refrigerated and centrifuged at 10,000 rpm for 10 min, and the supernatant was reserved for later use to obtain a crude enzyme solution (concentration: 7.3 mg / mL).

[0150] 2. Purify the crude enzyme liquid obtained in step 1 through a QSFF strong anion exchange column (1.0×10 cm), using an AKTA protein purification system (purchased from GE Healthcare, USA), and the sample loading flow rate is 0.5 mL / min.

[0151] 3. After completing step 2, set the flow rate to 1mL / min, and use buffer solution A to elute to the eluent OD 280 <0.05.

[0152] 4. After completing step 3, use buffer solution A containing 150mM NaCl to elute...

Embodiment 3

[0201] Embodiment 3, the application of recombinant phospholipase C AoPC in soybean crude oil degumming

[0202] 1. Screening of optimal conditions for recombinant phospholipase C AoPC in soybean oil degumming

[0203] 1. Recombinant phospholipase C AoPC phospholipid degumming and acid addition optimization

[0204] AoPC+Crtric acid: Weigh 300g of crude soybean oil (the oil that has not undergone post-degumming treatment) in a 100mL Erlenmeyer flask with a stopper and heat it to 70°C in a water bath, add 0.3mL of citric acid solution (prepared with water with a concentration of 0-50%) Citric acid solution, constant volume, w / w), pretreatment at 200rpm for 20min. After the treatment is completed, cool to the preset temperature (20-50°C, can be 25°C), add 0.6mL of 4% (g:ml) NaOH aqueous solution to adjust the pH to 8, then add 1% distilled water and a certain amount of The 14.8mg / mL recombinant phospholipase C AoPC pure enzyme solution obtained in the first step of the prepara...

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Abstract

The invention discloses aspergillus oryzae phospholipase C as well as an encoding gene and application thereof. The invention provides a protein, which is phospholipase C or recombinant phospholipaseC, and is a protein shown in the following 1) or 2) or 3) or 4): 1) a protein composed of an amino acid sequence shown in a sequence 2 in a sequence table; 2) a protein composed of amino acids from the 20th site to the 464th site of the sequence 2 in the sequence table; 3) a co-expression protein obtained by connecting a molecular chaperones label to the N terminal or / and C terminal of 1) or 2); and 4) a protein which is obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein in the step 1) or the step 2) or the step 3) and has the same function. The research aims to explore phospholipase C which is novel in source, high in enzyme activity and unique in property from aspergillus oryzae, and the aspergillus oryzae phospholipase C has a huge market prospect.

Description

technical field [0001] The invention belongs to the field of food biotechnology, and in particular relates to an Aspergillus oryzae phospholipase C and its encoding gene and application. Background technique [0002] Phospholipase C (phospholipase C, EC 3.1.4.3) is a specific action on glycerophospholipid C 3 Ester hydrolase on the glycerol phosphate bond to generate diacylglycerol (DAG) and phospholipid compounds (Sebastián, Industrial uses of phospholipases: current state and future applications. Applied Microbiology and Biotechnology (2019) 103: 2571 –2582). Phospholipase C has attracted widespread attention because of its great application prospects in food, medicine, health care products, bioenergy and other industrial fields. In the food industry, phospholipase C can improve the quality of dairy products by increasing lipid stability; hydrolyzing the inherent phospholipid components in bread during bread baking can improve the emulsifying properties of food; and can ...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N15/62C07K19/00C12N15/81C12N15/66C12N1/19C11B3/00C12R1/84
CPCC12N9/16C12N15/815C12N15/66C12Y301/04003C11B3/003C07K2319/35
Inventor 杨绍青江正强王灵闫巧娟相曼
Owner CHINA AGRI UNIV
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