Guava (Psidium guajava) 13-hydroperoxide lyase and uses thereof

Details Guava (Psidium guajava) 13-hydroperoxide lyase and uses thereof Guava (Psidium guajava) 13-hydroperoxide lyase and uses thereof

1998-05-13

2001-03-13

Stole, Einar (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Lyase

C435S252300, C435S252330, C435S254110, C435S254200, C435S325000, C435S410000, C435S320100, C536S023200, C536S023600, C536S023100

Reexamination Certificate

active

06200794

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to fatty acid 13-hydroperoxide lyase protein from guava (
Psidium guajava
) and the gene encoding the protein. The present invention also relates to the means for expressing guava 13-hydroperoxide lyase and methods of using guava 13-hydroperoxide lyase in the field of organic synthesis.
2. Background Art
Green notes, which include n-hexanal, hexan-1-ol, 2(E)-hexen-1-al, 2(E)-hexen-1-ol and 3(Z)- hexen-1-ol (also known as pipol), are used widely in flavors, particularly fruit flavors, to impart a fresh green character. Furthermore, green notes are essential for fruit aroma and are used extensively in the aroma industry. The demand for natural green notes has grown to exceed their supply from traditional sources such as mint (
Mentha arvensis
) oil. This has motivated research efforts toward finding alternative natural ways of obtaining these materials.
The synthesis of green note compounds starts from free (polyunsaturated) fatty acids such as linoleic (9(Z), 12(Z)-octadecadienoic) and &agr;-linolenic (9(Z), 12(Z), 15(Z)-octadecatrienoic) acids. In nature, these acids are released from cell membranes by lipolytic enzymes after cell damage. Fatty acid 13-hydroperoxides are formed by the action of a specific lipoxygenase (13-LOX) and are subsequently cleaved by a specific 13-hydroperoxide lyase (13-HPOL) into a C-6 aldehyde and a C-12 &ohgr;-oxoacid moiety. The aldehydes can subsequently undergo thermal isomerization and/or be reduced by dehydrogenase enzymes to give the other C-6 products (i.e., green notes) mentioned above (Hatanaka, 1993; Hatanaka, et al., 1987).
The enzyme 13-HPOL has proven difficult to study because it is membrane bound and is present in only small quantities in plant tissue. It was identified for the first time in banana fruits (Tressl and Drawert, 1973) and was subsequently studied in a number of different plant materials, including watermelon seedlings (Vick and Zimmerman, 1976), apple and tomato fruits (Schreier and Lorenz, 1982), tomato leaves (Fauconnier et al., 1997), cucumber seedlings (Matsui, et al, 1989), and soybean seedlings (Olias et al., 1990). The enzyme has been purified to apparent homogeneity from tea leaves (Matsui et al., 1991) and, more recently, from green bell pepper fruits (Shibata et al., 1995), tomato leaves (Fauconnier et al., 1997), and banana (European Patent Application, Publication No. EP 0801133 A2). The various characteristics of 13-HPOLs that have been studied are summarized in Table 1.
TABLE 1
Summary of the Properties of 13-HPOL from Different Sources
Native
Enzyme
Mass
Sub-Unit
pH
Source
(kD)
Structure
Structure
Optimum
pI
Cucumber
—
—
—
8.0
—
Green
170
55
Trimer
—
—
pepper
Soybean
240-260
62
Tetramer
6.0-7.0
—
seedlings
Tea leaves
—
53 and 55
—
7.5
—
Tomato
200
—
—
5.5
5.8-6.1
fruits
Watermelon
>250 
—
—
6.0-6.5
—
Tomato
216
73
Trimer
7.0
4.9
leaves
Guava has recently been identified as an excellent source of freeze-stable 13-HPOL for use in this synthetic pathway. Guava 13-HPOL is currently used in a industrial process for the production of green notes (U.S. Pat. No. 5,464,761). In this process, a solution of the required 13-hydroperoxides is made from linoleic or linolenic acid (obtained from sunflower and linseed oils, respectively) using freshly prepared soybean flour as a source of 13-LOX. This solution is then mixed with a freshly prepared puree of whole guava (
Psidium guajava
) fruit, as the source for 13-HPOL. The aldehyde products are then isolated by distillation. When the alcohols are required, fresh baker's yeast is added to the hydroperoxide solution before it is mixed with the guava puree. This yeast contains an active alcohol dehydrogenase enzyme that reduces the aldehydes as they are formed by 13-HPOL.
There are a number of disadvantages to this industrial process. The principal disadvantage is the requirement of large quantities of fresh guava fruit. Such a requirement means that the process has to be operated in a country where fresh guava fruit is cheaply and freely available. Even when such a site is found, availability is limited to the growing season of the fruit. Good quality guava fruit, for example, is only available for ten months of the year in Brazil.
A second disadvantage is that the desired enzyme activities are rather dilute in the sources employed. This means that relatively large amounts of soy flour (5%), guava puree (41%) and yeast (22%) have to be used in the process. The large volumes of these crude materials that are required for industrial production place physical constraints on the yields of green notes that can be achieved.
A third disadvantage is that it is a large-volume batch process, which, by its nature, does not make maximum use of the 13-HPOL enzyme's catalytic activity, is relatively labor intensive and generates a large amount of residual organic material. The residual organic material must subsequently be transported to a compost farm or otherwise discarded.
The present invention overcomes these limitations and disadvantages related to the source of guava 13-HPOL by providing purified and recombinant guava 13-HPOL proteins, nucleic acids, expression systems, and methods of use thereof
SUMMARY OF THE INVENTION
The present invention provides a fatty acid 13-hydroperoxide lyase (13-HPOL) and a nucleic acid encoding the lyase. In particular, it provides a guava-derived protein having 13-hydroperoxide lyase function and a nucleic acid encoding such protein. The present invention further provides a nucleic acid which specifically hybridizes with the nucleic acid encoding guava 13-hydroperoxide lyase under stringent conditions and which does not hybridize at the same stringent conditions to the nucleic acid encoding green pepper or banana 13-hydroperoxide lyase.
The present invention also provides means for expressing recombinant 13-hydroperoxide lyase. Specifically, a vector for the expression of a guava 13-hydroperoxide lyase comprising the nucleic acid of the present invention and cells containing the exogenous nucleic acid of the present invention are provided. Also provided is a method of expressing the recombinant protein produced by the transformed cells comprising optimizing active lyase function of the recombinant protein.
The present invention further provides methods of using recombinant 13-hydroperoxide lyase. Specifically, the present invention provides a method of cleaving a 13-hydroperoxide of linoleic acid into a n-hexanal and a C
12
-oxocarboxylic acid. Also provided is a method of preparing n-hexanal, 3-(Z)-hexen-1-al, 2-(E)-hexen-1-al, or their corresponding alcohols from 13-hydroperoxy-octadeca-9,11-dienoic acid or 13 hydroperoxy-octadeca-9,11,15-trienoic acid.


REFERENCES:
patent: 5464761 (1995-11-01), Muller, et al.
patent: 0801133 A2 (1997-10-01), None
patent: WO 00/00627 (2000-01-01), None
Fauconnier, M.L., Perez, A.G., Sanz, C., Marlier, M. (1997). Purification and Characterization of Tomato Leaf (Lycopersicon esculentumMill.) Hydroperoxide Lyase.J. Agric. Food Chem.45:4232.
Matsui K., Shibata Y., Kajiwara, T. and Hatanaka A. (1989). Separation of 13 and 9-hydroperoxide lyase activities in cotyledons of cucumber seedlings.Z. Naturforsch.44c:883-885.
Matsui K, Toyota H., Kajiwara T., Kakuno T. and Hatanaka A. (1991). Fatty acid hydroperoxide cleaving enzyme, hydroperoxide lyase, from tea leaves.Phytochemistry30:2109-2113.
Matsui K., Shibutani M., Hase T., and Kajiwara T. Bell Pepper Fruit Fatty Acid Hydroperoxide Lyase is a Cytochrome P-450 (CYP74B).FEBS Lett.394:21-24 (1996).
Olias J.M., Rios J.J., Valle M., Zamora R., Sanz L.C. and Axelrod B. (1990). Fatty acid hydroperoxide lyase in germinating soybean seedlings.J. Agric. Food Chem.38:624-630.
Schreier P. and Lorenz G. (1982). Separation, partial purification and characterization of a fatty acid hydroperoxide cleaving enzyme from apple and tomato fruits.Z. Naturforsch.37c:165-173.
Shibata Y., Matsui K, Kajiwara T. and Hatanaka, A. (1995). Purification and properties of fatty acid hydroperoxide l

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