Process for the preparation of nootkatone by laccase catalysis

Details Process for the preparation of nootkatone by laccase catalysis Process for the preparation of nootkatone by laccase catalysis

1999-09-08

2001-03-13

Padmanabhan, Sreeni (Department: 1621)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing oxygen-containing organic compound

C435S254100, C568S342000, C568S346000, C568S354000, C568S356000, C568S361000

Reexamination Certificate

active

06200786

ABSTRACT:

FIELD OF THE INVENTION
The invention is directed generally to production of natural flavors, and specifically to production of natural flavors by laccase catalysis.
BACKGROUND OF THE INVENTION
The food and beverage industries, as well as other industries such as the cosmetic industry and the health care industry, routinely use flavors to enhance their products' appeal. Natural flavors are especially desired by consumers for reasons of health, fitness, safety, aesthetics, etc. Worldwide consumer demand for natural flavors thus continues to increase. Natural flavors or natural flavorings have been defined at 21 C.F.R. §101.22 as the essential oil, oleoresin, essence or extractive, protein hydrolysate, distillate, or any products of roasting, heating or enzymolysis which contains the flavoring constituents derived from a spice, fruit or fruit juice, vegetable or vegetable juice, edible yeast, herb, bark, bud, root, leaf or similar plant material, meat, seafood, poultry, eggs, dairy products or fermentation products thereof, whose significant function in food is flavoring rather than nutrition.
One such flavor is nootkatone (4,4a,5,6,7,8-hexahydro-6-isopropenyl-4,4a-dimethyl-2(3II)-naphtalenone). Nootkatone is an important flavor constituent of grapefruit and is used commercially to flavor soft drinks and other beverages, as well as being used in perfumery. The conventional method for nootkatone preparation is by oxidation of valencene. However, nootkatone prepared by standard chemical oxidation methods cannot be considered a natural flavorant and thus has less commercial desirability. Additionally, the starting material valencene is expensive and thus methods that consume valencene are less commercially acceptable. Because of these drawbacks, there is still a need for commercially feasible methods to prepare natural nootkatone.
Several methods exist for the production of nootkatone, but each has limitations. For example, U.S. Pat. No. 5,847,226 discloses nootkatone production by oxidizing valencene to nootkatone, nootkatol or mixtures of nootkatone and nootkatol in the presence of an unsaturated fatty acid hydroperoxide. However, in the '226 patent the fatty acid hydroperoxide is generated by autooxidation, photooxidation, chemically catalyzed oxidation or by enzymatic oxidation using lipoxygenase, and then the fatty acid hydroperoxide catalyzes the autooxidation of valencene. Such an autooxidization is generally not a selective process. Previous attempts to use microbial or enzymatic transformations to prepare nootkatone have generally resulted in low yields. For example, Drawert et al. (1984) reported the biotransformation of valencene to nootkatone in cell suspension cultures but the starting level of valencene was too low to be practical. del Rio et al. (1991) studied the accumulation of nootkatone and valencene by callus cultures of Citrus spp. The level of nootkatone in nine-month old callus cultures was between 16-160 &mgr;g/100 g fresh weight. More recently, a Japanese patent disclosed biotransformation of valencene to nootkatone using a selected microbial fermentation, but the overall yield was low. In shake flask experiments with 50 ml working volume, only 2.5 mg of nootkatone was obtained from 500 mg of starting valencene (Okuda et al., 1994).
Thus, a commercially viable method to produce nootkatone utilizing laccase would be desirable.
SUMMARY OF THE INVENTION
The invention is directed to a process for producing nootkatone by reacting valencene and a composition having laccase activity, with valencene at a concentration greater than 0.1%, in the presence of an oxygen source to form valencene hydroperoxide. The hydroperoxide is degraded to form nootkatone, and the nootkatone is recovered. The hydroperoxide may be degraded by heating and/or by contacting a catalyst. Laccase may be from a microbial source, e.g., the fungi
Botrytis cinerea
61-34 and/or
Trametes versicolor
, or from a recombinant DNA source. A mediator and/or a solvent may be added to the valencene and the composition having laccase activity. The solvent is added at a concentration that maintains laccase activity.
The invention is also directed to a process for producing nootkatone by reacting valencene and a composition having laccase activity, wherein valencene is at a concentration of greater than 0.1%, in the presence of an oxygen source to form valencene hydroperoxide, then degrading the hydroperoxide to form nootkatone, and recovering the nootkatone. The composition having laccase activity may be a solution having a concentration of valencene up to about 64% by weight. Alternatively, laccase may be immobilized and used to oxidize valencene in the absence of a solvent.
The invention is further directed to a process for making nootkatone by reacting valencene and a composition containing laccase from either or both of
Botrytis cinerea
61-34 or
Trametes versicolor
, with the concentration of valencene greater than 0.1%, in the presence of a mediator and an oxygen source at a pH between about pH 3-7 to form valencene hydroperoxide. The valencene hydroperoxide is degraded to form nootkatone, and the nootkatone is recovered.
The invention is still further directed to a process for producing valencene hydroperoxide. Valencene and a composition having laccase activity are reacted in the presence of an oxygen source under conditions to form valencene hydroperoxide. The starting concentration of valencene is greater than 0.1%. A mediator and/or a solvent at a concentration to maintain laccase activity may be added.
The invention is additionally directed to a process for producing nootkatone by reacting valencene and immobilized laccase in the presence of an oxygen source to form valencene hydroperoxide, heating to degrade the valencene hydroperoxide to form nootkatone, and recovering the nootkatone. In this case, the concentration of valencene in the reaction may approach 100% without abolishing the laccase activity. A mediator and/or a solvent at a concentration to maintain laccase activity may be added to the valencene and the immobilized laccase.
The invention is additionally directed to a process for producing an essential oil having increased nootkatone. An essential oil containing valencene and a composition comprising laccase are reacted in the presence of an oxygen source to form a mixture of an essential oil containing nootkatone. The mixture is heated to increase the amount of nootkatone. The essential oil containing the increased nootkatone is recovered. The starting concentration of the essential oil is greater than about 0.5%. The essential oil may be orange oil, bitter orange oil, grapefruit oil, lemon oil, and/or tangerine oil. The essential oil may be fractionated, for example, to form an extract or a distillate.
An enzymatic process which can successfully produce natural nootkatone in commercially viable quantities and with relatively few byproducts is disclosed. Laccase was used to catalyze the oxidation of valencene. Laccase from
Botrytis cinerea, Trametes versicolor
or other microbial sources, as well as laccase purchased from commercial vendors and/or generated using recombinant techniques, could be used in either a reaction composition or in an immobilized form. The pH range is from pH 3 to pH 7, with pH 3.5 being optimum. One or more mediators and/or solvent at a concentration to maintain laccase activity could also be added. It was also found that the methods could be used to transform a series of natural flavor chemicals due to the broad substrate specificity of the enzyme.
The objectives and other advantages of this invention will be further understood with reference to the following figures, detailed description and examples.


REFERENCES:
patent: 4798799 (1989-01-01), Farbood et al.
patent: 5847226 (1998-12-01), Muller et al.
patent: 2181025 (1996-07-01), None
patent: 59-31728 (1982-08-01), None
patent: 6-303967 (1993-04-01), None
del Rio et al.,Accumulation of the sesquiterpenes nootkatone and valencene by callus cultures of Citrus paradisi, Citrus limonia and Ci

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