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A kind of aspergillus oryzae phospholipase c and its coding gene and application

A phospholipase and phosphatidylcholine technology, which can be used in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of low yield and poor stability of phospholipase C

Active Publication Date: 2022-05-03
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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  • A kind of aspergillus oryzae phospholipase c and its coding gene and application
  • A kind of aspergillus oryzae phospholipase c and its coding gene and application
  • A kind of aspergillus oryzae phospholipase c and its coding gene and application

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 an aspergillus oryzae phospholipase C, its coding gene and application. The present invention provides a protein, which is phospholipase C or recombinant phospholipase C, which is the protein described in the following 1) or 2) or 3) or 4): 1) the amino acid sequence shown in sequence 2 in the sequence listing Protein; 2) A protein comprising amino acids at positions 20 to 464 of Sequence 2 in the sequence listing; 3) A co-expressed protein obtained by connecting a chaperone tag to the N-terminus or / and C-terminus of 1) or 2); 4) combining 1) Or the protein in 2) or 3) obtained through substitution and / or deletion and / or addition of one or several amino acid residues and having the same function. This research is dedicated to discovering phospholipase C from Aspergillus oryzae with novel source, high enzyme activity and unique properties, which 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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Patent Type & Authority Patents(China)
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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