Method for improving phospholipase D transesterification activity and method for producing phosphatidylserine by using phospholipase D transesterification activity
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A technology of phosphatidylserine and phospholipase, applied in the direction of biochemical equipment and methods, enzymes, hydrolytic enzymes, etc., can solve problems such as neglect, and achieve the effect of simple and easy operation, low production cost and high conversion rate
Pending Publication Date: 2021-02-09
BONTAC BIO ENG SHENZHEN +1
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[0005] In the process of producing phosphatidylserine by using biological enzyme catalysis, the level of lipid-transferring activity of phospholipase D used for catalysis plays a key role in the conversion rate of the reaction. Over the years, people have been committed to developing a more active Phospholipase D, including searching for new sources of phospholipase D, and genetically modifying known phospholipase D, often ignores the process itself to improve the lipid-transferring activity of phospholipase D
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Embodiment 1
[0021] Comparative experiment of translipid activity
[0022] Add 100g of soybean lecithin with a phosphatidylcholine content of 58.6%, 200g of L-serine, and 20g of calcium chloride in 1L of pure water. Put into a constant temperature water bath at 42°C, stir at 300rpm, add 125uL phospholipase D (25U / g) to each group, and add 12.5mL ethanol (5% of water volume) and 25mL ethanol (5% of water volume) to groups 2-4, respectively. 10%), 12.5mL isopropanol (5% of water volume), use high performance liquid chromatography to monitor the conversion rate of PC→PS, the conversion rate measurement result after reaction carries out 2h, 4h, 6h and 8h is shown in the table below.
[0023]
[0024]
Embodiment 2
[0026] Enzyme-catalyzed production of phosphatidylserine
[0027] In 1L of pure water, add 100g soybean phospholipid with a phosphatidylcholine content of 53.6%, 200g L-serine, 20g calcium chloride, stir at 500rpm and heat up to 39-43°C, add 0.5mL phospholipase D (25U / g ), the conversion rates of PC→PS detected after 2h, 4h, and 6h were 50.9%, 77.9%, and 87.1%, respectively.
Embodiment 3
[0029] Enzyme-catalyzed production of phosphatidylserine
[0030] Add 100g phosphatidylcholine content in 1L pure water and be 53.6% soybean lecithin, 200g L-serine, 20g calcium chloride, 500rpm stir well and be warmed up to 39-43 ℃, add 50mL ethanol, 0.5mL phospholipase D ( 25U / g), the conversion rates of PC→PS were detected at 2h, 4h, and 6h to be 71.0%, 90.5%, and 94.4%, respectively.
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Abstract
Provided is a method for improving phospholipase D transesterification activity, comprising: adding an alcohol substance, which is a short-chain primary alcohol and/or secondary alcohol, to an enzymereaction system for producing phosphatidylserine by enzyme catalysis, the method being capable of accelerating the speed of an enzyme catalytic reaction and reducing the amount of enzyme used, while shortening at least half of the reaction time, universal applicability. The invention also provides a method for producing phosphatidylserine by enzyme catalysis, which comprises the following steps: mixing phosphatidylcholine-containing natural phospholipid with water, Lserine, calcium chloride, alcohol substances and phospholipase D, and stirring to react at 39-43 DEG C for 4-6 hours; wherein thealcohol substance is short-chain primary alcohol and/or secondary alcohol; the method has the advantages of short enzyme reaction time, high conversion rate and low production cost.
Description
technical field [0001] The invention relates to the technical field of biocatalysis, in particular to a method for improving the translipid activity of phospholipase D in the catalytic process in vitro and a method for producing phosphatidylserine by using it. Background technique [0002] Phospholipase D (Phospholipase D, English abbreviation PLD) belongs to the phosphatidyl diester synthetase, which can catalyze the transesterification reaction similar to ester condensation between the phospholipids in the substrate phase and the nucleophilic donor in the water phase. The transesterification activity of phospholipase D can modify phospholipids to prepare high-purity single phospholipids, rare phospholipids and a series of functional phospholipids. An important tool enzyme for phospholipids. [0003] Phosphatidylserine (Phosphatidylserine, English abbreviation PS), also known as serine phospholipid, diacylglyceryl phosphoserine, compound nervous acid, etc., is an important...
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