Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1998-12-23
2004-06-01
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S176000, C435S177000, C435S233000
Reexamination Certificate
active
06743609
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an isolated linoleate isomerase enzyme, an nucleic acid molecule encoding a linoleate isomerase enzyme, to immobilized cells containing a linoleate isomerase enzyme, and to a method for converting linoleic acid or linolenic acid to CLA 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. 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. None of the above-described methods, however, allow for the production of high purity, single isomer CLA.
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 activity, 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.
Therefore, there remains a need for purifying and identifying a linoleate isomerase enzyme and/or producing one by recombinant techniques. There also remains a need for finding and identifying an linoleate isomerase enzyme which does not require presence of a free carboxylic acid group in the fatty acid for isomerization. In addition, there remains a need for a method for producing CLA utilizing whole cells or isolated linoleate isomerase enzyme.
SUMMARY OF THE INVENTION
The present invention generally relates to isolated linoleate isomerase nucleic acid molecules, isolated linoleate isomerase proteins, immobilized bacterial cells having a genetic modification that increases the action of linoleate isomerase, and methods of using such nucleic acid molecules, proteins and cells to produce CLA.
One embodiment of the invention relates to an isolated linoleate isomerase. Included in the invention are linoleate isomerases from Lactobacillus, Clostridium, Propionibacterium, Butyrivibrio and Eubacterium, and particularly, from
Lactobacillus reuteri, Clostridium sporogenes, Propionibacterium acnes, Butyrivibrio fibrisolvens, Propionibacterium acidipropionici, Propionibacterium freudenreichii
and
Eubacterium lentum.
Particularly preferred linoleate isomerases include linoleate isomerases from
Lactobacillus reuteri, Clostridium sporogenes,
and
Propionibacterium acnes.
In one embodiment, an isolated linoleate isomerase of the present invention converts linoleic acid and linolenic acid to CLA, including (cis, trans)-9,11-linoleic acid and/or (trans, cis)-10,12-linoleic acid. A linoleate isomerase of the present invention includes linoleate isomerases having one or more of the following biochemical characteristics: a size of about 50 kDa or about 67 kDa; an optimum pH of about 6.8 or about 7.5; a specific linoleic acid isomerization activity of at least about 1000 nmoles mg
−1
min
−1
; a Km of about 8.1 &mgr;M for linoleic acid, a pH optimum of about 7.5, and a Ki of about 80 &mgr;M for oleic acid; and/or an initial velocity that decreases at about 60 &mgr;M linoleic acid. A linoleate isomerase of the present invention can be either a membrane bound or a soluble enzyme. The linoleate isomerase of the present invention can include lipid material.
In another embodiment, an isolated linoleate isomerase of the present invention includes an amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the complement of a sequence selected from the group of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:26. In another embodiment, the isolated linoleate isomerase is encoded by a nucleic acid molecule which includes a nucleic acid sequence having at least 24 contiguous nucleotides having 100% identity with nucleic acid sequence SEQ I
Deng Ming-De
Grund Alan D.
Rosson Reinhardt A.
Sanchez-Riera Fernando
Arkion Life Sciences LLC
Prouty Rebecca E.
Sheridan & Ross P.C.
Steadman David J
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