ES2380529B1 - NEW TIRAMINA OXIDASA, NUCLEIC ACID THAT CODIFIES AND USE THEMSELVES - Google Patents

Abstract

New tyramine oxidase, nucleic acid that encodes and use them. The new tyramine oxidase is part of the enzymes encoded by the genes present in the tyn and hpa gene clusters of Pseudomonas putida U, involved in a pathway of tyramine and / or dopamine degradation unknown until now. Said tyramine oxidase transforms tyramine or dopamine into 4-hydroxyphenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde, thereby reducing the amount of tyramine and / or dopamine in food. The invention also relates to nucleic acid molecules encoding the enzyme, vectors that allow its expression and, especially to recombinant microorganisms transformed with said vectors. In addition, the invention relates to the use of the enzyme, the nucleic acid molecules that encode it, the vectors that allow its expression, and, especially, the recombinant microorganisms that express said enzyme to reduce the amount of tyramine and / or dopamine in foods.

Description

NEW TIRAMIN OXIDASE, NUCLEIC ACID THAT CODIFIES AND USE OF

THE SAME

TECHNICAL FIELD OF THE INVENTION.

The technical field of the invention belongs to Biotechnology. The invention relates to a new tyramine oxidase that allows to reduce the content of tyramine and / or dopamine in a sample. It is based on a pathway of bacterial degradation of these compounds, unknown until now, which performs the transformation of tyramine and dopamine primarily into 4-hydroxyphenylacetic and 3,4-dihydroxyphenylacetic acids respectively, compounds that, when degraded by Enzymes from another complementary cluster are finally degraded in pyruvic acid and succinic acid. The invention relates to the tyramine oxidase protein involved in the pathway, the sequences that encode it, the vectors from which it can be expressed and the recombinant microorganisms in which said protein is expressed, as well as the use of any of them. to decrease the content of tyramine and / or dopamine in samples containing them, preferably food and beverages.

STATE OF THE TECHNIQUE Biogenic amines. General features

Amines are chemical compounds derived from ammonia that result from the replacement of the hydrogens of that molecule by alkyl radicals. As one, two or three hydrogens are substituted, the amines will be primary, secondary or tertiary. When they are originated as a consequence of the activity of living organisms and have biological activity (they perform important functions in cells) they are called biogenic or biogenic amines. Depending on the number of amino groups present in the molecule we can differentiate monoamines, diamines and polyamines. Aliphatic monoamines are widespread in nature where putrescine diamine is also abundant, while spermidine and spermine polyamides are produced by animals, plants and most bacteria (1).

Aromatic amines, caused by decarboxylation of amino acids, are the most common amines in food (histamine, 2-phenylethylamine, tyramine, etc.) and

they also have great importance as transmitters within the nervous system

central (dopamine, norepinephrine, epinephrine, serotonin, etc.).

We can make a distinction between endogenous biogenic amines, which are those that are synthesized in different tissues of higher organisms (such as adrenaline produced in the adrenal medulla or histamine in mast cells) and exogenous biogenic amines, which are ingested in the diet These exogenous biogenic amines may be present in foods of plant origin (fruits and vegetables), or they may appear in foods as a result of microbial activity during processing (cure of meats and cheeses) or during storage thereof . Because they can cause harmful effects in both man and animals, they are considered toxic substances.

The most important biogenic amines that can be found in food are histamine, putrescine, cadaverine, tyramine, tryptamine, phenylethylamine, spermine and spermidine; and the foods that contain them can be very varied (fish, meat, eggs, cheeses, fermented drinks, etc.) (2).

Fortunately, organisms have different natural detoxification systems (monoamine oxidase -MAO- or diaminooxidase -DAO-) that allow them to eliminate biogenic amines, avoiding the harmful effects caused by these compounds. However, there may be cases in which these systems do not work properly, or are inhibited by the action of certain drugs, so the presence of biogenic amines in food can pose a serious health problem.

For all these reasons, it is very interesting to select microorganisms that, when used in food processing processes (cured, fermentations, etc.), do not accumulate biogenic amines, or that do so in concentrations that are not dangerous to health. Genetic Engineering and Metabolic Engineering could contribute to obtaining this type of strains, ensuring, in addition, that another series of properties and characteristics that are necessary to maintain the standards of identity and quality of food are preserved. Biogenic amines as neurotransmitters

It has been known for decades that the catecholaminergic transmission is mediated by biogenic amines, including catecholamines (dopamine, norepinephrine and adrenaline) derived from the amino acid.

tyrosine; indolamine serotonin, synthesized from tryptophan; and histamine,

produced from the amino acid histidine. Catecholamines

Under the term catecholamines, all those biogenic amines derived from tyrosine that contain a catechol group and an amino group in its molecule are included. The first step in the synthesis of catecholamines is catalyzed by the enzyme tyrosine hydroxylase through a reaction that requires oxygen as a substrate and tetrahydrobiopterin as a cofactor, and allows dihydroxyphenylalanine (DOPA) as a final product (Figure 1). Therefore, the rate of tyrosine hydroxylase will be the limiting factor for the synthesis of the three catecholaminergic neurotransmitter amines (dopamine, norepinephrine and adrenaline).

Dopamine is produced by decarboxylation of L-DOPA. This reaction is carried out by the enzyme DOPA decarboxylase. The area of the brain where it is found in greater abundance is in the corpus estríatum, playing an essential role in the coordination of body movements (3). In patients suffering from Parkinson's disease, for example, degeneration of dopaminergic neurons has been observed, which will lead to the characteristic motor dysfunction associated with this disease (4).

Norepinephrine, also called norepinephrine, requires for its synthesis, from dopamine, the action of dopamine-3-hydroxylase. This catecholamine is mainly produced in neurons of the sympathetic ganglia and its action is related to sleep, wakefulness, attention and behavior.

Adrenaline, also called epinephrine, is present in the brain at lower levels than the other two catecholamines. The enzyme that synthesizes adrenaline, phenylethanolamine-N-methyltransferase, is located only in the secretory neurons of this catecholamine.

The most important enzymes in the catabolism of catecholamines are monoamine oxidase (MAO) and catechol O-methyltransferase (COMT) (5). These enzymes are found respectively in the mitochondria and cytoplasm of both neuronal and glial cells. Inhibitors of these enzymes are used clinically as antidepressants (6). Histamine

This biogenic neurotransmitter amine is produced by decarboxylation of histidine due to the action of histidine decarboxylase (Figure 2). Its metabolism involves both histidine methyltransferase and MAO. The highest concentration of this neurotransmitter is found in the neurons of the hypothalamus and its action is related to the alert and attention processes. Histamine is also released by macrophages in response to allergic reactions or tissue damage. Serotonin

This indolamine, also called 5-hydroxytryptamine, is synthesized in neurons from tryptophan ingested with food after being hydroxylated to 5-hydroxytryptophan by a reaction catalyzed by the enzyme tryptophan-5-hydroxylase. Subsequently, 5-hydroxytryptophan is decarboxylated by the action of a 5-hydroxytryptophan decarboxylase to give rise to serotonin (Figure 28). The main enzyme responsible for its degradation is MAO, as in the other biogenic amines. Serotonin is involved in the regulation of sleep and wakefulness.

In addition to neurotransmitter monoamines, there are other biogenic amines that have a similar molecular structure and act as neuromodulators or "false neurotransmitters." These endogenous amines, also called "trace" amines or microamines, are found in small amounts in the central nervous system and their study is becoming important in recent years. "Trace" amines

With the term "trace" amines or microamines, reference is made to a family of endogenous amino acids, structurally and metabolically related to dopamine, norepinephrine and serotonin (7-8). This group includes p-y m-octopamine, p-y m-tyramine, tryptamine and 3-phenylethylamine (Figure 3). All these molecules are heterogeneously distributed in the brain of mammals in very low concentrations (0.1-100 ng / g of tissue) (9), but they play an important role in coordinating the synaptic response mediated by neurotransmitter biogenic amines.

Recently, two specific receptors of these "trace" amino acids have been characterized that cannot be activated by neurotransmitter monoamines. These receptors are called TA1 and TA2, belong to the family of receptors associated with G proteins (GPCRs) and are located in the pre-and post-synaptic plasma membrane of receptor neurons. They are activated by tryptamine, p-tyramine and 3-phenylethylamine, as well as by amphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and other types of hallucinogenic drugs. (10) This

discovery has aroused great interest in these compounds to which, given

their physiological relevance, they are called "endogenous amphetamines" (11-12).

In addition to these recently discovered aspects, the co-transmitter function played by these "trace" amines in the neurotransmission systems mediated by dopamine, norepinephrine or serotonin (8) has been known for years. The structural similarity of these "trace" amines with neurotransmitter monoamines will allow them to act as substitutes or "false neurotransmitters" in the dopamine and noradrenaline systems. In addition, due to their functional similarity they are being used in the treatment of hepatic encephalopathy (13) or in Parkinson's disease (14).

Finally, "trace" amines can serve as neuromodulators in the central nervous system, but before explaining this activity, a clear distinction between neurotransmitter and neuromodulator should be made (15).

It is called a neurotransmitter, the molecule released by a neuron to the synaptic channel in response to an electrical activity and which will subsequently specifically bind to its post-synaptic receptors, causing the induction of a change in the excitability of the post-synaptic cell and thus, allow the passage of information.

A neuromodulator is also a molecule released by a neuron, but in this case it is not capable of causing a change in the excitability of the post-synaptic cell membrane by itself, since it needs the presence of a neurotransmitter. The release of a neuromodulator acts by modifying the action (increasing or decreasing it) of a coexisting neurotransmitter.

Therefore, it is not strange that "trace" amines have been implicated in the majority of neuropsychiatric disorders associated with dysfunctions in the catecholamine and indolamines systems, since these compounds modulate the signaling processes that occur in the post terminals. -and pre-synaptics of these systems. It is believed that alterations in the function of these "trace amines" are involved in the etiology of a wide variety of neuropathological disorders, including hallucinations, schizophrenia, depression, anxiety states, hyperactivity, bipolar disorder, etc. (11, 16-18). Presence of biogenic amines in food

Biogenic amines, in addition to being present in the central nervous system fulfilling neurotransmitter and neuromodulatory functions, are present in fermented foods and beverages, where they are generated by decarboxylation of their precursor amino acids. Their accumulation (especially that of aromatic biogenic amines) can make the intake of these foods harmful to health. The main biogenic amines that can cause toxicity when accumulated in food, are reflected in the figure

Four.

Amines and polyamines (PAs) are only found naturally in plant-based foods, since these molecules are found in plants and in their fruits, forming part of their cell walls or acting as a defensive system against attack of pathogens or predators (19). Due to their chemical nature, these compounds participate in numerous basic cellular processes, as well as in different events related to growth, development and the response of plants to certain stress conditions. PAs, in addition to being essential for plant growth, under appropriate conditions, can exercise specific morphogenesis control functions (20).

The predominant amine will depend on the type of fruit or plant considered; for example, in fruits such as lemon, tangerine and strawberry, putrescine predominates, while in raspberry and mushrooms, the predominant amine is tyramine (21). It has also been proven that between different varieties of the same fruit there may be a great variation in the levels of amines (22).

However, the biogenic amines present in many foods can also have an exogenous origin, being generated by decarboxylation of the precursor amino acids. Thus, they appear in a wide variety of foods, whether they are unfermented (fish, dairy products, meat, etc.) or those that have undergone some type of fermentation during their production (wine, beer, cheese, etc.). Its accumulation is an aspect to be taken into account due to the toxicological problems that its ingestion can generate.

There are factors that will limit the accumulation of biogenic amines (especially those that are due to microbial activity) in food. Thus, for example, the availability of substrate, the pH of the medium, the concentration of salts and the temperature will also have a great influence on the production of amines. Pyridoxal phosphate is a factor required for the decarboxylation of amino acids in most bacteria and, therefore, their presence or absence will be decisive for the synthesis of biogenic amines. Presence of amines in unfermented foods

The presence of biogenic amines in unfermented foods is a

Indicator of the presence of unwanted microbial activity, and therefore, the level of amines present can be used as an indicator of food spoilage by microorganisms. Normally, the amount of histamine, putrescine 5 and cadaverine increases during food spoilage, while spermine and spermidine levels decrease. Due to this characteristic, the Biogenic Amines Index (IAB), defined by Karmas (23) and expressed in mg / Kg, has been used to calculate the quality grade of a food. Currently, the detection and quantification of biogenic amines is performed using HPLC techniques, as

It will be described later in this work. IAB = [histamine] + [putrescine] + [cadaverine] / 1 + [spermine] + [spermidine]

Fish or meat with an IBA value below 1 are considered top quality, while values around 10 indicate a poor microbiological quality of the product.

15 Among the unfermented foods that accumulate endogenous amino acids, fish and meat, which are characterized by accumulating large concentrations of histamine and tyramine, respectively, are stored during storage, even if it is not prolonged. Moreover, it has been shown that the accumulation of amines as a result of microbial activity cannot

20 Avoid with vacuum packaging of the product (24). The only effective measure to prevent the accumulation of biogenic amines is the storage of products at low temperatures (24). Presence of amines in fermented foods During the preparation of fermented foods, the product

25 it is usually incubated for days, weeks and even months, until the necessary degree of fermentation or maturation is reached, so that a greater proliferation of microorganisms and, consequently, a greater presence of biogenic amino acids in these products can be expected. In addition, the development of these foods requires the participation of microorganisms that modify the properties of the

30 original raw material, so the elimination of these microorganisms would distort the quality, properties and characteristics of the products. This is what happens with foods as popular as cheese, sausages, sauerkraut or wine (25).

The most important amine that accumulates in cheese during ripening is tyramine (26) and, to a lesser extent, phenylethylamine. During this process, casein is slowly degraded by proteolytic enzymes,

thereby increasing the content of free amino acids that may be susceptible to serve as a substrate for specific bacterial decarboxylases, to give rise to the formation of CO2 and an amine.

On the other hand, the amine that accumulates mostly in sausages is histamine (21), but in this case its accumulation will depend on the manufacturing process, the type of meat used, its proportion and the quality of it, as well as of maturation time. In the case of sausages, the amount of accumulated amines in the final product can be greatly reduced by using starter cultures that contain the appropriate microorganisms to carry out the required fermentation, but do not produce these ami undesirable nas. This measure, which has meant a great advance in the regularization of fermentation processes, is not always effective, since the endogenous microbial flora (present in the original raw materials) may already be able to produce biogenic amino acids by itself.

A group of important products regarding the accumulation of biogenic amines are fermented beverages. Such is the case of beer and especially wine. The presence of amines in these drinks is responsible for the characteristic headache that is experienced after abusive consumption (26).

In the wine there are mainly histamine, tyramine and putrescine, in very variable amounts depending on the type of wine. The concentration of these amines is low during alcoholic fermentation and increases during malolactic fermentation. This explains why red wines have higher concentrations of these amines compared to white wines, since the latter do not suffer from malolactic fermentation. After this fermentation, the wine is usually sulphated to eliminate the populations of undesirable bacteria and yeasts from that moment, but even so, the concentration of biogenic amines continues to evolve and can reach up to 50 mg / I during aging (27 ). Although there is no definite regulation regarding the concentration of biogenic amines in wine, there are countries that have established import limits (Canada and Switzerland 10 mg / I, Netherlands 5 mg / I). This is due to the fact that the presence of amines in wine carries more risk than in other foods, since when alcohol interacts with them, the body's detoxification mechanisms will be affected and the chances of food poisoning are increased. Amnes.

The levels of biogenic amines in alcoholic beverages made by

Yeast fermentations are generally lower than those found in beverages in which lactic acid fermentation takes place (except yogurt), but still they can contain considerable amounts of putrescine, cadaverine, histamine and tyramine (2).

In summary, the main conclusions that can be drawn about the presence of biogenic amines in food are the following: Most foods are susceptible to deterioration due to the action of microorganisms capable of producing biogenic amines. High concentrations of certain amines in food can be harmful to health

Great importance should be given to the evaluation of the amines content of foods, as well as the presence of other agents that enhance their effect, such as other animals, alcohol, certain drugs.

High concentrations of biogenic amines in food can and should be avoided, through good manufacturing and storage practices (control of hygiene, contamination, temperature, etc.)

In the production of foods that require acid lactic fermentation, starter cultures of microorganisms that are amino acid negative decarboxylase should be used. In this sense, it would be very useful to prepare starter cultures that present in their composition microorganisms that not only do not produce these amines but are capable of degrading them. Toxicology of biogenic amines

As indicated previously, histamine, tyramine, tryptamine and 3-phenylethylamine are biologically active amines that can cause important physiological effects in humans, both psychoactive (neuromodulatory) and vasoactive. The consumption of foods with a high content of biogenic amines can cause a large number of pharmacological effects (Table 1) that characterize certain diseases, such as histamine poisoning or "cheese reaction" caused by tyramine intake. In addition, amines are currently being studied as precursors of carcinogenic compounds (21).

Table 1. Biogenic amines present in food and its effects on the organism.

IDI biogenic amine Pharmacological effects

ID

Histamine Adrenaline and norepinephrine release Stimulation of the uterine musculature, intestinal and respiratory system.

Stimulation of sensory and motor neurons.

Tyramine Increase in blood pressure. Control of gastric secretion. Peripheral vasoconstriction Increased heart and respiratory rate. Stimulation of lacrimation and salivation. Norepinephrine release Migraine.

Hypotension Putrescine and cadaverine Bradycardia. Enhancement of the effect of other amines.

3-Phenylethylamine Norepinephrine release. Increase in blood pressure. Migraine.

Tryptamine Increase in blood pressure.

Among all the biogenic amines present in food, it should be noted for its high toxicity (consequence of the greater number of physiological effects they cause), histamine and tyramine.

Histamine is a very biologically active amine, since it performs many actions within the body. Although mast cells and blood basophils contain large amounts of histamine, it is found

10 stored in characteristic granules and will not be released unless special reactions occur (allergic reaction). Histamine can stimulate the heart rhythm and this effect results in the release of adrenaline and norepinephrine by the adrenal glands, excitation of the uterine muscles and respiratory tract, stimulation of both motor and sensory neurons and control of gastric secretion (28) . Therefore, it is not surprising that histamine poisoning manifests skin symptoms such as hives and the appearance of edema or rashes, as well as gastrointestinal symptoms, such as nausea, vomiting and diarrhea. Other symptoms such as hypotension, headache or palpitations may also occur (29).

Despite the toxic nature caused by an excess of histamine, the presence of this amine in food does not have to be dangerous. There are many foods that contain small amounts of histamine and, therefore, will be easily tolerated by the body thanks to the existence of efficient detoxification systems in the digestive tract, which will metabolize both ingested histamine and formed histamine by the intestinal flora itself. This detoxification system is composed of two different enzymes: diamino oxidase and histidine-N-methyltransferase. These two enzymes are responsible for converting histamine into products without biological activity, and their efficiency is high when there is a normal consumption of dietary amine. However, these mechanisms are less effective if large amounts of amines are ingested, or in the presence of other amines that potentiate the toxic effect caused by histamine.

The presence of tyramine induces the release of norepinephrine from the sympathetic nervous system, which will cause an increase in blood pressure through peripheral vasoconstriction and an increase in heart rate. It can also cause pupil dilation, increased salivation, breathing and blood sugar levels (30).

Regarding the presence of tyramine in food, it is worth noting only its own toxicity, but also its harmful effects in the presence of monoamine oxidase (MAO) inhibitors, which can cause critical hypertension states (31-33). The MAO is responsible for the oxidative deamination of the amines derived from food, and they constitute the best endogenous defensive system against these toxic compounds, allowing their degradation before they pass into the bloodstream. The use of MAO inhibitor drugs during the treatment of certain mental illnesses (depression, schizophrenia, etc.) will cause the inhibition of this natural detoxification system and, as a consequence of this inhibition, the accumulation of tyramine in the blood, which will generate critical states of hypertension in patients. The first food that was associated with this process was cheese, so this increase in blood pressure is known as "cheese reaction" and can cause severe headaches, brain hemorrhages or heart failure (1).

In addition to being able to act as toxic agents, the involvement of the amino acids in the synthesis of derivatives that could act as mutagenic agents is currently being studied. By adding nitrates to foods as preservatives, and when they react with the amino acids present in these foods, Nnitrosamines are generated, which are carcinogenic compounds and constitute a serious risk to human health. Some examples of these processes are the reaction between tyramine and nitrites that are going to cause 3-diazothyramine, a compound that induces the appearance of cancer in the oral cavity of rats (30), and the reaction between tyramine and nitrates that when it occurs in conditions Acids gives rise to a mutagenic compound identified as 4- (2-aminoethyl) -6-diazo-2,4-cyclohexadienone (34). Production of biogenic amines in food

As we have already indicated, most of the amines present in food are generated by decarboxylation of their corresponding precursor amino acids, through the action of specific enzymes (amino acid decarboxylases) produced by the microorganisms present in those foods. These decarboxylases are present in a large number of species belonging to different bacterial genera.

For the formation of biogenic amines in food, the following requirements are needed: a) availability of free amino acids (generally caused by proteolytic action); b) presence of microorganisms possessing decarboxylase enzyme (positive decarboxylase); and c) that the appropriate physicochemical conditions exist that allow both bacterial growth and synthesis and decarboxylase activity. The positive decarboxylase microorganisms may be part of the endogenous flora of the food, or be introduced by contamination during the processing or storage processes. In the case of food and beverages that undergo fermentation processes, the introduction of starter cultures can affect the production of biogenic animals interacting, directly or indirectly, with both the endogenous flora and the contaminating flora (2).

Most decarboxylases maintain their activity even after the pasteurization process. This fact, together with the fact that most of the amines are thermostable, implies not only that the amount of amino acids already formed in the

food will not be eliminated with the pasteurization process, but even

will increase during storage. Amino acid decarboxylation

In the decarboxylation of amino acids, the removal of the acarboxyl group of the amino acid in question occurs to give rise to CO2 and the corresponding amine. This reaction is catalyzed by bacterial decarboxylases that are specific for each amino acid. Thus, for example, ornithine can be degraded to putrescine and lysine to cadaverine by the action of ornithine decarboxylase (35) and lysine decarboxylase (36) respectively. In the same way and provided that the appropriate conditions exist, histidine, tyrosine, tritophane and phenylalanine will be decarboxylated to histamine, tyramine, tryptamine and 3-phenylethylamine by the action of histidine decarboxylase (37), tryptophan decarboxylase (38), tyrosine decarboxylase (39) and phenylalanine decarboxylase (40), respectively. There is also an aromatic L-amino acid decarboxylase (AADC) that catalyzes the irreversible decarboxylation reaction of L-DOPA to dopamine, but which has a lower substrate specificity than the previous ones, since it is capable of carrying out the decarboxylation of both Tyrosine, such as phenylalanine, 5-hydroxytryptophan and tryptophan (41).

The amino acid decarboxylases have been widely studied in recent years (42). Most of them use pidoxal-5'-phosphate or pyruvate as coenzyme, and are dependent on vitamin B6. Currently there are a large number of sequences corresponding to amino acid decarboxylases that are collected in the different databases. Their comparative analysis has allowed them to be classified into four groups (Table 2) and identify functionally important regions within those sequences.

Two mechanisms of action for the decarboxylation of amino acids have been proposed, one is based on a pyridoxal phosphate-dependent reaction, and another requires a pyruvate molecule as a cofactor (43).

In pyridoxal-5-phosphate-dependent decarboxylation reactions, a Schiff base is formed due to the reaction of the aldehyde group of the pyridoxal with one of the amino groups belonging to a lysine located in the active center of the enzyme (internal aldimine) . The carbonyl group of pyridoxal-5-phosphate reacts easily with amino acids to form a new base of Schiff (external aldimine) that functions as an intermediate, and that allows these to be

subsequently decarboxylated to give rise to the corresponding amine and the

original pyridoxal phosphate molecule. In decarboxylation reactions not dependent on pyridoxal-5-phosphate, a pyruvate molecule (101) is involved. In this case, the piruvil group will be the

5 which covalently binds to the amino group of the amino acid forming a Schiff base, allowing them to be subsequently decarboxylated by a reaction very similar to the decarboxylation reaction dependent on pyridoxal-5-phosphate.

The decarboxylation of amino acids has an important energy function for bacteria in those nutrient-poor environments, since being an endothermic reaction, it constitutes an ATP generation system, at the same time leading to the synthesis of amino acids. On the other hand, the formation of amines will cause an increase in the pH of the medium, favoring bacterial growth. For all these reasons, the decarboxylation of amino acids can be considered as a very advantageous mechanism that allows the adaptation to the medium to a large number of

15 microorganisms

Table 2. pyridoxal-P-dependent amino acid decarboxylases with their known sequences (Sandmeier et a /., 1994).

I Enzyme

Group I Glycine decarboxylase (EC 1.4.4.2)

Group II Glutamate decarboxylase (EC 4.1.1.15)

Histidine decarboxylase (EC 4.1.1.22)

Access numbers in GenBank and sources

I

P23378, human; P15505, chicken; P26969,

Písum satívum

M84024, Escheríchía colí (GAD-a); M84025, Escheríchía colí (GADp); P20228, Drosophíla me / anogaster; mouse "; JH0423, rat (GAD65); P18088, rat (GAD67); P14748, cat; M74826, human (GAD65); M81883, human (GAD67)

P28577, Enterobacter aerogenes; P28578, K / ebsíella p / antíco / a; P05034, Morganella morganíí; X70644,

I Enzyme

Tyrosine decarboxylase (EC 4.1.1.25)

Amino acid decarboxylase aromatic (EC 4.1.1 .28)

Tryptophan decarboxylase (EC 4.1.1.17)

Group III Ornithine decarboxylase (EC 4.1.1.18)

Lysine decarboxylase (EC 4.1.1 .19)

Arginine decarboxylase (EC 4.1.1.17) Group IV Ornithine decarboxylase (EC 4.1.1.19)

Arginine decarboxylase (EC 4.1.1.20)

Access numbers in GenBank and sources

I

Drosophíla me / anogaster; P23738, mouse; P16453, rat; P19113, human.

M96070, Petroselínum críspurn '

S19796, Caenorhabdítís e / egans; P05031, Drosophíla me / anogaster;

P14173, rat; P22781; guinea pig,

P80041, pig P27718, bovine; P20711,

human

P17770, Catharanthus roseus

P21169, Escheríchía colí; P24169, Escheríchía colí (inducible)

P05033, Hafiía a / veí; P26934, Bacíllus subtitles; P23892, Escheríchía colí

P28629, Escheríchía colí

P28629, Escheríchía colí (biodegradative) P07805, Trypanosoma bruceí; P27116, Donovani Leíshmania; P27121, Neurospora crassa; P08432, Saccharomyces cerevísíae; P27120, Xenopus / aevís; P27118, chicken; P00860, mouse; P27119, Mus Paharí; P09057, rat; P14019, hamster; P27117, bovine P11926, human.

P21170, Escheríchía colí; P22220, Satin Oatmeal

Producing microorganisms of biogenic amines

Enzymes with decarboxylating activity have been found in a large number of bacteria, including species of enterobacteria, pseudomonadids, enterococci and lactobacilli, among others (44).

There are several enterobacteria with decarboxylastic activity (mainly related to the production of cadaverine and putrescine). Studies carried out in Vítro with Enterobacter c10acae and with different species of Serratia, as well as in Cítrobacter freundií and Enterobacter aerogenes, have demonstrated the ability of these microorganisms to form large amounts of putrescine and cadaverine (45). Other enterobacteria are characterized, however, by being large producers of histamine; such is the case of Klebsíella oxytoca (46), Escheríchía colí (47) or Morganella morganií (45). Although these enterobacteria are found in very low proportion in the final products, poor storage practices, or uncontrolled fermentation during processing, can cause them to proliferate significantly.

There are other microorganisms that produce biogenic amines of different nature. Such is the case of Pseudomonas putrefacíens, Aeromonas hydrophíla and Plesíomonas shígelloídes, microorganisms that are usually found in fish in poor condition (21).

Acid-lactic bacteria are used profusely in processes intended to obtain various foods (sausages, wine, cheeses, etc.) by fermentation, and although they cannot be considered toxic or pathogenic species, many of them are capable of producing biogenic amines. For example, some strains of Lactococcus and Leuconostoc produce appreciable amounts of tyramine and histamine.

(39) And strains of lactobacilli belonging to the species Lactobacíllus buchnerí, L. alímentaríus, L. plantarum, L. curva tus, L. farcímínís, L. bavarícus, L. homohíochií, L. reuterí and L. sakeí, are large producers of amines, especially tyramine (4850). Many of these lactic bacteria are used in the manufacture of cheeses, and are included in the starter cultures used by the dairy industry. Such is the case of Lactococcus lactís subsp. Lactis, Streptococcus. faecíum, S. mítís, Lactobacíllus helvetícus, L. caseí, L. acídófílus and L. arabínose, all of them identified as producing histamine (26).

When the accumulation of biogenic amines in meat is studied, it has been observed that species such as Carnobacterium. dívergens, C. píscícola and C. gallínarum

they are responsible for the presence of high concentrations of tyramine in it

(51).

Other studies have shown that Enterococcus faecalís is responsible for the accumulation of biogenic amines (3-phenylethylamine among others) in fermented foods (52).

The production of biogenic amines by fungi and yeasts in fermented foods has also been revealed. This is the case of Debaryomyces and Candida, two yeasts isolated from fermented meat, which have a greater histidine decarboxylase activity even than that observed in acid-lactic bacteria (50).

Analytical methods applied to the assessment of the production capacity of amino acids by microorganisms: Detection and quantification.

Due to the toxic effects that the amines present in food can cause, it has been necessary to design methods and techniques that allow the detection of amines producing capacities in the microorganisms used in the processes of manufacturing food products, as well as quantifying the presence of Amines in these foods. Methods used to detect microorganisms with aminobiogenic capacity

Several biochemical methods have been developed that allow the detection of strains producing histamine and tyramine from fermented meats and cheeses (45, 53-54). These methods are based on an assay that involves the use of a solid medium containing the amino acid precursor of the amine to be investigated and a pH indicator. Since the formation of amines from amino acids implies an elevation of the pH of the medium, if the study strain has the capacity to form amino acids, this will be reflected by a color change of the pH indicator present in the medium.

There are also molecular detection methods that are based on the design of specific primers for the sequences of the genes encoding decarboxylases responsible for the formation of amino acids. So far, three methods of gene detection involved in the production of amines have been developed:

Detection system of the hdc gene that encodes a histidine decarboxylase (54). For the design of the primers, the nucleotide sequences of the Lactobacillus sp30A hdcA gene, of Clostrídíum perfríngens and the histidine decarboxylase amino acid sequences of these two microorganisms were compared with

those of Lactobacíllus buchneri and Micrococcus. The analysis of the different sequences revealed the existence of a high degree of homology between the hdc genes of the different lactic bacteria, which allowed designing specific primers for the detection of this gene.

Detection system of the tdc gene encoding a tyrosine decarboxylase (55). For the design of the primers, the nucleotide sequences of the tdc gene of Enterococcus faecalís, Carnobacterium divergens and Lactobacíllus brevis were compared.

OCD detection system encoding an ornithine decarboxylase (56). The nucleotide sequence of the ornithine decarboxylase gene in an Oenococcus oeni strain was used for the design of the primers. Analytical methods used to detect amines in food

So far, several methodologies have been described that allow us to detect and quantify the presence of amino acids in food. Although the most commonly used method is high performance liquid chromatography (HPLC), the first assessment of amino acids was performed by thin layer chromatography and aimed at determining histamine in cat food (57). Currently, many specific methods of detection and quantification of amines in many foods have been described, all based on HPLC techniques (58-63). In recent years commercial ELlSA methods have appeared for the analysis of histamine in wines and other foods (64).

Some countries have set maximum values for the presence of amines in certain foods. Thus, for example, Switzerland has established a maximum concentration of 10 mg / L of histamine in wine as tolerable values for human health, while other countries such as Germany, Belgium and France recommend lower maximum values (2 mg / L, 5 -6 mg / L and 8 mg / L, respectively). No maximum limits have been imposed on the rest of biogenic amines, although given the importance of the effects of tyramine in the organism, the prompt appearance of recommendations or regulations on maximum permitted values for this amine is very likely. Degradation of biogenic amines

The presence of amines in the environment is increasing considerably in recent years as a result of industrial activity. Some of these amines, especially methylated ones, are very volatile and are involved in the formation of nitric oxide, an important gas causing the "greenhouse effect" (65-66). Instead, other amines that are present in large

variety of foods, can cause, as explained above,

toxic effects in the body.

The identification of the pathways and enzymes involved in the metabolism of these animals can be of great help for the conversion of these toxic compounds into other less harmful compounds and thus avoid their harmful effect on organisms and the environment. .

Many microorganisms can oxidize primary amines by generating products that can be used as a source of carbon and energy, as a source of nitrogen, or as both (67) and that are no longer toxic.

This mechanism of oxidation of primary amines is a widely distributed process in nature, since it has been identified in both eukaryotic and prokaryotic organisms and is catalyzed by different enzymes, including quinoproteins amino oxidases and quinoproteins or amino dehydrogenases quininoproteins ( 68). Both amino oxidases and amino dehydrogenases catalyze the conversion of amino acids into their corresponding aldehydes. The difference is that while amino oxidases (present in both eukaryotes and prokaryotes) produce toxic peroxides, amino dehydrogenases (present exclusively in bacteria) produce reduced equivalents that directly transfer the e-to the respiratory chain (69-70).

As for the location of these enzymes; it seems that they have a periplasmic location in the G- microorganisms; some are soluble and others are attached to the outer face of the cytoplasmic membrane. However, in G + microorganisms these enzymes are found in the cytoplasm or attached to the cytoplasmic membrane by their inner face. On the other hand, in certain yeasts, it has been found that its location is exclusively peroxisomal (68).

As mentioned above, the enzymes responsible for the oxidative deamination of the amino acids use different cofactors that are reduced after oxidation of the substrate and that participate in the transfer of one or two exogenous acceptors, such as cytochrome e, cupredoxins (azurine or amocyanine) or molecular oxygen. Depending on who is a natural acceptor of the natural distinction between amino oxidases and amino dehydrogenases can be made.

The majority of the enzymes involved in the oxidation of the different amino acids to their corresponding aldehydes are oxidoreductases that are characterized by using cofactors other than NAD (Pt or FAD + (although there are exceptions of amino oxidases flavoproteins that use FAD as cofactor and that they will explain later) These enzymes are called quinoproteins, because they use cofactors that have a quinone group (71) such as TPO (topaquinone), TTO (tryptophan tryptopilquinone), L TO (lysine tyrosylquinone) or CTO (cysteine tryptopilquinone). They are characterized in that each one is formed from one or two amino acids present in the enzyme itself after undergoing a post-transcriptional chemical modification (72-74).

Depending on the identity of the cofactor used, we can differentiate several types of enzymes that catalyze oxidative deamination reactions of amino acids: amino oxidases, amino dehydrogenases and quinoproteins amino dehydrogenases Ouinoproteins amino oxidases

These enzymes are also called copper dependent amino oxidases (O-AmO). As for the structure, they are generally homodimers formed by two identical subunits of the same size. They are characterized in that in each subunit they present a molecule of TPO, an enzymatic cofactor that is formed by post-transcriptional modification from one of the tryptophan residues existing in the enzyme. They also have a copper molecule, Cu (ll) that is coordinated with three histidine residues and two water molecules (75-76). Cu (ll) is necessary, both for the biosynthesis of the enzyme cofactor, and for enzymatic catalysis (76).

These enzymes are characterized in that they are the only ones that are widely distributed in both bacteria and higher organisms and catalyze the oxidation of a large number of primary amino acids. Thus, an O-AmO has been characterized in Klebsíella oxytoca and is related to the degradation of PhEtNH2 and tyramine (68). When E. colí (77) or Klebsíella aerogenes (K. pneumoníae) are grown in the presence of tyramine, it has also been observed that an O-AmO similar to that of Klebsíella oxytoca (68) is expressed. Likewise, other O-AmO have been identified, in G + bacteria and in yeasts. For example, in Arthrobacter globíformís an O-AmO containing copper and using as a substrate PhEtNH2 (75, 78) has been characterized, and in the yeast Hansenula polymorpha a methylamine oxidase implicated in the degradation of methylamine (76) has been identified.

The catalytic reaction carried out by these amino oxidases quinoproteins, takes place through a ping-pong transamination mechanism that can be divided

in two hemirreactions or stages, depending on the oxidation state of the cofactor (75,

78).

1. Reductive hemirreaction: Oxidative deamination of the substrate

the. The TPO cofactor participates in this stage by forming a covalent bond with the substrate (through the C5 carbonyl group), resulting in the formation of a Schiff base with the substrate.

lb. Schiff's base is then deprotonated by an aspartate residue near the active site and at the same time the cofactor is reduced. In this step another Schiff base is formed, but now it is the product that forms the Schiff base.

Ic. Subsequently, hydrolysis of this Schiff base occurs, releasing the aldehyde and leaving the aminoresorcinol form of the reduced TPO.

The stereospecificity of proton abstraction from the C1 position of the substrate has been studied in different enzymes, both bacterial and plant and animal. It has been found that the specificity varies depending on the nature of the enzyme and the substrates used. For example, in the case of dopamine, sometimes the pro-R proton and sometimes the pro-S are abstracted, depending on whether the reaction takes place in plants or animals (78).

11. Oxidative hemirreaction: reduction of molecular oxygen

In this stage there is the re-oxidation of the reduced TPO (TPOred) and the release of ammonium. The participation of Cu (ll) in this stage was pointed out after the discovery of the Cu (I) / semiquinone topa (TPOsq) form. Because the transfer of e-from TPOred to Cu (ll) to form Cu (I) / TPOseq is very fast, and because Cu (l) reacts easily with O2, the Cu (I) / state TPOseq has been proposed as a kinetically competent intermediary with Cu (l) that oxidizes directly to O2 • In this way, a balance is established between the Cu (II) / TPOred and Cu (I) / TPOsq form for intramolecular transfer of e - (75). Subsequently, the aldehyde generated after deamination of the substrate is oxidized to the corresponding acid. Ouinohemoproteins amino dehydrogenases.

They are heterotrimeric enzymes consisting of three subunits (al3v) that use CTO as a cofactor, which is formed from a cysteine residue present in the small subunit (V). The possibility that a fourth protein, whose function is still unknown, could intervene facilitating the formation of the cofactor is not ruled out.

These enzymes have, in addition to the quinone group, two heme c groups,

attached to one of the enzyme subunits, which act as active redox groups (69, 79). In this case, the e-released during the oxidation process are ultimately transferred to a cytochrome oxidase present in the transport chain, via the C550 cytochrome, as seen in Paracoccus denítrífícans; or via azurine as in P. putída (80-81).

To this group belongs the quinohemoprotein amino dehydrogenase (QHAmDH) of Pseudomonas putída U, responsible for the oxidative deamination of 2-phenylethylamine (82) and that of Paracoccus denítrífícans. The latter bacterium has two enzymes with amino dehydrogenase activity in its periplasm. A methylamine dehydrogenase (MADH) that recognizes methylamine and a QH-AmDH that recognizes aliphatic and aromatic primary amines, although it seems to show greater activity with n-butylamine and benzylamine (81, 83-84). Another QH-AmDH within this group has been purified from P. putida IFO 15633 and ATCC 12633 (69, 79-80).

The four genes encoding the QH-AmDH of P. putída and Paracoccus denítrífícans have been identified and sequenced. The ORF1 encodes the subunit a, which has two heme groups that act as redox groups during oxidation; ORF2 encodes a protein, whose function is unknown, but which plays an essential role in the oxidation of these compounds; ORF3 encodes the small subunit (to which the cofactor is attached); and ORF4 encodes subunit 13. These genes are transcribed together, so they constitute a single operon (69).

The mechanism of reactions catalyzed by this group of enzymes is as follows (80-81) (Figure 5):

The reaction is initiated by the nucleophilic attack of the nitrogen of the C6 amino group belonging to the carbonyl group of the CTQ cofactor, resulting in the formation of a carbinolamine intermediate (a).

Carbinolamine loses a water molecule and an imine (b) is formed.

Then an active site residue (probably ASP33y) abstracts a proton from the amine Ca to form the carbanionic intermediate ©. At the same time the CTQ is reduced after capturing an H20 molecule, giving rise to intermediate d.

The product resulting from this oxidation, the aldehyde (e), is finally released by hydrolysis of the new imine bond that had formed between Ca and the amino group.

It seems, therefore, that there is an intramolecular transfer of e-from

the complex generated by the reduced substrate-GTO to heme 1, from here to heme 11, and from it an intermolecular transfer to the exogenous acceptor occurs. This oxidative reaction will take place through two sequential reactions until it is

5 produces re-oxidation of the cofactor and heme groups.

It has been proven that P. putida OH-AmDH can be inhibited by pnitrophenylhydrazine. This compound binds to the active site of the enzyme, located between subunit 13 and v, in the same way as the substrate would. This union causes some important changes in the GTO cofactor and also conformational changes

In the side chains of the residues belonging to the amino acids of the active site, thus causing the modification of the active center and, thereby, the loss of enzymatic activity (80). Ouinoproteins amino dehydrogenases

These enzymes (O-AmDH) use as a cofactor the TTO that is formed from

15 of two tryptophan residues present in the small subunit of the enzyme. They have a structure 02132, where the cofactor is attached to subunits 13 (83).

This group includes methylamine dehydrogenase (MADH) of Methylobacteríum extorquens AM1 or Paracoccus denítrífícans (65) and amino

Aromatic amino acid dehydrogenase (AADH) described in Facalcal Alkalis and which seems to be involved in the catabolism of different primary amines such as PhEtNH2, tyramine and tryptamine (65).

These enzymes usually use blue copper proteins of type I (cupredoxins) as an e-acceptor. Thus, MADH use amycinin and AADH azurine 25 (83.85).

Regarding their genetic organization, nine genes that are transcribed in the same direction and apparently are involved in the degradation of amines (ORF1, aauBEDA, ORF2, ORF3, ORF4 and hemE) have been identified in Facalis Alcalgenes. The aauA and aauB genes encode the small subunit

30 and the AADH large one, respectively, and are homologues of the mauA and mauB genes that encode MADH. The aauE and aauD genes are homologues of mauE and mauD and, apparently, encode proteins that perform the same function (transport and folding of the small subunit in the periplasm, respectively). The homologous genes of mauF, mauG, mauL, mauM and mauN, which participate in the

35 biosynthesis of the TTO cofactor, are not found in the aau cluster. However, other ORFs have been identified, such as ORF2, which encodes a type c cytochrome monohemo, and ORF1, ORF3, and ORF4, to which no function has yet been assigned, but which appear to be essential for the degradation of certain amines, since their disruption is lethal in this bacterium (65).

The catalytic mechanism described for AADH and MADH is similar to that proposed for OH-AmDH, although these enzymes lack both heme groups.

In reactions catalyzed by O-AmDH, the transfer of 2e-from the reduced TTO cofactor to the external e-acceptor is produced by two consecutive reductions. The e-acceptor is azurine in the case of ADAs and amycinin in the case of MADH. Both physiological acceptors mediate the transfer of e-up to different types of soluble cytochrome c. In the case of ADHD, the reaction is favored when the ionic strength is high, while the activity of the MADH decreases when the ionic strength increases (85).

In both cases, after the release of the product, a quinol form of TTO is generated. For example, in the reduction of TTO by methylamine, formaldehyde is released but the amino group remains attached to the cofactor. Therefore, the reduction of the substrate involves the formation of an aminoquinol form of the TTO in which one of the oxygens of the carbonyl group is replaced by the amino group derived from the substrate. The release of the amino group occurs after the second transfer of eque which allows the regeneration of the quinone (85). Tyramine Deamination

The deamination of tyramine is carried out primarily by amino oxidases. These enzymes are widely distributed in all living organisms and allow the conversion, by oxidation, of tyramine and other primary amines into their corresponding aldehydes by releasing a molecule of hydrogen peroxide (hydrogen peroxide). Due to the high reactivity of these products (aldehyde and hydrogen peroxide), they must be rapidly transformed, by reactions mediated by a NAD-dependent phenylacetaldehyde dehydrogenase and by a cytoplasmic localization catalase respectively.

R-CH2NH2 + O2 + H20 RCHO + NH3 + H20 2

The amino oxidases (AOs) that carry out this tyramine deamination reaction can be classified into two subgroups: Flavoproteins amino oxidases (EC 1.4.3.4), which use FAD as a cofactor, and quinoproteins amino oxidases (EC

1.4.3.6) which, as explained above, are characterized by containing Cu (ll)

and TPO as cofactor.

Among the amino oxidases flavoproteins, it is worth mentioning the monoamine oxidases (MAOs) found in countless organisms, and such as the tyramine oxidase of Klebsíella aerogenes (86), that of Salmonella typhímuríum (87) or that of Sarcína lutea (88) , are capable of oxidizing tyramine, dopamine and norepinephrine. These enzymes are induced by the presence of tyramine and their genes are subjected to catabolic repression in the presence of glucose. But MAO is also present in higher organisms, and in them two isoenzymes have been described, MAOA and MAOB, this classification was made based on the specific inhibition of MAOA by the antidepressant clogiline and MAOB by deprenil (89) . Both isoforms are mitochondrial enzymes that play an important role in the inactivation of biogenic amines, such as adrenaline, norepinephrine, serotonin, dopamine and various "trace amines" (including tyramine).

These two isoforms of the MAO are distributed very heterogeneously in the different tissues of the human body, for example, in the liver the MAOB form is predominant, while in the mucosa of the duodenum the majority isoform is the MAOA, being this isoenzyme the responsible for the deamination of the animals introduced with the diet. For this reason critical hypertension processes will be caused in those patients who consume foods rich in amines and continue treatments with MAOA inhibitors. This is one of the reasons why MAO inhibitor drug treatments are currently aimed at inhibiting the activity of the MAOB isoform (90).

But tyramine deamination is not always catalyzed by amino oxidases flavoproteins. In many organisms, amino oxidases quinoproteins have been identified, (whose mechanism of action has already been explained above), responsible for tyramine deamination, such as Klebsíella oxytoca (68),

E. colí (77) and Euphorbía characías (91). In the latter case the enzyme is a soluble homodimer that contains in the active center a Cu (ll) ion and TPO as cofactor.

On the other hand, other G-bacteria deaminate tyramine through the activity of amino dehydrogenase enzymes. This is the case of Facalcal alkalis and Pseudomonas aerugínosa (68) that have a periplasmic amine dehydrogenase that uses TTO as cofactor.

Finally, it is worth mentioning the existence of another deamination system of

tyramine that is mediated by a peroxidase (EC 1.11.1.7) and that in the presence of H20 2 catalyzes, as a previous step to the oxidation of tyramine, the formation of an intermediate dimer (dithyramine). This peroxidase requires the presence of H20 2 to carry out its action and it has been proven that this enzyme works cooperatively with amino oxidases, the latter acting as donors of H20 2 • Subsequently, the intermediate generated will suffer oxidation mediated by the action of the corresponding (flavoprotein or quinoprotein) amino oxidase in the two amino groups, leading to the formation of a di-p-hydroxyphenylacetaldehyde. (91-93). It can also happen that this peroxidase uses aldehyde and H20 2 as substrates obtained as products of tyramine deamination by amino oxidases, giving rise to di-p-hydroxyphenylacetaldehyde without prior formation of dithyramine (Figure 6). Microbial degradation of tyramine

As indicated above, tyramine can trigger toxic effects in patients treated with MAO inhibitors, or in healthy people when their dietary intake is very high. Therefore, knowledge of the pathways responsible for the degradation of this compound by a microbial agent can have important applications in the field of public health. On the other hand, the transfer of the genes responsible for this pathway could provide the ability to degrade tyramine to organisms used in the food industry, thereby reducing the concentration of tyramine in foods (cheeses, wines, etc.). Finally, the fact that tyramine is an important "trace" amine with function in the central nervous system and that it has structural similarity with important neurotransmitters (for example, dopamine), makes the study of the metabolic pathway responsible for degradation of this compound may have interesting pharmacological applications, and may be used, for example, for the treatment of certain neurological diseases.

In certain microorganisms such as Klebsíella aerogenes (86), Mícrococcus luteus (67), Salmonella typhímuríum (87) and E. colí (62) the presence of enzymes with oxidic tyramine activity that are capable of oxidizing tyramine to 4- is described hydroxyphenylacetaldehyde. In addition, several of the genes and proteins responsible for the degradation of this compound have already been characterized (62). Thus, for example, in E. coli K12, the degradation of tyramine and other related aromatic amines (2-phenylethylamine, dopamine) requires the sequential action of a monoamine oxidase (member of the family of amino oxidases quinoproteins) encoded by the maoA gene, and a phenylacetaldehyde dehydrogenase, a product of the padA gene, that transforms the phenylacetaldehyde generated by the MAOA protein into 4-0HPhAc, which will later be degraded by a specific route (94). Upstream of the maoA gene is the maoB gene that is transcribed in the same direction and encodes a transcriptional regulator belonging to the AraC family. The product of the maoB gene activates the expression of maoA but not that of the padA gene, which is transcribed in the opposite direction. This indicates that, although these genes are physically close to the chromosome and are part of the same catabolic pathway, they do not constitute an operon (94). All microorganisms in which tyramine degradation has been studied use the same enzymes described in E. coli. Tyramine catabolism in P. putida U

Pseudomonas putida U (Spanish Culture Collection Type CECT 4848) is a bacterial strain capable of growing in numerous culture media with defined chemical composition (minimum media or MM), containing as carbon sources different molecules that are hardly degradable, some of which they may even be harmful or toxic to prokaryotes and eukaryotes (95-98). This metabolic versatility has made this bacterium the subject of numerous biochemical and genetic studies, which has led to the clarification of some of the catabolic pathways responsible for the degradation of these molecules. Thus, the routes involved in the degradation of phenylacetic acid and of various structurally related compounds have been identified (n-phenylalkanoic acids, phenylacetaldehyde, 2-phenylethanol, etc.) (82,96-98). Additionally, it has been proven that P. putida U is capable of efficiently degrading other aromatic compounds such as the amino acids phenylalanine, tyrosine and 3-hydroxyphenylacetic acid, using a pathway, characterized by our research group, which involves the conversion of all those compounds in homogetic which is subsequently degraded to fumaric acid and acetoacetic acid (99-100).

This bacterium is also capable of growing in MMs containing aliphatic amines (propyl, butyl, pentyl, hexyl, octal and nonylamines) and aromatic (2-phenylethylamine, tyramine and dopamine) as the only carbon sources. The routes responsible for the degradation of aliphatic amines and 2-phenylethylamine have been identified by our group (82), while the route responsible for the assimilation of tyramine and dopamine in this bacterium was unknown to date. In fact, many researchers proposed that the degradation of tyramine in bacteria belonging to the genus Pseudomonas was performed by the same enzymes described in E. coli, and involved the participation of enzymes encoded by the genes maoA, padA and maoB (94). However, the authors of the invention have reliably demonstrated that, surprisingly, P. putída U degrades tyramine and dopamine by a new catabolic pathway whose enzymes are encoded by the tyn genes as described below. This new catabolic route is the basis of the invention.

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SUMMARY OF THE INVENTION

The present invention relates to an alternative method for the degradation of tyramine and / or dopamine in samples containing them. It is based on the discovery of a new catabolic route, identified by the authors of the invention in Pseudomonas putída U, capable of degrading these two molecules to give rise to pyruvic acid and succinic acid (or succinic semialdehyde). The complete route involves the intervention of enzymes encoded in two clusters, tyn and hpa, which are contiguous in the genome of said bacterium. The enzymes of the first of the clusters, tyn, allow the transformation of both tyramine and dopamine into the corresponding 4-hydroxyphenylacetic acid (3,4-hydroxyphenylacetic acid in the case of dopamine), in two steps: the first involves the action of a tyramine oxidase composed of two subunits, TynA and TynB, capable of acting on both tyramine and dopamine, transforming them into the corresponding 4-hydroxyphenylacetaldehyde; the action on this compound of a dehydrogenase, the TynC enzyme, gives rise to the corresponding 4-hydroxyphenylacetic acid (3,4-hydroxyphenylacetic acid in the case of dopamine). The action on this compound of the enzymes encoded in the hpa cluster allows, through a sequence of catabolic reactions, its transformation into pyruvic acid and succinic acid (or succinic semialdehyde), two general metabolites, crossing of different metabolic pathways in any organism. The complete route needs a further step in the degradation of tyramine: while the action of the enzymes of the tyn cluster on dopamine directly gives rise to 3,4-dihydroxyphenylacetic acid, the substrate of a reaction catalyzed by the diopaxygenase HpaD (3,4- 2,3-dioxygenase dihydroxyphenylacetic), the tyramine transformation requires the action of a mono-oxygenase composed of two subunits, HpaB and HpaC (4-hydroxyphenylacetic monooxygenase) to transform the compound obtained after the action of the enzymes of the tyn cluster on tyramine, 4-hydroxyphenylacetic acid, in 3,4-hydroxyphenylacetic acid. From there, the catabolic procedure is common for both compounds.

Both the catabolic route itself and the enzymes that catalyze the reactions that are part of it (and, in particular, the first of the enzymes of the route, TynAB tyramine oxidase) are new and their nature was not obvious from the prior art, as previously mentioned, the route responsible for the assimilation of dopamine and tyramine in Pseudomonas putída U was unknown to date. The discovery of this route also demonstrates that the degradation of

Tyramine in Pseudomonas is not performed by the same enzymes described in E.

colí, as many authors proposed. That is why, both the tyramine and dopamine degradation process that starts from the tyramine oxidase of Pseudomonas putída (and that can be completed up to pyruvic and succinic acid by the action of the successive enzymes of the route), as well as the amino acid sequence of the enzymes that catalyze said reactions and the coding sequences of said enzymes, are part of the present invention. The expression vectors from which said enzymes can be synthesized (or enzymes with a high degree of homology with them, which are capable of catalyzing the same reactions), as well as the recombinant microorganisms that have been part of the invention are also part of the invention. transformed with said vectors.

Given the great interest that the application of the process of the invention may have in foods, to decrease its tyramine and / or dopamine content, additional use of microorganisms capable of synthesizing at least the first, a subset or all the enzymes necessary to complete the complete catabolic route described to reduce the content of tyramine and / or dopamine in foods that contain them or, even, the use of said microorganisms to act on raw materials to be transformed to be converted in foods or beverages ready for human or animal consumption and that either contain tyramine or dopamine naturally, or are likely to generate them during the process of transforming said raw materials into final foods. This is so for the microorganism that, as disclosed in the present application, naturally contains all the genes necessary to synthesize the enzymes that catalyze the reactions of the complete pathway (Pseudomonas putída U), as well as for possible recombinant microorganisms that have been transformed with some vector that allows the expression of at least one of the genes coding for the enzymes that catalyze the reactions of the catabolic process.

The tyn and hpa clusters also contain other open reading frames that encode polypeptides that appear to be involved in the regulation of the described catabolic pathway. Said polypeptides, the sequences that encode them, the embodiments of the procedure that are carried out in the presence of the regulatory polypeptides encoded in the hpa cluster and / or the regulatory polypeptides encoded in the tyn cluster are also included within the scope of the

invention, as well as expression vectors that include sequences coding for

the same and microorganisms transformed with said vectors.

Thus, the present invention provides both the alternative method described for the degradation of tyramine or dopamine in any sample, as well as the sequences of the proteins capable of catalyzing and regulating the degradation process of tyramine and / or dopamine to pyruvic acid and succinic acid. (or succinic semialdehyde), as well as the nucleotide sequences encoding said proteins. Expression vectors comprising sequences encoding at least one of said proteins and microorganisms transformed therewith also constitute aspects of the invention, as well as the use of Pseudomonas putída U or any of said recombinant microorganisms to decrease the tyramine content and / or dopamine in a sample of a food or beverage intended for human or animal use or in any intermediate product of the transformation of raw materials into the final food or beverage, including the raw materials themselves.

Therefore, a first aspect of the invention relates to nucleic acid molecules from which proteins capable of catalyzing and / or regulating the process of the invention can be synthesized, or encoding proteins with a high degree of homology with the same (defined either by the percentage of coincidence in the nucleotides (at least 60%), or by the ability to mate under strict hybridization conditions (the conditions under which complementary nucleic acid chains hybridize, which are easily identifiable by the person skilled in the art in each specific case, but which can be summarized as high temperature conditions (65 ° C, for example) and low salt concentration)), provided they are capable of carrying out the same activity. Specifically, they refer to the nucleic acid molecules from which the enzyme that catalyzes the essential stage of the degradation process, TynAB tyramine oxidase, or homologous proteins thereto that exhibit the same activity can be synthesized. Therefore, a first aspect of the present invention relates to an isolated nucleic acid molecule encoding a protein or protein complex capable of acting as tyramine oxidase, selected from the group consisting of:

a) a nucleic acid molecule comprising a first sequence that is at least 60% identical to the sequence represented by SEQ ID NO: 3 (corresponding to the DNA encoding the TynA TynA subunit) and a second sequence that it is identical, at least 60%, to the sequence represented by SEQ ID NO: 5 (DNA encoding the TynAB subunit TynAB);

b) a nucleic acid molecule comprising a sequence that

hybridize under strict conditions with the sequences of a);

c) a nucleic acid molecule comprising a first sequence encoding a polypeptide whose amino acid sequence is identical, at least 60%, to the sequence represented by SEO ID NO: 4 (TynA amino acid sequence) and a second sequence encoding a polypeptide whose amino acid sequence is identical, at least 60%, to the sequence represented by SEO ID NO: 6 (TynB amino acid sequence);

d) a nucleic acid molecule comprising a first sequence encoding a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEO ID NO: 4 and a second sequence encoding a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEO ID NO: 6, where the hybrid nucleic acid molecule, under strict conditions, with a DNA sequence comprising the sequences mentioned in a), or complementary sequences thereto.

In a preferred embodiment of the invention, the nucleic acid molecule comprises a first and a second sequence in which the homology percentage in alternative a) or in alternative c) is 100%.

In a further preferred embodiment, the isolated nucleic acid molecule also comprises a nucleic acid fragment from which the enzyme that catalyzes the next stage of the degradation process can be synthesized, TynC 4-hydroxyphenylacetaldehyde dehydrogenase, or a homologous protein thereto. Perform the same function. Thus, in said embodiment, the isolated nucleic acid molecule further comprises a sequence encoding a protein capable of acting as 4-hydroxyphenylacetaldehyde dehydrogenase, selected from the group consisting of:

a) a nucleic acid sequence that is identical, at least 60%, to the sequence represented by SEO ID NO :? (DNA encoding TynC); b) a nucleic acid sequence comprising a sequence that hybridizes under strict conditions with the sequence of a);

c) a nucleic acid sequence comprising a sequence encoding a polypeptide whose amino acid sequence is identical, at least 60%, to the sequence represented by SEO ID NO: 8 (TynC amino acid sequence);

d) a nucleic acid sequence comprising a sequence encoding a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEO ID NO: 8, where the nucleic acid molecule hybridizes, under strict conditions, with a sequence of DNA comprising the sequence mentioned in a), or a sequence complementary to it.

Again, it is especially preferred that the nucleic acid molecule be

corresponds to that of alternative a) or c), in which the homology percentage is

100%

Another additional embodiment, complementary to the previous ones, is that in which the nucleic acid molecule also comprises the coding sequences of the polypeptides that appear to have a regulatory function in the sequence of reactions catalyzed by the enzymes TynAB and TynC, which can be selected from TynR, TynD, Thus, another additional embodiment relates to an isolated nucleic acid molecule comprising, in addition to the sequence related to TynC and / or sequences related to TynAB, a sequence encoding a protein, selected from the group consisting of :

a) a nucleic acid sequence that is identical, at least 90%, to the sequence represented by SEO ID NO: 9 (TynR encoding DNA); b) a nucleic acid sequence comprising a sequence that hybridizes under strict conditions with the sequence of a);

c) a nucleic acid sequence comprising a sequence encoding a polypeptide whose amino acid sequence is identical, at least 90%, to the sequence represented by SEO ID NO: 10 (TynR amino acid sequence);

d) a nucleic acid sequence comprising a sequence encoding a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEO ID NO: 10, where the nucleic acid molecule hybridizes, under strict conditions, with a sequence of DNA comprising the sequence mentioned in a), or a sequence complementary to it.

Again, it is especially preferred that the nucleic acid molecule be

corresponds to that of alternative a) or c), in which the homology percentage is

100%

In another possible embodiment, the isolated nucleic acid molecule comprises, in addition to the sequence corresponding to TynAB, and, optionally, TynC and

TynR, a sequence encoding a protein, selected from the group consisting of: a) a nucleic acid sequence that is identical, at least 90%, to the sequence represented by SEO ID NO: 11 (TynD encoding DNA) ; b) a nucleic acid sequence comprising a sequence that hybridizes under strict conditions with the sequence of a);

c) a nucleic acid sequence comprising a sequence encoding a polypeptide whose amino acid sequence is identical, at least 90%, to the sequence represented by SEO ID NO: 12 (TynD amino acid sequence);

d) a nucleic acid sequence comprising a sequence encoding a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEO ID NO: 12, where the hybrid nucleic acid molecule, under strict conditions, with a sequence of DNA comprising the sequence mentioned in a), or a sequence complementary to it.

Again, it is especially preferred that the nucleic acid molecule be

corresponds to that of alternative a) or c), in which the homology percentage is

100%

In addition to the above combinations, the isolated nucleic acid molecule may also comprise the sequence encoding any of the remaining polypeptides identified in the tyn cluster: TynF, TynE and TynG, or combinations thereof. Thus, another possible embodiment is one in which the isolated nucleic acid molecule additionally comprises a sequence encoding a protein, selected from the group consisting of:

a) a nucleic acid sequence that is identical, at least 90%, to the sequence represented by SEO ID NO: 13 (DNA encoding TynF) and / or a nucleic acid sequence that is identical, at least in one 90%, to the sequence represented by SEO ID NO: 15 (DNA encoding TynE) and / or a nucleic acid sequence that is identical, at least 90%, to the sequence represented by SEO ID NO: 17 (DNA TynG encoder);

b) a nucleic acid sequence comprising a sequence that hybridizes under strict conditions with at least one of the sequences of a);

c) a nucleic acid sequence comprising a sequence encoding a polypeptide whose amino acid sequence is identical, at least 90%, to the sequence represented by SEO ID NO: 14 (TynF amino acid sequence) and / or a sequence encoding a polypeptide whose amino acid sequence is identical, at least 90%, to the sequence represented by SEO ID NO: 16 (TynE amino acid sequence) and / or a sequence encoding a polypeptide whose amino acid sequence is identical, at least 90%, to the sequence represented by SEO ID NO: 18 (amino acid sequence of TynG);

d) a nucleic acid sequence comprising a sequence encoding a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEO ID NO: 14 and / or a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEO ID NO: 16 and / or a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEO ID NO: 18, where the hybrid nucleic acid molecule, under strict conditions, with a DNA sequence comprising the less one of the sequences mentioned in a), or a sequence complementary to it.

Again, it is especially preferred that the nucleic acid molecule be

corresponds to that of alternative a) or c), in which the homology percentage is

100%

For the sequence of reactions catalyzed by the enzymes of the tyn cluster to be carried out in its entirety, it is particularly preferred that the isolated nucleic acid molecule comprises coding sequences of all polypeptides that appear to be encoded in the cluster: Thus, in a Particularly preferred embodiment of this aspect of the invention, the isolated nucleic acid molecule comprises the sequences represented by SEO ID NO: 3 (DNA TynA), SEO ID NO: 5 (DNA TynB), SEO ID NO:? (DNA TynC), SEO ID NO: 9 (DNA TynR), SEO ID NO: 11 (DNA TynD), SEO ID NO: 13 (DNA TynF), SEO ID NO: 15 (DNA TynE) and SEO ID NO: 1 ? (DNA TynG). One possibility would be for the nucleic acid molecule to comprise the sequence represented by SEO ID NO: 2, which corresponds to the tyn cluster of Pseudo monas putída U.

In a preferred embodiment, the isolated nucleic acid molecule comprises not only the open frame sequences identified in the tyn cluster, but also those of the enzymes capable of catalyzing the reactions that give rise to the transformation of the compound obtained by the action of the enzymes the tyn cluster on tyramine, transforming it into pyruvic acid and succinic acid (or succinic semialdehyde): the enzymes of the hpa cluster, preferably with the coding sequences of the additional polypeptides that appear to be also encoded in

that same cluster, and that seem to have regulatory function. As in the case of

tyn cluster, also within the scope of the invention are nucleic acid molecules from which proteins capable of catalyzing and / or regulating the sequence of reactions that results in the transformation of 4-hydroxyphenylacetic acid into syntheses can be synthesized. pyruvic acid and succinic acid, that is, molecules that encode proteins with a high degree of homology with those of the hpa cluster of Pseudomonas putída U and that show the same activity (catalytic or regulatory) as said proteins. Thus, in a particularly preferred embodiment of the nucleic acid molecule of the invention, it comprises,

In addition to the sequences encoding identical or analogous proteins in function and homologous in sequence to the Tyn delcluster, at least 13 sequences, of which each of which encodes a protein capable of catalyzing or regulating a stage of the transformation of a compound of Formula 111, COOH

tH2

15 in which R1 is H,

in pyruvic acid and succinic acid (or succinic semialdehyde) or any of its salts, following the following sequence of reactions:

CHM OMEO HMED

or

HsC - '~' - 'COCH QH {:: ---- GH ~ - · - · v. ~} -C;' ,.) () H

A ~ dQ Pin) vico S ~ miah.i ~~! Dc -5uo: dnico

where each of said sequences is selected from the group consisting of:

a) a sequence that is identical, at least 60%, to the sequence

represented by SEO ID NO: 19 (DNA hpaB), SEO ID NO: 21 (DNA hpaC), SEO ID

NO: 23 (DNA hpaD), SEO ID NO: 25 (DNA hpaE), SEO ID NO: 27 (DNA hpaF), SEO ID

NO: 29 (DNA hpaG1), SEO ID NO: 31 (DNA hpaG2), SEO ID NO: 33 (DNA hpaH), SEO

ID NO: 35 (DNA hpal), SEO ID NO: 37 (DNA hpaA), SEO ID NO: 39 (DNA hpaX), SEO

ID NO: 41 (DNA hpaR1), SEO ID NO: 43 (DNA hpaR2);

b) a sequence that hybridizes under strict conditions with at least one of

the sequences of a);

c) a sequence encoding a polypeptide whose sequence of

amino acids is identical, at least 60%, to the sequence represented by SEO

ID NO: 20 (hpaB), SEO ID NO: 22 (hpaC protein), SEO ID NO: 24 (hpaD protein),

SEO ID NO: 26 (hpaE protein), SEQ ID NO: 28 (hpaF protein), SEO ID NO: 30

(hpaG1 protein), SEO ID NO: 32 (hpaG2 protein), SEO ID NO: 34 (hpaH protein),

SEO ID NO: 36 (hpal protein), SEQ ID NO: 38 (hpaA protein), SEO ID NO: 40

(hpaX protein), SEO ID NO: 42 (hpaR1 protein), SEO ID NO: 44 (hpaR2 protein);

d) a sequence encoding a natural allelic variant of a polypeptide

which comprises the amino acid sequence represented by SEO ID NO: 20, SEO

ID NO: 22, SEO ID NO: 24, SEO ID NO: 26, SEO ID NO: 28, SEO ID NO: 30, SEO ID

NO: 32, SEO ID NO: 34, SEO ID NO: 36, SEO ID NO: 38, SEO ID NO: 40, SEO ID

NO: 42 or SEO ID NO: 44, where the hybrid sequence, under strict conditions, with

a DNA sequence comprising at least one of the mentioned sequences

in a), or a sequence complementary to it.

Fig. 7 shows the specific reaction catalyzed by each of the

mentioned enzymes. It is observed that the reaction catalyzed by the 4

Hydroxyphenacetic monooxygenase hpaBC is only necessary in the event that the

starting compound is tyramine, because the action of the enzymes of the tyn cluster

on dopamine already gives rise to 3,4-dihydroxyphenylacetic acid, the resulting product

of hpaBC activity.

Again, it is especially preferred that the nucleic acid molecule corresponds to that described in alternative a) or c), in which the homology percentage is 100%. Even more preferred is the case in which the nucleic acid molecule comprises the sequence represented by SEO ID NO: 45 (sequence representing the hpa cluster of Pseudomonas putída U).

Inclusion in any one of the nucleic acid molecules isolated from the

invention of a tyrosine decarboxylase coding sequence would allow,

by expressing the proteins encoded in it, decarboxylation could occur

of tyrosine present in the medium to give rise to tyramine, which then

could be degraded thanks to the other enzymes encoded also in the

isolated nucleic acid molecule: the compound to which it would reach would depend

of the particular isolated nucleic acid molecule chosen from among the possible

embodiments thereof previously described. Therefore, the case is preferred in the

that the isolated nucleic acid molecule also comprises a sequence

coding for a tyrosine decarboxylase. Particularly preferred is the case in the

that the tyrosine decarboxylase is the tyrosine decarboxylase A of Lactococcus lactís

(GenBank accession number: CAF33980, included in locus AJ630043).

Another aspect of the invention is the new enzymes and regulatory polypeptides identified, which serve to carry out the process of the invention: those corresponding to the tyn and / or hpa clusters of Pseudomonas putída U, as well as those that keep a high degree of homology with their protein sequences and that perform the same function (enzymatic or regulatory activity). Therefore, a further aspect of the invention relates to a purified protein polypeptide or complex that is selected from the group of:

a) a tyramine oxidase whose amino acid sequence is at least 60% identical to a first polypeptide sequence represented by SEO ID NO: 4 (TynA protein) and a second polypeptide sequence represented by SEO ID NO: 6 (TynB protein), which may be part of a single polypeptide sequence or may be forming a protein complex, associated by bonds other than the peptide bond, such as hydrogen bonds, disulfide bonds or van der Waals forces;

b) a hydroxyphenylacetaldehyde dehydrogenase, whose sequence is at least 60% identical to the polypeptide sequence represented by SEO ID NO: 8 (TynC protein);

c) a protein at least 60% identical to the polypeptide sequence represented by a sequence selected from the group of SEO ID NO: 10 (TynR protein), SEO ID NO: 12 (TynD protein), SEO ID NO: 14 (TynF protein), SEO ID NO: 16 (TynE protein) and SEO ID NO: 18 (TynG protein);

d) a hydroxyphenylacetaldehyde monooxygenase whose amino acid sequence is at least 60% identical to a first polypeptide sequence represented by SEO ID NO: 20 (hpaB protein) and a second polypeptide sequence represented by SEO ID NO: 22 (hpaC protein), hydroxyphenylacetaldehyde monooxygenase in which the sequence at least 60% identical to SEO ID NO: 20 and the sequence at least 60% identical to SEO ID NO: 22 may be part of a single polypeptide sequence or may be forming a complex protein, associated by bonds other than the peptide bond, such as hydrogen bonds, disulfide bonds or van der Waals forces;

e) a hydroxyphenylacetaldehyde dioxygenase, whose sequence is identical to

at least 60% to the polypeptide sequence represented by SEO ID NO: 24

(hpaD protein),

f) a dehydrogenase whose sequence is at least 60% identical to the

polypeptide sequence represented by SEO ID NO: 26 (hpaE protein),

g) an isomerase whose sequence is at least 60% identical to the

polypeptide sequence represented by SEO ID NO: 28 (hpaF protein),

h) a decarboxylase whose amino acid sequence is identical at least in

60% to the polypeptide sequence represented by SEO ID NO: 30 (protein

hpaG1);

i) a decarboxylase whose amino acid sequence is identical at least in

60% to the polypeptide sequence represented by SEO ID NO: 32 (protein

hpaG2),

j) a hydratase whose sequence is at least 60% identical to the

polypeptide sequence represented by SEO ID NO: 34 (hpaH protein),

k) an aldolase whose sequence is at least 60% identical to the sequence

polypeptide represented by SEO ID NO: 36 (hpal protein),

1) a protein at least 60% identical to the polypeptide sequence

represented by a sequence that is selected from the SEO ID group NO: 38

(hpaA protein), SEO ID NO: 40 (hpaX protein), SEO ID NO: 42 (hpaR1 protein),

SEO ID NO: 44 (hpaR2 protein),

or combinations thereof.

The case is preferred in which the polypeptide or protein complex comprises at least one sequence that is identical to that mentioned in one of sections a) to

one) .

Also included within the scope of the invention are compositions comprising at least one of the aforementioned protein polypeptides or complexes. Those compositions comprising at least the first enzyme of the process, which acts on tyramine or dopamine, are preferred: a tyramine oxidase whose amino acid sequence is at least 60% identical to a first polypeptide sequence represented by SEO ID NO: 4 ( TynA protein) and a second polypeptide sequence represented by SEO ID NO: 6 (TynB protein), which may be part of a single polypeptide sequence or may be forming a protein complex, associated by links other than the peptide bond, such as bridges of hydrogen, disulfide bridges or van der Waals forces; Among them, those that additionally comprise an enzyme capable of catalyzing the second stage of the process, a hydroxyphenylacetaldehyde dehydrogenase, whose sequence is at least 60% identical to the polypeptide sequence represented by SEO ID NO: 8 (TynC protein), are particularly preferred. ); more preferably, the composition will contain, in addition to those two enzymes, polypeptides identical or analogous to the remaining polypeptides encoded in the tyn cluster of Pseudomonas putída U, that is, at least one protein at least 60% identical to the polypeptide sequence represented by a sequence selected from the group of SEO ID NO: 10 (TynR protein), SEO ID NO: 12 (TynD protein), SEO ID NO: 14 (TynF protein), SEO ID NO: 16 (TynE protein) and SEO ID NO: 18 (TynG protein) and preferably all of them. The most preferred compositions are those comprising the aforementioned protein polypeptides or complexes and, additionally, identical or functionally identical protein polypeptides or complexes and sequence homologues (at least 60%) to those encoded in the hpa locus of Pseudomonas putida , that is to say:

-

a hydroxyphenylacetaldehyde monooxygenase whose amino acid sequence is at least 60% identical to a first polypeptide sequence represented by SEO ID NO: 20 (hpaB protein) and a second polypeptide sequence represented by SEO ID NO: 22 (hpaC protein), hydroxyphenylacetaldehyde monooxygenase in which the sequence at least 60% identical to SEO ID NO: 20 and the sequence identical to at least 60% to SEO ID NO: 22 may be part of a single polypeptide sequence or may be forming a protein complex, associated by links other than the peptide bond, such as hydrogen bonds, disulfide bonds or van der Waals forces;

-

a hydroxyphenylacetaldehyde dioxygenase, whose sequence is identical to

at least 60% to the polypeptide sequence represented by SEO ID NO: 24

(hpaD protein),

-

a dehydrogenase whose sequence is at least 60% identical to the

polypeptide sequence represented by SEO ID NO: 26 (hpaE protein),

-

an isomerase whose sequence is at least 60% identical to the

polypeptide sequence represented by SEO ID NO: 28 (hpaF protein),

-

a decarboxylase whose amino acid sequence is identical at least in

60% to the polypeptide sequence represented by SEO ID NO: 30 (protein

hpaG1);

-

a decarboxylase whose amino acid sequence is identical at least in

60% to the polypeptide sequence represented by SEO ID NO: 32 (protein

hpaG2),

-

a hydratase whose sequence is at least 60% identical to the sequence

polypeptide represented by SEO ID NO: 34 (hpaH protein),

-

an aldolase whose sequence is at least 60% identical to the sequence

polypeptide represented by SEO ID NO: 36 (hpal protein),

-

a protein at least 60% identical to each of the sequences

polypeptides represented by the SEO ID sequences NO: 38 (hpaA protein), SEO

ID NO: 40 (hpaX protein), SEO ID NO: 42 (hpaR1 protein), SEO ID NO: 44 (protein

hpaR2)

As in the previous cases, it is especially preferred that polypeptides

or protein complexes comprised in the composition comprise a sequence with homology with the sequence represented in the SEO ID group NO: 4 to SEO ID NO: 44 is 100%.

Another aspect of the invention is related to an expression vector comprising at least one of the possible embodiments of the nucleic acid molecule of the invention described above. In a preferred embodiment, said vector is a plasmid. Particularly preferred is the case in which said plasmid comprises a nucleic acid fragment comprising the coding sequences comprised in the Tyn cluster of Pseudomonas putída U. A particular embodiment refers to the case in which said nucleic acid fragment is inserted into the plasmid pK18 :: mob, which is a plasmid capable of remaining as an autonomous replicative form in some bacterial species and with integrative capacity in others.

Another preferred embodiment corresponds to the case in which said plasmid additionally comprises a nucleic acid fragment comprising the coding sequences present in the hpa cluster of Pseudomonas putida. I still know

prefers more the case in which the hpa cluster is inserted in the plasmid is

pK18 :: mob.

In another additional embodiment, the plasmid further comprises a sequence coding for a tyrosine decarboxylase. The case in which the tyrosine decarboxylase is tyrosine decarboxylase A from Lactococcus lactís is preferred.

In a further aspect of the invention, a host organism transformed with any one of the expression vectors described above is described. A preferred case is one in which the host organism is a bacterium, this being preferably a bacterium capable of transforming a sugar into lactic acid (acid lactic acid bacteria). These microorganisms are important because the formation of lactic acid in different raw materials results in denaturation and gelation of proteins, initiating their transformation into food derivatives such as cheese, yogurt, sausages, etc. they act as initiators of the transformation of many raw materials and, in some cases (yogurt), are responsible for their culmination; in others, such as cheese, particularly cured cheese varieties, favor the action of other microorganisms, molds in many cases, which are responsible, for example, for the appearance of characteristic slabid substances. Since in the process of transforming raw materials such as milk or meat into cheese or sausages, amounts of tyramine are often produced that could make it advisable to reduce the present content of said amine, to have lactic bacteria that, in addition to carrying out their function in the process of food transformation, have the enzymes to catalyze the catabolic route disclosed in the present application, which allows the degradation of tyramine and / or dopamine, would facilitate the reduction of the final levels of these biogenic amines present in the food final intended for human or animal consumption.

A possible embodiment of this aspect of the invention contemplates the case in which the expression vector is inserted into the host genome. In another alternative embodiment, said expression vector remains as an autonomous replicative form.

Another aspect of the invention, of great importance, relates to methods that employ the polypeptide sequences described above, to decrease the content of tyramine and / or dopamine in any sample, preferably food and beverages for human or animal consumption. , the raw materials from which they originate or the intermediate products for processing said raw materials in the final food or beverages. The procedure contemplates

all steps of transformation of tyramine and / or dopamine of the catabolic route

discovered by the inventors. All variants thereof comprising the first stage, the transformation of tyramine and / or dopamine into its corresponding 4-hydroxyphenylacetaldehyde are considered included within the scope of the invention. Thus, one aspect of the invention relates to a process for decreasing the content in a sample of a compound of Formula I

OH

Formula I

Wherein R1 is H (tyramine) or OH (dopamine) which comprises a step in which the compound of Formula I is transformed into a compound of Formula 11,

CHO

Formula 11

Wherein R1 is H or OH, characterized in that the transformation of the compound of Formula I into a compound of Formula 11 is catalyzed by a tyramine oxidase encoded by a nucleic acid molecule of claim 1 or 2 or by a tyramine oxidase whose sequence from

20 amino acids is at least 60% identical to a first polypeptide sequence represented by SEQ ID NO: 4 and at least 60% to a second polypeptide sequence represented by SEQ ID NO: 6, tyramine oxidase in which the sequence

at least 60% identical to SEO ID NO: 4 and the identical sequence in at least one

60% to SEO ID NO: 6 may be part of a single polypeptide sequence or may be forming a protein complex, associated by links other than the peptide bond, such as hydrogen bonds, disulfide bonds or van forces

5 der Waals.

A preferred embodiment refers to the case in which the tyramine oxidase is formed by two subunits, one of which comprises the amino acid sequence represented by SEO ID NO: 4 (TynA subunit of the TynAB tyramine oxidase of Pseudomonas putída U) and the second of which comprises the sequence

10 amino acids represented by SEO ID NO: 6 (TynB subunit of the TynAB tyramine oxidase of Pseudomonas putída U). In another embodiment, a step is included in which the compound of Formula II is transformed into a compound of Formula 111, COOH

tHz

15 Formula 111

wherein R1 is H or OH, in which the transformation of the compound of Formula II into a compound of Formula III is catalyzed by a 4-hydroxyphenylacetaldehyde dehydrogenase 20 encoded by a nucleic acid sequence selected from the group of: a) an acid sequence nucleic that is identical, at least 60%, to the sequence represented by SEO ID NO:?; b) a nucleic acid sequence comprising a sequence that hybridizes under strict conditions with the sequence of a);

C) a nucleic acid sequence comprising a sequence encoding a polypeptide whose amino acid sequence is at least 60% identical to the sequence represented by SEO ID NO: 8;

d) a nucleic acid sequence comprising a sequence encoding a natural allelic variant of a polypeptide comprising the 30 amino acid sequence represented by SEO ID NO: 8, where the nucleic acid molecule

hybridizes, under strict conditions, with a DNA sequence comprising the

sequence mentioned in a), or a sequence complementary to it.

or whose amino acid sequence is at least 60% identical to the SEO ID polypeptide sequence NO: 8

In the case where a 4-hydroxyphenylacetaldehyde dehydrogenase is involved in the process, it is preferred that it comprises a polypeptide sequence identical to the amino acid sequence of SEO ID NO: 8 (Pnudomonas putida U tynC protein).

It is particularly preferred that, in addition to the enzymes present that catalyze the transformation steps, at least one protein whose amino acid sequence comprises at least 60% identical sequence to the polypeptide sequence represented by a sequence that is represented in the sample select from the SEO ID group NO: 10, SEO ID NO: 12, SEO ID NO: 14, SEO ID NO: 16 and SEO ID NO: 18 or combinations thereof (the remaining polypeptides synthesizable from the sequences that form part of the tyn cluster, and they seem to have regulatory activity). The case in which the homology percentages are 100% is preferred. It is particularly preferred that at least one protein comprising the amino acid sequence represented by SEO ID NO: 1 O (TynR) be present.

It is particularly preferred that the process does not culminate in obtaining the compound of Formula 111, but continues until its degradation in pyruvic acid and succinic acid (or succinic semialdehyde), especially following the sequence of reactions catalyzed by the enzymes of the hpa cluster of Pseudomonas putida U: When the starting compound is tyramine (R1 = H), the process requires a step of transforming 4-hydroxyphenylacetic acid into 3,4-dihydroxyphenylacetic acid, while, when the starting compound is dopamine (R1 = OH), the compound of Formula III obtained after the reactions catalyzed by the enzymes of the tyn cluster is already 3,4-dihydroxyphenylacetic acid; from there, the reactions are identical both if it has split from tyramine or if it has split from dopamine. Therefore, a further embodiment of the process of the invention refers to the case in which the compound of Formula III obtained is that in which R1 is H, which comprises a subsequent stage in which said compound is transformed into the compound of Formula 111, wherein R1 is OH, by a reaction catalyzed by an enzyme with 4-hydroxyphenylacetaldehyde monooxygenase activity. Within this embodiment, it is preferred that the 4-hydroxyphenylacetaldehyde monooxygenase capable of catalyzing that reaction has a high degree of homology with the corresponding Pseudomonas putída U enzyme, HpaBC, an enzyme composed of two subunits. Thus, in that case, it is preferred that the amino acid sequence of the 4-hydroxyphenylacetaldehyde monooxygenase be at least 60% identical to a first polypeptide sequence represented by SEO ID NO: 20 (HpaB) and a second polypeptide sequence represented by SEO ID NO : 22 (HpaC), said sequences may be part of a single polypeptide sequence or may be forming a protein complex, associated by links other than the peptide bond, such as hydrogen bonds, disulfide bonds or van der Waals forces. More preferably, 4-hydroxyphenylacetaldehyde monooxygenase is formed by two subunits, one of which comprises a polypeptide sequence identical to the amino acid sequence of SEO ID NO: 20 and the second of which comprises a polypeptide sequence identical to the sequence of SEO ID amino acids NO: 22.

As mentioned, embodiments of the process in which the compound of Formula 111, wherein R 1 is OH (3,4-dihydroxyphenylacetic acid), is transformed into pyruvic acid and succinic acid or succinic semialdehyde, or any of its salts, following a reaction sequence analogous to that catalyzed by the enzymes of the hpa cluster of Pseudomonas putída U. In said embodiment, the process comprises the following steps:

a) transforming 3,4-dihydroxyphenylacetic acid into 5-carboxymethyl-2-hydroxy uconic semialdehyde; b) transform 5-carboxymethyl-2-hydroximuconic semialdehyde into 5-carboxymethyl-2-hydroximuconic acid, c) transform 5-carboxymethyl-2-hydroxyconic acid into 5-oxo-pent-3ene-1,2,5-tricarboxylic acid, d) transform 5-oxo-pent-3-ene-1,2,5-tricarboxylic acid into 2-hydroxyhept-2,4-diene-1,7-dioic acid, e) transform 2-hydroxy-hept-2,4 acid -diene-1,7-dioic acid in 2-oxo-hept-3ene-1,7-dioic acid, f) transform 2-oxo-hept-3-ene-1,7-dioic acid into 2,4-hydroxy acid -hept-2ene-1,7-dioic acid, g) cleaving 2,4-hydroxy-hept-2-ene-1,7-dioic acid in pyruvic acid and succinic acid or semialdehyde succinic acid, where any of the acids mentioned may be in the form of any of

Your salts

In a preferred embodiment, the enzymes used in the respective steps

they are enzymes with an activity analogous to those of the hpa cluster and with a high degree of homology with them (at least 60%), that is: a) a hydroxyphenylacetaldehyde dioxygenase whose sequence is at least 60% identical to the polypeptide sequence represented by SEO ID NO: 24, b) a dehydrogenase whose sequence is at least 60% identical to the polypeptide sequence represented by SEO ID NO: 26, c) an isomerase whose sequence is at least 60% identical to the polypeptide sequence represented by SEO ID NO: 28, d) a decarboxylase whose amino acid sequence is at least 60% identical to the polypeptide sequence represented by SEO ID NO: 30, e) a decarboxylase whose amino acid sequence is at least identical 60% to the polypeptide sequence represented by SEO ID NO: 32, f) a hydratase whose sequence is at least 60% identical to the polypeptide sequence represented by SEO ID NO: 34, g) an aldolase whose sequence is identical At least 60% of the polypeptide sequence represented by SEO ID NO: 36. Even more preferred is the case in which the said homology percentages are 100%.

More preferably, in the embodiments of the process comprising these steps, carried out by the described enzymes, in addition to the enzymes that catalyze the sequence of reactions, at least one protein analogous in function and with high homology is present in the sample with at least one of the remaining polypeptides encoded in the hpa cluster, that is, a protein whose amino acid sequence comprises a sequence at least 60% identical to the polypeptide sequence represented by a sequence that is selected from the SEO ID group NO : 38, SEO ID NO: 40, SEO ID NO: 42, SEO ID NO: 44, or combinations thereof. Even more preferred is the case in which the said homology percentages are 100%.

As previously mentioned, in a possible further embodiment of the process of the invention the compound of Formula I is one in which R1 is H (tyramine) and the process comprises a previous stage in which the compound of Formula I is the result of a tyrosine decarboxylation reaction. The case in which the decarboxylation of tyrosine is catalyzed by the tyrosine decarboxylase A of Lactococcus lactís is preferred.

In one embodiment of the process of the invention, the enzymes that catalyze

The procedure is added to the sample as part of a composition of the invention.

In another embodiment, the enzymes that catalyze the steps of said process are synthesized in the sample from one of the expression vectors of the invention described above.

The embodiment is preferred in which the enzymes that catalyze the reactions of the process of the invention are synthesized by a microorganism present in or added to the sample. In that case, one possibility is that the coding sequences of the enzymes synthesized by the microorganism are naturally present in its genome: this is what happens with Psedomonas putída U (CECT 4848), whose presence in the sample in which it is wanted Reducing the tyramine and / or dopamine content is one of the possible alternatives to perform the process of the invention.

Another possible embodiment, for which particular preference is also taken, corresponds to the case in which the microorganism is one of the recombinant host organisms of the invention: this gives many possibilities to choose the exogenous genes introduced therein and, thereby, the steps of the process of the invention. On the other hand, the choice of the microorganism also grants a lot of versatility to choose the additional characteristics that the sample is to be endowed with, in addition to the decrease in tyramine and / or dopamine; This characteristic is of special interest for its application in the food industry.

Accordingly, embodiments of the process of the invention are of particular interest, those in which the process of the invention is carried out within the framework of the food industry, the sample in which it is desired to decrease the content of the compound of the Formula I (tyramine or dopamine) a food or beverage intended for consumption by humans or animals, a starting raw material for obtaining said food or drink or an intermediate product in obtaining the food or drink.

A preferred case is one in which the food, raw material or intermediate product is selected from a dairy derivative, milk or milk mixture from any mammal, or an intermediate product from the transformation of milk or milk mixture and the compound of the Formula I that is to be transformed is that in which R1 is H (tyramine), since dairy derivatives, and cheeses in particular, are products in which tyramine concentrations may appear that advise their decrease to make them more appropriate for human consumption, avoiding possible side effects. Therefore, in a preferred embodiment of the above, the food, raw material or intermediate product is selected from cheese, milk from cow, sheep or goat (the species of which usually comes from the milk used as raw material in the preparation of cheese), a mixture of milk from at least two of the above species; or an intermediate product of the transformation of milk or milk mixture into cheese. The case is preferred in which the enzymes that catalyze the process steps are synthesized by a microorganism present in the sample or added thereto. A specific case is one in which the microorganism is a recombinant host organism described above, preferably a bacterium capable of transforming a sugar into lactic acid, even more preferably belonging to the genus selected from Lactobacillus; Lactococcus, Leuconostoc, Pedíococcus, Streptocuccus. This provides the advantage, on the one hand that the transformation is carried out with a microorganism of a species that naturally intervenes in the process of food transformation; on the other, if the microorganism is added from the beginning, as an initiating culture, it will be present from the beginning of the process to control the tyramine that is produced. Thus, another additional embodiment refers to the described process, in which the microorganism acts as an initiator (starter) of the transformation of milk into a dairy derivative.

Another alternative embodiment of the process of the invention is that in which it is carried out in an alcoholic beverage, in which the beverage, raw material or intermediate product is selected from must, barley, malt, wine, beer, an intermediate product of the transformation of barley in beer, an intermediate product of the transformation of must into wine, any other alcoholic beverage that requires alcoholic fermentation by yeasts or any intermediate product of its transformation. Again, the case is preferred in which the enzymes that catalyze the process steps are synthesized by a microorganism present in or added to the sample, which is a recombinant host organism of the invention. In a particularly preferred embodiment, the beverage is wine and the microorganism is added to the wine during malolactic fermentation.

Another alternative embodiment describes a process, in which the food, raw material or intermediate product is selected from a sausage; an intermediate product of the transformation of meat into sausage or beef; porcine

or deer or mixtures thereof intended for the preparation of a sausage.

Another alternative embodiment is that relating to a process, in which the food, raw material or intermediate product is selected from sauerkraut; the raw material thereof or an intermediate product of obtaining sauerkraut.

Even more preferred is an embodiment in which in the last two processes mentioned, the enzymes that catalyze the process steps are synthesized by a microorganism present in the sample or added thereto which is a recombinant host organism described above. The case in which the microorganism acts as an initiator (starter) of the transformation of the raw material into the sausage or sauerkraut is preferred.

Because of its importance in the application in the food industry, a further aspect of the invention relates to the use of microorganisms that synthesize the enzymes suitable for carrying out the process of the invention to decrease the content of tyramine and / or dopamine in a food or drink intended for human or animal consumption. A first possibility is that the organism synthesizes these enzymes naturally; Therefore, a possible embodiment is the use of Pseudomonas putída U to reduce the content of tyramine or dopamine in a food or beverage intended for human or animal consumption, in the raw material used to obtain it or in an intermediate product of the transformation of the raw material in the food or drink.

The second possibility is that the microorganism is a recombinant microorganism, in which the appropriate genes have been introduced to synthesize the desired regulatory enzymes and polypeptides. Thus, a final aspect refers to the use of a recombinant host organism of the invention, to decrease the content of tyramine or dopamine in a food or beverage intended for human or animal consumption, in the raw material used for obtaining it or in an intermediate product of the transformation of the raw material into the food or drink.

The control of starter microorganisms that initiate the processes of transformation of certain raw materials into foods or beverages (dairy products, sausages, alcoholic beverages, other foods in which fermentation processes such as sauerkraut, pickles or olives) It has gained great interest in recent years in the food industry, as it allows to control the process conditions, provide the final products with desired characteristics and, in particular, facilitates the obtaining of final products whose characteristics are more homogeneous and identifiable by the consumer as the expected characteristics in a certain brand, made more difficult to achieve when starting from the initiating organisms that contain the raw material naturally. The use of initiators (starters) capable of carrying out the tyramine and / or dopamine transformation process of the invention is a further advantage within the process control, allowing the control of the concentration of these biogenic amines from the beginning of the process. , as they are produced and, even, as the case may be, that they are the same microorganism that can produce them that allow their elimination. That is why a preferred embodiment of the use of the invention is that in which the host organism acts as an initiator (starter) of the process of transformation of the raw material into the food or beverage intended for human or animal consumption. It is particularly preferred that the food is a dairy derivative (more preferably cheese), a sausage, or a food during which fermentation such as sauerkraut, pickles or olives occurs.

Detailed description of the invention

The bacterium P. putida U (CECT 4848) has been characterized by a new catabolic pathway responsible for the degradation of the biogenic amines tyramine and dopamine. The different genes that encode the enzymes that make up such a route, have been identified by isolating different Pseudomonas putída U mutants unable to grow in media that contained as sole sources of tyramine or dopamine carbon. The mutagenic agent used was the transposon Tn5, which acts by randomly integrating itself into the chromosome of the bacteria (98,105). Through this procedure, eleven different mutants were isolated that were grouped into three types based on their ability to degrade different carbon sources. Type 1 mutants included those that were unable to grow in chemically defined media when they contained tyramine or dopamine as the only carbon sources. However, these mutants effectively degraded 4-hydroxyphenylacetic and 3,4-dihydroxyphenylacetic acids, as well as many other carbon sources. The second type (type 2) included a group of mutants that did not grow in those media that contained as sole sources of tyramine or 4-hydroxyphenylacetic carbon, but that they did in those others to which dopamine or other susceptible carbon sources had been added if used by the parental strain (P. putida U CECT 4848). The third group of mutants, those included in type 3, were characterized in that they could not grow in those culture media where tyramine, dopamine, 4-hydroxyphenylacetic or 3,4-dihydroxyphenylacetic carbon was used as sole sources of carbon. However, these mutants grew effectively in those same media when these compounds were replaced by other carbon sources that could be assimilated by the wild strain.

All these results indicated that in type 1 mutants, the transposon had been integrated into a DNA sequence belonging to one of the genes necessary for the transformation of tyramine and dopamine into 4-hydroxyphenylacetic and 3,4-dihydroxyphenylacetic acids. respectively (Fig. 7), or the transposon had been integrated into an area that affected the expression of any of these genes. In addition, the fact that these mutants grew well in media supplemented with 4-hydroxyphenylacetic and 3,4-dihydroxyphenylacetic, suggested that in them the transposon was affecting genes that had to do with the deamination of these two amines but not with subsequent catabolic stages. .

In type 2 mutants, the transposon must have been integrated into a DNA sequence corresponding to one of the genes encoding enzymes required for the catabolism of tyramine and 4-hydroxyphenylacetic acid, but not for the degradation of dopamine. Taking into account that there is only one catalytic stage whose alteration justifies this metabolic behavior, these mutants should be affected in the gene coding for 4-hydroxyphenylacetic hydroxylase (HpaBC in Fig. 7).

Type 3 mutants cannot catabolize tyramine, dopamine, 4-hydroxyphenylacetic or 3,4-dihydroxyphenylacetic acid, which indicates that in them the transposon is affecting the expression of some of the genes that encode the enzymes responsible for the transformation of 3,4- dihydroxyphenylacetic in the final products (pyruvic acid and succinic acid) (Fig. 7).

The identification of the transposon insertion point in each of the mutants and the sequencing of adjacent areas, has allowed us to identify all the genes necessary for the degradation of tyramine, dopamine, 4-hydroxyphenylacetic and 3,4-dihydroxyphenylacetic acid in P. putída U. All of them are located in a 25132 base pair DNA fragment that includes two consecutive gene clusters (Fig. 8). The tyn cluster (12339 base pairs) groups the genes required in P. putída U for the transformation of tyramine and dopamine of 4-hydroxyphenylacetic and 3,4-dihydroxyphenylacetic acids respectively, this being the first description of this set of genes ( tynABFECGRD) and, in addition, the first evidence that shows that this new route is involved in the deamination of these two compounds. Next to the tyn cluster is the hpa cluster (12722 base pairs), which contains all the genes (hpaR1TetRBCIHXFDEG2G1AR2) necessary for the catabolism of 4-hydroxyphenylacetic acid (including its 3,4-dihydroxyphenylacetic acid derivative) in this bacterium (Fig. 8) . The sequences of all those genes (tyn and hpa) are included in Fig. 9.

In summary, we have shown that in P. putída U the transformation of tyramine and dopamine into pyruvic acid and succinic acid requires the genes corresponding to the tyn and hpa clusters, and that the degradation of tyramine in this bacterium implies the participation of a higher number of genes than those described for other microorganisms (68,77, 86-88, 91, 94). Another important difference is that in P. putída U the deamination of tyramine and 2-phenylethylamine is carried out by the action of different enzyme complexes (82). On the other hand, the genes responsible for the degradation of 4-0H-PhAc in P. putída U, have an organization very similar to that described for the same route of E. eoli.

The identification of the tyn genes can have important biotechnological implications since, as we have indicated in the "State of the Art" section, the consumption of foods with a high tyramine content can cause a large number of pharmacological effects. Therefore, obtaining a genetic construct that allowed carrier organisms to degrade this compound could be used to transform those microorganisms involved in fermentation. Thus preventing the accumulation of tyramine in these foods.

These recombinant strains capable of degrading tyramine could even be used in the development of starter cultures, giving them greater efficiency, since the starters, although they usually consist of non-producing strains of biogenic amines, not they can prevent the accumulation of the amines produced by the microbial flora present in the original raw materials. On the other hand, if these starters had the presence of bacteria capable of degrading tyramine and dopamine, they would avoid the accumulation of these compounds in food, regardless of the raw materials used in their preparation.

In addition, analyzed from a strictly economic point of view, the possibility of providing bacterial strains with interest for the food industry with the capacity required to degrade tyramine, may be essential for the development of new products and the opening of new markets since Many countries (Canada, Switzerland or the Netherlands, among others) are setting limits for the concentration of biogenic amines in imported foods, especially wine. The presence of amines in wine carries more risk than in other foods, since by containing alcohol, the body's detoxification mechanisms will be affected, increasing the chances of poisoning by amine intake.

By transfer of the tyn and hpa gene clusters as isolated or tandem genetic cassettes (tyn and hpa clusters) or identical genes, whose sequences are similar and fulfill the same function as the tyn and hpa genes of

P. putída U 4848 we could confer to any bacterium, both G + and G-, of the capacity required to degrade tyramine and dopamine, thus avoiding the accumulation of this compound in those foods in which the bacterium was involved in the manufacturing process. The transfer of the tyn and hpa clusters or identical genes, whose sequences are similar and that fulfill the same function as the tyn and hpa genes of P. putída U 4848 to strains of Lactococcus lactis would prevent the accumulation of tyramine (generated by decarboxylation of the tyrosine by tyrosine decarboxylase present in this organism) in those cheeses and derivatives whose production process involves this bacterium.

Additionally, the fact that the enzymes encoded in the tyn and hpa clusters are also capable of degrading dopamine will allow for genetically engineered organisms that can assimilate this important neurotransmitter, which could have important applications in food and gene therapy. that some of the tyn genes could be used for the treatment of certain degenerative diseases or those related to mental disorders caused by high concentrations of these amines.

Finally, through Metabolic Engineering, we have managed to establish a new useful route for the degradation of the amino acid L-tyrosine through the joint participation of the enzymes encoded by the tyn and hpa clusters of P. putída U and the tdcA gene that encodes the tyrosine decarboxylase of Lactococcus lactís (see Example 5). An interesting application of the confluence in the same microorganism of the genes tyn and hpa and tdcA, which allow to catabolize tyrosine through the tyramine intermediate, could be the preparation of foods with low content in this amino acid. In order to obtain these, starter cultures containing explicitly designed microorganisms could be used to express the enzymatic activities Tyn, Hpa and TdcA. In this way we would have food that could be suitable for the consumption of those patients suffering from alkaptonuria (metabolic disease characterized by the absence of the enzyme homogentisate dioxygenase that causes the blockage of the degradative pathway of phenylalanine and tyrosine and that imply the accumulation of homogetic acid in the tissues causing degeneration of the affected tissue) and other diseases related to the existence of an excessive accumulation of tyrosine catabolites in different tissues (99-100).

EXAMPLE 1. Identification of the genes responsible for the degradation of tyramine and dopamine in P. putida U

Pseudomonas putída U (CECT 4848) is a bacterium that can grow using tyramine (5 mM) or dopamine (5mM) as the only carbon sources, when grown in a medium of defined chemical composition that contains (in giL) KH2P04 (13, 6); (NH4hS04.7H20 (0.25); FeS04.7H20 (0.0005). If the culture medium was solid, 2.5% (w / v) agar was also added. The incubations were performed in a orbital shaker, at 30 ° C and 250 rpm, using 500 mL Erlenmeyer flasks containing 100 mL of medium (Fig. 10), when P. putída U was mutated with the transposon Tn5, following the methodology described by us in other publications (82 , 95, 98100, 1005), we isolated several mutants incapable of degrading tyramine and dopamine (Fig 10) but that, however, grew well when other carbon sources (4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, were added to the medium) phenylacetic, benzoic, octanoic, glutamic, succinic, 2-phenylethylamine.) Sequence localization

(98) of the transposon on the chromosome of the different mutants, allowed us to verify that this mobile genetic element had been inserted into two open reading frames or open reading frames (ORFs) (tynA and tynB genes) that encode two proteins (TynA and TynB) (Fig 7 and Fig 8) which, according to these results, were essential for the tyramine and dopamine cartilage in P. putída U.

The additional sequencing of the areas adjacent to these genes allowed us to identify the ORFs indicated in Figure 8 as tyn and whose sequences are included in Fig. 9. All those genes (tynABFECGRD) are close to another known cluster (hpa) , responsible for the transformation of 4-hydroxyphenylacetic acid (and its derivatives) into general metabolites (pyruvic and succinic acids) (Fig 8 and Fig. 9). EXAMPLE 2. Identification of the minimum functional genetic unit required for oxidative deamination of tyramine and dopamine in P. putida U

The functional analysis of the genes that make up the tyn cluster was performed by interrupting each of them following a procedure that involves a simple recombination event, and that is based on the use of an internal fragment of each gene as it is He has described in different publications (82, 98-100). When the disruption of any of these genes involved lack of function (absence of growth in media supplemented with tyramine or with dopamine), a wild copy of the gene that had been affected in each mutant was cloned into a replicative plasmid in Pseudomonas (pBBR1 MCS- 3, abbreviated pMC) (102) And it was expressed, in trans, in that mutant, establishing whether or not the effect observed after the mutation of that particular gene was reversed. Following this method, we found that in P. putída U the genes tynA, tynB, tynC and tynR were essential for the deamination of tyramine and dopamine to occur and for the further oxidation of the aldehydes generated to 4-hydroxyphenylacetic acids and 3,4-dihydroxyphenylacetic acid, respectively (Fig. 7).

In other cases, the disruption of certain genes (tyn F, tynE, tynG and tynD) did not affect the ability of different mutants to degrade tyramine and dopamine, so we conclude that, at least in P. putída U, these are not indispensable for assimilating these amines (probably because there are other genes in their genome that encode homologous enzymes).

These results indicate that the genetic construction that has the minimum information necessary to catalyze the oxidative deamination of tyramine and dopamine in P. putída U, is tynABCR. EXAMPLE 3. Obtaining a genetic construct that can degrade tyramine and dopamine in other bacteria

In order to have a genetic construct that could be transferred to different microorganisms in such a way as to confer the ability to partially or totally degrade tyramine and dopamine, the genes that integrate the entire tyn cluster into plasmids pK18 were cloned: mob (replicative in E. colí and integrative in Pseudomonas). (82, 98-100,103) (Fig 11). This plasmid has no origin of replication in Pseudomonas. The process of obtaining the tyn cluster was carried out by recombination of a DNA fragment (cloned in pK18 :: mob) homologous to the region of one end of the cluster, by subsequent digestion to all of the DNA with Xbal and by religion end of digested DNA. The selection of clones containing cloned the tyn cluster was performed by selecting the plasmid marker (resistance to km). With them, E. coli W14 (a mutant incapable of degrading phenylethylamine and tyramine was transformed because it lacked the maoA and maoB genes) (62, 94) and P. putída KT2440 a wild strain that is very similar metabolically and genetically to P. putída U, but that lacks the tyn and hpa genes. The results presented in Fig. 12 reveal that the recombinant E. coli strain (E. colí W14 pKtyn), unlike the parental strain (E. colí W14 or of this strain transformed with the plasmid without insert -E. Coli W14 pK18 :: mob-) grew in minimal media supplemented with tyramine or dopamine. However, P. putída KT2440 pKtyn could not do it unless another source of carbon was supplemented to the crops (Fig 13). In this case, after growing, the analysis of the culture broths by HPLC (see conditions at the end of the example) revealed that P. putída KT2440 pKtyn had transformed both tyramine and dopamine into 4-hydroxyphenylacetic and 3.4 -dihydroxyphenylacetic acid respectively and that this conversion was not carried out by the wild strain (P. putída KT2440) or that same strain transformed with the plasmid without insert (P. putída KT2440 pK18 :: mob). These results indicate that in the presence of another carbon source, the recombinant strain P. putída KT2440 pKtyn expresses the tyn genes, but, since it does not possess the hpa genes, it cannot continue degrading the products generated (4-hydroxyphenylacetic, 3,4- dihydroxyphenylacetic) so it excretes them by accumulating them in the broth. In the absence of an additional carbon source,

P. putída KT2440 pKtyn cannot grow because it cannot obtain energy either from tyramine or from dopamine (Fig 13). However, E. coli W14 pKtyn degrades both compounds since E. coli W14 has an hpa cluster that allows it to continue degrading the 4-hydroxyphenylacetic and 3,4-dihydroxyphenylacetic atoms generated through the Tyn path (Fig. 12) .

Additionally, we found that the expression in E. coli W14 of a construct that contained all the tyn genes except tynD was unable to grow using tyramine or dopamine as the only carbon sources at 37 ° C, while at 30 °. These results suggest that the tynD gene encoding an alleged tyramine deaminase could constitute a catalytic subunit that facilitates or accelerates the deamination rate at restrictive temperatures for P. putída U.

Analysis of the culture broths by high performance liquid chromatography (HPLC).

The consumption of tyramine, dopamine, 4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid and other compounds, as well as the accumulation of the catabolic intermediates accumulated by the different mutants when they were grown in liquid medium, was carried out by means of HPLC analysis. To do this, samples were taken from the culture broths (1 ml) at different times. These were centrifuged

(31,000 x g, 20 minutes) to remove cell debris and were filtered through Millipore filters (pore size 0.2 IJm). Different aliquots of 20 111 were analyzed by HPLC (Beckman System Gold Mod 126. Programmable Solvent Module) equipped with a UV / visible variable wavelength detector (Beckman System Gold Diode Array Detector Module Mod 168), an integrator with the system Waters Millenium 32 computer analysis and a reverse phase column (Nucleosil C18, 250 x 4.6 mm internal diameter) microparticulate (particle size 10 IJm, pore size 1 IJm) (Phenomenex Laboratories USA) with an Analytical Guard Cartridge pre-column System, (KJO-4282).

The mobile phase used contained KH2 P04 (50 mM, pH 4) and acetonitrile (CH3 CN), in a 99: 1 ratio (vol / vol). The flow was maintained at 2.5 ml / min and the eluent was monitored at 210 nm. Under these conditions, the retention times for dopamine, tyramine, 2-phenylethylaminePhEtNH2, 3,4-dihydroxyphenylacetic acid, 4-hydroxyphenylacetic acid, and phenylacetic acid were 3; 4.5; 9.6; 13; 24 and 54 minutes, respectively. EXAMPLE 4. Obtaining and using a genetic construct that contains all the tyn and hpa genes and that gives the recipient organism the ability to assimilate tyramine and dopamine

Since the use of the tyn genes allowed only those microorganisms that had a functional hpa route to grow at the expense of tyramine or dopamine, we proceeded to design a genetic construct that allowed us to transfer both clusters (tyn and hpa) to other organisms, conferring them the ability to degrade these two biogenic amines to pyruvate and succinate.

To achieve this, we followed the procedure outlined in Fig. 14 and which involved: (i) the cloning of the genes adjacent to the tyn and hpa clusters in two plasmids (pK18 :: mob and pJQ200KS) (104); (ii) two recombination events, simple and independent, through which both plasmids were inserted into the bacterial chromosome (one on each side, see Fig. 14); (iii) digestion of the entire bacterial chromosome with Xbal or Hindlll restriction endonucleases, and (iv) the release of a DNA fragment carrying plasmid pK18 :: mob and an insert of just over 25 kilobases containing the clusters tyn and hpa (Fig. 14). The transformation of P. putída KT2440 with this integrative plasmid by triparental mating (98, 105) gave the recombinant bacteria the ability to grow in media containing tyramine or dopamine as the only carbon sources (Fig 13). EXAMPLE 5. Design of a metabolic drift protocol that gives different microorganisms the ability to degrade the amino acid L-tyrosine via the tyramine route

The catabolism of the amino acid L-tyrosine (a proteinogenic amino acid

precursor of many amines with extraordinary biological importance) has been studied in numerous living things. In certain bacteria (such as Pseudomonas) and in all eukaryotic cells, degradation takes place through a well-known route that involves its deamination (transamination) to phydroxyphenypruvic; decarboxylation and intramolecular rearrangement of this compound to give homogenetic (2,5-dihydroxyphenylacetic); the opening of the benzene ring causing maleilacetoacetic acid; its isomerization to fumarylacetoacetic acid and, finally, the hydrolysis of this compound to give fumaric and acetoacetic acid (99). However, many bacteria, as in the case of the E. colí paradigm, do not have a catabolic pathway to transform that amino acid into general intermediaries and therefore cannot grow in those media where there is only a source of tyrosine carbon. Other bacteria only partially degrade tyrosine by reactions that involve deamination or decarboxylation (see prior art).

We have developed a procedure that allows combining the tyn and hpa genes of P. putída U and tdcA, which encodes the tyrosine decarboxylase of Lactococcus lactís, so that the expression of one or the other in different microorganisms (depending on their catabolic capacity ) confer on the recipient microorganism the ability to degrade the amino acid L-tyrosine, via tyramine, to pyruvate and succinate.

P. putída U is capable of degrading L-tyrosine via the homogentysic route (99), however a mutant in which the hmg cluster (99) has been deleted cannot completely catabolize tyrosine, since it is unable to degrade the Homogentisic acid, a compound that accumulates in the broth and that in contact with the oxygen in the air darkens (as is the case with alkaptonuric patients). However, this P. hmg mutant has the functional Tyn and Hpa pathways. Therefore, if that bacterium were able to decarboxylate tyrosine to tyramine, it could grow at the expense of this carbon source. To verify this, we cloned the Lactococcus lactis tdcA gene into plasmid pBBR1 MCS-3 (pMC) and that construct was used to transform P. putida U L1hmg. The recombinant strain obtained P. putída U L1hmg pMCtdcA) was able to efficiently degrade L-tyrosine when grown in different media containing this amino acid as the only carbon source (Fig. 15). In addition, it was observed that the metabolic derivation to tyramine was very high since homogentisate hardly accumulated in the culture medium. When is

same construction (pMCtdcA) was used to transform a mutant of P. putída U in which in addition to the hmg cluster, the hpd gene encoding the enzyme 4-hydroxyphenylpyruvate dioxygenase (responsible for the synthesis of homogetic from 4-hydroxyphenylpyruvic) had been deleted , the recombinant strain obtained (P. putida 5 L1hmgL1hpd pMCtdcA) degraded the tyrosine even more efficiently than the one that lacked only the hmg cluster (Fig. 15). This effect can be explained by assuming that, since the degradative path is blocked at a stage prior to the formation of homogentisic acid, the loss of excretable catabolic intermediates is lower and, consequently, a higher percentage of L-tyrosine will be decarboxylated and degraded.

Through the Tyn and Hpa tracks.

As we have indicated previously, E. colí W14 has the hpa cluster, so this bacterium can degrade 4-hydroxyphenylacetic and 3,4-dihydroxyphenylacetic, but cannot assimilate L-tyrosine or tyramine (all strains of E. colí lack the homogentísico route and this particular strain has lost, after suffering an event of

15 deletion, the maoA and maoB genes). Therefore, so that E. colíW14 can grow in L-tyrosine using tyramine as an intermediate, it requires a tyrosine decarboxylase activity that generates tyramine, and the enzymes encoded by the tyn genes to transform that amine into 4-hydroxyphenylacetic.

The transformation of E. colí W14 with the constructions pKtyn and pMCtdcA we

20 allowed to obtain a recombinant strain (E. coli W14 pKtyn pMCtdcApMCtdcA) that grew in defined composition media containing L-tyrosine or L-phenylalanine as the only carbon sources (Fig. 16). Brief Description of the Figures Figure 1 shows the biosynthetic pathway of neurotransmitters

25 dopaminers. In this route, the amino acid tyrosine is hydroxylated by tyrosine hydroxylase in dihydroxyphenylalanima (L-DOPA), which in turn is decarboxylated to dopamine by the enzyme DOPA decarboxylase. Dopamine, by hydroxylation with dopamine hydroxylase, gives rise to norepinephrine, and this, in turn, by the action of a phenylethanolamine N-ethyltransferase, gives rise to adrenaline.

Figure 2 is the schematic representation of the synthesis of histamine (A) from histidine by the action of a histidine decarboxylase, and serotonin (B) from tryptophan, thanks to two enzymes: tryptophan 5-hydroxylase and 5-dihydroxytryptophan decarboxylase. Figure 3 shows the structure of the most important "trace" amines. The ~

Phenylethylamine, tyramine and tryptamine are synthesized by decarboxylation of

corresponding precursor amino acids by the action of an aromatic L-amino acid decarboxylase. Octamine is synthesized by hydroxylation of tyramine by the action of a tyramine-~ -hydroxylase.

Figure 4 represents the main biogenic amines present in food and their precursor amino acids.

Figure 5 is a schematic representation of the reaction mechanism used by the QH-AmDH to oxidize the primary amines (modified figure of Sun et al, 2003, ref. 81).

Figure 6 is a schematic representation of tyramine oxidation in

Euphorbia charades.

Figure 7 is the schematic representation of the metabolic steps responsible for the degradation of tyramine, dopamine, 4-hydroxyphenylacetic acid and 3,4-dihydroxyphenylacetic acid (homoprotocathetic) in P. putída U and the enzymes that catalyze each of them. The different abbreviations correspond to: 4-0HPhAc (4-hydroxyphenylacetic acid), 3,4-diOH-PhAc (homoprotocatechic acid), CHMS (5-carboxymethyl-2-hydroxyconic acid semialdehyde), CHM (5-carboxymethyl-2-hydroxyconic acid), OPET (5-oxo-pent-3-ene-1, 2,5-tricarboxylic acid), HHDD (2-hydroxy-hept-2,4-diene-1,7-dioic acid), OHED (2-oxo-hept acid -3-Jan-1,7-dioic) and HHED (2,4-dihydroxy-hept-2-ene-1,7-dioic acid). The enzymes are: TynAB (tyramine oxidase), TynC (4-0H-Phenylacetaldehyde dehydrogenase), HpaBC (4-0H-PhAc monooxygenase), HpaD (3,4-diOH-PhAc 2,3-dioxygenase), HpaE (CHMS dehydrogenase ), HpaF (CHM isomerase), HpaG1 G2 (OPET decarboxylase), HpaH (hydratase) and Hpal (HHED aldolase). This scheme also shows the organization of the genes involved in this catabolic pathway in P. putída U. The metabolic block point in the different types of mutants is also indicated in the figure.

Figure 8 is the scheme of the genetic organization of the two c1usters (tyn and hpa) involved in the degradation of tyramine and 4-0H-PhAc in P. putída. In this scheme, the insertion point of the Tn5 transposon is also indicated in each of the mutants, as well as some of the restriction cuts used for the elaboration of different genetic constructs.

Figure 9 is the nucleotide sequence of the DNA fragment containing the tyn and hpa genes in Pseudomonas putída U (CECT 4848). The regions of the sequence that have a secondary structure of type loop (loop) are boxed.

In Figure 10, the curve diagram on the left represents the curves of

growth measures such as (A54onm) in MM + dopamine (5mM) medium corresponding to the P. putída U CECT 4848 (wild) (.), P. putída U tynR :: pK18mob (.6.) strains and one of the type I (Aa) (e), type II (A2) (o) and type III (A7) (o) mutants. The 5 graphs on the right represent the residual concentration of dopamine in the culture broth of the different strains grown in MM + dopamine medium (5Mm). The behavior of the different mutants included in the different groups was similar to each of those indicated in the figure.

Figure 11 is a schematic representation of genetic construction

pKtyn-In this construction, the genes that integrate the entire tyn cluster into plasmids pK18 :: mob (replicative in E. colí and integrative in Pseudomonas) were cloned. (82, 98-100,103) (Fig 11).

In Figure 12, the curve diagram on the left represents the growth curves measured as (A54onm) measured in the E. colíW14 strain strain pKtyn (e, o) and the E. colíW14 control strain pK18 :: mob (. , o) when grown at 30 ºC (e, •) and at 37 ºC (o, o) in a medium containing tyramine (5mM) as the sole source of carbon. The panel on the right represents the residual concentration of tyramine in the culture broth of E. colí W14 pKtyn grown at 30 ° C and at 37 ° C. Similar results were obtained by culturing said strains in minimal medium supplemented with dopamine.

Figure 13 depicts the growth curves corresponding to the P. putída KT2440 pKtynhpa Xbal (o, e) and P. putída KT2440 pKtyn (or,.) Strains when grown in a medium containing tyramine (5mM) (or, or ) or 4-0H-PhAc (5mM) (e, •). Growth was measured as A540nm.

Figure 14 shows the schematic representation of the strategy followed to obtain the pKtynhpa Hindlll and pKtynhpa Xbal constructs that contain the genetic information necessary to degrade tyramine, dopamine, 4-0HPhAc and 3,4-diOH-PhAc.

Figure 15 represents the growth curves measured as (A54onm) corresponding to the strains: P. putida U. 6.hmgABC pMCtdcA (e); P. putída U .6.hpd .6.hmgABC pMCtdcA (or) and their respective control strains: P. putída U .6.hmgABC pMC (.) And P. putída U .6.hpd .6.hmgABC pMC (or )., when grown in a medium of defined composition containing L-tyrosine (5mM) as the sole source of carbon.

Figure 16 Growth curves measured as (A54onm) corresponding to strain E. colí W14 pKtyn pMCtdcA (e) and its respective control strain E. colí

W14pKtyn pMC (.), When grown in a medium of defined composition containing L-tyrosine (5mM) as the sole source of carbon.

Claims (20)

1. An isolated nucleic acid molecule that encodes a protein or protein complex capable of acting as tyramine oxidase, selected from the group consisting of: a) a nucleic acid molecule comprising a first sequence that is identical, at least 60%, to the sequence represented by SEO ID NO: 3 and a second sequence that is identical; at least 60%, at sequence represented by SEO ID NO: 5; b) a nucleic acid molecule comprising a sequence that hybridize under strict conditions with the sequences of a); c) a nucleic acid molecule comprising a first sequence encoding a polypeptide whose amino acid sequence is identical, at least 60%, to the sequence represented by SEO ID NO: 4 and a second sequence encoding a polypeptide whose amino acid sequence is identical, at least 60%, to the sequence represented by SEO ID NO: 6; d) a nucleic acid molecule comprising a first sequence encoding a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEO ID NO: 4 and a second sequence encoding a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEO ID NO: 6, where the hybridized nucleic acid molecule, under strict conditions, with a DNA sequence comprising the sequences mentioned in a), or complementary sequences thereto. 2. Isolated nucleic acid molecule according to claim 1, selected from the group consisting of: a) a nucleic acid molecule comprising a first sequence that is identical to the sequence represented by SEO ID NO: 3 and a second sequence that is identical to the sequence represented by SEO ID NO: 5; b) a nucleic acid molecule comprising a first sequence encoding a polypeptide whose amino acid sequence is identical to the sequence represented by SEO ID NO: 4 and a second sequence encoding a polypeptide whose amino acid sequence is identical to the sequence represented by SEO ID NO: 6.

3.

Nucleic acid molecule according to claim 1, comprising the sequences represented by SEO ID NO: 3 and SEO ID NO: 5.

Four.

A purified protein polypeptide or complex with tyramine oxidase activity comprising two amino acid sequence fragments: a first fragment whose amino acid sequence is at least 60% identical to a first polypeptide sequence represented by SEO ID NO: 4 and a second fragment whose amino acid sequence is at least 60% identical to a second polypeptide sequence represented by SEO ID NO: 6, tyramine oxidase in which the sequence is at least 60% identical to SEO ID NO: 4 and the identical sequence at least 60% to SEO ID NO: 6 may be part of a single polypeptide sequence or may be forming a protein complex, associated by links other than the peptide bond, such as hydrogen bonds, disulfide bonds or van der Waals forces .

5.

Purified polypeptide or complex with tyramine oxidase activity according to claim 4, comprising two amino acid sequence fragments: a first fragment whose amino acid sequence is identical to a first polypeptide sequence represented by SEO ID NO: 4 and a second fragment whose sequence of Amino acids are identical to a second polypeptide sequence represented by SEO ID NO: 6, tyramine oxidase in which the sequence identical to SEO ID NO: 4 and the sequence identical to SEO ID NO: 6 may be part of a single polypeptide sequence or may be forming a protein complex, associated by bonds other than the peptide bond, such as hydrogen bonds, disulfide bonds or van der Waals forces.

6.

An expression vector comprising a nucleic acid molecule according to any one of claims 1 to 3.

7. Expression vector according to claim 6, which is a plasmid.

8.

Expression vector according to claim 7, wherein the nucleic acid molecule of any one of claims 1 to 3 is inserted into plasmid pK18 :: mob.

9.

A host organism transformed with an expression vector according to any one of claims 6 to 8.

10. Host organism according to claim 9, which is a bacterium.

eleven.

Host organism according to claim 10, which is a bacterium capable of transforming a sugar into lactic acid.

12.

Host organism according to any one of claims 9 to 11, wherein the expression vector of any one of claims 6 to 8 is inserted into the host genome.

13.

Host organism according to any one of claims 9 to 11, wherein the expression vector of any one of claims 6 to 8 remains as an autonomous replicative form.

14.

A composition comprising the polypeptide or protein complex of claim 4 or 5.

fifteen.

Use of the nucleic acid molecule of any one of claims 3, of the expression vector of any one of claims 6 to 8, of the host organism of any one of claims 9 to 13, of the purified polypeptide or complex of a any of claims 4 or 5 or the composition of claim 14 to decrease the content of tyramine or dopamine in a food or beverage intended for human or animal consumption, in the raw material used for obtaining it or in an intermediate product of the transformation of the raw material into the food or drink.

16.

Use according to claim 15, wherein a host organism of any one of claims 9 to 13 is used.

17.

Use according to claim 16, wherein the host organism acts as an initiator (starter) of the process of transformation of the raw material into the food or beverage intended for human or animal consumption.

18.

Use according to claim 17, wherein the food is a dairy derivative.

19.

Use according to claim 18, wherein the dairy derivative is a cheese.

Use according to claim 17, wherein the food is a sausage. 21. Use according to claim 17, wherein the food is a food in which fermentation occurs in its manufacturing process, which is selected from sauerkraut, pickles or olives. SEQUENCE LIST <110> BIOGES STARTERS S.A. <120> NEW TIRAMINA OXIDASA, NUCLEIC ACID THAT CODIFIES AND USES ITSELF <130> P-101090 <160> 45 <170> PatentIn version 3.3 <210> 1 <211> 25132 <212> DNA <213> Pseudomonas putida U <220> <221> misc feature <222> (1) .. (25132) <223> Sequence containing the tyn and hpa clusters <400> 1 tcaggcgaaa cgctcgaagc ggtacggtga cgggtcgatc agcggggtgg cctgggccac 60 caggtctgcc gccagctggc cagcagcagg cgaggtgccg aagccatgcc cggaaaagcc 120 ggtggccagg gtcaggcccg gaatactggc caccgggccg atgaccgggt tggagtcggg 180 ggtgacgtca atcgtgccgg cccaggcgct ggcgatacgg acaccggcca gcctgttcga 240 aggttgcgca ggccgctttc tggcctcgtc gttgagggcc gggttggcgt gcgggtcttg 300 tacccgtaca cgctcgaagg gggttacatc cgttgccttc cagcgccggg ccagggccag 360 gtccttgaag aagtacttgc caaagctgat gcgcaaaaag tcccgctggg cacgcagctg 420 cgcttgccca gggcaggtaa gcagcaggtg atcgagggtg aggaaggcgt ccagcgcgcc 480 gcgctgggtg atgatgtagc cgccgtcctt gtgcttgcgg aaggaaaaat ctggtgcgcc 540 cacggcgatg tcggttggcc cgtccatggg ctctgtgcgc agcacggaac aggtcagcgg 600 caaggtcggc aggttgatgc ccaggttgcc gaggaacttg cgcgaccaca ggccaccggc 660 cagcaacacc tggtcgcagc ggatttcacc ttgctcggtg accaccccgc tgacacggcc 720 ggctgcggtg accagcgtgc gcaccgcgca gttctccact accactgcac ctttggcgat 780 cgccgcccgg gcgatggcgc tggcggccag ggtcggttcg gcgcgggcgt cggagggggt 840 gaagatgcca cc tgcccaat ccgcccgacc acccggcacc atccgggtga tttcccgcgt 900 gctcagcagg cgcgaatcca ggcccagcgc ctcgacgctt ttcagccagc cttcatgcat 960 gcccatctgc gtgtcgttac ggccgatgaa catgatgccg gcttgccgat agccaacgtc 1020 cgtgcgggca gctgccaacc cagccgatca tctcggccca gccgccagtg ccaggggaat 1080 gtcatgggcg tggcggttgg tcttgcgcac ccagcccagg ttgcgcgacg actgctcccc 1140 agcgatgcgc cccttctcca gcaccaccac cggtatgttg cgttcggcga ggctcagtgc 1200 ggcggtgagg ccgataatgc cgccaccgat gatcaccacg gtagtggcgt cggggtggcg 1260 ggtgctggtt tgcacagggg cgatcgtggg agacatggct ttactctttg ttgtgcgtgc 1320 ttcagcgcca agggggagtg tcactggcca gccagcagcc aggcggatca gggtcacttg 1380 cgcttgcccc gcaccgcggt aggcggtgac ctccagctcg accttgtaaa cggtggagcc 1440 cagcggcggg caggtgaccg tggtggccgg gtcgatgccg cggaacttct cgccgatcac 1500 gtccatgacc cgtggtacat cggcagggtc ctggatgaac acgcgcgagt tgatgacatc 1560 ggccaggctg gcatcgactg cggccagcgc ggtttcgatg ttggcgaaca cctggtgggt 1620 ctgttcgatg acgtcctctg gaatgacctg ggtctgcggg ttgcgtccgg cggtgttgga 1680 cagttgtcca gacgtgaatc ccgccaccag gcgggagtag ctggccatgg cttcgaactt 1740 ggagccggtt ttcagtttga tgatctgtgt catgggcttt gccttgttat ccggttgcgg 1800 ggatcagctg agaacggggg tttcccagag gttgagcttt acgccgatgc cttgctcgag 1860 cgccttgcgg tacaccacgg tgccccaggc cacgtcttcg acgggcatgc cgcccaccga 1920 catcaggatg atttcgtcgt catgcaggcg gcccggtgcg tcgccgctga tgatcttgcc 1980 gatgtcttcc acctgctcgg cggccagcgt atcatgtcca gccttcggca tgaagcgcac 2040 cgccggccgg gaataccacc tggccttcat gctggttgcg ggtgttcggg gtcttgacga 23340 acaacaccgg cttgaccggc agttgcttgt acggtgcttc cacgaacgcc gcttggtgct 23400 gctgcagcaa accctggtag ttcagcgcga cgccgaacag ggtgccgctg gcaacgtcaa 23460 gcagggcatg gctcatgctc ttctcctggc agtgcagggc ggtggccgtc ctgcggattt 23520 cgttaatgtg ttaatgttat agttaatatg ttaacgatgg tcaaggggtg gccagtggcg 23580 aggcaaggca cctgccggca tcgtcaacag ccatgggcca tttgcgagca ggtcaagcga 23640 agcagccatg agcgaccggc atccgatacc gaacatcaac attggccagg tttacgacca 23700 gcgctacagc gacagcgagg tgcattacga ccggctgggc aacctggcgg gctttttcgg 23760 gcgcaacatg ccggtgcacc ggcatgaccg gtttttccag gtgcattacg tgaagtcggg 23820 cacagtacgg gtgtatctgg atgaccagca gtacatcgag gccgggccga tgttcttcct 23880 cacgccaccc acggtggcgc acgcgttcgt caccgaagct gacagcgacg ggcatgtgct 23940 gacggtgcgc cagcaactgg tgtggcaatt gatcgaagcc gacgccagcc tgctgccggc 24000 gggcatgcag gtgcagccag cctgtgtggc gctgggcaac ctgccggccg aatacaaggc 24060 cgaggcgcag cgcctgcaag gctggctgga cgcgttgagt gacgagtttg ccacgcagc to 24120 accgggtcgc gaggcggcgt tgcagtcgct gacccgcctg atcatgatca gcctgctgcg 24180 gctgtgcccc aactcgctgg aatcgacccc ggcgcggcat gaagacctga agatcttcca 24240 ccgtttcaat gccctgatcg aagcgcatta ccttgagcat tggccgctgg cccgctacgc 24300 gcagcagatt ggcgtgaccg aggcacggct gaacgatgtg tgccggcgca tcgccgactt 24360 gccatccaag cgcctggtgc tggaacggct gatgcaggag gccaagcgtt tgctgttgtt 24420 ttccggcagc acggccaacg aaatctgtta ccagctcggc ttcaaggatc cggcctattt 24480 cagccgcttc ttcaaccgct acgccaagct cacacccggg gagtaccgcc agcggcaggc 24540 agaattgcag tgaaatggcc atggcggctc acccgggtgc tgttgttgtt tacagcggat 24600 ggtcgcagcc cgcgcgccgg gcttgaatgg gttttccgtg gaacagattg cactttccat 24660 cgtgcatgcc cttaaattcg aaaagccaca tgaattgaga ggtttgacca tgaccaagac 24720 ctcacgctaa gcaaccttcg gcctgttgca ggcccgagaa gccgcgatgg catttttcag 24780 gccgctgttg aaccagcacg acctgaccga gcagcaatgg cgggtaatcc gcatcctcaa 24840 gcagcacggc gagctggaga attatcagtt ggcggaactg gcctgcatcc tcaagccgag 24900 catgaccggg gtactggggc gcctggagcg gtgcggcggc to agacgggctg gaaggccgc 24960 gcaggaccag cgacgggtgt tcgtcagcct gaccgaaaga ggggaggcgt gctttgcctc 25020 gatgaaggaa ggcatggagg ccaactacca gaagattcag gcgcagtttg gtgaagagaa 25080 gctgcggcgtgg <210> 2 <211> 12339 <212> DNA <213> Pseudomonas putida U <220> <221> misc feature <222> (1) .. (12339) <223> Cluster tyn <400> 2 tcaggcgaaa cgctcgaagc ggtacggtga cgggtcgatc agcggggtgg cctgggccac 60 caggtctgcc gccagctggc cagcagcagg cgaggtgccg aagccatgcc cggaaaagcc 120 ggtggccagg gtcaggcccg gaatactggc caccgggccg atgaccgggt tggagtcggg 180 ggtgacgtca atcgtgccgg cccaggcgct ggcgatacgg acaccggcca gcctgttcga 240 aggttgcgca ggccgctttc tggcctcgtc gttgagggcc gggttggcgt gcgggtcttg 300 tacccgtaca cgctcgaagg gggttacatc cgttgccttc cagcgccggg ccagggccag 360 gtccttgaag aagtacttgc caaagctgat gcgcaaaaag tcccgctggg cacgcagctg 420 cgcttgccca gggcaggtaa gcagcaggtg atcgagggtg aggaaggcgt ccagcgcgcc 480 gcgctgggtg atgatgtagc cgccgtcctt gtgcttgcgg aaggaaaaat ctggtgcgcc 540 cacggcgatg tcggttggcc cgtccatggg ctctgtgcgc agcacggaac aggtcagcgg 600 caaggtcggc aggttgatgc ccaggttgcc gaggaacttg cgcgaccaca ggccaccggc 660 cagcaacacc tggtcgcagc ggatttcacc ttgctcggtg accaccccgc tgacacggcc 720 ggctgcggtg accagcgtgc gcaccgcgca gttctccact accactgcac ctttggcgat 780 cgccgcccgg gcgatggcgc tggcggccag ggtcggttcg gcgcgggcgt cggagggggt 840 gaagatgcca cc tgcccaat ccgcccgacc acccggcacc atccgggtga tttcccgcgt 900 catcatctgg aaggcatagg tgtttatagt gcgtgcgtgc agctgcggct ccagcttgtt 11580 gaggaagctg tcgcacgccc acagcagctt gctggcgcgt accgagccac ggccggtgcg 11640 taccgtgatg cgctcgccgt aggtcacttc cagggccggg ctgtgttcga agatgcgcgc 11700 accatggccc accagtgcct gcgcttcgcc cagcagcagg ttcagggaat gcacatggcc 11760 accgcccatg tgcatcaggg cgctgctgta ggcgttgctg ccgatgatct ggcgcacttc 11820 gctgccaccg agaaaacgga tctcgtcgcg ggtattgatc gccttgaacg ccttctccca 11880 tttgcgcagg gtctgttcct ggcggcggtt gaagcccatg tagccatagc cgtggcagaa 11940 gtcggcgtcg atggcgtagc gggcgatgcg gtccttgatg atgccggcgc ccagttcgct 12000 atatccctca gatttcgaaa cgccctgatc accgacgctg ctgcggatct tctccaggtc 12060 gtggccgatg cccgccatga tctgcccgcc gttgcgcccg ctaccgccgt agcccagata 12120 acggccctcg agcacgacga tattggtcac gccttgttcc gccagctcca gggcggtgtt 12180 aatgccggag aaaccgccac cgatcaccac gacatcggcc tcgatgtcgc gttccagggt 12240 tgggaagctc aggttgtact tcttggtcgc cgagtagtag gtggggctct cgagggtgat 12300 12339 catgacgccg cctgctgact ggaaatgggt agaaatcat <210> 3 <211> 1296 <212> ONA <213> Pseudomonas putida U <220> <221> COS <222> (1) .. (1296) <223> TynA coding sequence <400> 3 atg tct cee acg ate gcc cct gtg caa acc agc acc cgc falls cee gac 48 Met Ser Pro Thr 11e Wing Pro Val G1n Thr Ser Thr Arg His Pro Asp 1 5 10 15 gcc act acc gtg gtg ate ate ggt ggc ggc att ate ggc etc acc gcc 96 Ala Thr Thr Val Val 11e 11e G1y G1y G1y 11e 11e G1y Leu Thr Ala 20 25 30 gca ctg agc etc gcc gaa cgc aac ata ccg gtg gtg gtg ctg gag aag 144 Ala Leu Ser Leu Ala G1u Arg Asn 11e Pro Val Val Val Leu G1u Lys 35 40 45 ggg cgc ate gct ggg gag cag tcg tcg cgc aac ctg ggc tgg gtg cgc 192 Gly Arg 11e Wing G1y G1u G1n Ser Ser Arg Asn Leu G1y Trp Val Arg 50 55 60 aag acc aac cgc falls gcc cat gac att cee ctg gca ctg gcg gct gat 240 Lys Thr Asn Arg His Wing His Asp 11e Pro Leu Wing Leu Wing Asp Wing 65 70 75 80 cgg ctg tgg gcc gag atg cee gca cgg gtt ggc agc gac gtt ggc tat 288 Arg Leu Trp Wing G1u Met Pro Wing Arg Val G1y Ser Asp Val G1y Tyr 85 90 95 cgg caa gcc ggc ate atg ttc ate ggc cgt aac gac acg cag atg ggc 336 Arg G1n Wing G1y 11e Met Phe 11e G1y Arg Asn Asp Thr G1n Met G1y 100 105 110 atg cat gaa ggc tgg ctg aaa agc gtc gag gcg ctg ggc ctg gat tcg 384 Met His G1u G1y Trp Leu Lys Ser Val G1u Ala Leu G1y Leu Asp Ser 115 120 125

att gae gte Ile Asp Val 370

aee thr cee gae Pro Asp tee aae Ser Asn 375 eeg gte Pro Val ate gge Ile Gly 380 eeg gtg Pro Val Gee Ala agt be 1152

att eeg Ile Pro 385

gge gly etg Leu aee thr etg Leu 390 Gee Ala aee thr gge gly ttt tee Phe Ser 395 ggg gly eat his gge gly tte Phe gge Gly 400 1200

aee thr

teg eet get Ser Pro Ala get Ala 405 gge gly eag Gln etg geg Leu Ala Gea Gae Wing Asp 410 etg gtg Leu Val Gee Ala eag Gln 415 Gee Ala 1248

aee thr

eeg Pro etg Leu ate gae Ile Asp 420 eeg Pro tea eeg Ser Pro tae ege Tyr Arg 425 tte Phe gag Glu egt Arg tte gee Phe Ala 430 tga 1296

<210> <211> <212> <213>

4 431 PRT Pseudomonas putida u

<400>

4

Met 1

Be pro Thr Ile Wing 5 Pro Val Gln Thr 10 Be Thr arg His Pro Asp 15

To

Thr Thr Val 20 Val Ile Ile Gly Gly Gly 25 Ile Ile Gly Leu 30 Thr wing

To

Leu Be 35 Leu  To Glu Arg Asn 40 Ile Pro Val Val Val 45 Leu Glu Lys

Gly Arg 50

Ile Ala Gly Glu Gln 55 Be Be Arg Asn Leu 60 Gly Trp Val Arg

Lys 65

Thr asn Arg His Wing 70 His Asp Ile Pro Leu 75 To Leu To To Asp 80

Arg

Leu Trp Wing Glu 85 Met Pro Ala Arg Val 90 Gly Be Asp Val Gly Tyr 95

Arg

Gln To Gly 100 Ile Met Phe Ile Gly Arg Asn 105 Asp Thr Gln 110 Met Gly

Met

His Glu 115 Gly Trp Leu Lys Ser Val 120 Glu To Leu Gly 125 Leu Asp Be

Arg

Leu 130 Leu Be Thr arg Glu 135 Ile Thr Arg Met Val 140 Pro Gly Gly Arg

Wing Asp Trp Wing 145

Gly Gly 150 Ile Phe Thr Pro Be Asp Wing Arg Wing 155 Glu 160

Pro

Thr Leu To Wing 165 It will be the Ile Ala Arg Wing Wing 170 Ile Ala Lys 175 Gly

Wing Val

Val Val Glu Asn Cys  Wing Val Arg Thr Leu Val Thr wing To

180 185 190 Gly Arg Val Ser Gly Val Val Thr Glu Gln Gly Glu Ile Arg Cys Asp 195 200 205 Gln Val Leu Leu Wing Gly Gly Leu Trp Ser Arg Lys Phe Leu Gly Asn 210 215 220 Leu Gly Ile Asn Leu Pro Thr Leu Pro Leu Thr Cys Ser Val Leu Arg 225 230 235 240 Thr Glu Pro Met Asp Gly Pro Thr Asp Ile Val Val Gly Ala Pro Asp 245 250 255 Phe Ser Phe Arg Lys His Lys Asp Gly Gly Tyr Ile Ile Thr Gln Arg 260 265 270 Gly Ala Leu Asp Ala Phe Leu Thr Leu Asp His Leu Leu Leu Gly Lys 275 280 285 Arg Tyr Leu Pro Gln Leu Arg Ala Gln Arg Asp Phe Leu Arg Ile Ser 290 295 300 Phe Gly Lys Tyr Phe Phe Lys Asp Leu Ala Leu Ala Arg Arg Trp Lys 305 310 315 320 Wing Thr Asp Val Thr Pro Phe Glu Arg Val Arg Val Gln Asp Pro His 325 330 335 Wing Asn Pro Wing Leu Asn Asp Glu Wing Met Arg Asn Leu Lys Wing Wing 340 345 350 Trp Pro Val Phe Glu Gln Wing Arg Ile Wing Being Wing Trp Wing Gly Thr 355 360 365 Ile Asp Val Thr Pro Asp Being Asn Pro Val Ile Gly Pro Val Ala Ser

370

375 380

Ile Pro

Gly Leu Thr Leu To Thr Gly Phe Be Gly His Gly Phe Gly

385

390 395 400

Thr

Be pro To To Gly Gln Leu To Asp wing Leu Val To Gln To

405

410 415

Thr

Pro Leu Ile Asp Pro Be pro Tyr arg Phe Glu Arg Phe Ala

420

425 430

<210> 5 <211> 1140 <212> ONA <213> Pseudomonas putida U <220> <221> COS <222> (1) .. (1140) <223> TynB <400> 5

tae tyr

eeg Pro etg gtg Leu Val ege ege Arg Arg 245 gag Glu tgg gte Trp Val aag Lys 250 eet Pro gge geg Gly Ala tte Phe etg Leu 255 Gee Ala 768

atg Met

cea gee Pro Ala eeg Pro 260 tge agt Cys Ser ate gae gee Ile Asp Ala 265 gge atg Gly Met gag Glu eag Gln gae gae gtg Asp Asp Val 270 816

ege aag Arg Lys

gtg gtg Val Val 275 gae aae Asp Asn aee thr gge Gly 280 Leu etc tae tyr gag Glu Gee Ala tgg Trp 285 tte Phe Gaa Glu gag Glu 864

etg Leu

cee Pro 290 aag Lys eet geg Pro Ala His falls aae Asn 295 His falls gta Val eeg Pro etg gta ggt gtg Leu Val Gly Val 300 ege Arg tte Phe 912

atg Met 305

gae Asp atg Met att gee Ile Ala Gaa Glu 310 gge gly aeg Thr etg gee Leu Ala Gee Wing 315 gag Glu eag Gln gtg Val Gaa Glu gae Asp 320 960

ate gge aag Ile Gly Lys

ate Ile ate age Ile Ser 325 gge gae gea Gly Asp Ala eeg Pro 330 gge ege Gly Arg etg Leu eat his gae gae Asp Asp 335 1008

Gaa Glu

ate Ile ate etg atg teg gtg Ile Leu Met Ser Val 340 gge gge atg Gly Gly Met 345 cee gte Pro Val  Gaa Glu gae gtg Asp Val 350 Gee Ala 1056

tgg gge Trp Gly

aee gtg gtg Thr Val Val 355 tae ege aag Tyr Arg Lys 360 geg wing Leu etc gag Glu eaa Gln gge Gly 365 ate gge gta Ile Gly Val 1104

aag Lys

etc Leu 370 aae Asn Leu etc tgg trp Gaa Glu aee Thr 375 cee gtt Pro Val Leu etc age Ser tga 1140

<210> <211> <212> <213>

6 379 PRT Pseudomonas putida u

<400>

6

Met 1

Thr Leu Asp Thr Arg 5 Ile Asp Phe Ile Tyr 10 Leu Be Glu Gln 15 Asp

Met

Ile Arg Ala 20 Gly val Thr asp Met 25 Pro wing Cys Val Asp 30 Thr met

Glu

Glu

Met 35 Phe Gly Leu Leu Tyr 40 Gln Gly Asp Tyr Arg 45 Met To Gly

Pro Asn 50

Be asp Be his Gly Ala 55 Met Ile Thr Phe 60 Pro Glu His Be

Pro 65

Phe Pro Asn Met Pro 70 Lys Pro Thr Wing Asp Arg Arg 75 Met Met 80 wing

<210> <211> <212> <213>

7 1488 ONA Pseudomonas putida u

<220> <221> <222> <223>

COS (1) .. (1488) TynC

<400> 7 atg age gae Met Ser Asp 1

ate aee 11e Thr 5 Leu etc eta Leu eet gee gte Pro Ala Val 10 aeg gee Thr Ala tte Phe etg gee ege Leu Ala Arg 15 48

gag Glu

eat his gge gtg G1y Val 20 tte ate falls Phe 11e His gge G1y eag G1n 25 His falls Leu etc Gee Ala age eag Ser G1n 30 teg teg Be Being 96

teg aae Ser Asn

att gee gtg gte 11e Ala Val Val 35 aae Asn eeg gee aae Pro Ala Asn 40 gge G1y eag G1n aee Thr 45 ate gee 11e Ala His falls 144

ate gee gae gee aae 11e Ala Asp Ala Asn 50

eag gee gat gte G1n Ala Asp Val 55 gae Asp eat his gee gte Ala Val 60 age Ser tee be teg be 192

ege Arg 65

eaa gge ttt G1n G1y Phe aee gee Thr Ala 70 tgg teg falls Trp Ser His aee thr age cee gee gee ege gee Ser Pro Ala Ala Arg Ala 75 80 240

gea gtg etg Ala Val Leu

tte aag Phe Lys 85 etg gee gae Leu Ala Asp etg etg gaa gee aae Leu Leu G1u Wing Asn 90 ege Arg gaa gaa G1u G1u 95 288

etg geg Leu Ala

eag etg gaa aee G1n Leu G1u Thr 100 ttg Leu eaa tee gge aag G1n Ser G1y Lys 105 Leu etc ate 11e gge G1y 110 att 11e tee be 336

egt geg Arg Ala

tte gaa gta Phe G1u Val 115 eag G1n eag G1n Gee Geg Ala Wing 120 His falls tte Phe etg Leu ege tae tae gee Arg Tyr Tyr Ala 125 384

gge tgg geg Gly Trp Wing 130

aee thr aag Lys ate aee 11e Thr 135 gge G1y eag G1n aee thr ate aee 11e Thr 140 eeg Pro teg etg Ser Leu cee Pro 432

teg tte gee Ser Phe Ala 145

ggt G1y gag G1u ege tae Arg Tyr 150 age gee Ser Ala tte Phe aee Thr 155 etg Leu ege Arg gag eeg G1u Pro att 11e 160 480

gge gtg gtg gtg Gly Val Val Val

gge ate gtg G1y 11e Val 165 eeg Pro tgg aae Trp Asn 170 tte gee Phe Ala age atg Ser Met ate gee 11e Wing 175 528

ate tgg aag 11e Trp Lys

etg gee Leu Ala 180 teg gee Ser Ala etg Leu aea Thr 185 aee thr gge tge G1y Cys age Ser att 11e 190 ate 11e Leu etc 576

aee aae Thr Asn

gae Asp 435 etg gge aag Leu Gly Lys Gee Ala atg Met 440 ege atg Arg Met ate eeg Ile Pro eaa Gln 445 ate eag Ile Gln Gee Ala 1344

ggt gly

aee Thr 450 etg tgg gte Leu Trp Val aae Asn atg Met 455 His falls aee thr etg Leu Leu etc gae Asp 460 eeg get Pro Ala gta Val eeg Pro 1392

ttt ggg gge Phe Gly Gly 465

ate aag Ile Lys get Ala 470 tee be gge gly att gge ege Ile Gly Arg 475 gag Glu tte Phe gge gly teg gee Ser Ala 480 1440

tte ate gat gae Phe Ile Asp Asp

tte Phe 485 aee thr gag Glu Leu etc aag Lys teg gtg atg Ser Val Met 490 ate ege tae Ile Arg Tyr 495 tga 1488

<210> <211> <212> <213>

8 495 PRT Pseudomonas putida u

<400>

8

Met 1

Be asp Ile Thr 5 Leu Leu Pro wing Val 10 Thr wing Phe Leu Wing 15 Arg

Glu

His Gly Val 20 Phe Ile His Gly Gln 25 His Leu To Be Gln 30 Be Be

Be asn

Ile Wing 35 Val Val Asn Pro Wing 40 Asn Gly Gln Thr 45 Ile Ala His

Ile Ala Asp Ala 50

Asn Gln Asp Val 55 wing Asp His Wing Val 60 Be Be Be

Arg 65

Gln Gly Phe Thr Ala 70 Trp Be his Thr Be Pro Wing Wing 75 Arg Wing 80

Wing Val

Leu Phe Lys 85 Leu Asp wing Leu Leu 90 Glu Asn wing Arg Glu 95 Glu

Leu

To Gln Leu 100 Glu Thr  Leu Gln Be 105 Gly Lys  Leu Ile Gly 110 Ile Be

Arg wing

Phe 115 Glu Val Gln Gln Wing wing 120 His Phe Leu Arg Tyr Tyr Ala 125

Gly Trp Wing 130

Thr Lys Ile Thr 135 Gly Gln Thr Ile Thr 140 Pro Be Leu Pro

Be Phe Ala 145

Gly Glu Arg Tyr 150 It will be the Phe Thr 155 Leu Arg Glu Pro Ile 160

Gly val

Val Val Gly 165 Ile Val Pro Trp Asn 170 Phe Ala Be Met Ile Wing 175

Ile Trp Lys

Leu To It will be the Leu Thr Thr Gly Cys Be Ile Ile Leu