Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2000-04-28
2004-03-16
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S233000, C435S235100, C435S325000, C435S348000, C435S419000, C435S252300, C435S254110, C435S254200, C435S252310, C435S252330, C435S257200, C435S174000, C435S320100, C536S063000, C536S023700, C536S024300
Reexamination Certificate
active
06706501
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an isolated (trans,cis)-10,12-linoleate isomerase enzyme, to a nucleic acid molecule encoding a (trans,cis)-10,12-linoleate isomerase enzyme, to immobilized cells containing a linoleate isomerase enzyme, to an immobilized (trans,cis)-10,12-linoleate isomerase enzyme, and to a method for converting linoleic acid or linolenic acid to CLA or derivatives thereof using the isolated linoleate isomerase enzyme, nucleic acid molecule and/or immobilized cells.
BACKGROUND OF THE INVENTION
The term “CLA” is used herein as a generic term to describe both conjugated linoleic acid and conjugated linolenic acid. The CLA compounds (cis,trans)-9,11-linoleic acid and (trans,cis)-10,12-linoleic acid are recognized nutritional supplements and effective inhibitors of epidermal carcinogenesis and forestomach neoplasia in mice, and of carcinogen-induced rat mammary tumors. CLA has also been shown to prevent adverse effects caused by immune stimulation in chicks, mice and rats, and has been shown to decrease the ratio of low density lipoprotein cholesterol to high density lipoprotein cholesterol in rabbits fed an atherogenic diet. CLA also reduces body fat in mouse, rat, chick and pig models. CLA has also been shown to be effective in treating skin lesions when included in the diet.
CLA occurs naturally in various amounts in virtually all foods. The principle natural sources of CLA are dairy products, beef and foods derived from ruminant animals. In the U.S., beef, beef tallow, veal, lamb (3-4 mg CLA/g fat; 84% cis-9,trans-11) and dairy products (3-7 mg CLA/g fat; 80-90% cis-9,trans-11) have the highest concentration of CLA. CLA concentrations 2-3 times higher are found in Australian dairy products and pasture-fed beef and lamb. Very low concentrations of CLA (0.1-0.7 mg CLA/g fat; ca. 40% each cis-9,trans-11 and trans-10,cis-12) are found in commercial vegetable oils.
CLA is a normal intermediate of linoleic acid metabolism. In cows, (cis,trans)-9,11-CLA produced by natural bacterial flora that is not further metabolized is incorporated into lipids and then into host tissues and milk. Animals take up and incorporate CLA into normal tissue and milk from dietary sources such as milk, milk products or meat containing CLA, or from CLA dietary supplements.
CLA can be synthetically obtained from alkaline isomerization of linoleic or linolenic acid, or of vegetable oils which contain linoleic acid, linolenic acid or their derivatives. Heating vegetable oil at about 180° C. under alkaline conditions catalyzes two reactions: (1) fatty acid ester bonds from the triglyceride lipid backbone are hydrolyzed, producing free fatty acids; and (2) unconjugated unsaturated fatty acids with two or more appropriate double bonds are conjugated. Commercial CLA oils available at the present time, typically made from sunflower oil, are sold without further purification. They contain a mixture of CLA isomers as well as other saturated and unsaturated fatty acids. Generally, chemical synthesis produces about 20-35% (cis,trans)-9,11-CLA and about 20-35% (trans,cis)-10,12-CLA, and the balance as a variety of other isomers. The presence of the non-active, non-natural isomers introduces the need to purify (cis,trans)-9,11-CLA and/or (trans,cis)-10,12-CLA, or to demonstrate the safety and seek regulatory approval of these non-beneficial, non-natural isomers for human use. It is not feasible economically, however, to isolate single isomers of CLA from the CLA made by alkaline isomerization. Using a fractional crystallization procedure, it is possible to enrich 9,11-CLA relative to 10,12-CLA and vice versa. U.S. Pat. No. 6,015,833, issued Jan. 18, 2000, to Saebø et al. describes the chemical production of CLA compositions from seed oils with a total CLA content of at least 50%, and with less than 1% contaminating octadecadienoic acid isomers. Another approach, described in WO 97/18320 to Loders Croklaan B. V. uses lipases to selectively esterify 10,12-CLA and thus enrich the 9,11-CLA fraction. The above-described methods, however, do not typically allow for the production of high purity, single isomer CLA, and if single isomer production is achieved on a large scale level, such a process is expected to be expensive.
One method of overcoming the shortcomings of chemical transformation is a whole cell transformation or an enzymatic transformation of linoleic acid, linolenic acid or their derivatives to CLA. It is well known that a biological system can be an effective alternative to chemical synthesis in producing a desired chemical compound where such a biological system is available. The existence of linoleate isomerase enzyme to convert linoleic acid to CLA has been known for over thirty years, however, no one has yet successfully isolated the enzyme. And because it has not yet been isolated, the linoleate isomerase enzyme has not been sequenced.
In many microorganisms, the linoleate isomerase enzyme converts linoleic acid to CLA as an intermediate in the biohydrogenation step. Kepler and Tove have identified this enzyme in
Butyrivibrio fibrisolvens
(Kepler and Tove,
J. Biol. Chem
., 1966, 241, 1350). However, they could not solubilize the enzyme; i.e., they were unable to isolate the enzyme in any significantly pure form (Kepler and Tove,
J. Biol. Chem
., 1967, 242, 5686). In addition, earlier studies have indicated that only compounds which possess a free carboxyl group and a cis-9,cis-12 double bond moieties are isomerized by linoleate isomerase. See Kepler and Tove,
Methods in Enzymology
, 1969, 14, 105-109, and Kepler et al.,
J. Biol. Chem
., 1970, 245, 3612.
Another research group, Park and colleagues, published an article in
J. Food Science Nutrition
(Vol. 1: 244-251, 1996), describing the purification of a protein which Park et al. believed to be the
Butyrivibrio fibrisolvens
linoleate isomerase. However, based on the initial characterization of the enzyme's activity by Kepler and Tove (see above) and the present inventors' purification, sequencing and characterization of three demonstrated linoleate isomerases, the present inventors believe that it is very unlikely that the protein that was purified and described by Park et al. is actually a linoleate isomerase. More particularly, it is well established in the art that for successful purification of particulate enzymes, such enzymes must first be converted into a soluble form. Although Park et al. demonstrate that the
Butyrivibrio fibrisolvens
linoleic acid isomerase is membrane bound, Park et al. describe no such solubilization of the enzyme. Instead, an isolated protein pellet was simply resuspended in phosphate buffer, a procedure that will generally not solubilize any membrane protein, and therefore raises significant doubts about the described purification, particularly in view of previously described purification attempts by Kepler and Tove (
J. Biol. Chem
. 242:5686-5692, 1967). Indeed, as discussed above, Kepler and Tove had described their extensive but unsuccessful efforts using well accepted solubilization methods (e.g., chelators, organic solvents, high salt, detergents) to attempt to solubilize the isomerase. Furthermore, in contrast to the 19 kD molecular weight of the putative isomerase that was eventually reported by Park et al., the main isomerase activity eluted quite early from the column during purification, indicating an apparent molecular weight of several hundred kD, and not 19 kD. When this initial material was applied to a phenyl sepharose 4B column, multiple broad peaks of activity were observed. This is not typical, and again indicates that the isomerase preparation was heterogeneous, had not been solubilized properly, and was undoubtedly associated with other membrane proteins. One of these activity peaks was then applied to a Superose 6 gel filtration column, yielding a single 19 kD band on gel electrophoresis. Finally, this sample was assayed by Park et al. for isomerase activity using HPLC, which is not appropriate for detection of CLA, since it does not resolve th
Deng Ming-De
Grund Alan D.
Rosson Reinhardt A.
Arkion Life Sciences LLC
Prouty Rebecca E.
Sheridan & Ross P.C.
Steadman David J
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