GB2501671A

GB2501671A - Smoking article

Info

Publication number: GB2501671A
Application number: GB1204124.0A
Authority: GB
Inventors: Endry N Prasetyo; Yahia Lemmouchi; Georg Guebitz; Gibson S Nyanhongo
Original assignee: British American Tobacco Investments Ltd; British American Tobacco Co Ltd
Current assignee: British American Tobacco Investments Ltd
Priority date: 2012-03-08
Filing date: 2012-03-08
Publication date: 2013-11-06
Also published as: GB2501671A8; GB201204124D0

Abstract

A smoking article, the article comprises an oxidase enzyme. Preferably one or more redox mediators of an enzyme-redox mediator system are incorporated into the smoking article in addition to one or more co-substrates of the oxidase enzyme. The oxidase enzyme may be immobilized with respect to a solid support. Preferably, the oxidase enzyme is incorporated into a filter or filter element of the smoking article and may be contained inside an additive release component such as a breakable capsule. Preferably, the oxidase enzyme removes one or more chemical substances from tobacco smoke without modifying the concentration of nicotine contained in the tobacco smoke. The oxidase enzyme may be a laccase enzyme and may be derived from tobacco. Advantageously the smoking article is capable of removing chemical substances from an aerosol generated by the smoking article.

Description

Smoking Article

Field of the Invention

The present invention relates to the incorporation of enzymes into smoking artides and particularly, although not exclusively, to smoking articles capable of removing chemical substances from an aerosol generated by the smoking article.

Background

As used herein, the term "smoking article" includes smokeable products such as io cigarettes, cigars and cigarillos whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco or tobacco substitutes and also heat-not-burn products (i.e. products in which flavour is generated from a smoking material by the application of heat without causing combustion of the material) and other articles capable of generating tobacco derived aerosols. Typically, smoking articles are provided with filters for removing constituents from the gaseous flow.

The tobacco smoke inhaled by the user of a smoking artide consists of many different chemical substances and it is often desirable for one or more of these chemical substances to be removed. Therefore, there are various strategies that have been developed for this purpose.

For example, the removal of chemical substances from tobacco smoke may be achieved by chemical or physical adsorption of chemical substances to a material of the smoking article. An adsorbent material, such as activated carbon, may be incorporated into the smoking article for this purpose.

Summary

According to a first aspect, there is provided a smoking article comprising an oxidase enzyme.

The oxidase enzyme incorporated into the smoking article may directly or indirectly lead to the chemical modification and/or removal of one or more chemical substances from the tobacco smoke. One or more of these chemical substances may be undesirable for human inhalation.

The concentration of one or more chemical substances may therefore be reduced in the tobacco smoke inhaled by the user of the smoking article.

Different oxidase enzymes catalyse the chemical reaction of their substrate by utilizing different reaction mechanisms. In some embodiments, the oxidase enzyme incorporated into the smoking article catalyses the oxidation of its substrate via a one-electron oxidation mechanism. Such an enzyme may be a laccase enzyme.

In some embodiments, one or more redox mediators of an enzyme-redox mediator io system are incorporated into the smoking article in addition to the oxidase enzyme.

In some embodiments, one or more co-substrates of an oxidase enzyme are incorporated into the smoking article in addition to the oxidase enzyme.

The oxidase enzyme may be immobilized with respect to a solid support in the smoking artide, wherein the oxidase enzyme is bonded to, or trapped by, said solid support.

Preferably, the oxidase enzyme is incorporated into a filter or filter element of the smoking article.

The oxidase enzyme may be contained inside an additive release component. The additive release component may be a breakable capsule.

Preferably, the oxidase enzyme removes one or more chemical substances from the tobacco smoke without modiing the concentration of nicotine contained in the tobacco smoke.

The oxidase enzyme may be derived from a range of different sources. For example, the oxidase enzyme may bc dcrivcd from tobacco.

According to a second aspect, an oxidase enzyme is used to direcfly or indirecfly ead to the chemical modification and/or removal of one or more chemical substances from the tobacco smoke of a smoking artide, wherein one or more of these chemical substances are undesirable for human inhalation.

According to a third aspect, there is provided a filter or filter element that comprises an oxidase enzyme.

Brief Description of the Drawings

Embodiments of the invention will now be described, by way of example only, with reference to accompanying drawings, in which: Figure 1 shows the UV-Vis absorption spectra collected for three different nicotine-containing samples; I0 Figure 2 features the HPLC chromatogram obtained for two nicotine-containing samples; Figure 3 shows the mass spectrum obtained by HPLC-MS following the incubation of a sample containing nicotine in the presence of thccase enzymes; Figure 4 ilbistrates seven graphs, each of which shows how the concentration of dioxygen was found to decrease when one of seven different compounds were separately exposed to laccase enzymes; Figure 5 depicts a graph of reaction rate as a function of substrate concentration for the laccase-catalysed oxidation of hydroquinone, which follows Michaelis-Menten kinetics; Figure 6 shows the HPLC-MS spectrum obtained after the incubation of methylhydroquinone and 3-aminobiphenyl in the presence of laccase enzymes. Also pictured is the molecule which would be formed by a coupling reaction between oxidised methylhydroquinone and 3-aminobiphenyl; Figure 7 shows the HPLC-MS spcctrum obtained after the incubation of hydroquinone and benzo[a]pyrene in the presence of thccase enzymes. Also pictured is the molecule which woifid be formed by a coupling reaction between oxidised hydroquinone and benzo[a]pyrene; Figure 8 features the HPLC-Uvchromatograms obtained following the separate incubation of three different non-substrate compounds in the presence of laccase enzymes and hydroquinone; and Figure 9 shows a graph of formaldehyde consumption as a function of time for samples containing different size fractions of lignosulfate compounds.

Detailed Description

One or more oxidase enzymes may be incorporated into a smoking article and each of these oxidase enzymes may lead to the removal of one or more of the chemical substances contained in tobacco smoke.

Preferably, when an oxidase enzyme is incorporated into the smoking article it has an io activity between 10 -10,000 Units/mi, more preferably 10 -5,000 Units/mi, and most preferabiy 10 -1,000 Units/mi.

Nicotine is a constituent of tobacco smoke that often contributes to the positive experience had by the user of a smoking article, so it may be not desirable for the oxidase enzyme to remove this m&ecu e. Preferably, nicotine is not one of the chemical substances removed by the oxidase enzyme. In contrast, it is desirable for the oxidase enzyme to remove chemical substances which do not contribute to the positive experience had by the user of a smoking article, and which may potentially have deleterious effects towards human health. One or more of the chemical substances removed from tobacco smoke by the oxidase enzymes may be undesirable for human inhalation.

Oxidase enzymes may be incorporated into any part of the smoking article, for instance those parts exposed to smoke or another aerosol produced by the smoking article, and are preferably incorporated so that they are able to lead to the removal of one or more chemical substances from tobacco smoke or other aerosol generated during use of the smoking article. They may be stored on the outer surface of, and/or contained inside, the smoking article. Preferably, the oxidase enzymes are incorporated into a filter or ffltcr clcmcnt of thc smoking articlc.

The oxidase enzymes incorporated into the smoking artide are preferably immobilized.

The oxidase enzymes may be immobilized, for exampie with respect to part of the smoking article, such as a solid support in the smoking article. The enzymes maybe immobilized by any suitable mechanism of immobilization, so that the oxidase enzymes are bonded to, or trapped by, a part or component of the smoking article. Suitable mechanisms of immobilization include, but are not limited to: chemisorption (covalent or ionic bonding), physisorption (van der Waals bonding), supramolecular entrapment (physical trapping of enzymes on the molecular scale), and membrane confinement (confinement of enzymes inside an enzyme-impermeable membrane).

Preferably, the oxidase enzymes are immobilized by covalent chemisorption. The enzymes are consequently likely to be more exposed to their substrates than they would be if immobilized by supramolecular entrapment or membrane confinement. Covalent bonding results in particularly strong attachment of the enzymes for the purpose of immobilization, making it less likely that the enzymes will become detached, and less io likely that the enzymes will be inhaled during use of the smoking article.

The solid support with respect to which the enzymes may be immobilized may be a polymer, which may be cross-linked. Preferably, the enzymes are immobilized with respect to a solid support in a filter or filter element of the smoking article, and most preferably, the enzymes are immobilized with respect to cefitilose acetate in a filter or filter element of the smoking artide.

A cross-linker may be utilized to immobilize the enzymes with respect to the solid support, wherein a cross-linker is a molecule that is attached to the solid support which provides the necessary chemical functionality to immobilize the enzymes by any of the aforementioned mechanisms of immobilization. This can be particularly useful when the solid support does not comprise chemical functionality to immobilize the enzymes itself. Preferably, the cross-linker is covalently attached to the solid support and covalently attached to the enzymes.

Suitable cross-linkers for covalently bonding to the solid support and to the enzymes include, but are not limited to: glutaraldehyde; molecules comprising a glyoxyl functional group; and molecules comprising an anhydride functional group, such as 2-mcthylmalcic anhydride and polyalkencoxide-co-malcic anhydridc.

Cross-Bnkers can be particularly useful for immobilizing enzymes with respect to ceflullose acetate. This is because cellifiose acetate does not have very reactive functionality for immobilizing the enzymes itseff. Preferably, the cross-Unker utilized to immobilize enzymes with respect to cellulose acetate is covalently bonded to cellulose acetate and covalently bonded to the enzymes.

A suitable cross-linker maybe bonded to cellulose acetate and bonded to the immobilized enzymes using any suitable series of chemical reactions. For example, ceflulose acetate may first undergo activation reaction(s), which provide suitably reactive functiona' groups on the p&ymer m&ecule for covalently bonding to the cross-linker molecule. These functional groups may then covalently bond to the cross-linker, which is itself attached to the enzymes for immobilization. Activation of cellulose acetate may also provide suitably reactive functional groups for directly bonding to the enzymes without a cross-linker.

io There are many different ways in which cellulose acetate may be activated for covalent couphng. For example, the p&ymer may first undergo partial deacetyhution before undergoing oxidation. Partial deacetylation results in at least a proportion of the acetyl groups of the molecule being reduced under any suitable reducing conditions to hydroxyl groups. These hydroxyl groups may then undergo chemoselective oxidation in the presence of any suitable oxidising agent, such as a periodate salt, to form aldehyde groups. These aldehyde groups are highly electrophilic and so prime the m&eciile for covalently coupling to a cross-linker or enzyme that comprises sufficiently nucleophihc functionality to react with the aldehyde. The aldehyde groups may react with an amine of the cross-linker or enzyme to form an imine bond, for example, which may be reduced to an amine if a more stable covalent bond is required.

The oxidase enzymes may be contained inside an additive release component incorporated into the smoking article, wherein an additive release component is anything which is capable of retaining an oxidase enzyme and releasing it as and when desired. The additive release component may be a breakable capsule, and preferably, the additive release component is a breakable capsule and is positioned in a filter or filter element of the smoking article.

An oxidase enzyme incorporated into a filter or filter clement of a smoking article is able to remove one or more chemical substances from the mainstream tobacco smoke as it is drawn through the smoking article by the user. This is surprising since tobacco smoke passes through the filter or filter eh�^ment of a smoking article with such high speed. Importanfly, this resdts in a very short time during which the chemical substances of the tobacco smoke are exposed to the oxidase enzymes; in fact, the residence time for smoke in a typical 27 mm-long cigarette filter, during standard measurements of tar content, is of the order of milliseconds. Yet, despite this, it has been found that chemical substances may be removed from mainstream tobacco smoke during use of a smoking article comprising oxidase enzymes. Moreover, it has been found that chemical substances that are not direct substrates of the incorporated oxidase enzymes maybe removed.

An oxidase enzyme may be incorporated into the smoking article as part of a composition comprising additional chemical substances. These additional chemical substances may be added for a variety of different purposes, which include, but are not limited to: enhancing the stability of the native state of the oxidase enzyme, providing io conditions which promote catalysis by the oxidase enzyme, and modifying the composition of the tobacco smoke. For example, the composition may comprise a deodoriser, a diluent, an adsorbent, or any other substance that is capable of modifying the tobacco smoke. Where local regulations permit, the composition may comprise a flavourant, such as menthol.

As used herein, the term "flavour", "flavouring", and "flavourant" refer to materials which, where local regifiations permit, may be used to create a desired taste or aroma.

Preferred flavourants include extracts (e.g., licorice, hydrangea, Japanese white bark magnolia leaf, chamomile, fenugreek, clove, menthol, Japanese mint, aniseed, cinnamon, herb, wintergreen, cherry, berry, peach, apple, Drambuie, bourbon, scotch, whiskey, spearmint, peppermint, lavender, cardamon, celery, cascarilla, nutmeg, sandalwood, bergamot, geranium, honey essence, rose oil, vanilla, lemon oil, orange oil, cassia, caraway, cognac, jasmine, ylang-ylang, sage, fennel, piment, ginger, anise, coriander, coffee, or a mint oil from any species of the genus Mentha), flavour masking agents, bitterness receptor site blockers, receptor site enhancers, sweeteners (e.g., sucralose, acesulfame potassium, aspartame, saccharine, cyclamates, lactose, sucrose, glucose, fructose, sorbitol, or mannitol), and other additives such as chlorophyll, minerals, botanicals, or breath freshening agents. They may be imitation, synthetic or natural ingredients or blends thereof.

The flavour may be a tobacco flavour. Where the flavour is dehvered in liquid form the tobacco flavour could be derived from tobacco extract. Where the flavour is derived from a solid product, the product co&d be tobacco leaf in shredded, partic&ate or granular form, or in the form of reconstituted tobacco sheet material.

The Enzyme Commission (EC) numbers referenced throughout the specification are used to classify enzymes based on the chemical reactions they catalyse. The EC number for each enzyme-catalysed chemical reaction can be found in "Enzyme Nomenclature", which was published in 1992 for the International Union of Biochemistry and Molecular Biology (IUBMB) by Academic Press, Inc. An oxidase enzyme is an oxidoreductase (EC 1): it catalyses a redox chemical reaction.

More specifically, an oxidase enzyme catalyses a redox reaction in which the substrate of the enzyme is the electron donor and dioxygen is the electron acceptor in the redox reaction.

An oxidase enzyme incorporated into the smoking article can directly or indirectly remove a chemical substance from the tobacco smoke. Direct removal: the chemical substance is removed because it is a substrate of the oxidase enzyme and may undergo enzyme-catalysed oxidation. Indirect removal: the chemical substance removed does not act as a substrate of the enzyme but instead undergoes a non-enzyme-catalysed reaction with a product of enzyme catalysis.

One way in which an oxidase enzyme can indirectly remove a chemical substance from tobacco smoke is by first catalysing the oxidation of its substrate, before the product of this reaction undergoes non-enzyme-catalysed reactions with non-substrates of the oxidase enzyme in tobacco smoke. In this case, such a substrate which undergoes enzyme-catalysed oxidation before undergoing a non-enzyme-catalysed reaction with one or more further compounds may be referred to as a "co-substrate".

One or more chemical substances contained in the tobacco smoke may act as co-substrates. Alternatively or in addition, one or more chemical substances may be added to the smoking article which can act as co-substrates of the incorporated oxidase enzyme. The addition of co-substrates to the smoking article may facilitate the removal of a larger number of chemical substances from the tobacco smoke, since the co-substrates may undergo enzyme-catalysed oxidation before the formed products may undergo non-enzyme-catalysed reactions with chemical substances contained in the tobacco smoke.

Preferably, the oxidase enzyme incorporated into the smoking article catalyses the oxidation of its substrate by a one-electron oxidation mechanism. In a one-electron oxidation mechanism a single electron is abstracted from the enzyme's substrate, and the removal of a single electron is likely to result in the formation of a radical. The formation of a radical by an oxidase enzyme can lead to the indirect removal of a particularly arge number of chemical substances from the tobacco smoke, and it is for this reason that it is advantageous to incorporate an oxidase enzyme that catalyses the oxidation of its substrate by a one-electron oxidation mechanism.

The radical may be formed by the enzyme-catalysed oxidation of a co-substrate of the enzyme or a redox mediator of the enzyme, for example. I0

A co-substrate which forms a radical through enzyme catalysis is likdy to resifit in the reaction and removal of a large number of non-substrate chemical substances from tobacco smoke. The formed radical is likely to be thermodynamically and kinetically unstable, and so is likely to react with a large number of different chemical substances in tobacco smoke during use of the smoking artide. Furthermore, when a radical does react it often forms another radical in a propagation step, which can itself react to form another radical, and so on. Many iterations of these non-enzyme-cat&ysed reactions may take place, which can result in a "cascade" of reactions, leading to the removal of a large number of chemical substances from the tobacco smoke and the formation of a range of different products. In some cases, a sequence of these reactions can result in the formation of one or more (co-)oligomer or (co)polymer molecules.

A redox mediator which forms a radical through enzyme catalysis is also likely to result in the reaction and removal of a large number of non-substrate chemical substances from tobacco smoke. The formed radical of a redox mediator for an oxidase enzyme that catalyses oxidation by a one-electron oxidation mechanism has a higher redox potential than the enzyme, and so may oxidise chemical substances in the tobacco smoke for which it would not otherwise be thermodynamically favourable to undergo oxidation. Thc radical of the redox mcdiator is also likdy to be unstable, making reactions between the redox mediator radical and other chemical substances ldneticaily favoured as weff In the process of oxidising another chemical substance, the redox mediator radical wifl be reduced back to the oxidation state it had prior to undergoing enzyme-catalysed oxidation. The redox mediator is thus recycled and returns to the chemical form in which it is a substrate of the oxidase enzyme. The above-detailed enzyme-catalysed and -10-non-enzyme-catalysed reactions may therefore proceed iteratively as the redox mediator shuttles between its oxidised and reduced states, and potentially lead to the removal of a particularly large number of chemical substances from the tobacco smoke.

A system comprising an oxidase enzyme that catalyses oxidation by a one-electron oxidation mechanism, a redox mediator, and a chemical substance which the redox mediator can oxidise, may be referred to as an enzyme-redox mediator system. One or more chemical substances contained in the tobacco smoke may act as redox mediators in an enzyme-redox mediator system. Alternatively or in addition, one or more io chemical substances maybe added to the smoking article which can act as redox mediators. The addition of these redox mediators to the smoking article may facilitate the removal of a larger number of chemical substances from the tobacco smoke.

A redox mediator and/or co-substrate incorporated into the smoking article may be obtained by isothtion from a natural source, by isothtion from a natural source followed by chemical modification, or by chemical synthesis.

There are many types of oxidase enzyme that may catalyse a redox reaction by a one-electron oxidation mechanism. Some examples are: laccases (EC 1.10.3.2), bilirubin oxidases (EC 1.3.3.5), and catechol oxidases (EC 1.10.3.1). Preferably, laccase enzymes are incorporated into the smoking article. Advantageously, laccase enzymes often have low substrate-specificity and thus may catalyse the oxidation of many different chemical substances by a one-electron oxidation mechanism.

Laccases are copper-containing oxidase enzymes that may be derived from many different sources, such as plants, bacteria and fungi (including filamentous fungi and yeast). There are many known examples of genera of living organism which comprise species from which suitable laccase enzymes may be derived.

There are many known examples of genera of fungus which comprise species from which suitable laccase enzymes may be derived. These include, but are not Umited to: Aspergillus, New'ospora (e.g. N. crassa), Podospora, Botrytis (e.g. B. cinerea), collybia, Fomes, Lentinus, Plew'otus, Trametes (e.g. T. hirsuta, T. modesta T. vi/Losa and T. versicolor), Rhizoctonia (e.g. R. soLani), coprinus, (e.g. C. plicatiLis and C. cinereus), Psatyrella, Myceliophthora (e.g. M. thermophila), Scierotium, ScytaLidium (e.g. S. thermophilum), Polyporus (e.g. P. pinsitus) , Phiebia (e.g. P. radita), Coriolus, (e.g. C. hi rsutus) , Rhus (e.g. R. vernicjfera), Pyricularia, CorioLopsis, and Pyenoporus -11 - (e.g. P. cinnabarious). In particular, a suitable laccase enzyme may be derived from a species of the fungus genera Trametes, Myceliophthora, Scytalidium or Polyporus.

There are many known examp'es of genera of bacteria which comprise species from which suitable laccase enzymes may be derived. These include, but are not limited to: Azospirillurn, Bacillus, Pseudomonas, and Streptomyces.

In some embodiments, the laccase enzyme incorporated into the smoking article is derived from tobacco. I0

The optimum temperature at which a laccase enzyme catalyses the oxidation of its substrate varies from one laccase enzyme to another. The optimum temperature for many different laccase enzymes has been investigated in the art, and it has been found that the optimum temperature for laccase-enzyme catalysis usually falls within the range 40°C -80°C. This is advantageous since the temperature which a laccase enzyme is exposed to in the filter or filter dement of a smoking article, where the laccase enzyme is preferab'y incorporated, is Bkdy to be the same as, or sbghtly higher than, ambient temperature during use of the smoking article. An incorporated laccase enzyme is therefore unlikely to undergo denaturation during use of the smoking article.

In preferred embodiments, a laccase enzyme incorporated into the smoking article has an optimum temperature that is close to, or equal to, the temperature which the laccase enzyme is exposed to in the smoking article. In a filter or filter element of the smoking article, where the laccase enzyme is preferably incorporated, the temperature is likely to be the same as, or slightly higher than, ambient temperature. Therefore, a laccase enzyme incorporated into the smoking article preferably has an optimum temperature for catalysis that is slightly higher than, or equal to, ambient temperature.

The optimum pH at which a laccase enzyme catalyses the oxidation of its substrate varies from one thccase enzyme to another. The optimum pH for many different thccase enzymes has been investigated in the art. It has been found that the optimum p11 for a laccase enzyme derived from a species of bacteria usuafly falls within the range 7-9, and that the optimum pH for a laccase enzyme derived from a species of fungi usuay falls within the range 3-5. Advantageously, therefore, there are a number of different laccase enzymes from which to choose a laccase enzyme with a pH optimum that is close to, or equal to, the pH that the enzyme is likely to be exposed to in the smoking article.

-12 -In preferred embodiments, the laccase enzyme incorporated into the smoking article has an optimum pH that is dose to, or equal to, the pH which the laccase enzyme is exposed to in the smoking article. This can be achieved in two ways: First, a laccase enzyme may be selected for incorporation into the smoking article due to possessing a certain optimum pH. For example, if a laccase enzyme is incorporated into a smoking article in which it is exposed to an alkali pH, a laccase enzyme derived from a species of bacteria is preferred; however, if a laccase enzyme is incorporated into a smoking artide in which it is exposed to an acidic pH, a thccase enzyme derived from a species of fungi is preferred.

Alternatively or in addition, the pH to which the incorporated laccase enzyme is exposed may be controlled by suitably modiing the composition of the smoking artide. For example, the composition as part of which the enzyme may be incorporated into the smoking artide may comprise one or more acidic compounds to provide an acidic pH, or one or more alkali compounds to provide an alkali pH. Also, for examp'e, the composition of the smokeable filler material of the smoking article may be modified so that it undergoes combustion to produce a tobacco smoke with an acidic or alkali pH.

The redox potential of a laccase enzyme varies from one laccase enzyme to another. The redox potential of many different laccase enzymes has been investigated in the art, and it has been found that the redox potential (measured relative to the Normal Hydrogen Electrode [NHE]) of a laccase enzyme usually falls within the range E° = 0.45 V -o.8o V. In preferred embodiments, a laccase enzyme incorporated into the smoking article has a high redox potential, making the catalysed oxidation of a greater number of different chemical substances in tobacco smoke thermodynamically favorable, potentially resulting in the removal of a greater number of chemical substances from the tobacco smoke. Preferably, a laccase enzyme incorporated into the smoking article has a redox potential that falls within the range F° = o.6o V -0.80 V. Most preferably, a laccase enzyme incorporated into the smoking article has a redox potential that falls within the range E° = 0.70 V -0.80 V. -13 -A laccase enzyme derived from a species of fungi usually has a redox potential within the range: E° = 0.45 V -0.80 V. A laccase enzyme derived from a species of bacteria usuay has a redox potential within the tower range: E° = 0.45 V -0.54 V. Preferably, therefore, a accase enzyme incorporated into the smoking artide is derived from a species of fungi rather than a species of bacteria. Most preferably, the laccase enzyme incorporated into the smoking article is derived a species of fungi with a particularly high redox potential, such as Tram etes villosa, Pycnoporous cinnabarinus or Botrytis cinerea.

io Most substrates oflaccase enzymes are organic compounds, many of which are aromatic and often comprise an amine and/or a phend group or similar. Some substrates of laccase enzymes are often found in tobacco smoke and these chemical substances may therefore undergo enzyme-catalysed oxidation during use of a smoking article in which laccase enzymes are incorporated. Examples of chemical substances often contained in tobacco smoke that are substrates of thccase enzymes include, but are not limited to: diphenols, such as catechol, methylcatechol, hydroquinone, methylhydroquinone, and trimethylhydroquinone; methoxy-substituted monophends; aromatic amines, such as 2-napthylamine; and PolyAromatic Hydrocarbons (PARs), such 2-methylnapthalene.

One or more of the co-substrates that may be incorporated into the smoking article may be co-substrates of laccase enzymes in particular. One or more of these co-substrates may be hydroquinone and/or methylhydroquinone, which experiments have shown to be examples of effective co-substrates of laccase enzymes for the removal of chemical substances found in tobacco smoke. Hydroquinone has been shown to undergo enzyme-catalysed oxidation before coupling to benzo[a]pyrene; methylhydroquinone has been shown to undergo enzyme-catalysed oxidation before coupling to 3-aminobiphenyl. Details of supporting experimental evidence are provided below.

An enzyme-redox mediator system for a laccase enzyme is known in the art as a Laccase Mediator System (LMS), and the redox mediator of an LMS is herein referred to as an LMS mediator. One or more of the redox mediators that maybe incorporated into the smoking article may be LMS mediators.

Any suitable LMS mediator may be incorporated into the smoking article. Examples of LMS mediators include, but are not Bmited to: NO-and/or NOH-containing compounds, which may comprise a cycloaliphatic and/or heterocyclic and/or aromatic ring, such as i-hydroxybenzotriazole, N-hydroxypthalimide, TEMPO, N-hydroxyacetanifide, and Viohirie acid; hetereocyclic compounds; phenolic compounds, such as p-Coumaric acid, Ferufic acid, Sinapic acid, Acetosyringone, Syringaldehyde, and Acetovanillone; nitroso compounds; polyoxometalates; 2,4-pentanedione; phenothiazin; ABTS; and derivatives of any of the above.

An LMS mediator and/or laccase co-substrate incorporated into the smoking article may be obtained by isolation from a natural source, by isolation from a natural source io foflowed by chemical modification, or by chemical synthesis.

A radical species formed during any of the aforementioned series of chemical reactions may be detected using a variety of different analytical techniques, such as by the use of different types of spectroscopy. For example, Electron Spin Resonance (ESR) spectroscopy could be used, which is a very sensitive and higUy specific method for the detection of radicals. Alternatively or in addition, a radical species may be identified using UV-Vis spectroscopy if the TJV-Vis spectrum of the radical species can be detected and the obtained UV-Vis spectrum is distinguishable from other chemical species in the measured sample.

An oxidase enzyme incorporated into a smoking article maybe of any origin. For example, an oxidase enzyme may be derived from prokaryotic or eukaryotic organisms, such as animals, plants or micro-organisms.

The term "derived" in the context of this invention may mean that the enzyme has been isolated from a living organism, such that the amino acid sequence of the isolated enzyme corresponds to the amino acid sequence of the living organism's native enzyme.

Alternatively or in addition, the term "derived" may mean that the enzyme has been isolated from a living organism after being synthesised by protein synthesis from a DNA sequence generated by genetic recombination. In this ease, the amino acid sequence of the is&ated enzyme may be the same as that of the native enzyme or may be a modified version thereof. This modified amino acid sequence may be a fragment of the native amino acid sequence or may result from the addition, substitution or insertion of amino acids into the native amino acid sequence. The term "derived" also encompasses enzymes that have been modified in vivo, such as by the process of glycosylation or phosphorylation, or have been modified in vitro.

-15 -Oxidase enzymes may also be obtained by any suitable process of chemical synthesis.

An oxidase enzyme may be obtained from a micro-organism by using any suitable technique. For example, an oxidase enzyme may be obtained by the fermentation of a suitable micro-organism in a suitable medium before subsequent isolation of an oxidase enzyme from the sample. The oxidase enzyme may be isolated as part of a composition comprising additional chemical substances.

io The process of obtaining an oxidase enzyme from a micro-organism may first comprise the insertion of an oxidase-enzyme-coding DNA sequence into the micro-organism.

This may be achieved by the host micro-organism undergoing transformation with a suitable recombinant DNA vector, which may be a plasmid. This DNA vector may comprise DNA sequence(s) which code for the protein synthesis of the amino acid sequence of oxidase enzyme(s), a'ong with DNA sequence(s) necessary to facilitate protein synthesis of oxidase enzyme(s) in the host micro-organism. The DNA vector or parts thereof may be incorporated into the genome of the host cdl after transformation.

The DNA of the DNA vector may be of genomic, cDNA or synthetic origin or any combination of these, and maybe isolated or synthesised in accordance with methods known to the skilled person.

Experimental Work A series of experiments were conducted to investigate the effect of laccase enzymes in vitro towards certain tobacco smoke components. The disclosed experimental work is not intended to limit the scope of the invention.

There were four parts to the study: 1. Effect of Laccasc Enzymes towards Nicotine Evidence was collected to indicate whether thccase enzymes decrease the concentration of nicotine in tobacco smoke.

The concentration of nicotine was measured after a period of incubation in the presence of laccase enzymes, and the concentration of nicotine was measured after a period of incubation in the absence of laccase enzymes (control). Three analytical techniques were used to determine the concentration of nicotine following the incubation period: UV-Vis spectroscopy, HPLC and HPLC-MS.

The results collected by each anay6cal technique support the conclusion that the concentration of nicotine is not affected by the presence of laccase enzymes in vitro.

Figure 1 illustrates the UV-Vis absorption spectra collected from three different samples, which are labelled on the graph. The spectrum of each sample comprises a peak at approximately the same wavelength ( 270 nm), which corresponds to Xm for a molecule of nicotine under the adopted experimental conditions. Each spectrum has approximately the same absorbance magnitude at this wavdength, suggesting that the nicotine concentration is approximately the same in each sample, thus further suggesting that the concentration of nicotine was not affected by the presence of laccase enzymes.

Figure 2 illustrates the resdts obtained by HPLC. The same peak, which corresponds to nicotine, is observed after the incubation period for the sample comprising luccase enzymes and for the sample not comprising laccase enzymes. The height of the peak is approximately the same for each sample, suggesting that the concentration of nicotine after incubation is approximately the same in each sample, thus further suggesting that the concentration of nicotine was not affected by the presence of laccase enzymes.

Figure 3 illustrates the mass spectrum obtained by JJPLC-MS for the sample comprising nicotine and laccase enzymes, following the incubation period. The highest peak has an m/z ratio of 163.1, thus indicating that nicotine was present in high concentration in the measured sample (a single molecule of nicotine has a mass of 162.2 Da). Further support is thus provided that the concentration of nicotine was not affected by the presence of laccase enzymes.

2. Enzyme-catalysed Oxidation of Smoke Comoonents Evidence was collected to indicate whether or not, and the rate at which, a number of different tobacco smoke components undergo oxidation in the presence of laccase enzymes. The tested smoke components were: methylhydroquinone, hydroquinone, trimethyihydroquinone, 4-methylcatechol, 2-napthylamine, 2-methylnapthylamine, and quinoline. -17-

In order to measure the rate of oxidation for each tested smoke component, the concentration of dioxygen was measured over time. Laccases are oxidase enzymes, and so by definition dioxygen m&ecu]es act as the electron acceptor in the cataysed redox reaction. Therefore, a decrease in dioxygen concentration corresponds to a laccase-catalysed oxidation reaction taking place, and correlates with a decrease in the concentration of the tested smoke component.

For each tested smoke component, the concentration of dioxygen was measured over time for two samples. The first sample served as a control, consisting of the smoke Jo component and a buffer, whHe the second samp'e consisted of the smoke component, a buffer and thccase enzymes.

In Figure 4 there are presented seven graphs, each of which shows how the concentration of a particular smoke component was found to change over time in the presence and absence (contr&) of laccase enzymes.

For each of the four dipheno] compounds the concentration of dioxygen is shown to decrease over time in the presence of laccase enzymes but to remain constmt in the absence of laccase enzymes. This suggests that the concentration of each of these smoke components also decreases over time in the presence of laccase enzymes, and so indicates that enzyme-catalysed oxidation is taking place. Therefore, these data suggest that the tested diphenol smoke components are good substrates of the tested laccase enzymes.

For the PAHs and derivatives, the concentration of dioxygen is shown to decrease at a slower rate compared to the tested diphenol compounds in the presence of laccase enzymes. This suggests that the concentration of each of these smoke components does not decrease significantly over time in the presence of laccase, and so indicates that the enzyme-catalysed oxidation reaction is slow. Therefore, these data suggest that the tested PAl-Is and derivatives are poor substrates of the tested hiccase enzymes.

g. Kinetics of Enzvme-catalvsed Oxidation of Phenolic Comnounds The kinetics of the enzyme-cata]ysed oxidation reaction for the the foflowing phenofic compounds was investigated: hydroquinone, methylquinone, trimethylquinone, catechol, and methylcatechol. The laccase enzymes were shown to follow Michaelis-Menten kinetics for each of the tested substrates. -18-

Figure 5 provides an example of a laccase following Michaelis-Menten kinetics. The graph depicts the rate of reaction as a function of substrate concentration for the laccase-catalysed oxidation of hydroquinone and features the familiar hyperbofic curve representative of enzymes-catalysed reactions proceeding by Michaelis-Menten kinetics.

The Km and Vmax parameters were determined for laccase enzymes in the presence of each of the aforementioned substrates molecules. The results are detailed in Table 1 io below. The optimum conditions for laccase enzyme catalysis were found to be independent of the substrate undergoing oxidation; the optimum pH was approximately 4.5 and the optimum temperature was approximately 30°C.

Table i:

Substrate e / mM-lcm-1 Km / mM Vmax / gMs-' Hydroquinone 13.403 0.135 2.525 Methythydroquinone 9.946 0.092 3.898 Trimethylhydroquinone 9.018 0.089 3.245 Catechol 0.925 0.236 12.333 Methylcatechol 0.779 0.134 13.111 From the experimental data provided in Table 1, it may be deduced that the tested laccase enzyme has the greatest binding affinity for trimethylhydroquinone, since the smallest Km value was calculated for this substrate. It may also be inferred that the greatest potential catalysis of oxidation by laccase is towards methylcatechol, since the greatest Vmax value was calculated for this substrate.

4. Indirect Removal of Non-Laccase-Substrate Molecules Experiinens were conducLed Lo invesL.igae wheLher cerLain accase enzyme subsLraes may act as as co-substrates and react with with non-laccase-substrate compounds often found in tobacco smoke.

The results described earlier suggest that laccase enzymes are able to effectively catalyse the oxidation of diphenol molecules. Therefore, the two diphenol compounds hydroquinone and methylhydroquinone were tested as co-substrates in this part of the 3° study The first compound, methyihydroquinone, was trialled as a co-substrate in the presence of 3-aminobiphenyl, a molecule for which laccase-catalysed oxidation is very stow. The two molecules were incubated together for a period of time in the presence oflaccase enzymes.

HPLC-MS analysis followed to determine the chemical composition of the sample after the incubation period. The obtained HPLC-MS spectrum is pictured in Figure 6, which includes is a peak with an m/z ratio of 290.1, thus suggesting that a molecule with a mass of approximately 290.1 Da was present in the measured sample. Furthermore, this is the peak of highest intensity in the spectrum, therefore indicating that the formed chemical species was in high concentration.

Figure 6 also pictures the molecule which would be formed by a coupling reaction between oxidised methythydroquinone (both hydroxy groups of hydroquinone have been oxidised to carbonyl groups) and 3-aminobiphenyL The mass of this m&ecule is 289.3 Da, and thus is responsible for the measured m/z ratio of 290.1.

Evidence is thus provided that methylhydroquinone may act as a co-substrate and undergo enzyme-catalysed oxidation before undergoing a non-enzyme-catalysed reaction with the non-substrate molecule 3-aminobiphenyl.

The second tested compound, hydroquinone, was trialled as a co-substrate with a number of different non-substrate compounds. The same procedure as described above for methylhydroquinone in the presence of 3-aminobiphenyl was used for hydroquinone in the presence of benzo[a]pyrene.

Figure 7 illustrates the obtained HPLC-MS spectrum, which includes is a peak with an m/z ratio of 391.2, thus suggesting that a molecule with a mass of approximateiy 391.2 Da was present in the measured sample. Furthermore, this peak is of high intensity, indicating that the formed chemical species was in high concentration.

Figure 7 also pictures the mo'ecule which woffid be formed by a coupling reaction between oxidised hydroquinone (both hydroxy groups of methylhydroquinone have been oxidised to carbonyl groups) and benzo[a]pyrene. The mass of this molecule is 390.4 Da, and thus is responsible for the measured m/z ratio of 391.2.

Evidence is thus provided that hydroquinone may act as a co-substrate and undergo enzyme-catalysed oxidation before undergoing a non-enzyme-cataysed reaction with the non-substrate m&ecue benzo[a]pyrene.

Hydroquinone was further tested as a co-substrate with the following tobacco smoke components: 3-(1-nitrosopyrrolidin-2-yfl pyridine (NNN), formaldehyde, 4-(meth1-nitrosamino)-1-(3-pyridy)-1-butanone (NINK), crotonaldehyde, 4-aminobipheny and acrylonitrile.

For these non-substrate compounds HPLC-UV analysis foflowed incubation with accase enzymes; the chemical substances present in the sample after the period of incubation were separated by reversed phase HPLC, and detected by the absorption of UV-frequency radiation.

Resifits suggest that every one of the tested non-substrate compounds reacted wfth, and coup'ed to, the oxidised product ofhydroquinone. Figure 8 shows the HPLC chromatograms obtained for three of the tested non-substrate compounds. Each of these indudes a peak corresponding to a molecule formed through coupling between oxidised hydroquinone and a non-substrate compound.

Therefore, results suggest that hydroquinone serves as a very effective co-substrate for the remova' of a large number of tobacco smoke components. The m&ecule is a good substrate of hiccase enzymes and after enzyme-catalysed oxidation is able to couple to a arge number of different compounds.

Finally, the the ability of lignosulfonate compounds to act as co-substrates for the removal of formaldehyde was investigated. The decrease in formaldyde concentration was measured over time for different sampks, each of which consisted of a different size fraction of lignosulfonate compounds.

Figure 9 shows a graph of the resdts, from which it maybe deduced that Bgnosulfonate does indeed act as a good co-substrate for the remova' of fomi&dehyde. It &so indicates that the smaller the mass of the lignosulfonate compound, the greater the rate at which formaldyhde is removed from the sample.

In order to address various issues and advance the art, the entirety of this disclosure shows by way of illustration various embodiments in which the claimed invention(s) may be practiced and provide for superior removal ofchemica substances from smoking article aerosols. The advantages and features of the disdosure are of a representative sample of embodiments only, and are not exhaustive and/or exclusive.

They are presented only to assist in understanding and teach the claimed features. It is to be understood that advantages, embodiments, examples, functions, features, structures, and/or other aspects of the disclosure are not to be considered limitations on the disclosure as defined by the claims or limitations on equivalents to the claims, io and that other embodiments may be utilised and modifications may be made without departing from the scope and/or spirit of the disdosure. Various embodiments may suitably comprise, consist of, or consist essentially of, various combinations of the disclosed elements, components, features, parts, steps, means, etc. In addition, the disclosure includes other inventions not presently claimed, but which may be claimed in future. -22-