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Phospholipase C

A technology of phospholipase and enzyme solution, applied in the field of enzyme degumming, can solve the problems of high enzyme activity, inability to completely remove phospholipids, and inability to find pH range, etc., and achieve the effect of cost saving and wide hydrolysis substrate characteristics.

Active Publication Date: 2015-05-20
WILMAR SHANGHAI BIOTECH RES & DEV CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Purifine (DSM company), which has been successfully commercialized and developed, has a working pH range of 6-7 and is a neutral PC-PLC that can hydrolyze PE and PC, but cannot hydrolyze PI, so the complete removal of phospholipids cannot be achieved.
In addition, in deep enzymatic degumming, it is necessary to add PC-PLC and PLA1 together to achieve complete removal of phosphorus, and the working pH of PLA1 is 4-5, so when Purifine and PLA1 are used in combination, it is impossible to find a suitable pH range, allowing both enzymes to work simultaneously with higher enzyme activity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0151] Embodiment 1, PFSMC expression and purification

[0152] The sequence shown in SEQ ID NO: 2 (its coding sequence is shown in SEQ ID NO: 1) was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., and cloned in the expression vector pET24a(+).

[0153] The pET24a (+) with this nucleotide sequence is transformed into Escherichia coli BL21 (DE3) to induce expression (refer to "Molecular Cloning Experiment Guide", the third edition, Science Press 2002, [US] J. Sambrook Written by D.W Russell, translated by Huang Peitang, etc.). The specific process is as follows:

[0154] Pick positive transformants and culture overnight at 37°C in LB medium, inoculate the seed culture solution into the expression medium at 1% inoculum size, and culture at 37°C, 220 rpm until OD600=0.6-0.8.

[0155] After cooling to 20°C, add IPTG to 0.1mM for induction, and express overnight at 20°C and 190rpm. After induction of expression, the cells were collected by centrifugation. Resuspend t...

Embodiment 2

[0158] Embodiment 2, PFSMC substrate preference

[0159] Mix phospholipid substrate (15.75g / L soybean powder phospholipid (a mixture of phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine phosphatidic acid, etc.), 5mM CaCl into 10ml 2 ), add 0.5ml PFSMC (2.9mg / ml) to another tube of 10ml phospholipid mixed substrate, add 0.5ml water as a control, react at 35℃, 200rpm for 18 hours, add 10ml chloroform, shake and mix, and centrifuge at 10,000g for 10 minutes , the solution is divided into upper and lower layers.

[0160] Take 1ml of the upper aqueous phase, lyophilize and resuspend in 1ml of heavy water. Take 1ml of the lower chloroform phase, dry it in an ultra-clean bench, resuspend in 3ml of NMR analysis buffer, and conduct 31P-NMR analysis on the two phases respectively.

[0161] The NMR results of the phospholipid substrate chloroform phase added with water are shown in Picture 1-1 , Visible, phospholipid substrate mainly contains thr...

Embodiment 3

[0163] Part enzymatic properties of embodiment 3, PFSMC

[0164] In a 200 μL reaction system, containing soybean powder phospholipids 0.5% (w / v), buffer system (25mM Tris-Cl, pH7.5), 5mM CaCl 2 , add phospholipase PFSMC 20μl (0.097mg / ml), react for 30min, add 200μL of chloroform, shake and mix for 30s, carry out centrifugation at 12,000rpm for 1min, take 80μL supernatant and add it to the phospholipase reaction system, the final volume is 200μL, the system Also contains 50mM Tris-Cl (pH9.0), 10mM MgCl 2 , CIAP 10 U / μL. The reaction was carried out in a 37 °C water bath for 30 min. Add 740 μL of deionized water, 20 μL of 10% (w / v) ascorbic acid, and 40 μL of 2.5% (w / v) ammonium molybdate solution. Develop color at 37°C for 10 minutes. The solution after color development was taken for absorbance detection at 700nm. Combined with the standard curve detected by phosphomolybdenum blue, the data is analyzed and regressed, and after multiplying by the dilution factor, the activ...

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PUM

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Abstract

The invention provides a phospholipase C as well as a coding sequence, production and an application thereof. The phospholipase C can be used for hydrolyzing phosphatidylcholine and phosphatidyl ethanolamine, has the capability of effectively hydrolyzing phospholipid nearby an acidic pH region or a neutral pH region or an alkaline pH region, has wide tolerance to the pH regions, has a certain degree of thermal stability, is sensitive to zinc ions, and exerts the hydrolysis ability without depending on zinc. The phospholipase C is significant for fermentation production of enzyme and industrialized application.

Description

technical field [0001] The invention relates to the field of enzyme degumming, in particular to a phospholipase C. Background technique [0002] Enzymes that catalyze the hydrolysis of phospholipids can be called phospholipases. They are widely distributed in nature and have played a variety of functions, from the toxicity of snake venom (Janssen et al.1999) to signal transduction (Singer et al.1997) to human Digestion is a biological characteristic and process in which phospholipases are involved (De Maria et al.2007). Although the substrates of phospholipases are all phospholipids, they can act on different sites on phospholipid molecules. According to different hydrolysis sites, phospholipases are divided into five types: phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase B (PLB), phospholipase C (PLC) and phospholipase D (PLD). For example, PLA1 and PLA2 can hydrolyze the fatty acid acyl ester bonds at the sn-1 and sn-2 positions of glyceryl, respectively....

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N15/70C12N1/21C11B3/00
Inventor 丛芳李芳许骏
Owner WILMAR SHANGHAI BIOTECH RES & DEV CENT
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