Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2001-08-08
2003-10-21
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S132000, C435S128000, C435S198000, C435S197000, C435S168000, C424S094600
Reexamination Certificate
active
06635456
ABSTRACT:
SUBJECT OF THE INVENTION
The present invention concerns a process for the preparation of pure phosphatides starting from mixtures of natural phosphatides, or their single components, such as soybean or egg lecithin or animal phospholipids, or from synthetic phosphatides by reacting them with phospholipase D, with transphosphatidylation activity, in aqueous medium alone in the presence of defined substrates containing a primary or a secondary alcoholic group. The invention also refers to the preparation, purification and characterisation of the phospholipase D used in the process.
BACKGROUND OF THE INVENTION
The synthesis of pure phospholipids, particularly on an industrial scale, is a particularly widespread problem. Indeed, there have been numerous scientific publications and patents, including some very recent ones that describe various methodologies. Generally, said methods exploit the transphosphatidylation properties of phospholipase D to obtain optically active phosphatides. One of the main problems is the fact that each of these methods is suited to the preparation of one specific phosphatide alone and cannot be adapted for the synthesis of the whole class of compounds. Generally, the most widely studied phospholipid is phosphatidylserine (PS), as it is widely used in the preparation of pharmaceutical compositions, in the preparation of liposome formulations and food supplements. Relatively little or nothing is reported concerning the synthesis of sphingophospholipids.
One limitation of all the methods reported in both the scientific and patent literature consists in the fact that the reaction of transphosphatidylation occurs in diphasic water/organic solvent systems. This presents a series of technical problems linked with the use of large quantities of solvent, especially when the industrial process is of a chemical nature, aimed at obtaining a quality product. In patent application No. DE 19917249 A1, a method is described that actually employs the aqueous phase alone, but neither the yield nor degree of purity of the PS obtained, nor the type of the utilised enzyme is reported. Moreover, there is no mention of whether it is possible to obtain other phospholipids besides PS by using the same technique and starting from the substrates used, or whether in the conditions described other phospholipids can act as reaction substrate. Japanese Patent Publication No. 5/42917 (JP 2130088) also discloses a method employing a medium comprised of water alone or a mixture of water and an organic solvent. However, this patent states that the water content is favored to be 10% by weight or less to prevent a side reaction. This reference, therefore, appears to suggest that using an aqueous environment alone is not favorable. In fact, the examples therein disclose only processes employing a biphasic mixture of water and ethyl ether.
The generic nature of the information given or absence of teaching in the aforesaid prior art concerning the applicability of the transphosphatidylation reaction of phospholipase D in aqueous phase alone could in no way have led an expert in the field to think that this was the solution to the problem. Moreover, the importance of removing impurities deriving from the use of organic solvents in processes for the manufacture of products for use in the alimentary and pharmaceutical fields has only become known in recent years, following the limitations dictated by the United States Pharmacopoeia (USP) and the European guidelines (CPMP/CH/283/95).
Another critical point that has not been investigated in-depth, either in the scientific literature or in patents, concerns the peroxidation of products caused by the use of heterogeneous phases of water/solvent in emulsion during the transphosphatidylation reaction, the reaction conditions used and the consequent need to perform numerous steps of the process (re-precipitation, washings and possibly also chromatography) to obtain products with a high degree of purity. Many of the solvents used for these reactions in the heterogeneous phase do not guarantee the absence of radicalic precursors typical of the initiation of the peroxidation reaction. Moreover, the shaking/stirring required to achieve a reaction in the heterogeneous phase increases the likelihood of there being contact with oxygen in the atmosphere and consequent triggering of oxidative phenomena. This peroxidation acts like a chain reaction, when even negligible initial primer steps can give devastating results over time, even though the triggering conditions have been eliminated or minimised. The peroxidation of a “fatty” substance such as triglycerides (oils or fats) and phospholipids too, causes the fatty acids to become “rancid”, with the consequent formation of an unpleasant smell and taste. It is particularly important that the products should have a high degree of palatability (smell and taste) when they, particularly PS, are used to prepare food supplements (nutraceuticals) with particular formulations such as granules or the so-called “functional foods”, to which the product is added to enrich their content. Consequently, it is important that the products obtained, particularly PS, should be clearly characterised in chemical terms, both with regard to the chemical composition of the fatty acids, and for the extent of their peroxidation and consequent palatability.
Lastly, it should be pointed out that the phospholipase D enzymes available on the market mainly have transphosphatidylation activity on the phosphatidylcholine fraction of the phospholipid mixture. Therefore, the other components such as phosphatidylethanolamine (PE) undergo hydrolysis to phosphatidic acid, thus reducing both the yield and the degree of purity of the finished product.
DETAILED DESCRIPTION OF THE INVENTION
Surprisingly, the Applicant has discovered that by using a purified fraction of phospholipase D from
Streptoverticillium hachijoense
(n/k/a
Streptomyces hachijoense
) it is possible to obtain a transphosphatidylation reaction with various alcoholic receptors starting from a mixture of natural phosphatides or their purified fractions in a 100% aqueous environment with high yields of product, a high degree of purity and a peroxide index (degree of peroxidation) of less than 5, established according to the European Pharmacopoeia—Suppl 2000, page 41 (method A), by one single reaction step and one single precipitation.
A second aspect of the present invention consists in the fact that this particular enzymatic preparation has a transphosphatidylation activity with high yields even on substrates other than phosphatidylcholine (lecithin), thus enabling the process to be used with low-cost, unpurified raw materials too.
The importance of the strain used for the preparation of the Phospholipase D and its fractionation can be deduced from the following table, where a comparison with different commercial enzymatic preparations is reported for the purified enzymatic fraction prepared according to Example 1.
Preparation of phosphaditylserine
Strain
Company
Product
PS
PA
PC
Streptomyces sp.
Sigma
P4912
50%
45%
5%
type VI
Streptomyces sp.
Asahi Chemical
PL-DP
50%
45%
5%
Industry Co., Ltd
Streptomyces
Sigma
P8023
40%
40%
20%
chromofuscus
type VI
Actinomadura sp.
Meito Sangyo Co.,
No code
45%
40%
15%
Ltd
Streptoverticillium
Fidia
FRS
95%
5%
—
hachijoense
pure lecithin, phosphatidylcholine content 95%, as substrate; reaction conditions as in example 1
Another advantage of the actual enzyme preparation and of the reaction conditions described in the instant invention is the practically complete conversion of the substrate to PS as compared to the conversion rate of not more than 70% achieved in JP 213008's diphasic water/organic solvent system.
A third aspect of the present invention concerns the preparation of pharmaceutical and cosmetic compositions and food and dietary supplements based on phospholipids, obtained according to the process described above, having a degree of peroxidation of less than 5 and a high degree of palatability, such as to make th
Kirschner Guenter
Menon Giampaolo
Vaccaro Susanna
Birch & Stewart Kolasch & Birch, LLP
Fidia Farmaceutici S.p.A.
Marx Irene
LandOfFree
Procedure for the preparation of pure phosphatides with... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Procedure for the preparation of pure phosphatides with..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Procedure for the preparation of pure phosphatides with... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3136782