A new type of phospholipase

A technology of phospholipase and nucleic acid molecules, applied in the field of new phospholipase and its coding gene, can solve the problems of low efficiency

Active Publication Date: 2021-05-28
WILMAR SHANGHAI BIOTECH RES & DEV CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, PC and PE in phospholipids are mixed and difficult to distinguish, so it is difficult to prepare pure GPE. Generally, sodium methoxide is used to catalyze the alcoholysis of PC and PE mixture, and then GPE is separated by ion exchange resin, which is very inefficient.

Method used

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  • A new type of phospholipase
  • A new type of phospholipase
  • A new type of phospholipase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053]Inoculate Schizochytrium sp.ATCC 20888 into 50ml YPD liquid medium (1% yeast powder, 2% peptone, 2% glucose) and cultivate for 48h, collect the bacteria by centrifuging at 4000rpm for 5min at 4°C and wash with deionized water for 2 The cells were ground in liquid nitrogen, and then the genomic DNA was extracted using the MiniBEST Universal Genomic DNA Extraction Kit from Takara Company.

[0054] Synthesize primers gU (SEQ ID NO: 3), gD (SEQ ID NO: 4), use this pair of primers to amplify the phospholipase gene from the Schizochytrium genome, and make it 5' with NdeI and 3' with NdeI Contains Not I restriction enzyme sites. The PCR system is 50 ul, and the composition is: 13.5 ul of water, 25 ul of 2×GC I buffer, 6 ul of dNTPs, 2 ul of each primer (20 μM mother solution), 0.5 ul of LA-Tag (Takara Company), and 1 ul of template. The PCR amplification program is: denaturation at 98°C for 5 minutes; 12 cycles of thermal asymmetric PCR: 95°C for 30s, 63°C for 30s, 72°C for 1m...

Embodiment 2

[0071] Example 2: Activity Analysis

[0072] Inoculate the positive recombinant bacteria g1-9k-1# and the control 9k into 50ml of YPD culture medium, and cultivate overnight at 28°C on a shaker at 200rpm. Centrifuge at 400rpm for 2min to remove the supernatant, resuspend the bacteria in 50ml BMMY culture medium (1% yeast extract, 2% peptone, 100mM potassium phosphate pH6.0, 1.34% YNB, 4×10-5% biotin, 0.5% methanol ), cultured on a shaker at 200 rpm at 28°C, and induced by adding 250 μl of anhydrous methanol every 24 hours. After methanol-induced expression for 96 hours, the fermentation supernatant was collected by centrifugation at 12,000 rpm at 4°C for 10 minutes, filtered through a 0.22 μm membrane, and replaced with buffer by ultrafiltration using Millipore’s 10K ultrafiltration membrane, and then 50ml of the fermentation broth was concentrated to 5ml.

[0073] 1. Hydrolyzed phospholipid experiment:

[0074] Use 5% soybean mixed phospholipids, lecithin, and cephalin as s...

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Abstract

The invention relates to a new phospholipase and its coding gene. It also relates to vectors and host cells comprising said genes. The phospholipase provided by the invention comprises or consists of a sequence selected from: (a) the amino acid sequence shown in SEQ ID NO: 2, and (b) substituted by the sequence described in (a) , a sequence obtained after deletion or addition of at least one amino acid, and said sequence still maintains the function of SEQ ID NO:2. The phospholipase provided by the invention has the ability to hydrolyze phosphatidylethanolamine acyl ester but not to hydrolyze phosphatidylcholine.

Description

technical field [0001] The invention relates to a novel phospholipase and its coding gene. It also relates to vectors and host cells comprising said genes. Background technique [0002] Phospholipases are enzymes that hydrolyze phospholipids, and are divided into several categories according to the location of hydrolysis. Phospholipase A1 (PLA1) hydrolyzes the free fatty acid at the sn-1 position; phospholipase A2 (PLA2) hydrolyzes and releases the fatty acid at the sn-2 position; fatty acid; phospholipase C (PLC) hydrolyzes the glycerol-phosphate bond to release diacylglycerol and phosphate; phospholipase D (PLD) hydrolyzes the phosphate-alkaline ester bond to release phosphatidic acid and base (choline, ethanolamine or muscle alcohol). Phospholipases are widely used in phospholipid removal and phospholipid modification. [0003] Lecithin (phosphatidylcholine, Phosphatidyl choline, PC) is the most commonly used phospholipid. It is a natural surfactant with many importa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/63C12N1/19C12R1/84
CPCC12N9/16
Inventor 戴小军苏斐牛其文
Owner WILMAR SHANGHAI BIOTECH RES & DEV CENT
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