Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing organic compound containing a metal or atom other...
Reexamination Certificate
1992-02-03
2001-09-04
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing organic compound containing a metal or atom other...
C435S128000, C435S188000, C435S196000, C435S197000, C435S212000
Reexamination Certificate
active
06284501
ABSTRACT:
TECHNICAL FIELD
This invention relates to a process for modifying a phospholipid by specific exchange of the acyl group in the sn-2 position.
BACKGROUND ART
For some applications of phospholipids it is desirable to specifically incorporate fatty acyl groups into the sn-2 position of the molecule, e.g. in order to modify the emulsification properties, to increase the stability against oxidation or to improve the physiological or nutritional value of the phospholipid.
It has been described that exchange of acyl groups in diacylphospholipids can be catalyzed by native or derivatized lipase (JP-A 63-185,391, JP-A 63-105,686). These lipases are known to possess activity against primary alcohols or primary esters, so during phospholipid interesterification a significant fraction of the incorporated fatty acids is incorporated into the sn-1 position of the phospholipids.
It is known from U. Z. Muratova et al., Biochemistry (USSR), 52(7), 919-922 (1988) that purified phospholipase A
2
from rat liver mitochondria membranes catalyzes ester exchange between phospholipid and fatty acid, but practical use of this finding has never been considered. The enzyme is very unstable, especially in purified form, it has a low specific activity as a hydrolytic enzyme, and does not appear amenable to economical production.
H. P. Franck et al., Z. Naturforsch. 23b, 439-448 (1968) reported that under appropriate conditions, snake venom phospholipase A
2
could be used to remove the acyl group from the sn-2 position of a phospholipid (to form lysophospholipid) and then to reinsert the fatty acid. However, J. Goerke et al., Biochim. Biophys. Acta, 248, 245-253 (1971) reported that this finding was due to misinterpretation of results.
Apart from these erroneous conclusions of H. P. Franck et al., commercially available phospolipases A
2
from snake or bee venoms or of pancreatic origin have never been described as being capable of interesterifying phopholipids although these enzymes are very well described with regard to hydrolytic properties, of which for instance their strict specificity towards the sn-2 position of phospholipids are well known.
It is the object of the invention to provide a process for modifying a phospholipid by specific exchange of the acyl group in the sn-2 position, using a readily available enzyme with high specific activity, such that a high degree of incorporation of a desired fatty acid can be obtained.
STATEMENT OF THE INVENTION
Very surprisingly, it has been found that it is possible to achieve the desired exchange reaction of phospholipid by reacting it with a free fatty acid in the presence of an extracellular phospholipase A
2
. In contrast to mitochondrial phospholipase A
2
these enzymes are known to have high specific activity and to be extremely stable even at temperatures above 70° C.
Accordingly, the invention provides a process for modifying a phospholipid by specific exchange of the acyl group in the sn-2 position, characterized by reacting the phospholipid with a free fatty acid in the presence of an extracellular phospholipase A
2
.
Phospholipid
The process of the invention may be applied to any desired kind of glycero-phospholipid containing a fatty acyl ester group in the sn-2 position, particularly to 1-alkyl-2-acyl-phospholipid (ether-phospholipid) and to diacyl-phospholipid.
Fatty acid
The exchange reaction of the invention may be used to incorporate any desired fatty acid into a phospholipid. Some examples of fatty acids that may be of particular interest are:
Long-chain (C
18
-C
22
) polyunsaturated fatty acid, such as linoleic, arachidonic, alpha-linolenic, eicosapentaenoic, docosahexaenoic or gammalinolenic acids. These may be incorporated to improve the physiological or nutritional value of the phospholipid, especially a diacyl-phospholipid.
C
2
-C
18
saturated fatty acids. These may be incorporated to modify emulsification properties, to modify the physiological value or to improve oxidation stability of a phospholipid, especially a diacyl-phospholipid.
Acetic acid may be incorporated into an ether-phospholipid to prepare compounds with hormonal activity.
Phosgholipase A
2
preparation
The extracellular phospholipase A
2
to be used is preferably a venom enzyme (especially from bee venom or snake venom) or a digestive enzyme (especially from pancreas, e.g. porcine pancreas). To ensure the specific incorporation into the sn-2 position of a phospholipid the phospholipase A
2
preparation should be essentially free of phospholipase A
1
, phospholipase B or lipase activity.
An example is Lecitase™ (product of Novo Nordisk a/s), a preparation of porcine pancreatic phospholipase A
2
containing virtually no lipase activity.
A suitable dosage of phospholipase for obtaining a high degree of exchange in a reasonable time is generally in the range 5,000 to 100,000 IU/g of phospholipid. The units of activity (IU), mentioned in this specification are measured as described in G. H. de Haas et al., Biochim. Biophys. Acta, 159, 103-117, (1968).
For the practice of the invention the phospholipase may be precipitated or immobilized on a suitable carrier, e.g. formed by precipitation on silica (celite) particles or by adsorption on a suitable carrier, e.g. an adsorbent resin of the acrylic type an example of which is Lewatit E 2001/85 (product of Bayer). The catalysts are typically loaded with 10,000-100,000 IU per g (dry weight) of catalyst.
Process conditions
The interesterifying process should be carried out under conditions at which both the phospholipid and the fatty acid are miscible in a fluid phase, e.g. solubilized in an organic solvent that also allows the enzyme catalyst to be active. The solvent may be hexane, heptane, petroleum ether or chlorinated hydrocarbons. Alternatively, the phospholipid may be solubilized directly in the fatty acid.
The process temperature should be chosen after considering thermostability of the phospholipase. Generally 20-80° C. will be suitable.
The process may be carried out as a batch reaction, where the ingredients are stirred gently throughout the reaction period. The amount of phospholipase preparation in the reaction mixture will typically be 1-10% w/w, and the reaction time will generally be ½-72 hours, preferably ½-24 hours.
Alternatively, the process may be carried out continuously by letting the substrate mixture (and solvent, if used) pass through a fixed bed column of phospholipase catalyst. The residence time will typically be 1-12 hours.
The amount of water in the reaction system should be controlled, since a certain water activity is required to activate the immobilized phospholipase, but too high water content may cause complete hydrolysis of the phospholipid into lysophospholipid. A suitable water content is generally 0.01-1% (w/w) of the total reaction system.
Furthermore the water should contain some Ca
++
e.g. provided as CaCl
2
, as Ca
++
is an essential cofactor for venom and pancreatic phospholipases A
2
. A suitable Ca
++
concentration in the water phase is 1 mM-1 M.
In a batch system the water containing Ca
++
may be provided by hydrating the phospholipase A
2
catalyst with a solution of Ca
++
before reaction, preferably to 0.5-15% water by weight. In a continuous column system, water containing Ca
++
may be introduced by hydrating the catalyst as above, and further by having some water dissolved in the substrate.
In processes where polyunsaturated fatty acids are to be incorporated into phoapholipids, it may be essential to protect the fatty acids from oxidation. This can be done by running the reaction under a blanket of an appropriate non-oxidizing gas like nitrogen, helium or argon.
After the reaction, the modified phospholipid may be recovered by conventional methods.
REFERENCES:
patent: 4382035 (1983-05-01), Eibl
Muratova et al., Biochem., vol. 52, No. 7, pp. 919-922 (1988).
Franck et al., Z. Naturforch, vol. 23 b, pp. 439-448 (1968).
Georke et al., Biochim. Biophys. Acta, vol. 248, pp. 245-253 (1971).
Garbell Jason I.
Lambiris Elias J.
Lilling Herbert J.
Novozymes A/S
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