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LIPASES AND PHOSPHOLIPASES IN PHOSPHOLIPID SYNTHESIS

Virto, Carmen LU (1999)
Abstract
The synthesis of phospholipids with defined fatty acid and polar head composition catalysed by lipases and phospholipases was investigated. Lipases from <i>Rhizopus arrhizus</i> and <i>Candida antarctica</i> were used for the direct acylation of DL-a-glycerophosphate and L-a-glycerophosphatidylcholine, with different fatty acids. Esterification reactions can be carried out in systems with low water activity, in which the equilibrium is favoured towards synthesis, and hydrolysis is minimised. For optimisation of the processes, it is important to consider various reaction parameters, principally the water content of the media. In the acylation of glycerophosphate and glycerophosphorylcholine, fatty acid vinyl esters... (More)
The synthesis of phospholipids with defined fatty acid and polar head composition catalysed by lipases and phospholipases was investigated. Lipases from <i>Rhizopus arrhizus</i> and <i>Candida antarctica</i> were used for the direct acylation of DL-a-glycerophosphate and L-a-glycerophosphatidylcholine, with different fatty acids. Esterification reactions can be carried out in systems with low water activity, in which the equilibrium is favoured towards synthesis, and hydrolysis is minimised. For optimisation of the processes, it is important to consider various reaction parameters, principally the water content of the media. In the acylation of glycerophosphate and glycerophosphorylcholine, fatty acid vinyl esters gave better yields (>95%) than free fatty acid, in the formation of lysophosphatidic acid and phosphatidic acid or lysophosphatidylcholine and phosphatidylcholine. Solvent free systems were chosen for both reactions, but in some cases conversions could be easily improved by the addition of a small amount of solvent, such as <i>t</i>-butanol.



The choline polar head group of lysophosphatidylcholine was exchanged with glycerol in a reaction catalysed by a phospholipase D from Savoy cabbage, in an aqueous micellar system. Characterisation of this reaction and of the hydrolysis showed that both reactions are similar in their Ca<sup>2+</sup> ion dependence, kinetic parameters and pH optima. Contrary to the action of the enzyme towards phosphatidylcholine, phospholipase D does not need detergents or solvents to display optimum activity, with lysophosphatidylcholine as substrate.



A combination of two phospholipases was used for the formation of lysophosphatidic acid and the analogue, 1-acyl-dihydroxyacetonephosphate, in diethyl ether-aqueous two-phase systems. Microbial phospholipase D from <i>Streptomyces</i> sp. was used in the transesterification of egg phosphatidylcholine with 1-lauroyl-glycerol or 1-lauroyl-dihydroxyacetone, to form two complex phospholipids, 1-lauroyl-phosphatidylglycerol and 1-lauroyl-phosphatidyldihydroxyacetone, respectively. The Km values for the alcohol acceptors were very low, 23±7 and 104±5 mM for 1-lauroyl-glycerol and 1-lauroyl-dihydroxyacetone, respectively. Phospholipase C from <i>Bacillus cereus</i> selectively hydrolysed the obtained products, giving the desired 1-acyl-glycerophosphate and 1-acyl-dihydroxyacetonephosphate in stoichiometric amounts. The facile recovery of the products from the ether phases in both PL D and PL C catalysed reactions, makes this a very attractive system.



Finally, the direct esterification of dihydroxyacetone and lauric acid in solvent systems at controlled water activity is described. High yields (>90%) were easily achieved at low water activities of 0.06-0.11. The main product of this reaction was 1-lauroyl-dihydroxyacetone under most of the conditions tested, but the formation of the 1,3-dilauroyl-dihydroxyacetonephosphate could easily be increased by selection of a more hydrophobic solvent, or with the use of vinyl laurate as acyl donor. Using the latter approach, sole formation of 1,3-dilauroyl-dihydroxyacetone was achieved if the reactions were carried out in hermetically closed reactors. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Haraldsson, Gudmundur, Institute of Science, University of Iceland, Reykjavik, Iceland
organization
publishing date
type
Thesis
publication status
published
subject
keywords
transphosphatidylation, acylation, vinyl ester, Phospholipid synthesis, lysophospholipid, kinetics, Biotechnology, Bioteknik
pages
138 pages
publisher
Carmen Virto, Dept. of Biotechnology, Lund University
defense location
Kemicentrum, Hörsal C, Sölvegatan 39, Lund
defense date
1999-10-29 13:15
external identifiers
  • Other:ISRN: LUTKDH/TKBT--99/1042--SE
ISBN
91-628-3798-2
language
English
LU publication?
yes
id
bc21e271-c732-4ffc-b563-c19d0d5eb02e (old id 39994)
date added to LUP
2007-10-14 17:30:58
date last changed
2016-09-19 08:45:09
@misc{bc21e271-c732-4ffc-b563-c19d0d5eb02e,
  abstract     = {The synthesis of phospholipids with defined fatty acid and polar head composition catalysed by lipases and phospholipases was investigated. Lipases from &lt;i&gt;Rhizopus arrhizus&lt;/i&gt; and &lt;i&gt;Candida antarctica&lt;/i&gt; were used for the direct acylation of DL-a-glycerophosphate and L-a-glycerophosphatidylcholine, with different fatty acids. Esterification reactions can be carried out in systems with low water activity, in which the equilibrium is favoured towards synthesis, and hydrolysis is minimised. For optimisation of the processes, it is important to consider various reaction parameters, principally the water content of the media. In the acylation of glycerophosphate and glycerophosphorylcholine, fatty acid vinyl esters gave better yields (&gt;95%) than free fatty acid, in the formation of lysophosphatidic acid and phosphatidic acid or lysophosphatidylcholine and phosphatidylcholine. Solvent free systems were chosen for both reactions, but in some cases conversions could be easily improved by the addition of a small amount of solvent, such as &lt;i&gt;t&lt;/i&gt;-butanol.<br/><br>
<br/><br>
The choline polar head group of lysophosphatidylcholine was exchanged with glycerol in a reaction catalysed by a phospholipase D from Savoy cabbage, in an aqueous micellar system. Characterisation of this reaction and of the hydrolysis showed that both reactions are similar in their Ca&lt;sup&gt;2+&lt;/sup&gt; ion dependence, kinetic parameters and pH optima. Contrary to the action of the enzyme towards phosphatidylcholine, phospholipase D does not need detergents or solvents to display optimum activity, with lysophosphatidylcholine as substrate.<br/><br>
<br/><br>
A combination of two phospholipases was used for the formation of lysophosphatidic acid and the analogue, 1-acyl-dihydroxyacetonephosphate, in diethyl ether-aqueous two-phase systems. Microbial phospholipase D from &lt;i&gt;Streptomyces&lt;/i&gt; sp. was used in the transesterification of egg phosphatidylcholine with 1-lauroyl-glycerol or 1-lauroyl-dihydroxyacetone, to form two complex phospholipids, 1-lauroyl-phosphatidylglycerol and 1-lauroyl-phosphatidyldihydroxyacetone, respectively. The Km values for the alcohol acceptors were very low, 23±7 and 104±5 mM for 1-lauroyl-glycerol and 1-lauroyl-dihydroxyacetone, respectively. Phospholipase C from &lt;i&gt;Bacillus cereus&lt;/i&gt; selectively hydrolysed the obtained products, giving the desired 1-acyl-glycerophosphate and 1-acyl-dihydroxyacetonephosphate in stoichiometric amounts. The facile recovery of the products from the ether phases in both PL D and PL C catalysed reactions, makes this a very attractive system.<br/><br>
<br/><br>
Finally, the direct esterification of dihydroxyacetone and lauric acid in solvent systems at controlled water activity is described. High yields (&gt;90%) were easily achieved at low water activities of 0.06-0.11. The main product of this reaction was 1-lauroyl-dihydroxyacetone under most of the conditions tested, but the formation of the 1,3-dilauroyl-dihydroxyacetonephosphate could easily be increased by selection of a more hydrophobic solvent, or with the use of vinyl laurate as acyl donor. Using the latter approach, sole formation of 1,3-dilauroyl-dihydroxyacetone was achieved if the reactions were carried out in hermetically closed reactors.},
  author       = {Virto, Carmen},
  isbn         = {91-628-3798-2},
  keyword      = {transphosphatidylation,acylation,vinyl ester,Phospholipid synthesis,lysophospholipid,kinetics,Biotechnology,Bioteknik},
  language     = {eng},
  pages        = {138},
  publisher    = {ARRAY(0x95ece20)},
  title        = {LIPASES AND PHOSPHOLIPASES IN PHOSPHOLIPID SYNTHESIS},
  year         = {1999},
}