MXPA00011426A - Oxygenase enzymes and screening method - Google Patents

Abstract

A method for detecting the presence of an oxygenated compound which is produced when a substrate is reacted with an oxygenase for the substrate. The method involves reacting a coupling enzyme with the oxygenated compound to form a polymeric oxygenated compound which is fluorescent or luminescent. Measurement of the fluorescence or luminescence of the polymeric oxygenated compound provides indirect detection of the oxygenated compound produced by reaction of the oxygenase with the substrate. The method is carried out in a whole cell environment wherein the cell is transformed to express both the oxygenase being screened and the coupling enzyme. The method can be used to measure the activity of monooxygenases and dioxygenases on aromatic substrates. The method is amenable to large scale screening of enzyme mutants to isolate those with maximum oxygenase activity.

Description

OXYGENASE ENZYMES AND SELECTION METHOD The Government has certain rights for this invention under Concession No. N0014-96-1 -0340, assigned by the United States Navy. This application claims priority of the E.U.

No. 60 / 094,403 filed July 28, 1998; No. 60 / 106,840 filed on November 3, 1998; No. 60 / 086,206 filed on May 21, 1998; No. 60 / 106,834 filed on November 3, 1998.

BACKGROUND OF THE INVENTION Field of the Invention The invention relates to enzymes, called oxygenases, which are biologically active proteins that catalyze certain oxidation reactions that include the addition of oxygen to a substance. Oxygen transfer from an oxygen donor compound, such as molecular oxygen (O2) and hydrogen peroxide (H2O2), to any one of millions of useful aromatic or aliphatic substrate compounds, is important in organic chemistry and in many biochemical reactions. Typical oxidation reactions include hydroxylation, epoxidation and sulfoxidation, which are widely used in the production of chemicals that include pharmaceutical compounds and others in medicine. Enzymes that catalyze or improve oxidation reactions are useful in science and iirt T i i rTfcM ^^ tüüÉÉi = ^ i industry. The invention relates to novel oxygenase enzymes having improved properties. The invention also relates to methods for selecting oxygenase enzymes, and more particularly, to methods for identifying oxidation enzymes that exhibit catalytic activity with respect to the insertion of oxygen into aliphatic or aromatic compounds. The selection method includes introducing an organic substrate compound to an oxygen donor compound in the presence of a test enzyme. Oxygen donors Exemplary include dioxygen or molecular oxygen (O2) and peroxides such as hydrogen peroxide (H2O2) and t-butyl peroxide. Exemplary substrates include naphthalene, 3-phenylpropionate, benzene, toluene, benzoic acid and anthracene. An oxygenated product is formed when the test enzyme has Oxidation activity, particularly oxygenase activity, under test conditions. A coupling enzyme is used to gather molecules of the oxygenated product into larger polymers or molecules that absorb UV light, produce a color change or are fluorescent or luminescent. Exemplary coupling enzymes include peroxidases from various plant and microbial sources, such as horseradish peroxidase (HRP), cytochrome peroxidase, tulip peroxidase, lignin peroxidase, carrot peroxidase, peanut peroxidase, peroxidase. soybean, Novozyme® 502 peroxidase, as well as such laccasas ? .l, l «? .. r, illl r fgfrt i .i m i ?? f ^^^^^ l ^ í ^ ^ ^ am ^ a? ? l? as laccasa fungal. The presence and degree of a change in absorbency, color, fluorescence or luminescence can be detected or measured and indicates the presence of oxygenated product. The detection can be increased by a chemiluminescent agent, such as luminol. These techniques provide a reliable indication of oxygenase activity, that is, the production of oxygenate by the reaction of the oxygen donor with the substrate in the presence of (and medal for) the enzyme. The method is preferably carried out in a complete cell environment. A host cell is transformed, using genetic engineering techniques, to express a selected oxygenase, and can also be engineered to express a coupling enzyme. The method is receptive to large-scale selection of enzyme mutants to isolate those with desired oxygenase activity, for example maximum activity under certain conditions or towards a particular substrate compound. The method is also receptive to selecting libraries of genes isolated from nature (50). Oxygenase enzymes typically utilize molecular oxygen, in the presence of co-factors, coenzymes and / or ancillary proteins, to add oxygen to a substrate. Oxygen is a highly reactive chemical element. In pure molecular form, if a gas that is a main component of air, and is stable as a combination of two oxygen atoms (O2). Appears in water (H2O) in rocks and minerals, in many organic compounds, and is active HIMMÉB in many physiological and biochemical processes. Some enzymes that use O2 can use other oxygen donors, for example, peroxides (according to a reaction scheme called the peroxide deviation path), but do so deficiently, with low activity and low production of oxygenated product. In addition, certain coenzymes, cofactors or auxiliary proteins may still be required, although the peroxide deviation does not require the difficult coenzymes, for example, NAD (P) H, associated with trajectories using O2 as a substrate. The improved oxygenase enzymes of the invention are capable of efficiently catalyzing reactions where oxygen is added to a substrate, using oxygen donors instead of molecular oxygen, and without requiring certain cofactors, coenzymes or auxiliary redox proteins. These new enzymes have significantly more activity than native enzymes. For example, they are at least twice as active, and typically are ten or more times as active as a wild type enzyme towards a particular substrate or under particular reaction conditions.

Description of Related Art The publications and reference materials observed herein and in the appended Bibliography are each incorporated by reference in their entirety. Reference is made numerically in the text and in the Bibliography below. Catalysts, Enzymes and Oxygenases. An enzyme is a > - 5 - biological catalyst, typically a protein, that promotes a biochemical reaction. A catalyst allows a chemical reaction to proceed at a faster rate or under different conditions that occurred otherwise. Usually, a catalyst by itself does not change at the end of the reaction, although oxidative enzymes can be slowly deactivated during these reactions. Oxygenase enzymes that are able to catalyze the insertion of oxygen into chemical compounds (open-chain) aliphatic and (ring-containing) aromatics, and other chemical compounds or substrates can have many potential applications in the manufacture of pharmaceuticals, in the production of chemicals and also in medicine. The dioxygenases introduce two oxygen atoms, for example, both oxygens of a donor such as molecular oxygen (O2). Monooxygenases, also called mixed function oxygenases, add an oxygen atom to a substrate compound. In these reactions a second oxygen of the oxygen donor can be combined with hydrogen (H +) in an attached reaction, to form water (H2O). Compounds other than molecular oxygen, such as peroxides, can also donate oxygen to a substrate in the presence of various oxygenases. Common monoxigenation reactions include hydroxylation or epoxidation. In a hydroxylation reaction, oxygen is introduced to a substrate as a hydroxide (OH) group. In an epoxidation reaction, oxygen is introduced as a bridge through two other atoms, typically in place of a double bond between two carbon atoms. This can form a reactive or activated group having a ring of three members of one oxygen atom and two carbon atoms. A common deoxygenation reaction is sulfoxidation. In a sulfoxidation reaction, two oxygen atoms are added to a sulfur atom that binds to two other atoms, typically two carbon atoms, each of which is part of a hydrocarbon chain. The introduction of oxygen to a compound can change its biochemical activity or functionality, and can activate the compound so that it can participate in additional chemical reactions. The oxygenated substrates can be used organically or industrially in the synthesis of useful compounds of starting materials or intermediates. Oxygenation may also be useful in the disintegration of compounds, to provide starting materials and intermediates for other reactions. For example, bacteria use oxygenases to digest aromatic compounds. Problems Directed by the Invention. Among the problems addressed by the invention are the significant disadvantages of many known enzyme systems. These problems have avoided the commercial use and exploitation of such systems. Many oxygenases, like other enzymes, require costly coenzymes (eg, NADPH) and ancillary proteins (eg, a reductase enzyme) and can often be used in reactors or cells - 7 - complete with recycled coenzymes, to keep costs down of the coenzyme. Known enzymes are also relatively inefficient or unstable under industrial conditions, and can be undesirably deactivated by reaction products or by-products, or for other reasons. These types of enzyme systems, particularly when used in whole cell reactions, are also prone to competitive reactions that can decrease selectivity and production. In this way, enzymes that do not require coenzymes, use fewer coenzymes, or use less expensive coenzymes are desirable. Enzymes that are more efficient, more stable, or that work under different conditions are also desirable. It would also be desirable to provide enzymes that are not adversely affected by competitive reactions. Enzymes that promote the oxidation of different substrates, or use different oxygen donor compounds, may also be desirable, as would enzymes that are more or less specific than the enzymes known to catalyze certain reactions. For example, hydrogen peroxide or other peroxides are good choices of oxidant for the manufacture of fine chemicals, since their use would require less specialized equipment, and lower total cost, than molecular oxygen due to the greatly simplified catalyst system. A suitable selection method for oxygenases is also desirable, and would provide an important tool in the discovery and identification of new and improved oxidation enzymes. Enzymatic oxygenation reactions are particularly intriguing, because the oxyfunctionalization of deactivated organic substrates remains a largely unsolved challenge for synthetic chemistry. This is especially true for regiospecific reactions, where oxygenation at a specific position of a substrate only occurs in one of two or more possible ways. For example, the regiospecific hydroxylation of aromatics by purely chemical methods is remarkably difficult. Reagents for another or o-hydroxylation of ring compounds, at positions in the ring that are adjacent or adjacent to each other, are described in the literature. Reagents are also available for para or p-hydroxylation, at positions in the ring in which they are opposite each other. However, the specific oxygenation of enantiomers (mirror image forms of a compound) is difficult and not well understood. In these reactions, an enantiomer is preferably oxygenated, but the mirror image enantiomer of the same polymer is poorly oxygenated, or not oxygenated at all. Similarly, it is difficult to oxygenate a substrate with high enantiospecificity, that is, to create a particular enantiomeric form against another. In this way, oxygenation to form a particular enantiomer is difficult. Consequently, oxidation enzymes that facilitate particular enantiospecific or regiospecific reactions would be desirable, or in - they are done more efficiently or in some better way. Oxidation enzymes. Several native mono- and dioxygenase enzymes are known from different human, plant and animal sources. These include enzymes such as chloroperoxidase (CPO), large numbers of citrochrome P450 (P450) enzymes, methane monoxigenases (MMO), toluene monooxygenases, toluene dioxygenases (TDO), biophenyl dioxygenases and naphthalene dioxygenases (NDO). These enzymes have demonstrated the ability to catalyze hydroxylation and many other useful and interesting oxidation reactions. However, they are generally unsuitable for the industry due to their inherent complexity, low stability and low productivity under industrial conditions (for example, in the presence of organic solvents, high concentrations of reagents, etc.). One class of known oxidation enzymes is the cytochrome P450 enzymes. These heme proteins have iron-containing heme groups and are important monooxygenase enzymes included in, among other reactions, detoxification of toxic or foreign materials (xenobiotics), drug metabolism, carcinogenesis, and steroid biosynthesis (5 and 6). An exemplary P450 enzyme P450Cam, from Pseudomonas putida, whose natural substrate is camphor, is also capable of regiospecific hydroxylation of a variety of substrates including, at a low activity level, naphthalene (C10H8), a T-10 aromatic compound - bicyclic (7) However, the catalytic change of this enzyme requires the reduced form of nicotinamide adenine dinucleotide (NADH) as a coenzyme and two auxiliary proteins. One of these proteins is putidaredoxin, an iron-sulfur protein (also called ferrodoxin) that acts as an electron carrier to move electrons from NADH. The other auxiliary protein is enzyme putidaredoxin reductase, a flavoprotein that catalyzes the transfer of hydrogen atoms from one substrate to another (8). This requirement for two redox proteins and NADH makes P450cam and other P450 catalysis highly expensive and difficult to use in industrial and laboratory applications. It would be desirable to provide a more economical and simpler hydroxylation and catalyst system type P450, in particular a system that requires less or no coenzyme or ancillary proteins. P450 enzymes typically use dioxygen (O2) as the oxygen donor for hydroxylation, adding an oxygen to a substrate compound, such as naphthalene, and forming water with hydrogen and other oxygen as a by-product. They are more efficient when using dioxygen with expensive coenzymes, such as the reduced forms of nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADHP), collectively "NAD (P) H". Auxiliary proteins may also be needed for the efficient activity of the enzyme. However, several P450s (and possibly some MMOs) are capable of catalyzing the hydroxylation of an organic substrate using a MMI → peroxide, such as hydrogen peroxide or alkyl peroxides, through the so-called peroxide deviation path (9). Peroxides are compounds, other than molecular O2, in which the oxygen atoms are bonded together. Other oxygen donors include peroxyacids, NalO, NaCIO2, and yodosyl benzene. Nordblom et al. (11) studied the hydroxylation of hydroperoxide-dependent substrate by liver microsomal P450 in liver microsomes. It was shown that a variety of substrates were attacked by the enzyme in the presence of eumeno hydroperoxide. Using benzfetamine as the substrate, it was also shown that other peroxides, including hydrogen peroxide, peracids and sodium chlorite, could be used in place of oxygen (11). Rahimt? La et al. (12) showed that eumeno hydroperoxide is capable of supporting the hydroxylation of several aromatic compounds (biphenyl), benzypyrene, coumarin, aniline) by the cytochrome P450 in hepatic microsomes. Unfortunately, native cytochrome P450 is rapidly deactivated by peroxides and other oxidants. Enzyme Chloroperoxidase (CPO) from Caldariomyces fumago has an active site whose structure is similar to cytochrome P450 enzymes. CPO will catalyze several oxidation reactions, including enatioselective hydroxylation, epoxidation and sulfoxidation, using peroxides. This enzyme uses peroxide efficiently but can not use molecular oxygen because it does not have the coenzyme machinery of the P450 enzymes. CPO also provides an example of an enzyme that is inactivated by M * M ^^ r ^ ÍHMiiífeti reactive intermediaries. Heme alkylation by the epoxide product in the CPO catalyzed epoxidation of 1 -alkenes results in deactivation of CPO. Heme oxygenases such as P450s and heme peroxidases, which are peroxidase enzymes that contain the heme prosthetic fraction, are generally prone to deactivation through the oxidation of the porphyrin ring in the heme substrate, by the reaction with the so-called suicide inhibitors. formed during the catalysis, and also by formation of Compound III (for peroxidases). Compound III is an intermediate enzyme-substrate-oxygen-iron complex, sometimes referred to as an oxyperoxidase. For example, enzyme horseradish peroxidase (HRP) is deactivated during the oxidation of phenol compounds, for example, six-membered hydrocarbon ring structures containing one or more hydroxyl (OH) groups. In theory, this could be due to the formation of phenoxy radicals that react with oxygen to form a kind of reactive peroxy radical. Compound III is formed in the presence of excess hydrogen peroxide and is not included in the reaction cycle. However, its accumulation reduces the amount of active enzyme. The stability of Compound III in turn depends on the specific enzyme. The velocities of all these deactivation trajectories depend on the structure of the protein, i.e. the particular proteins, structures and conditions included. Therefore, they are receptive to improvement through mutations. This includes oxygenases that are more suitable for functioning in the presence of high concentrations of hydrogen peroxide, or other peroxides or agents that donate oxygen. Enhanced oxygenates also include those that are more resistant to deactivation, do not require enzymes or use them more efficiently, operate under different conditions or with different specificities, or that hydroxylate different substrates or a variety of substrates or that they do so in a more efficient manner. efficient. As an example, it would be desirable to make modified P450 enzymes that are functionally similar or equivalent to CPO, or that share desirable CPO characteristics. An improved P450 enzyme of this class, for example, would have the ability to oxygenate a substrate or substrates using a peroxide, for example, hydrogen peroxide, without costly coenzymes, and with high efficiency and improved deactivation resistance. Modification of Enzyme, The limits observed in the use of native enzymes are thought to be a consequence of evolution. Enzymes have been included in the context and environment of a living organism, to carry out specific biological functions under conditions conducive to life, not laboratory or industrial conditions. In some cases, evolution may favor or even require less than optimally efficient enzymes. For example, detoxification enzymes, such as cytochrome P450 enzymes, function to help convert foreign chemical compounds (xenobiotics) into other compounds that an organism can use, which are nontoxic, or that occur in non-toxic amounts. In order to deal with environmental conditions or foreign compounds an organism has not been found before, that the detoxification enzymes can attack a relatively large number of substrates, and can accidentally produce products that are as or more toxic than the substrate. In this way, maximizing the flow of potentially harmful foreign substrates to process, for example, using a fully efficient catalyst, may not be the best evolutionary strategy. This is particularly true when there is a time-dependent xenobiotic profile, which means that the organism can only safely handle both foreign material at a time (2). In this situation, a less than maximally active enzyme that balances appropriately to the particular needs of the organism and its environment would be a better evolutionary goal. In a laboratory or industrial establishment, it is desirable to provide enzymes that are more active, and process more substrates more quickly. In this way, the result, efficiency, working conditions, stability and other properties of known enzymes are not thought to be altered, nor are they limitations that are seen as essential to the nature of these catalysts as proteins. It is possible that these native catalysts may be included in vitro, or that the analogous catalysts may otherwise be developed, to alter or increase the properties of the enzyme, for example to obtain much more efficient industrial or laboratory oxidative catalysts. The selectivity of enzyme and substrate specificity can also be altered to match the need for the synthetic chemical. The improved catalysts can also be obtained by selecting cultures of native organisms or libraries of expressed gene (3). A technique that can be applied to the discovery of improved catalytic enzymes is direct evolution. Direct evolution is a procedure by which the evolutionary process is accelerates in vitro to produce mutant enzymes that have certain desired characteristics. An example of the use of direct evolution to identify and isolate para-nitrobenzyl esterases is set forth in the U.S. Patent. No. 5,741, 691. See also US Patent. No. 5.81 1, 238 (13). Other techniques, such as mutagenesis random, can also be used to obtain new enzymes. The improved enzymes can also be discovered in nature. According to a preferred embodiment of the invention, the directed evolution or random mutagenesis can be used to produce a set of efficient catalysts that can be run oxidations using agents other than dioxygen (O2) as the oxidant. For example, peroxides such as hydrogen peroxide (H2O2) can be used. Directed evolution can also be used to alter the properties of oxidative enzymes that use molecular oxygen. A variety of such enzymes, including P450s of cytochrome, other monooxygenases, and ^ m * M? dioxigenases such as toluene dioxygenase, facilitate the useful oxygenation reactions. It is desirable to alter the reactivities, selectivities and stabilities of these enzymes to produce improved enzymes. An important tool to find improved oxidation biocatalysts in nature, by directed evolution, by random mutagenesis, or by other means, is a sensitive, accurate and rapid selection method. In accordance with the above, there is a need to develop new and improved breeding methods that are well suited for use in connection with directed evolution procedures.

SUMMARY OF THE INVENTION According to the invention, a method is provided for selecting oxidation enzymes or oxygenases. New and improved oxidation enzymes are also provided. More particularly, the presence of oxygenates that are produced by the action of an oxygenase on a particular substrate is detected. The invention is particularly well suited to select large numbers of both naturally occurring and mutated oxygenates to determine their activity with respect to a wide range of substrates, including aliphatic or aromatic compounds. It was discovered that the detection of oxygenated compounds produced by the action of an oxygenase can be improved by reacting the oxygenated compound with a coupling enzyme to form a polymeric oxygenated compound that absorbs UV light, produces a color change, or is luminescent, i.e. , phosphorescent or preferably fluorescent. The presence and quantity of oxygenated compounds in a sample can be indicated by detecting, observing or measuring the presence, and if desired, the degree of light absorption, color change, fluorescence, or luminescence. It was also discovered that the luminescence and detection of the polymeric oxygenate compound can be further increased by creating the polymeric oxygenate in the presence of a chemiluminescent agent, such as luminol, to increase the intensity of chemiluminescence and / or life time. Other agents can also be used to increase the reactions of color change or color development (44). The invention is particularly well suited for complete cell selection procedures wherein a host cell, such as the E. coli bacterium, is transformed with a suitable vector to express an oxygenase to be selected. The transformed cell is created with a substrate, such as naphthalene, for a sufficient time to allow an oxygenated compound, eg, hydroxylated naphthalene, to form. A coupling enzyme, such as horseradish peroxidase (HRP), is provided and allowed to react with the oxygenate, to form a polymeric oxygenate compound that exhibits increased levels of UV light absorption, luminescence, or fluorescence in the case of naphthalene hydroxylated polymer. The fluorescence generated by the polymeric oxygenate is measured by known means to provide indirect detection of oxygenase activity, for example, the amount of oxygenate produced by the reaction of oxygenase with the substrate. The coupling enzyme can be produced accidentally and added to the cell culture, or in a preferred embodiment, it can be produced intracellularly, ie, by or within the same cell that produces the oxygenase. In this manner, a complete cell selection system is provided, wherein a suitable host cell is transformed with suitable vectors to provide co-expression by the transformed cell of both an oxygenase and a coupling enzyme. As a result, the infusion of the substrate into the cell results in the contemporary generation of oxygenates due to the action of oxygen on the substrate and the formation of polymeric oxygenates resulting from the action of the coupling enzyme on the oxygenates. When desired, one or more cofactors, coenzymes or auxiliary proteins can be used to improve the activity of the oxidation enzyme or increase the oxygenation reaction. The invention is particularly well suited for selecting a large number of naturally occurring or mutated oxygenases to determine relative activities of the enzyme with respect to a substrate, and in particular to establish which enzymes show the highest activity with respect to a given substrate or which insert oxygen at a different site on the substrate ^^ MaM ^ riMÉ ^^ HattÜ (shows different regiospecificity). The invention is applicable to both monooxygenases and dioxygenases and can be used to detect oxygenated compounds formed by hydroxylation or epoxidation. The invention can also be applied to sulfoxidation reactions. Hydroxylation enzymes are preferred. The invention is also suitable for selecting libraries of oxygenase catalysts that are not enzymes, for example, compounds generated by combinatorial chemistry (43, 48, 49). The addition of oxygen by such catalysts can be analyzed by the addition of a coupling enzyme under conditions suitable for the coupling reaction. For example, conditions can be modified after the oxygenation reaction to place the coupling reaction. However, it may not be necessary to significantly modify the reaction conditions for some coupling enzymes. As an example, it is known that horseradish peroxidase works through a wide range of conditions and in aqueous medium and in a wide variety of non-polar organic solvents. The above features and other attendant advantages of the invention will be better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of a reaction path of an exemplary embodiment of the invention. Figure 2 is a map of an exemplary vector used to express cyanochrome wild type P450cam oxygenase and its mutants in E. coli. Figure 3A shows a nucleotide coding sequence for wild type cytochrome P450cam monooxygenase [SEQ. ID. DO NOT. 1]. Figure 3B shows an amino acid sequence for wild type citrochrome P450cam monooxygenase [SEQ. ID. DO NOT. 2]. Figure 4A is a pictorial representation of an exemplary 96-well plate analysis according to the invention. Figure 4B is a diagrammatic representation of a reaction scheme according to the invention. Figure 5A is a tabular representation of the cavities in the 96-well plate, in which different media and components are used to evaluate the effect on P450cam activity of E. coli host cells. Figure 5B is a graphical representation of the activity of P450cam as measured by an analysis according to the invention. Each column of the graph represents the total P450cam activity in each corresponding cavity of the 96-well plate, as a measure of the fluorescence produced by the oxygenated, polymerized reaction products of hydroxylated naphthalene by hydrogen peroxide in the presence of the enzymes HRP and P450cam - Figure 6 is a pictorial representation of an exemplary analysis according to the invention. Figure 7 is a pictorial representation of how the simultaneous expression of the oxygenase and coupling enzyme in E. coli leads to the generation of fluorescent cells. Figure 8 shows the development of fluorescence, over time, in whole cells transformed to co-express the coupling enzyme HRP and oxygenase P450 with naphthalene substrate and peroxide oxygen donor (•); without naphthalene substrate (Y); and without oxygen donor. For comparison, fluorescence was evaluated in whole cells transformed to express HRP without P450 (A), P450 without HRP (B) and host cell that does not transform (+). Figure 9 shows the effect of inducing levels on coexpression and production of P450 enzyme and HRP enzyme in E. coli host cells, according to a preferred embodiment of the invention. Figure 10 shows the fluorescence of E. coli host cell colonies transformed to co-express HRP and P450 enzymes, in the presence of hydrogen peroxide and naphthalene. Figures 1 1 A-1 1 D show the computer aided image analysis of a group of fluorescent cell colonies in a P450 / HRP whole cell analysis according to the invention. Figures 12A-12F show the results of image analysis, in graphic form, of the fluorescence displayed by the E. coli host cell hills (control), and the same host cells transformed to express the P450 enzyme, HRP enzyme, or both, and under different assay conditions (with or without substrate and oxygen donor). Figure 13 represents a method for automatically detecting positive colonies (fluorescence) of whole cells that produce active oxygenase enzyme according to the invention. Colonies of the host cells are placed on the plate, and the plate is conceptually divided by a mill into rectilinear compartments, each of which can be scanned by conventional image analysis equipment (Figure 13A). Each compartment is screened for fluorescence colonies (Figure 1B) and the number of positive (fluorescent) colonies counted. Fluorescence intensity is also measured (Figure 13C). The colonies containing improved oxygenase (fluorescence above a certain level) can be identified and selected. This technique can be automated. Figure 14A shows the results of an experiment using coumarin as a substrate for oxygenation, in an analysis of the invention. Figure 14B shows the results of an experiment using 3-phenyl propionate as the substrate. Figure 15 shows a 96 well plate analysis according to the invention, in which the fluorescence of the oxygenated 3-phenyl propionate substrate and polymerized in a co-expressing cell system of P450 / HRP E. coli is amplified using luminol. A comparison is shown with a host cell control, and with cells transformed to express P450 enzyme without HRP and HRP enzyme without P450. The results using ultraviolet (UV) irradiation are shown in Figure 1A. The results without irradiation are shown in Figure 1B. Figure 16 shows the cytochrome peroxidase c expression vector of yeast (CCP) pet-26b (+) CCP. Figure 17 shows the detection of fluorescence in an embodiment of the invention in which cytochrome peroxidase C (CCP) is used as a coupling enzyme that is co-expressed with P450 enzyme in a whole cell system. Comparisons with an E. coli host cell control, without substrate, and with cells transformed to express CCP without P450 and P450 without CCP, are also shown, Figure 1.8 shows the toluene dioxygenase (TDO) expression vector pXTD14 . Figure 19A shows the results of digital scanning of a section of a plate containing fluorescent mutant P450cam colonies. Figure 1B shows the results of -32,000 clones of a digital scan of -200,000 plate clones containing fluorescent mutant P450cam colonies. Figures 1C-19F show a graphical representation of the activities of the P450 enzyme from a sample of fluorescent mutant P450Cam colonies as measured by fluorescence, in an analysis of the invention. Figure 20 shows the results of measuring the fluorescence of 96 randomly selected clones of the large mutant library (-20,000 colonies) on a screen according to the invention. Figure 21 is a map of an exemplary vector, pETpelBHRP, formed by inserting the HRP gene into the pET-22b (+) plasmid, which contains a T7 promoter and a pelB signal sequence. The resulting vector was used as the starting point for mutagenesis to express horseradish peroxidase in E. coli host cells. Figure 22 shows the coding sequence of pelB signal peptide ([SEQ ID NO: 14]). Figure 23 shows an amino acid and 15 nucleotide sequence encoding a variant HRP enzyme designated HRP1 A6 ([SEQ ID NO: 16 and SEQ ID NO: 17]). Figure 24 is a map of the expression vector pETpelBHRP1 A6 Figure 25 is a map of the expression vector pYEXS I-20 HRP containing a coding sequence for HRP cloned in the secretion plasmid pYEX-S 1.

DETAILED DESCRIPTION OF THE INVENTION The invention concerns oxidation enzymes and a general method for selecting enzymes that are capable of ^ uÉtíj ^^ SáJu ^^ * - 25 - oxygenate several substrates. In particular, the invention is especially well suited for evaluating the activity of enzymes that are capable of oxygenating aromatic substrates. Definitions: The term "substrate" means any substance or compound that is converted or wants to be converted into another compound by the action of an enzyme catalyst. The term includes aliphatic and aromatic compounds, and includes not only a single compound, but also combinations of compounds, such as solutions, mixtures and other materials containing at least one substrate. Aromatic substrates are preferred. Non-limiting and exemplary aromatic substrates include naphthalene, 3-phenylpropionate (3-PPA), coumarin, benzene, toluene, and benzoic acid. Preferred substrates, particularly in connection with the screening method of the invention, are naphthalene and 3-phenylpropionate. An "oxidation reaction" or "oxygenation reaction", as used herein, is a chemical or biochemical reaction that includes the addition of oxygen to a substrate, to form an oxidized or oxygenated product or substrate. An oxidation reaction is typically accompanied by a reduction reaction (hence the term "redox" reaction, for oxidation and reduction). A compound "oxidizes" when it receives oxygen or loses electrons. A compound "reduces" (loses oxygen and gains electrons). According to the invention, the oxidation reactions are preferably oxygenation reactions that add oxygen to a substrate. Oxygen typically donates electrons in ionic form such as OH "or O22". Conceptually, electrons (negatively charged subatomic particles), for example as hydrogen ions (H + + H22 +). An "ion" is an atom or molecule with a net negative or positive charge, that is, it has excess electrons (a negative charge) or no electrons (a positive charge). In this way, oxidation reactions can also be called "electron transfer reactions" and include the loss or gain of electrons (eg, oxygen) or protons (eg, hydrogen) of a substance. Preferred oxidized compounds of the invention are those that are "oxygenated," which means they have received oxygen. The term "enzyme" means any substance composed completely or largely of protein that catalyzes or promotes, more or less specifically, one or more biochemical reactions. A protein is a polypeptide (one or more peptides), which means it is a chain of chemical building blocks called amino acids that are linked together by chemical bonds called peptide bonds. A protein, which includes enzymes, can be "native" or "wild type", meaning that it occurs in nature; or it may be a "mutant", "variant" or "modified" meaning that it has been elaborated, altered, derived, or is somehow different or changed from a native protein. A "test enzyme" is a substance that contains protein that is tested • PHYSIOLOGY to determine if it has the properties of an enzyme. The term "enzyme" can also refer to a catalytic polynucleotide "eg, RNA or DNA." The "activity" of an enzyme is a measure of its ability to catalyze a react, and can be expressed as the rate at which the product For example, the activity of the enzyme can be represented as the amount of product produced per unit time, per unit (for example, concentration or weight) of enzyme.The "stability" of an enzyme means its ability to work through time in a particular environment or under particular conditions One way to assess stability is to analyze your ability to withstand a loss of activity over time, under given conditions. The stability of the enzyme can also be varied in other ways, for example, by determining the relative degree to which the enzyme is in a folded or unfolded state. In this way, one enzyme is more stable than another, or has improved stability, when it is more resistant than the other enzyme to a loss of activity under the same conditions, is more resistant to deployment, or is more durable by any suitable measure. An "oxidation enzyme" is an enzyme that catalyzes one or more oxidation reactions, typically by adding, inserting, contributing or transferring oxygen from a source or donor to a substrate. Such enzymes are also called oxidoreductases or redox enzymes, and comprise oxygenases, hydrogenases or aMe b reductases, oxidases and peroxidases. An "oxygenase" is an oxidation enzyme that catalyzes the addition of an oxygen to a substrate compound. A "dioxygenase" is an oxygenase enzyme that adds two oxygen atoms to a substrate. A "rnonooxygenase" adds an oxygen atom to a substrate. An "oxidase" is an oxidation enzyme that catalyzes a reaction, in which molecular oxygen (dioxygen or O2) is reduced, for example by donating electrons to (or receiving protons from) hydrogen. Preferred oxidation enzymes of the invention include without limitation, oxygenases (dioxygenases or monooxygenases), including hydroxylase, epoxidases and sulfoxidases, which respectively catalyze hydroxylation, epoxidation and sulfoxidation reactions. Of these, monooxygenases, hydroxylases and dioxygenases are preferred. Exemplary oxidation enzymes include, without limitation, modified or native chloroperoxidase (CPO), cytochrome P450s, methane monooxygenases (MMOs), toluene monooxygenase, toluene dioxygenases (TDO), naphthalene dioxygenases (NDO) and biphenyl dioxygenases. A preferred oxidation enzyme is P450 of modified or native cytochrome. In the terms "oxygen donor", "oxidation agent" and "oxidant" means a molecule or compound substance that donates oxygen to a substrate in an oxidation reaction. Typically, the oxygen donor is reduced (accepts electrons). Exemplary non-limiting oxygen donors include dioxygen or molecular oxygen (O2) and peroxides, including alkyl peroxides such as t-butyl peroxide, and more preferably hydrogen peroxide (H2O2). A peroxide is any compound that has two oxygen atoms bonded together. The term "coupling enzyme" means an enzyme that catalyzes a chemical or biochemical reaction in which an oxygenated product or substrate reacts to form a detectable complex, aggregate, polymer, other reaction product. An enzyme of The preferred coupling catalyzes the formation of a reaction product having a color change or increased, UV absorbance or luminescence (eg, fluorescence). For example, a suitable coupling enzyme catalyzes the formation of a fluorescent polymer by joining two or more substrate molecules oxygenated each other. According to one embodiment of the invention, the fluorescence of the polymerized oxygenate is more readily detectable than the fluorescence, if any, of the oxygenated substrate that has not been polymerized. A coupling enzyme may or may not be an oxidation enzyme, stipulating that it works to catalyze the formation of a detectable oxygenated reaction product. Exemplary coupling enzymes include, without limitation, peroxidases from various plant and microbial sources such as horseradish peroxidases (HRP), cytochrome peroxidase c, tulip peroxidase, lignin peroxidase, peroxidase carrot, peanut peroxidase, seed peroxidase »" - "- -m- soybean, Nobozyme® 502 peroxidase as well as laccases such as fungal lacass. HRP and laccase are the preferred coupling enzymes. As used herein, a "luminescent" substance means any substance that produces detectable electromagnetic radiation, or a change in electromagnetic radiation, most notably visible light, by any mechanism, including color change, UV absorbance, fluorescence and phosphory. Preferably, a luminescent substance according to the invention produces a detectable fluorescence or UV absorbance color. The term "chemiluminescent agent" means any substance that increases the detectability of a luminescent signal (eg, fluorescent), for example by increasing the intensity or lifetime of the signal. A preferred and exemplary chemiluminescent agent is 5-amino-2,3-dihydro-1,4-eftalazine-clione (luminol) and the like. Other chemiluminescent agents include 1, 2-dioxetanostalcomotetramethyl-1,2-dioxetane (TMD) 1,2-dioxetatanones and 1,2-dioxetadiones. The term "polymer" means any substance or compound that is composed of two or more building blocks ('mers') that are repetitively linked to each other, for example, a "dimer" is a compound in which two building blocks have been joined. building. The term "cofactor" means any non-protein substance that is necessary or beneficial for the activity of an enzyme. * -'- a - - * «- 31 - A coenzyme means a cofactor that interacts directly with and serves to promote a reaction catalyzed by an enzyme. Many coenzymes serve as vehicles. For example, NAD + and NADP * carry hydrogen atoms from one enzyme to another. An "auxiliary protein" means any protein substance that is necessary or beneficial for the activity of an enzyme. The term "host cell" means any cell of any organism, which is selected, modified, transformed, grown or used or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gene , a DNA or RNA sequence, a protein or an enzyme. "DNA" (deoxyribonucleic acid) means any chain or sequence of the chemical building blocks of adenine (A), guanine (G), cytosine (C) and thymine (T) called nucleotide bases that bind together in a structure of deoxyribose sugar. DNA can have a strand of nucleotide bases, or two complementary strands that can form a double helix structure. "RNA" (ribunucleic acid) means any chain or sequence of chemical building blocks ele adenine (A), guanine (G), cytosine (C) and uracil (U) called nucleotide bases that are bonded together in a structure of Ribose sugar RNA typically has a strand of nucleotide bases. A "polynucleotide" or "nucleotide sequence" is a series of nucleotide bases (also called "nucleotides") in DNA and RNA and means any chain of two or more nucleotides. A nucleotide sequence typically carries genetic information including the information used by the cellular machinery to make proteins and enzymes. These terms include double or single strand genomic and cDNA, RNA, any genetically manipulated or synthetic polynucleotide 5, and both sensitive and antisensitive polynucleotides (although only the sensitive strands are represented herein). These include double or single filament molecules, ie, DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as "protein nucleotide acids" (PNA) formed by conjugating the bases in an amino acid structure. It also includes nucleic acids containing modified bases for example thio-uracil, thio-guanine and fluoro-uracil. The polynucleotides herein may be flanked by natural regulatory sequences or may be associated with heterologous sequences including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'-encoded regions and the like. The nucleic acids can also be modified by many means known in the art. Non-limiting examples of such Modifications include ventilation, "covers", substitution of one or more of the naturally occurring nucleotides with an analogue, and internucleotide modifications such as, for example, those with uncharged bonds (eg, methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc) and with links charged (eg, phosphorothioates, phosphorodithioates, etc.). The The polynucleotides may contain one or more additional covalently linked residues, such as for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-licin, etc.), intercalators (e.g. , acridine, weightralene, etc.), chelators (for example, metals, radioactive metals, iron, oxidative metals, etc.) and alkylators. The polynucleotides can be derived by the formation of an ethyl or methyl phosphodiester or an alkyl phosphoroamidate linkage. In addition, the polynucleotides herein may also be modified with a tag capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin and the like. The proteins and enzymes are made in the host cell using instructions in DNA and RNA according to the genetic code. Usually, a DNA sequence that has instructions for a particular enzyme or protein is "transcribed" into a corresponding RNA sequence. The RNA sequence is "translated" into the sequence amino acids that make up the protein or enzyme. An "amino acid sequence" is any chain of two or more amino acids. Each amino acid is represented in DNA or RNA by one or more triplets of nucleotides. Each triplet forms a codon corresponding to an amino acid. For example, the amino acid licin (Lys) can be encoded by the nucleotide triplet or AAA codon or by the AAG codon. (The genetic code has some redundancy, also called degeneration, which means that most amino acids have more than one corresponding codon). Because the nucleotides in the DNA and RNA sequences are read in a group of three for protein production, it is important to start reading the sequence in the correct amino acid, so that the 5 correct triplets are read. The manner in which a nucleotide sequence is grouped into codons is called the "reading structure". A "coding sequence" or a "sequence encoding" a polypeptide, protein or enzyme is a nucleotide sequence which, when expressed, results in the production of that polypeptide, protein or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. The term "gene" also called a "structural gene" means a DNA sequence that encodes or corresponds to a particular sequence of amino acids that comprise all or part of one or more enzymes or proteins and may or may not include regulatory DNA sequences such as promoter sequences, which determine, the conditions under which the gene is expressed. Some genes, which are not structural genes, can be transcribed from DNA to RNA but do not move in an amino acid sequence. Other genes can function as regulators of structural genes or as regulators of DNA transcription. The terms "express" and "expression" mean allowing or causing the information in a gene or DNA sequence to manifest, for example, by producing a protein by activating the • HIMÉIHkÉtdiM ÉiH ^^ MHiMIíatt H cellular functions influenced in the transcription and translation of a corresponding gene or DNA sequence is expressed in or through a cell to form an "expression product" such as a protein. The expression product itself, for example the resulting protein, can also be said to be "expressed" by the cell. An expression product can be characterized as intracellular, extracellular or segregated. The term "intracellular" means something that is inside a cell. The term "extracellular" means something that is outside of a cell. A substance is "secreted" by a cell if it appears to a significant extent outside the cell, from somewhere or within the cell. The term "transformation" means the introduction of an "alien" gene (ie, extrinsic or extracellular), DNA or RNA sequence to a host cell, so that the host cell will express the sequence or gene introduced to produce a desired substance , typically an enzyme or protein encoded by the introduced sequence or gene. The introduced sequence or gene can also be called a "cloned" or "foreign" sequence or gene, it can include control or regulatory sequences, such as start, stop, promoter, signal, secretion, or other sequences used by a genetic machinery of the cell. The sequence or gene may include non-functional sequences or sequences with no known function. A host cell that receives and expresses RNA or DNA has been "transformed" and is a "transformant" or a "clone". The DNA or RNA introduced into a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species. The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a sequence of DNA or RNA (eg, a foreign gene) can be introduced into a host cell to transform the host and promote the expression (for example, transcription and translation) of the introduced sequence. The vectors typically comprise the DNA of a transmissible agent, into which the foreign DNA is inserted. A way The common thread of inserting a segment of DNA into another DNA segment includes the use of enzymes called restriction enzymes that divide DNA into specific sites (specific groups of nucleotides) called restriction sites. Generally, foreign DNA is inserted into one or more restriction sites of the vector DNA and Then it is carried by the vector in a host cell together with the transmissible vector DNA. A segment or DNA sequence having added or inserted DNA, such as an expression vector, can also be called a "DNA construct". A common type of vector is a "plasmid," which is usually a self-contained molecule of double-stranded DNA usually of bacterial origin, which can readily accept additional (extraneous) DNA that can be easily introduced into a suitable host cell. A plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for insert foreign DNA. The coding DNA is a DNA sequence rtvaa i áá k * »* - J. which encodes a particular amino acid sequence for a particular enzyme or protein. The promoter DNA is a DNA sequence that initiates, regulates or otherwise mediates or controls the expression of the coding DNA. The DNA promoter and the coding DNA can be from the same gene or from different genes, and can be or different organism. A large number of vectors, including fungal and plastic vectors have been described for replication and / or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include plasmids pKK (Clonetech), pUC plasmid, pET plasmids (Novagen, Ine, Madison, Wl), pret plasmids or pREP plasmids, (Invitrogen, San Diego, CA), or pMAL plasmids (New England Biolabs, Beverly, MA), and many appropriate host cells, using methods described or cited herein or otherwise known to those skilled in the relevant art. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, for example antibiotic resistance and one or more expression cassettes. Preferred vectors are described in the Examples, and include without limitation pcWori, pET26b, pXTD14, pYEX-S1, pMAL, and pET22. The term "expression system" means a host cell and compatible vector under suitable conditions, for example for the expression of a protein encoded by the foreign DNA carried by the vector and introduced into the host cell. Common expression systems include E. coli host cells and plasmid vectors, and insect host cells and baculovirus vectors. The terms "mutant" and "mutation" mean any detectable change in genetic material, for example DNA, or any process, mechanism or result of such change. This includes gene mutations, in which the structure (DNA sequence) of a gene is altered, any gene or DNA that originates from any mutation process, and any expression product (eg, protein or enzyme) expressed by a DNA or gene sequence modified. The term "variant" can also be used to indicate an altered or modified gene, DNA sequence, enzyme, cell, etc. , that is, any kind of mutant. The "conservative sequence variants" of a polynucleotide sequence are those in which a change of one or more nucleotides at a given codon position results in no alteration in the amino acid encoded at that position. The "conservative variants of function" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the function and total conformation of the Polypeptide, including, but not limited to, replacement of an amino acid with one that has similar properties (such as, for example, acidic, basic, hydrophobic, and the like). Amino acids with similar properties are well known in the art. For example, arginine, histidine, hilicin, are basic amino acids hydrophilic and can be interchangeable. Similarly, Isoleucine, a hydrophobic amino acid can be replaced with leucine, methionine or valine. Amino acids other than those indicated as conserved may differ in a protein or enzyme such that the percent similarity of the amino or protein sequence between any of the two proteins of similar function may vary and may be from 70 to 99% , as determined according to an alignment scheme such as the Cluster Method, where the similarity is based on the MEGALIGN algorithm. A "conservative variant of function" also includes a polypeptide or enzyme having at least 60% amino acid identity as determined by the BLAST or FASTA algorithms preferably at least 75%, more preferably at least 85% and even more preferably at less 90% and that has the same or substantially similar properties or functions as the enzyme or protein origin or native with which it is compared. The "isolation" or "purification" of a polypeptide or enzyme refers to the derivation of the polypeptide by removing it from its original environment (for example, from its natural environment if it occurs naturally, or from its host cell if it is produced by DNA methods). recombinant). Methods for polypeptide purification are well known in the art, including without limitation, preparative disk gel electrophoresis, isoelectric focusing, HPLC, reverse phase HPLC, gel filtration, ion exchange and division chromatography, and countercurrent distribution. . For some purposes, it is preferable to produce the polypeptide in a recombinant system in which the protein contains an additional sequence tag that facilitates purification such as but not limited to a polyhistyrin sequence. The polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid phase matrix. Alternatively, antibodies raised against the protein or against peptides derived therefrom can be used as purification reagents. Other purification methods are possible. A purified polypeptide or polynucleotide may contain less than about 50%, preferably less than about 75%, and more preferably less than about 90% of the cellular components with which it was originally associated. A substantially pure enzyme indicates the highest degree of purity that can be achieved using conventional purification techniques known in the art. The polynucleotides are "hydribizable" to each other when at least one strand of a polynucleotide can be hardened to another polynucleotide under conditions of defined accuracy. The hybridization accuracy is determined, for example, by a) the temperature at which hybridization and / or rinsing is carried out, and b) the polarity and ionic strength (eg, formamide) of the hybridization and rinsing solutions, as well as other parameters. Hybridization requires that the two polynucleotides contain substantially complementary sequences, depending on the accuracy of hybridization, however, inequalities can be tolerated.

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Typically, hybridizing two sequences to high accuracy (such as, for example, in an aqueous solution of 0.5X SSC at 65 ° C) requires that the sequences show some degree of complementarity over their entire sequence. Conditions of intermediate accuracy (such as, for example, an aqueous solution of 2X SSC at 65 ° C) and low accuracy (such as, for example, an aqueous solution of 2X SSC at 55 ° C), require correspondingly less total complementarity between the hybridization sequences. (1 X SSC is 0.1 5 M NaCl, 0.015 M Na citrate). The polynucleotides that "hybridize" to the polynucleotides herein can be of any length. The general genetic engineering techniques and tools described herein, including transformation and expression, the use of host cells, vectors, expression systems, etc., are well known in the art. The Selection Method The analysis or selection method of the invention is applicable to a variety of enzymes, and is especially suitable for selecting oxygenases (monooxygenases or dioxygenases) which are capable of hydroxylating a substrate. In a broad aspect, the selection method comprises combining, in any order, substrate, oxygen donor, test oxidation enzyme, and coupling enzyme. The components of the analysis can be placed in or in any suitable medium, vehicle or support, and combine under predetermined conditions.

The conditions are chosen to facilitate, adapt, promote, investigate or test the oxidation of the substrate by the oxygen donor in the presence of the test enzyme, and can be modified during the analysis, for example to facilitate the action by the coupling enzyme. The coupling enzyme provides a way to detect and measure successful oxidation, that is, the formation of an oxygenated product of the substrate. In some embodiments, one or more cofactors, coenzymes and auxiliary or additional proteins can be used to promote or increase the activity of the oxidation enzyme, the coupling enzyme or both. In a preferred embodiment of the invention, the test enzymes are provided by host cells that have been transformed by genetic engineering techniques, so that they express the test oxidation enzyme. The test enzyme can be produced and retained within the cell, or it can be secreted out of the cell. In any case, the test enzyme can be recovered from the host cells for use in in vitro analysis or "test tube", where the enzyme is combined with the other ingredients of the assay. The enzyme that is secreted outside the cell can usually be recovered in a non-destructive manner, by collecting it from the growth medium, usually without interrupting the cells, or in a plate from which the cells grow. When the enzyme remains inside the cell, it is typically recovered by forcing the cells so that the enzyme can be released and separated from the cell medium and debris.

In a more preferred embodiment, it is not necessary to recover the test enzymes from the host cells, because the host cells are used in the selection method, in an analysis so called "whole cell". In this embodiment, the substrate, oxygen donor, and coupling enzyme are delivered to transformed host cells or to the support growth medium for the cells. In a preferred form of this approach, the test enzyme is expressed and retained within the host cell, and the substrate, oxygen donor, and coupling enzyme are added to the solution or plate containing the cells. Substrates, donors typically cross the cell membrane and enter the cell. If so, the substrate and the donor find the enzyme test. The oxygenated products resulting from this discovery can cross the cell membrane (leave the cell) and react in the direction of the coupling enzyme to form a detectable reaction product. Although it is less desirable, any component of the analysis that does not cross the cell membrane can be introduced directly into the cell by known methods. These techniques are particularly useful when the coupling enzyme produces a signal that can be observed from outside the cell, such as a luminescence reaction product, or when the co-expression of the coupling enzyme is difficult and interferes with the reactivity of the enzyme. the test enzyme. Such measurements are not destructive, and allow isolation and additional work with the cells that produce active enzymes. When a fluorescent signal is used, for example, transformed host cells that produce more active oxidation enzymes are "illuminated" in the assay and can be easily identified, and distinguished or separated from cells that do not "light up" as much and which produce enzymes inactive, less active enzymes or no enzyme. The oxygenated substrate that is secreted by the cell can interact with the coupling enzyme in the cell medium to form a detectable extracellular reaction product. If the host cells grow on a solid support, a fluorescent signal can be identified as a ring that "lights up" around the cells that produce the active oxidation enzyme. Depending on how cells grow too close, this method can allow active and non-active host cells to be distinguished, but is probably less reliable than an intracellular method. In embodiments where all host cells in or in a particular medium produce the same test enzyme, the choice of intracellular or extracellular approach is likely to be determined as a matter of convenience, unless other circumstances favor or require one technique over the other . In a particularly preferred embodiment, the host cells are transformed to produce both a test enzyme and a coupling enzyme. The substrate and the donor are added to the cell medium and taken up by the cells. The active enzyme * - 45 - produces an oxygenated substrate, which is converted into a reaction product detectable by the coupling enzyme. A preferred detectable reaction product is luminescent, for example fluorescent. This can be achieved, for example, by using a coupling enzyme, such as a lacquer or HRP, which forms fluorescent polymers of the oxygenated substrate. A chemiluminescent agent, such as luminol, can also be used to increase the detectability of the luminescent reaction product, such as fluorescent polymers. The detectable reaction products also include color changes, such as colored materials that absorb measurable UV light. The method of the invention is indirect in that it does not measure the presence of an oxygenated compound that is produced by the action of an oxygenase or a substrate. Instead, the invention detects or measures the reaction product that is made by the action of a coupling enzyme on a successfully oxygenated substrate. In a preferred embodiment, an oxygenated substrate is reacted in the presence of a coupling enzyme to form dimers or polymers of the oxygenated substrate. More particularly, a luminescence that is characteristic of the oxygenated substrate or its polymer is observed or measured. More typically, the polymers are fluorescent, and can be detected by known methods. This is advantageous, because the oxygenated substrate may be impossible or very difficult to detect directly. For example, the oxygenated substrate may not show fluorescence or any other convenient marker, it may do so at very low levels that are difficult to detect, or it may do so at a wavelength there are large interferences from other components of the test mixture. In this way, the invention serves to mark or amplify the oxygenated product or substrate so that it can reliably detect or measure. The invention is sensitive to the activity of the enzyme and in addition is sensitive to the position of oxygenation or hydroxylation of the enzyme, that is, the regioselectivity of the enzyme. For example, different colors can be produced and detected depending on where the enzyme has introduced oxygen. A schematic representation of the chemical reactions used in a preferred embodiment of the invention is shown in Figure 1. An aromatic substrate, for example, benzene and substituted benzene or naphthalene is hydroxylated by an oxidation enzyme. Suitable enzymes include chloroperoxidase (CPO), cytochrome P450s (P450), methane monooxygenases (MMO), toluene monooxygenases, toluene dioxygenases (TFO), biphenyl dioxygenases and naphtha leno (NDO) dioxigenases, or any other the many mono- and di-oxygenases. An oxygenated product is formed, in which one or more hydroxyl (OH) groups have been substituted at one or more positions on the ring of the aromatic substrate, for example, instead of hydrogen. These oxygenated products usually do not fluoresce, or show a very small change in fluorescence and can be difficult to detect or measure. Treatment with a coupling enzyme, such as laccase or peroxidase (eg, HRP) under appropriate conditions produces dimers or polymers of the oxygenated product that are colored or fluorescent, and can be easily detected. A chemiluminescent agent, such as luminol, can be used in addition to the coupling enzyme, to further increase the detection and measurement of fluorescent oxygenate compounds. Production of Test Enzymes (Host Cells and Vectors) In one aspect of the invention, a method is provided of whole cell selection, in which a test oxidation enzyme is produced by a transformed host cell using a suitable expression system. The types of host cells and expression systems that are suitable for use according to the invention are those that are capable of expressing enzymes of oxidation. Host cells are preferred which can also express coupling enzymes. E. coli is a preferred exemplary cell. Other exemplary cells include other bacterial cells such as Bacillis, Pseudomonas, yeast cells, insect cells and filamentous fungi such as any species of Aspergillus cells. For some applications, it may be preferred such as toxicity screening of certain compounds, plant, human, mammalian or other animal cells. Suitable host cells can be transformed, transfected or infected as appropriate by any method jjytj? ^ suitable that includes electroporation, CaCI2 mediated DNA uptake, fungal infection, microinjection, microprojectile transformation, archabacteria, fungi, especially yeast, and plant and animal cells. Of particular interest are E. coli and Saccharomyces cerevisiae. Any of the well known procedures for inserting expression vectors into a cell for the expression of a given protein or peptide can be used. Suitable vectors include plasmids and viruses, particularly those known to be compatible with host cells expressing oxidation enzymes and oxygenases. The invention is especially well suited for selecting large numbers of mutant oxygenates, wherein the cells are transformed with a number of different vectors expressing different mutant oxygenates. The mutant oxygenase genes can be prepared using methods such as DNA redistribution, as shown for example in the U.S. Patent. No. 5,605,793 (16) or by random mutagenesis for example using error-prone polymerase chain reactions (PCR). See for example, Patents of E.U. Nos. 5,741, 691 and 5,81 1, 238 (13) and PCT Application No. PCT / US98 / 05956 (17). Once the host cell has been transformed with the desired vector expressing the oxygenase to be tested, the cell line is maintained and grown under conditions that promote the expression of oxygenase within the cell. In general, oxygenase remains inside the cell and is not removed. After the transformed cells have been cultured for a sufficient time to generate oxygenase, the cells are contacted with or otherwise treated with the substrate of interest. This results in the generation of oxygenated compounds within the cell. In most cases, the oxygenate will diffuse from the cell where it can be reacted with a coupling enzyme to form polymeric oxygenates. See, figure 1. After the reaction with the coupling enzyme, the oxygenate forms dyes or polymers that are colored or fluorescent. The dimer or polymer is detected to provide a measurement of the oxygenase activity. If desired, luminol or other luminescent or color enhancing material can be added to augment the signal or provide polymers with longer chemiluminescent lifetimes. Preferred cells for these applications are bacterial cells such as E. coli and Bacillus and yeast cells, for example, S. cerevisiae in which libraries of different mutants can be made (dozens or more, and typically thousands). Exemplary coupling enzymes that can be used according to the invention include peroxidases and laccases. Exemplary specific enzymes include horseradish peroxidase (HRP), cytochrome peroxidase c, and several other peroxidases from various plant and microbial sources such as soybean seed peroxidase., tulip peroxidase, lignin peroxidase, carrot peroxidase, Nobozyme® 502 peroxidase, etc. as well as fungal laccasa. Although it is possible to add coupling enzyme for its reaction with an oxygenated compound that diffuses from the host cells, it is preferred that the coupling enzyme be coexpressed within the cells to provide an intracellular selection system. The transformation of the cell to express the coupling enzyme is carried out in a manner similar or analogous to the transformation of the cell to express oxygenase. The result is a cellular system that is provided for the indirect detection of the presence of oxygenated compounds that are produced within the cell when a substrate is reacted with an expressed oxygenase within the cell. The co-expression of the coupling enzyme provides an easily available source of enzymes to polymerize the oxygenate to form fluorescent, chemiluminescent or colored products that can be detected within the cell. In general terms, one embodiment of the complete cell selection method includes the following stages: 1) HRP added to cells expressing oxygenase. Host cells expressing a test oxidation enzyme grow under conditions that will promote functional expression of oxygenase activity. The substrate to be oxygenated is added and the oxidation reaction is allowed to proceed under appropriate conditions, the desired conditions (temperature, substrate, solvent, etc.) to select which ones reflect the desired properties of the oxygenase. The cells can also be forced to release the test release enzyme in the medium. To detect the formation of oxygenated products, a coupling enzyme (a peroxidase such as horseradish peroxidase) is added to the reaction mixture (typically the cell growth medium), together with an oxygen donor such as hydrogen peroxide. The substrate can be added before peroxide and horseradish peroxidase, or it can be added at the same time. In some cases, the substrate may be added later, but this may be less efficient or otherwise less desirable. In some circumstances (for example when the substrate is sensitive to peroxide) it is preferable to add the substrate before the other components of the analysis. The advantage of adding substrate, oxygen donor and coupling enzyme in a contemporary manner is that the analysis can then follow the kinetics of the oxidation reaction catalyzed by oxygenase. The color or fluorescence that indicates the formation of an oxygenated reaction product will accumulate in the cell culture and can be detected by any number of media. The addition of appropriate compounds (eg, luminol) may allow the product to be detected by chemiluminescence. 2) HRP co-expressed with oxygenase (intracellular reaction). In this embodiment, an oxygenase and coupling enzyme (eg, HRP) are both expressed by the host cell, so that the coupling enzyme need not be added separately. Cells that express both oxygenase and HRP grow under conditions that will promote the functional expression of both activities. The substrate is added, and the reaction is allowed to proceed under appropriate conditions (conditions desired for selection). The color or fluorescence will accumulate on its own in the cell culture, or both, and can be detected by any number of media. As above, the addition of appropriate compounds (eg, luminol) during the reaction may allow the product to be detected by chemiluminescence. Examples are provided for practicing the invention and it is understood that they are only exemplary and do not limit the scope of the invention or the appended claims. A person of ordinary skill in the art will appreciate that the invention can be practiced in many forms according to the claims and descriptions therein. EXAMPLE 1 Complete Cell Selection for Hydroxylation of Naphthalene by Cytochrome P450cam with Added Horseradish Peroxidase (HRP) This example establishes an activity analysis of the fluorogenic complete cell, exemplary for the hydroxylation of naphthalene by a mutant cytochrome P450cam enzyme. East * M ~ * - ~ "• * • -" »*» - 53 - simple complete cell selection procedure avoids problems associated with analyzes that require the interruption of cells or centrifugation stages. The example demonstrates that large libraries of enzyme mutants can be rapidly selected and effectively utilize the methods of the invention. Naphthalene, an aromatic hydrocarbon, shows weak fluorescence. When E. coli expressing the P450cam of oxygenase is taken by host cells, the naphthalene is hydroxylated by the enzyme to produce an oxygenated product with a weak but characteristic fluorescence emission (em) at a wavelength of 430-465 nm. When the hydroxylized naphthalene diffuses out of the cell, the activity of P450cam is determined fluorometrically by amplifying the weak fluorescence. According to the invention, the HRP-catalyzed polymerization of the hydroxylated product results in a large increase in fluorescence intensity and this is used for high yield selection of catalysts. Although the hydroxylated naphthalene shows blue fluorescence at high concentration levels, the colonies, which have a low intracellular concentration of hydroxylated naphthalene are only weakly fluorescent. With intensification of fluorescence aided by HRP, very low levels of P450cam activity can be detected. Therefore, there is significant benefit in terms of sensitivity to select the enzyme mutants for improvements in activity by this method. Cells, enzyme and chemicals. All analytical grades of chemicals were used. Horseradish peroxidase (type II, EC 1.1.1.7, oxidoreductase) was purchased from Sigma Chemical Co. Naphthalene and its hydroxylated derivatives, 1-naphthol and 2-naphthol, were purchased from Sigma and Aldrich. ABTS [2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid]] and 30% hydrogen peroxide solution were purchased from Sigma, Isopropyl-beta-d-thiogalactopyranoside (IPTG) was purchased from ICN Biomedicals, Ine, (Aurora, OH) Thiamine, glycerol and delta-aminolevulinic acid (ALA) were purchased from Sigma The regulators were prepared from analytical grade reagents (pH 9: 100 mM dibasic disodium phosphate buffer, pH 7.45: 100 mM Tris-HCl buffer, pH 7.0: 1000 mM potassium phosphate buffer.) The E. coli host cells used herein were strains designated E.coli XL10 Gold and BL21 (DE3). Obtained from Stratagene, La Jolla CA (Catalog Nos. 200317 and 200131) Provision of the Indication Element: 1 liter of HCl solution (90% v / v of distilled water: concentrated HCl) containing 0.5 g of MgCl, 30 of FeCl2 .6H2O, 1 g of ZnCI2.4H2O, 0.2 g of CoCl2.6H2O, 1 g of Na2MoO4, 2H2O, 0.5 g of CaCl2.2H2O, 0.1 g of CuCI2 and 0.2 g of H2BO3. Wastes, cells, enzymes, chemicals and provision of the indicia element were used, or may be used, in each of the examples. Where any significant difference in the subsequent Examples is observed. A. Expression of Optimization of Recombinant P450nam in E. coli An E. coli expression system was engineered to provide host cells that are transformed by plasmid vectors containing DNA encoding mutant P450 oxygenases. The resulting transformants each express a mutant P450 enzyme as a test oxidation enzyme for use in the invention. As shown, expression conditions that reproducibly promote high expression of P450cam in E. coli were identified. The determination of other appropriate conditions, including selective modification of the expression conditions to adapt the particular needs of the analysis, are well within the experience of the subject. Expression was started with XL-10 Gold E. coli cells (from Stratagene) transformed with the expression vector pCWori (+) P450cam. See FIG. 2. The plasmid structure of pCWori) +) contains a replication origin of pBR322, the lac Iq gene, Ampr, and a replica bacteriophage origin. The plasmid also contains a lac UV5 promoter and a double Ptac promoter region followed by a translation initiation region. The DNA sequence inserted into the plasmid structure comprises the structural gene of P450cam. A nucleotide sequence encoding this enzyme is set forth in FIG. 3A [SEQ. ID. DO NOT. 1]. This gene produces the P450cam oxidation enzyme native of P. putida when cloned into the E. coli host cell using the plasmid pCWori (+) as an expression vector. The amino acid sequence of this enzyme is shown in FIG. 3B [SEQ. ID. DO NOT. 2]. Host cells transformed with this vector can serve as a control or comparison for other P450 enzymes, or other oxidation enzymes, or can be used to produce test enzymes for use in the selection method of the invention. For example, other P450 genes, including new strains of native P450 or P450 gene mutants can be transformed into E. coli host cells using the same or a similar plasmid system. 10 The experiments were carried out in both the scale of culture flasks and on a smaller scale, in 96-well microtiter plates, with LB medium, brutal broth (TB) and modified animal medium (M9, containing 20% glucose or glycerol). All these media were evaluated for induction optimization, using 1.0 mM of IPGT as the inducer to activate the synthesis (transcription) of the P450 oxidation enzyme in E. coli. For the optimization of expression levels, the ultra-competent XL-10 Gold E. coli cells (Stratagene, La Jolla, CA) transformed with pCWori + _P450cam grew in LB / Amp plates at 37 ° C at night. The single isolated colonies of transformed E. coli cells were then seeded in 2 ml volume of LB / amp culture medium. After 18 hours they grew at 37 ° C, an aliquot (0.5 ml) of this culture was used to inoculate a volume of 50 ml of each different culture medium: minimum media LB / amp, TB / Amp and modified M9 (glucose or glycerol) / amp. One hundred microliters of ^^^ "- 57 - provision of the pre-prepared cue element (vitamin B1) were added to each flask of the 50 ml culture medium. After 8-12 hours of growth (8 hours for TB, 12 hours for M9), the flask cultures were cooled to 30 ° C. ALA was added (it is unstable at higher temperatures) and the cells were induced with IPTG for 24 hours. For growth and selection of the 96-well plates, a complete cycle of single colonies was collected from the origin plate and transferred directly into TB / amp or M9 medium (glycerol glucose) / zampe incubated at 37 ° C in wells of a plate 96 cavities microtitre. All the additives were added in the growth medium and the induction conditions are the miasma for the flask culture conditions described above. The hydroxylation activity mediated by P450cam was estimated to be naphthalene (NP9 as the substrate) Horseradish peroxidase (HRP), as a purified form, was used as a coupling enzyme.The conversion rate of MPH, which is provided to the The total amount of wild-type P450 enzyme expressed was found to be influenced by the additives used.For example, full-cell hydroxylation activity increased dramatically when ferrous chloride (FeCl2) and thiamine (vitamin B1) were added for all media tested At least 60 times higher activity was obtained even under minimal conditions of M9 (glucose M9 and glycerol M9), compared to media that did not contain these two additives, however, the addition of ALA (0.5 - 1 .3 mM) resulted in small increase (20-25%) in # ** activity of P450cam, compared to the addition of FeCI2 and thiamine, which appeared 24-48 hours in the induction period, reached a maximum at -24 hours, and declined later. Control cultures using the same plasmid pCWori (+) _ P450cam transformed into an E. coli strain that received no addition of those cofactors (thiamin, FeCl2, ALA) produced little or no P450cam activity for at least 48 hours of culture. The formulation of the medium that has been found to be the most useful for obtaining the highest cell activity in 96-well plates and flask cultures was Brutal Broth (TB). An increase of 1.5 times in the activity of the whole cell was obtained when the cells grew in Brutal broth (TB) containing 1.3 mM of AL, in comparison with the M9 medium. One or more of these 15 activities can also be used in other modalities. The reaction conditions and mds for the analysis of whole cell activity in a 96-well microplate are shown in FIG. 4A. H.H. Purification of P450ram using maltose binding affinity tag 20 To verify if wild-type P450ca can catalyze the hydroxylation reaction of naphthalene using hydrogen peroxide as the oxygen donor, P450ca.n was expressed and purified using the fusion vector Maltose (BP) linkage 25 C2 from NE Egland Biolabs (Beverly, MA). The P450cam gene of the vector • • - * ^ - ^^ - > - pCWori (+) _ P450can, was cloned in the Xmm I and Hind lll sites of the MBO expression vector at the 3 'end of the wrong division site E-factor Xa- the vector pCWori (+) _ P450cam was linearized with Ndel (contains initial codon of P450cam, ATG), obtuse terminated with Klenow (5-3"5 exo-incubated with 2.5 mM of free salt dTTP Li) and nucelase of Mung seed After the Hind III cut, the P450cam gene fragment was purified by using agarose gel extraction and then ligating the BP vector.The BP expression vector contains an ampicillin marker gene and a lacZ alpha fragment.

E. coli (DHdalfa) was carried out using CaCl2 and thermal shock (45 seconds at 42 ° C). For selection of ampicillin resistance and complete gene insertion, cells grown on LB / amp agar plate were transferred to a fresh medium containing 20 ug / ml X-gal. For the purification of P450ca, a transformant was cultured in 500 ml of TB / amp liquid medium. Except for the addition of 2.35 g / l of glucose, all protein and induction conditions were the same as described in section A above. (Expression of optimization of recombinant P450cam in E. coli). Affinity separation using an amylose column was done as described by Riggs (1990) (37). The final concentration of the purified BP-P450cam fusion protein (c.a. 88 kDa) was approximated by the intensity ratio of coomassie blue dye with a standard protein marker after polyacrylamide gel electrophoresis of SDS. Concentration '-' * * .-- end of BP-P450cam is estimated to be 5 x 10"8 M (Mw 89,000) C. Analysis of P450cam hydroxylation using purified protein and whole cells P450cam activity in E. coli it was verified by measuring the conversion of naphthalene (NP) to a hydroxylated product (eg, 1-naphthol, 2-naphthol) that emits a blue fluorescence (Imax fl: 465 nm with 350 nm excitation) when the HRP exogenously added polymerizes the product.The hydroxylated NO presumably diffused out of the cells, and the fluorescence was intensified by the addition of HRP and hydrogen peroxide. The cells were grown in 96-well microplates or flasks that were carefully cultured and resuspended in 0.1-1 ml of dibasic sodium phosphate buffer (pH 9.0, 100 mM). 50 μL of this solution was then added in the same regulator (total 200 μl) containing reaction mixtures. A rinsing step of the cell is optional in both cases (however, this step reduces the level of background fluorescence). The procedures and reaction conditions for the analysis of complete cell activity in a microplate of 96 cavities is shown in FIG. 4A. In Step I, the individual colonies showing fluorescence in the first selection are each loaded into a well of a 96-well plate containing 100 μl of TB medium. In Stage II, the colonies are allowed to grow overnight at 37 ° C. Then, in step III, they are induced for 24 hours at 30 ° C with a volume of 120 μl of IPTG and elements "- ^ of indication (0.5-1 mM of IPTG, 1 mM of thiamin, 0.5-1.3 mM of ALA and 0. 5 Provision of Indication Elements per 1 mL of medium. This induces the expression of the enzyme P450cam- In Stage IV, a substrate test solution and an oxygen donor are introduces, to provide reagents for the oxidation reaction catalyzed by P450cam- The test solution contains: 50 μL culture broth (flask or 96-well culture) 1 00 μL dibasic sodium phosphate buffer (50 mM, pH 9 ) 1 0 μL pure ethanol 10 substrate 20 μL supply of naphthalene (saturated; 1 g / 1 3 ml in pure ethanol) oxidizer 10 μL provision of hydrogen peroxide (100 mM) coupling enzyme 1 0 μL provision of HRP (1400 units) / 10 ml) 200 μL The characteristic blue fluorescence generation within the cells was measured in a Parker Elmer HTS 7000 96-well microplate fluorescence reader (emission at 465 nm with excitation at 350 nm). A 96-well white microplate (Nunc, VWR) was used to reduce the background fluorescence of the reaction chamber during detection and integration time (20 ms). The substrate was 20 μl of a saturated solution of naphthalene (NP) in ethanol (EtOH). The oxygen donor was a final concentration of 5 mM hydrogen peroxide (H2O2) and the * -J Enzyme coupling was 1.0 μL of HRP. The volume was adjusted to 200 μL with culture broth, regulator and ethanol. The oxygenation reaction, as an indication of P450cam activity, was measured using a Parker Elmer HTS 7000 96-well microplate fluorescence reader (emission at 465 nm with excitation at 360 nm; 5 Gain 24; measuring time 32 minutes). The reaction reaction scheme is shown in the diagram in FIG. 4B. A substrate of naphthalene and a hydrogen peroxide (H2O2), oxygen donor are introduced into whole cell cultures of E. coli host cells transformed with plasmid pCWori (+) _ P450cam. The plasmid contains DNA encoding the P450cam enzyme. The substrate and oxygen donor enter the cells, where the substrate is oxygenated in an oxidation reaction measured by the P450cam enzyme. This results in the oxygenation of the naphthalene substrate, to produce a hydroxylated compound or reaction product that exhibits a still weak characteristic fluorescence. In the presence of the horseradish peroxidase (HRP) enzyme and additional hydrogen peroxide, the oxygenated compound forms a highly fluorescent polymer, which can be accurately detected. The purified BP-P450cam was also used to carry out this reaction. In this case, the hydroxylation activities of naphthalene were measured in 200 μL reactions in a 96-well microplate. 5.28 x 10'9 M BP-p450 fusion protein (a tenth dilution of the purified protein) was added to the dibasic sodium phosphate buffer containing 7 units of peroxidase - "" j - "- ^ - '" - - - * - - -,. - j. . ... , .. .-.. - .. , ..... to. A - ^, LAa »a?» J horseradish in purified form, and 10 mM naphthalene. The reaction was started after the addition of hydrogen peroxide (2.5 mM and 5 mM). The increase in fluorescence (RFU, fluorescence measurement unit) was measured at the same emission and excitation using the microplate fluorescence reader. D. 96-well Plate Analysis Results A screening experiment conducted on a 96-well plate is shown diagrammatically or tabularly in FIG. 5A. The results of this selection, using the embodiment of the described 96-well plate of the invention, are shown in FIG. 5B. In this analysis, the activity of the whole cell for hydroxylation of naphthalene by P450cam and hydrogen peroxide is evaluated for different media (TB, glucose M9 and glycerol M9) with different concentrations of ALA (0.5 and 1.3 mM) in each of the series of cavities in the 96-well plate. See FIG. 5A. As described above, each reaction is induced by 1 mM of IPTG, 1 mM of thiamine, and 0.5-1.3 mM of ALA, with trace elements. Columns A-D of the 96-well plate contained transformed E. coli host cells to produce P450cam (pCWori (+) vector: P450cam was used). The E-H columns with the E. coli cells that were not transformed for produced P450cam (control strain (XL-10 Gold)). Rows 1-3 of the 96-well plate contained Brutal Broth (TB) and 0.5 mM ALA. Rows 4-6 contained M9 glycerol medium and 0.5 mM ALA. The first row of each group of three ri ^ b_i_ ^ iiuaH ^ «* * - 64 - rows used 200 μl of cultivation volume. The other two rows of each group of three used 100 μL of cultivation volume. Fluorescence in each well was measured using a microplate fluorescence reader [Perkin Elmer, HTS 7000]. The degree of fluorescence provides a still accurate, indirect indication of oxygenated substrate, which in turn provides a measure of P450cam activity. As shown in FIG. 5B, the lower P450 activity was observed for the cultivation volume of 200 μL, longer containing a smaller concentration of a substrate and oxygen donor, compared to the lower (more concentrated) cultivation volume of 100 microlites. (Compare Rows 1, 4, 7 and 10 (200 μL volume) with the other Rows (100 μL volume)). This shows that the observed fluorescence, and degree of fluorescence, actually registers the P450 enzyme reaction and oxygenation of interest. The results also show that TB is a significantly more favorable medium than any of the M9 media tested, and the highest concentration of ALA (1.3 mM) is marginally more favorable than the lower concentration tested (0.5 mM). ALA is an important heme synthesis intermediate (P450 is a heme protein) and the synthesis of P450 in the host cell cytoplasm is regulated in part by the concentration of the synthesized heme. A high level of P450cam protein expression (total activity: 430 RFU / min) was obtained using TB and 1.3 mM ALA, 100 μL volume.

The experiment shows that the host cells can be effectively transformed to express and activate the P450 enzyme that can be used to catalyze the oxygenation of a substrate of a substrate in a complete cell analysis adapted for high throughput selection, for example, in a format of 96-well plate. Fluorescence produced by oxygenated substrate such as hydroxylated naphthalene can be detected and measured reliably, particularly when amplified by a coupling enzyme such as HRP. To support these results, the use of P450cam peroxide deviation path was verified using the purified BP-P450cam enzyme. The considerable increase of the poly (naphthol) fluorescence was observed: 6.8 ± 0.5 a. or. (RFU) / min / nmol with 2.5 mM H2O2; and 19.7 ± 0.5 a. or. (RFU) / min / nmol with 5 mM H2O2, in the absence of NADH and two auxiliary electron transfer proteins (putidaredoxin and reductase). This supports the discovery that P450can. You can use hydrogen peroxide as an oxygen donor in this reaction. EXAMPLE 2 Selection of Complete Cell for Naphthalene Hydroxylation Image of Analysis and Co-expression of P450cam Without Horseradish Peroxidase (HRP) This example demonstrates that the co-expression of HRP with monooxygenase P450 leads to the accumulation or fluorescence within the cells, which can be monitored by digital image analysis. In EXAMPLE 1, above, HRP was added to whole cells transformed to express P450cam. In this Example, the E. coli host cells are transformed to express both enzymes, HRP and P450cam- Thus, it is not necessary to add HRP in a separate analysis step, nor is it necessary to monitor the growth medium for changes in fluorescence that indicates oxygenation and activity of P450cam- In transformed host cells to produce both enzymes, the analysis reaction occurs within the cells when the substrate and the oxygen donor are provided, for example, hydrogen peroxide and naphthalene. The fluorescence of, within, and / or around the cells that produce an oxygenated compound and polymer (mediated by the two enzymes) can be detected and measured. The detailed methods used in this example are given below: A. Co-expression of HRP1A6 and P450 a Recombinant in E. coli Genes and Plasmids. A HR P mutant gene that produces active HRP in E. coli was prepared as described in EXAMPLE 9 and in U.S. Provisional Application Serial No. 60 / 094,403 filed July 28, 1998. This gene encodes a mutant HRP identified as "HRP1 A6". The gene for HRP1 A6 is limited to pETpe1 BHRP 1 A6 and cloned into the kanamycin resistant vector pET26b (+) (Novagen, Madison Wl), producing pETpe1 BHRP1 A6. Except for the antibiotic marker, this vector is identical to ^^? k? ^^ Jigfl? S pETpe1 BHRP1A6 set forth in FIG. 24. The expression vector pCWori (+) _ P450cam was prepared as set forth in EXAMPLE 1. pCWori (+) _ P450cam and pETpelBHRP1A6Kan transformation. The chemical transformation using CaCl2 (60 mM) 5 and thermal shock (45 seconds at 42 ° C) was used to introduce the plasmid pETpelBHRP1 A6Kan in BL21 E. coli (DE3). Successful transformants were identified by selection of LB / kan agar plates (6-30 μg / ml kanamycin). The positive clones were then made chemically competent and transformed with the second plasmid, pCWori (+) _ P450cam. The identification of BL21 E. coli (DE3) clones containing both genes was identified by growth on LB / kan plates (30 μl / ml) amp (100 μg / l amp). The abbreviation "amp" indicates the antibiotic ampicillin, and "kan" indicates the antibiotic kanamycin. The cells containing the Kan or Amp DNA fragments will grow in the medium containing the respective antibiotic. This can be used as a so-called "selection marker", according to well-known techniques, to identify and isolate different groups of cells with different properties using the ability or non-ability to resist the antibiotic as a brand. B: Cell Growth and aaar plate reaction for image analysis In this procedure, pure cultures of E. coli (containing (pCWori (+) _ P450cam and pETpelBHRP1A6Kan) were seeded on TB / agar plates (Falcon, # 1007 or Q-bot) supplemented with 100 ^ ^ ^ ^ ^ ^ - * - • *** »•" "" • • • - • - "μg / ml ampicillin, 30 μg / ml kanamycin, 100 μg / ml of the indicator element supply solution, 0.25 mM thiamine , 1 mM of ALA and 0.5 mM of IPTG, and grew at 37 ° C for 6 hours, in which the point of incubation temperature was lowered to 30 ° C to obtain even and small colony size distribution (< 0.8 mm diameter) for detection of exact hydroxylation activity.The growth temperature deviating from 37 ° C (after 6 hours) to 30 ° C is preferred for uniform growth control, which facilitates the image analysis. that cells growing at 37 ° C for 24 hours generally contained both small and large cells that can not be easily used for image analysis.After 16 hours incubation of simultaneous cell growth and protein expression, the hills formed on the origin plates were copied (to make a replica) and transferred on a nitrocellulose membrane, and then incubated on an agar plate / M9 / 10% (w / v) glucose / 5% (v / v) ethanol containing 6 mM naphthalene and 10 mM hydrogen peroxide for selection by fluorescence image analysis. The optimum temperature and time for this hydroxylation of naphthalene was estimated at 30 ° C and 12 hrs. The detailed methods are described in FIG. 6. Analysis of P450cam hydroxylation using HRP and P450 of complete cell co-expression. Host cells transformed to express P450cam and HRP were grown in 10 ml TB / amp / kan (100 μg / ml ampicillin, 30 μg / ml kanamycin) containing 0.2 mM thiamine. 1 mM of ALA, and 20 μL of indicator element supply solution. The grown cells were harvested and carefully resuspended in 1 rnl of dibasic sodium phosphate buffer (pH 9.0, 100 mM). After the addition of 10 μl of naphthalene (0.5 g / 13 ml of pure ethanol at 25 ° C), 10 μl of ethanol, and 10 μl of total peroxide solution (provision: 100 mM) to 170 μL of solution of cell suspension (total 200 μl of total reaction volume), the characteristic blue fluorescence generation within the cells was measured by a 96-cavity Perkin Elmer HTS 7000 fluorescence reader (emission at 465 nm with excitation at 350 nm ). A 96-well white microplate (Nunc, VWR) was used to reduce the antecedent fluorescence of the reaction chamber during detection and integration time (20 ms). See, EXAMPLE 1 and FIG. 4A. Analysis of HRP activity. The peroxidase activity expressed in BL21 (DE3) E. coli transformed using the vector, pETpelBHRP1A6Kan, described above, was estimated colorimetrically using ABTS (2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid). were cultured by centrifugation (Beckman CS 6R) at 3350 rpm and resuspended in 1 ml of 100 mM potassium phosphate buffer (pH 7.5 at 25 ° C.) An aliquot of 50 μL of this mixture is added to 40 μL of 6.4 mM of ABTS solution The ABTS oxidation was monitored at 405 nm using a thermoset spectrometer (Perkin Elmer UV / VIS Lambda20) at 25 ° C.

C. Selection results of co-expression of P450 am and C of mutant HRP in E. coli. This embodiment of the analysis can be illustrated as shown in FIG. 7. A host cell such as E. coli (for example, strain BL21 (DE3)) is transformed by two expression vectors: (1) the plasmid pCWori (+) _ P450cam; and (2) the plasmid pETpelBHRP1A6Kan. Transformed cells can be cultured from individual cells or colonies, to produce a source of transformant for use in an analysis of the invention. A substrate (e.g., naphthalene) and oxygen donor (e.g., hydrogen peroxide) are introduced to the transformed host cells under favorable conditions (e.g., conditions that induce P450 expression and / or activity). These reagents, together with any added co-factor or coenzyme, enter each cell, where find the P450 enzyme that is produced there. P450 catalyzes substrate hydroxylation (addition of oxygen in the form of a hydroxide group, OH) to form, for example, hydroxylated naphthalene. In the presence of a coupling enzyme also produced within the cell, such as HRP, the hydroxylated naphthalene forms polymers and oxygenated dimers, which are highly fluorescent and have a characteristic fluorescence profile that can be easily detected. Typically, polymeric oxygenates do not leave the cell. Therefore, the accumulation of hydroxylated product in the transformed cells provides significant advantages for detection and measurement of • U ^ &? Á. fluorescence, and for identification of cells, which successfully produce the P450 enzyme, and which does so at relatively high levels. As coumarin shown, 3-phenylpropionate and other substrates can be used in place of naphthalene. The construction of this selection system is based on inducible prokaryotic expression vectors that allow active coexpression of both enzymes, P450cam and HRP, in the same host strain. As described, a pCWori + vector, which contains the p450cam gene was inserted into E. coli B21 (DE3). This resulted in E. coli host cells that express the introduced P450 gene, and produce a functional P450 enzyme that can be used to catalyze the first reaction in the analysis, the oxygenation reaction described above. For the second enzymatic reaction, the coupling reaction, an active HRP mutant gene inserted into the expression vector pET26b was also transformed into E. coli BL21 (DE3) as described above. Alternatively, cytochrome peroxidase C (CCP) can be used as a coupling enzyme, and a functional gene expressing this enzyme can be transformed into E. coli or into a yeast host cell using similar means. The yeast can also be used as the host cell for the expression of P450 enzymes or co-expression of oxygenase and coupling enzyme. The characteristics of these expression vectors are summarized in TABLE 1.

TABLE 1 Characteristics of the plasmids used for the co-expression of P450 with HRP and coupling enzymes Gene insertion; Type Marker Origin 5 vector promoter antibiotic replica P450 cam from P. putida Ptac Ptac PBR322 Ampr * RBS and l .C (ATCC17453); space: 3bp pC or¡ + HRP1 A6: T7 PBR322 Kanr pET-26b (+) pET-26b (+) contains pe. B leader Peroxidase c of T7 PBR322 Kanr ** without pe1 B cytochrome of S. leader cerevisiae; PET- 26b (+) * 3 bases of space between ribosomal binding site and codon of initiation, ** sequence of pe1 B leader was removed from the original pET-26b (+) vector to avoid protein secretion. The experimental time course of fluorescent product generation using this co-expression system is illustrated in FIG. 8. With the HRP / P450cam double vector system, more than 370% increase in absolute cell fluorescence level was observed after 30 minutes of reaction with 5 mM hydrogen peroxide. A study of the effects of IPTG concentration more above the 0.1 mM range indicated that 0.5 mM of IPTG is optimal ^ UM &l ^ Mgí ^ for the co-expression system P450ca coupling / HRP E.coli BL21 (DE3). As shown in FIG. 9, this concentration of IPTG induces the highest activity for P450 enzyme in the presence of an appropriately high HRP activity. In this way, the co-expression of P450cam and HRP at appropriate levels (IPTG -0.5 mM) resulted in marked intensification of the intracellular fluorescence level. This co-expression system is advantageous in that the fluorescence remains associated with the cavity (not diffusible).

The background intensities either remain constant over time (host strain as a negative control) or show small increases over time (cells without hydrogen peroxide or naphthalene). As a result, the expression intracellular HRP with activity of P450cam in BL21 (DE3) showed that it is a complete selection system and effective self-content to detect hydroxylation reactions using the peroxidase deviation path. Although P450cam by itself also produces fluorescent naphthols, the total measured intensity was lower than the HRP / P450carn co-expression system. 0 D. Image Acquisition, Processing and Analysis Full-cell fluorescence images on agar plates were scanned using an Eye of Needle II and a 350 nm ultraviolet illuminator mounted on top (Stratagene, La Jolla, CA). The images were analyzed digitally 5 using the Optimas 5.0 software package (Óptimas Corporation, • * - • * - * - - • - * * * WA). The gray-level fluorescent colonies were filtered using a blue fluorescence band path filter with excitation at 350 nm to remove the background fluorescence. The establishment parameters for the acquisition of the fluorescent signals using this system BL21 (DE3) are as follows: blue band pass filter (range of 430-470 nm), zoom lens level = 4 x, exposure time of the fluorescence image = 1/1 0 seconds. The selected gray level colonies were analyzed with a coupled charge device (CCD) and subsequent computer aided image analysis. A weight score of 255 was used, with zero as the lowest value and 255 as the highest fluorescence intensity. (This configuration can be modified as appropriate). The mean background fluorescence intensities of the E. coli strain BL21 (DE3) (plasmid-free control strain) were estimated to be 0 to 5. The fully automated segmentation algorithm (pattern recognition and subsequent propagation algorithm) for the colony recognition and size measurement was adapted to avoid manual reference subjective and time sensitive contours of the colony. Measurement of individual colony size and single isolated colony detection were derived from computer-determined colony boundaries and fluorescence differences with different sets of threshold levels. The estimated total analysis time was approximately 5 seconds during 1 05 colonies.

The blue fluorescence is derived from the products synthesized by the enzymatic reactions coupled by P450cam and HRP. The fluorescent images explored gave a clear result that correlates with the hydroxylation activity of specific P450cam 5. It is suggested that the smaller individual colonies are better for fluorescence image analysis. The maximum size limit of the colony for this image analysis was estimated to be 0.8 mm in diameter. The typical dimensions of the imagined colonies were -0.4 to 8 mm and there were approximately 9 to 17% of fluorescence deviations within the size distribution. The scanned images (FIG 10) are further processed by configuring the total threshold, geometry recognition, intensity quantization, local and global segmentation, and edge cut to reduce the background fluorescence. A major consideration was the separation of the coating colonies in the two-dimensional cell fluorescence image. The scanned original fluorescence image was preferably complex and included many unclear edge cut-outs to analyze (FIG.1 1A).

By imposing the sequential combinations of bitmap fingering Bolean and passing through a uniform luminescence enhancement algorithm (Extensis, Extensis Co.) the images could be turned fine for further evaluation (mainly, cutting the edge by size reduction, limit erasure, and division) in the OPTIMAL analyzer. See FIGS. 1 1 B-1 1 D. During the j ^ g ^^ j ^^ processing of the second image, the colonies that touch each other were selected first by the semiautomatic algorithm provided by this package and after a limit detection algorithm was passed to erase any colony that struck 5 the limit. FIG. 1 1 B still shows the regions of two or three cells in contact, and FIGS. 1 1 C and 1 1 D show improved fluorescence images after several processing cycles. Using these image analysis techniques, FIG. 12 shows a comparison of fluorescence intensities in 10 BL21 (DE3) E. coli for the following combinations: A. co-expression of P450cam / HRP with the addition of naphthalene and H2O2; B. P450Cam with the addition of naphthalene and H2O2; C. HRP with the addition of naphthalene and H2O2; 15D. Host strain of BL21 (DE3) E. coii untransformed as a negative control; E. co-expression of P450cam / HRP with H2O2 without naphthalene; F. co-expression of P450cam / HRP with naphthalene and without H2O2; The lateral images on the left are original fluorescent colonies of two explored dimensions. The histograms on the right side, seen in the lunar landscape, are the fluorescent intensities resulting from the individual colonies. The co-expression of P450cam / HRP (Combination A) gave the highest fluorescence intensity among the tested cells.

There was an increase of 3 times the absolute fluorescence level between the cells that host the P450cam / HRP vectors (A) and the P450cam expression vector alone (B), as estimated in the lunar landscape view. Due to the low level of fluorescence generated, approximately less than a quarter of the colonies could be counted with the cells harboring only the P450cam expression vector- The background fluorescence levels tested with the other four cases (host strain HRP, BL21 (DE3 ), and in the presence or absence of naphthalene and H2O2) were much lower and clearly distinguishable from the fluorescence generated by the co-expression system. The qualified fluorescence intensities of these control cases (FIGS 12C, 12D, 12E and 12F) almost all fell between 0 to 5. None of these four cases (C, D, E, F) scored one hit during the analysis of image. Therefore, cells expressing oxygenase and peroxidase enzymes can be identified by plaque-based image analysis as active in the hydroxylation reaction. FIG. 13 shows a technique for automatically detecting fluorescent colonies and the result of fluorescence intensity analysis. A total of 843 cells in scanned area of 5 x 5 square cm were counted (out of 20,000 colonies counted in an entire 25 x 25 cm square plate). The individual positive colony fluorescence intensities could be integrated into the explored area. The E. coli cells exposed to naphthalene in the plate survived for 24 hours of incubation. The computer-assisted techniques described above can also be used in connection with a high speed or automatic mode. The results of image analysis are consistent with the data obtained from analyzes carried out on 96-well plates (see FIG 8). In these experiments it is shown that cells having P450cam only resulted in relatively low fluorescence formation (almost three times lower absolute fluorescence level), compared to the approach of two enzymes. Even though the ñafióles show fluorescence, the intensities estimated were very low at nanomolar concentration levels (200-250 a.U. PARA 10 - 1 00 nmol / ml). In this way, fluorometric observation of the complete direct cell of the hydroxylation reaction of P450cam can be performed, and coexpression with HRP leads to significant selection advantages. An important advantage of this enzyme-coupled analysis system is that it generates an amplified fluorescence signal that provides for the formation of the oxygenated product. This enhanced signal allows the selection of large numbers of host cells expressing oxygenase enzymes, for example, by fluorescence digital imaging or fluorescence activity cell (FACS) selection. The greater the signal amplification provided by this selection process, the lower the oxygenase activity that can be identified by this selection process. In addition, false-false positives and false-negative ones will be identified during the selection ? A ü kt fi fi fi ^ para para para para para para para para para para para para para para para para para para para. EX EMPLO 3 Selection of the Complete Cell for P450 Activity of Cytochrome Towards Other Substrates by Image Analysis and Coexpression of P450cam with Horseradish Peroxidase (HRP) In this example, coumarin and 3-phenylpropionate are used as substrates instead of naphthalene. The co-expression of HRP with the monooxygenase of P450 leads to the generation of fluorescence for coumarin and 3-phenylpropionate as substrates of the hydroxylation reactions. All experimental conditions are the same as those described in EXAMPLE 2, except for the substrate concentrations used. The final concentrations of 3-phenylpropionate and coumarin were 1.2 g / l and 6x102 g / l, respectively. Coumarin and its hydroxy derivatives, 7-hydroxycoumarin and 4-hydroxycoumarin, were purchased from Sigma Chemical Co. (St. Louis, MO). 3-phenylpropionate and its derivatives of 2- / 4-hydroxy (3- (2-hydroxyphenyl) propyanate and 3- (4-hydroxyphenyl) propionate) were also purchased from Aldrich and Sigma Chemical Co. The maximum values of characteristic hydrocoumarin or hydroxy (3-phenylpropionate) were detected using a fluorimeter with a broadband trajectory fluorescence emission filter (465 +/- 30 nm), ie a Perkin Elmer HTS 7000. The whole cell reaction system that uses the co -expression of P450cam with horseradish peroxidase was used. At pH 9, 4- and 7-hydroxycoumarin it also shows a characteristic blue-green fluorescence (emission at 450-495 nm, excitation at 350 nm). This fluorescence is hard to detect, however, because coumarin itself shows too intense fluorescence, which leads to high background fluorescence under these conditions. FIG. 14 shows the results of the intensification of fluorescence aided by HRP for two substrates, coumarin (FIG.14A) and 3-phenylpropionate (FIG.14B) Each bar graph named shows, respectively, the results of: (1) the -expression of P450cam and HRP1AL in BL21 host cells (DE3) E. coli; (2) expression of P450cam in BL21 (DE3) host cells E. coli; (3) expression of HRP1A6 in BL21 (DE3) host cells E. coli; and (4) BL21 (DE3) host cells E. coli. With coumarin, which is already too fluorescent, some intensification was found: 35% higher than with the expression of P450ca alone. In the case of 3-phenylpropionate, the HRP assistance gave 300% higher fluorescence intensity, compared to the controls (P450cam in BL21 (DE3); HRP in BL21) DE3), and the host strain). EXAMPLE 4 Analysis of hydroxylation based on chemiluminescence light augmentation This example demonstrates the use of chemiluminescence detection to monitor the formation of hydroxylated products, using horseradish peroxidase as the coupling enzyme. See FIG. 15. In this example, the coupling enzyme is coexpressed with the oxidation enzyme in bacterial cells, as shown for example in EXAMPLE 2. In a 96-well plate analysis, as previously described, the signal provided when using a P450 monooxygenase and the HRP coupling enzyme was measured (column A in FIG.15A) and compared to the signal from the cells that do not have the coupling enzyme (column 5), does not carry out the hydroxylation reaction ( column 6) and does not carry out any reaction (column 7). Rows E and F contain the substrate (3-phenylpropionate), while rows G and H contain no substrate. Oxidation of the chemiluminescent agent, luminol, by peroxidase catalysis leads to a colored product, but generates nothing (or very weak) chemiluminescence in the presence of only hydrogen peroxide (without coupling enzyme). The photocurrent (intensity) of the chemiluminescence light generated was measured using an Alphalmager system (Alphalmager 2000, see 3.3, Alphalmager Corporation). This luminometer includes a multi-chamber cabin for luminescence detection (a light-tight case with a matt black interior), photocurrent analysis software (Alphaimager 2000, see 3.3) and a CCD detector for light intensity detection. The light emitted from the individual cavities of a white "Nunc" fluoroplate of the 96-well type was measured in the chamber luminometer. Luminol was purchased from Molecular Probes. { Eugene, OR) and 3-phenylpropionate and 30% hydrogen peroxide were purchased from Sigma. The reaction mixture (200 μl) contained 0.1 mM of 8.6 borate regulator with sodium perborate (3 mM), luminol (60 and 120 uM), and 3-phenylpropionate (0.5 mM). The co-expression conditions of P450cam / HRP and cell growth are the same as in EXAMPLE 2. After 24 hours of induction to produce the hydroxylation of P450cam of 3-phenylpropionate was carried out for 20 minutes. The conditions of the hydroxylation reaction are as described in EXAMPLE 2. Hydrogen peroxide (5 mM) was added. The reaction conditions and results are shown in FIG. 15. Luminescence measurements show that P450cam-catalyzed hydroxylation of 3-phenylpropionate increases the generation of luminescence light. The chemiluminescence of the cells containing P450camo and expressed HRP (Line 4, Row F of FIG.15B) was increased up to 98 times, compared to the luminol reaction itself (Line 4, Rows G and H). Therefore, hydroxylated 3-phenylpropionate leads to a significant increase in light emission and can be monitored using this approach. The integrated light emission from the strain that coexpresses both enzymes shows more than 1000 fold increase in the first 40 seconds after the reaction starts, compared to other background levels. The prolonged and intense light emission of the reaction increased by the incorporation of the additional hydroxylated aromatic phenol lasted more than 7 minutes. This is particularly useful for the selection of enzymes with relatively weak hydroxylation activities, for example, in connection with particular substrates. In addition, multiple colonies can be analyzed rapidly and simultaneously using color image analysis. The abbreviation "ILDV" (for example, FIG.15B) indicates 5 the density value of] uz integrated, as a unit of chemiluminescence intensity. The results under UV epi conditions show that the hydroxylation of 3-phenylpropionate can also be detected using the combined forms of light intensities (a kind of light energy amplification), which were generated by light emission as fluorescence and chemiluminescence, by separated (FIG 15A). Although the absolute light density was increased, in this case, the double-mode detection obtained increased the antecedent. EXAMPLE 5 Monitoring Oxidation by Co-Expression with Cytochrome Peroxidase C This example demonstrates the use of another peroxidase, cytochrome peroxidase C (CCP), a yeast gene that is expressed in E. coli as the coupling enzyme to select hydroxylation catalyzed by P450carn. The yeast CCP enzyme is expressed in the functional form in E. coli host cells. A. Construction of the Cytochrome Peroxidase C Vector The Cytochrome Peroxidase C (CCP) gene of S. cerevisiae of pT7CCP (donated by Dr. David Goodin, The Scripps Research Insititue, La Jolla, CA) was recloned in sites? / Of I and Bam Hl expression vector pET-26b (+) kanamycin resistant - - - - * j "-" - * ~ (purchased from Novagen, Inc., Madison, Wl). pT7CCP carries a gene for CCP, in which the N-terminal sequence has been modified for the Met-Lys-Thr amino acids, as described in Goodin et al. (1990) (39), and Fitzgerald e to (40). First, the pT7CCP vector containing the CCP gene was linearized with Pvu II, obtuse terminated with Mung seed nuclease to give a ligation site between the 3 'end of this gene and the Bam Hl site in pET-26b (+). Then, this gene fragment was cut using Nde I for ligation of the 5 'end with the vector. The N-terminal pe1 B signal sequence located in the upstream region (224-289) of the pET-26b (+) vector was removed by digestion of Nde I and BamH I to allow intracellular CCP expression. Bam Hl then cuts the obtuse end for further ligation with the CCP gene fragment. FIG. 16 shows the vector map pET26b + CCP. fí. Complete cell selection for cytochrome P450 activity using co-expression of CCP Expression was taken with BL21 (DE3) E. coli cells transformed with pCWori (+) _ P450cam and pET26b + CCP vectors, as previously described. The plasmid structure of pET-26b (+) contains a T7 promoter in region 361-377, plasmid f1, and a kanamycin coding region. For the co-expression of both enzymes, P450cam and CCP, 1 mM of IPTG and 50 μg / ml of kanamycin were used. The results are shown in FIG.17. The coexpression of P450cam / CCP gave highly intensified fluorescence signals when naphthalene hydroxylation was tested. A 3.2-fold increase in the absolute fluorescence level, compared to the P450cam reaction alone, was observed. EXAMPLE 6 Use of Laccase as a Coupling Enzyme In this example, a laccase enzyme was used as a coupling enzyme, instead of a peroxidase. When laccase is used, there is no need to add hydrogen peroxide, since this enzyme can catalyze the oxidative coupling reaction using molecular oxygen. This is useful when oxidative enzymes that do not require peroxide are selected for the reaction. Laccases are copper-containing enzymes that catalyze the oxidation of a variety of substrates, such as phenols, mono-, di- and poly-phenols, substituted phenols of methoxy and aromatic amines. The laccases coupled four of these oxidations from an electron to the reduction of four irreversible electrons from dioxygen to water. Each reaction of an electron generates a free radical. The aryloxy radicals formed by laccases are unstable and typically pass a second reaction. This reaction can be an enzymatic oxidation (converting phenol to quinone in many cases), a non-enzymatic reaction such as hydration, disdosification, or oxidation / reduction, or the radical can be coupled to other phenolic structures in a polymerization reaction that produces products that they are often colored and / or highly fluorescent.

Laccase was purchased from Sigma Chemicals as a crude acetone powder of Rhus vernicia fungus. The laccase powder had a minimum of 50 units per mg, with the unit activity defined as) A530 of 0.001 / min at pH 6.5, 30 ° C, 3 mL of solution with syringaldazin as substrate. HRP type II (RZ approximately 2.0) and the other chemicals were purchased from Sigma. The fluorescence readings were taken with a Perkin-Elmer HTS 7000 plate reading fluorometer. The excitation and emission wavelengths were 360 nm and 465 nm, respectively. The experiments were carried out at 25 ° C in the opaque white Nunc 96-well plate cavities with a total liquid of 200 μL. Except where otherwise stated, each cavity contained 10% pure ethanol to improve the solubility of substrates and products. Where HRP was used for comparison, approximately 2.3 units of HRO and 5 mM of H2O2 were in each cavity. Each experiment was run at pH values of 6.5, 7.5 and 9.0 using phosphate regulators (10 mM and 100 mM, depending on the experiment), which does not show fluorescence. The Tris regulator was not used because it increases the background fluorescence. Except where it is established, the results are given of conditions at pH 9.0, which are either the best results or hardly distinguishable from the other conditions. In order to evaluate laccase in a complete cell analysis useful for identifying the formation of hydroxylated aromatic compounds by oxidative enzymes (such as cytochrome P450cam and toluene dioxygenase), laccase was added to soons containing cells, naphthalene, and naphthol. P450cam was expressed in BL21 (DE3) strain E. coli using pCWori + plasmid. The BL21 (DE3) cells with or without the expressed protein grew in the Brutal Broth and after 8 hours were induced with 1 mM of IPTG for 24 hours. 50 μL of each type of cell soon (with or without plasmid) was added to twelve cavities (six cavities for each type). The pH 9 regulator was added to each well so that the final volume after all additions would be 200 μL. Approximately, 15 laccase units were added to each cavity, and the mixtures were allowed to pre-incubate for approximately 45 minutes to remove any background activity between the laccase and the components of the cell soon. In a set of six cavities, three cells harboring the plasmid with untransformed cells, 10 μL. of saturated naphthalene in ethanol was added to each soon. 1-Naphthol was then added to one of each type of cavity (with or without naphthalene; and with or without P450cam) at a concentration of 1000 uM. Similarly, 2-naphthol was added to four other cavities. These cavities in which naphthol is added (in addition to naphthalene) stimulated situations in which P450cam is capable of producing naphthol of naphthalene at high concentrations (1 00 uM). All the cavities contained 10% ethanol. For comparison, the same twelve cavities were prepared using HRP and H2O2 instead of laccase. These same experiments were performed at pH 6.5. ^^ ^^^^^^ a Mm In all the cavities containing 1-naphthol (100 uM) and either HRP or laccase a pale yellow color change from the cell solution to a dark brown color occurred. The change was faster with HRP (approximately 1 minute compared to approximately 1 hour). In the case of 2-naphthol, the change in color to a light orange occurs, although this is pronounced less and is slower. The difference in color formation between comparable solutions can produce intense color changes, in theory because the HRP preparation by itself already has a slightly brown color. It is relatively difficult to discern a difference in color between comparable solutions with and without added naphthalene or with or without P450 expression, indicating that the level of hydroxylation of naphthalene by the enzyme is very low at the level of expression in this experiment, and in its activity towards this substrate under the test conditions. No color change was observed for any of the cavities that do not contain naphthol. These results indicate that whenever naphthol is produced in a sufficiently high concentration by the hydroxylation enzyme, laccase can be used as a coupling enzyme for identification of naphthol in a colorimetric complete cell analysis. EXAMPLE 7 Detection of Catechols formed by Dioxigenation Catalyzed by Toluene Dioxygenase (TDO) of a Substituted Benzene This example demonstrated the use of horseradish peroxidase to detect the formation of the products (catechols) of dioxygenation catalyzed by TDO of chlorobenzene followed by dehydrogenation . A host cell, E. coli in this example, is transformed with a vector having a functional TDO gene, and transformed cells grow under conditions suitable for the expression of TDO. The host cells in this example are also transformed to express the enzyme dihydrodiol dehydrogenase, and can be transformed to express HRP, as described in other EXAMPLES herein. The total reaction used in the analysis of this example is shown below. fluorescent products ocokx.adoi The first set of reactions (catechol formation) is catalyzed by plasmid pXTD14 of plasmid containing alpha DH5 E. coli, containing the genes todC1 C2BA (for toluene dioxygenase) and todD (for dihydrodiol dehydrogenase). A map of this construction is shown in FIG. 18. For plasmid construction, alpha DH5 £. coli was used as a host and the transformants grew in LB which contains 50 μg / ml of ampicillin at 37 ° C. The expression vector of pTrc99A was purchased from Pharmacia Biotech (Uppsala, Sweden). A wild-type 2.1 kb todC1-todC2 fragment was produced by PCR in tuned pDTG601 (provided by D. Gibson, University of Iowa) (41), using the following primaries. A forward primary T DO-5F: 5'- GATCATGAATGAGACCGACACATCACCTATC-3 [SEQ. ID. DO NOT. 3]; and A reverse primary TDO-2R: 5'- ACGAATTCTAGAAGAAGAAACTGAGGTTATTG-3 [SEQ. ID. DO NOT. 4]. The fragment was directed with fisipHI and EcoRI and subcloned into the? / Col-EcoRI site of pTrc99A to construct pXTD2. The restriction sites in the primaries are underlined. A DNA fragment containing todC2-todB-todA-todD genes was amplified from pDTG602 (provided by D. Gibson, University of Iowa) (41) by PCR using the following primaries: A primary forward labeled by Bam II TDO-9F (the Bam Hl restriction sequence is underlined),, 5'-TTGGATCCGGTGGACCTTGTCCATTTG-3 '[SEQ. ID. DO NOT. 5]; and A TDO-14R reverse primer (restriction sequence Xba I is underlined) (5'-GCTCTAGATCAACCGAAGTGCTTGTCGAC-3 '[SEQ ID NO: 6].) The resulting 3.0 kb fragment was digested with Bam Hl and Xba I and was purified by the QIAquick PCR Purification Kit (QUIAGEN) This fragment was cloned into the Bam Hl-Xoa I site of pTrc99A to produce plasmid pXTDI O. Then pXTDI O was digested with EcoRI and FíamHI and ligated to a fragment of todC1 wild type of 1.2 kb digested with EcoRI and BamHl This wild type todC1 fragment was produced by PCR using: A forward primary TDO-12F: 5'- CGGAATTCTAGGAAACAGACCATG-3 '[SEQ. ID. DO NOT. 7]; and A primary inverse TDO-13R: 5'- CCGGATCCAACCTGGGTCGAAGTCAAATG-3 '[SEQ. ID. DO NOT. 8] of the hardened DNA pXTD2. The restriction sites in the plasmids are underlined. The resulting plasmid is pXTD14. The E. coli DHE alpha strain was transformed with pKK223-3 (Amersham Pharmacia Biotech, Uppsala, Sweden) was used as control. In this example, a chlorobenzene substrate was oxygenated by the addition of two hydroxyl groups, through TDO, and the resulting annular structure of the substrate is stabilized to a double bond through dihydrodiol dehydrogenase. The oxygen donor in this reaction is molecular oxygen (O2), obtained by the E. coli O2 host dissolved in the medium. In another reaction, the dihydroxylated product is reacted in the presence of HRP and hydrogen peroxide, to form fluorescent or colored products. Thus, in this example, the substrate is chlorobenzene (or any suitable aromatic substrate), the oxidation enzyme is toluene dioxygenase (TDO), the oxygen donor is molecular oxygen, and the coupling enzyme is horseradish peroxidase. (HRP). The following procedure was used to prepare supernatant containing catechol produced by TDO: 1) Add 0.5 μL of each seed culture overnight to two flasks containing 20 mL of LB-Amp and stir for three hours at 37 ° C. 2) Add 200 μL of 100 mM IPTG to each flask, and shake at 30 ° C for two hours. 3) Centrifuge the cultures at 300 rpm for 10 minutes and discard the supernatant. 4) Resuspend the tablet in 4 mL of 50 mM phosphate buffer, pH 7.4, containing 1 0 mM chlorobenzene and 0.2% glucose 5) Incubate at 30 ° C for two hours (2 mL in 1 5 mL of tube ) 6) Add 12 mL of 50 mM phosphate buffer, pH 7.4 and centrifuge at 3000 rpm for 10 minutes. 7) Transfer of supernatant containing catechol to a fresh tube To detect the catechol products, 1 μL of 2mg / mL of HRP and 10 μL of 1 M H2O2 were added to 200 μL of supernatant. A two-fold dilution of the supernatant was also analyzed. In the case of E. coli containing pXTD14, the solution became red porco after the addition of HRP and H2O2 to the supernatant containing catechol. The 2X dilutions were subjected to spectrum-photometric analysis. The baseline was taken to be the supernatant (pKK223-3) of the control cultures with only added H2O2. The absorbance profile of pKK223-3 with HRP and H2O2 was essentially flat. The absorbance profile of the strain expressing TDO (pXTD14) showed a small maximum value at 281.5 * "" * • - - nm which, on the basis of previous experiments, corresponds to the presence of chlorocatechol. When HRP is added to the supernatant of pHTD14, the absorbances appeared around 340 nm and 500 nm corresponding to the polymer formed when the chlorocatechol was oligomerized by HRP. EXAMPLE 8 Identification of Enhanced Mutants of P450cam An important aspect of this invention is to identify mutants in a high throughput selection of mutagenized gene libraries. A screening strategy with high throughput fluorescence image analysis has been implemented, in order to identify bacterial clones expressing improved hydroxylation enzymes. Mutants of P450cam with enhanced activity of naphthalene and hydrogen peroxide (peroxide deviation path) have been identified. These mutants are also more active in a related substrate, 3-phenylpropionate. In general, the method uses polymerase chain reaction (PCR) techniques to generate a library of oxygenase mutants, using DNA sequences (e.g. as primaries and / or tests) of a known enzyme or initiation as a template. In this example, the P450cam mutants were derived from the P450cam gene above treated. A. Mutagenesis of the P450cam gene The mutagenic PCR procedure of Cadwell and Joyce (1992) (15) was used with some modifications. For a reaction Ma? - - of 100 μl, the following was included: 10 μl of 1 0 X regulator (Boehringer Mannheim, Germany, PCR reaction regulator) 1 00 mM Tris / HCl, 500 mM KCl, pH 8.3 at 20 ° C ) 28 μl MgCl2 (25 mM supply solution) 0.2 μl dATP (100 mM supply) 0.2 μl dCTP (100 mM supply) 1 μl dTTP (100 mM supply) 0.7 mM MnCl2 10 1. 5 μl of feed primary (9.8 pmol / ul) 1 μl of reverse primary (14.0 pmol / ul) 1 μl (5 units) of Taq polymerase (Boehringer Mannheim) 15 0.01% gelatin (from 1 0x supply) 20 fmol of tempered pCWori (+) _ P450cam 42.1 μl of ddH2O error prone PCR was run on a programmable thermocycle (PTC200, MJ Research) for 30 cycles (denaturation 94 ° C, 30s; softening 45 ° C, 30s; elongation 72 ° C, 2 min). The sequences of the inverse primary (25-mers) and the forward (24-mers) used were: [SEQ. ID. DO NOT. 9] 5'-CATCGATGCTTAGGAGGTCATATG- 3 \ and 25 [SEQ. ID. DO NOT. 10] 5'-TCATGTTTGACAGCTTATCATCGAT- 3 'where the Nde I restriction site is underlined. The size of the total insertion gene to be amplified between the two primaries is 1.4 kb. B. Purification, cloning and expression of DNA The Qiaex team (Qaigen, Germany) was used for the purification of PCR product. The purified PCR product was redissolved in TE buffer (10 mM Tris-HCl, pH 8.0) and subjected to electrophoresis in 1% agarose gels to verify purity. After digestion with Nde I (10 u) and Hind lll (10 u) for 12 hours at 37 ° C, the Nde \ -Hind lll fragment was purified again by gel extraction and inserted into pCWori + shuttle vector. The ligation was carried out at 16 ° C for 9 hours with 200 U of T4 DNA ligase (Boehringer Mannheim). The ligation mixture was then used to transform ORO cells BL21 (DE3) E. coli (Stratagene) which also has pETpelBHRP1 A6Kan introduced as in other examples herein. For the selection of cells containing two different plasmids, a TB / amp plate (100 ug / ml) / kan (30 ug / ml) was used for cell growth and simultaneous protein expression. The E. coli strain containing pCWori (+) _ P450cam and pETpelBHRP1 A6Kan grew at 37 ° C for 6 hours, then induced for the expression of HRP and P450cam by changing the incubation temperature at 30 ° C. After 16 hours, the colonies were stamped on nitrocellulose membranes and transferred onto fresh plates containing hydrogen peroxide and naphthalene for fluorescence image analysis, using the procedure of EXAMPLE 2. C. Results of Selecting the Activity of P450 Mutant in a Complete Cell System Approximately 55,000 mutant P450cam clones in 3-Q-bot plates were selected in naphthalene as a substrate using digital fluorescence imaging. The selected highly fluorogenic mutant colonies identified by digital imaging were transferred to a 96-well plate for confirmation by more detailed measurements, as described in EXAMPLE 2. FIG. 19A shows the results of a digital scan of sections of the plates containing fluorescent mutant P450cam colonies. The fluorescence values of the colony are plotted in descending order. Adjusting the threshold level to the point where the wild type fluorescence is close to or below the detection limit allows one to see (count) only the colonies expressing P450cam activity as compared to or greater than the wild type levels . This demonstrates to one the advantages of using imaging methods to select, in comparison with, for example, microprincipal plate analyzes. In the microprincipal plates the deficiently active or inactive colonies must be counted (measured) along the active ones.

^ ^ ^ ^ ^ ^ ^ ¿A large number of colonies (-20%) shows activity that is comparable to or higher than the activity of wild-type P450cam. The level of wild type is -320 units of fluorescence. The highest mutant activity showed 1830 fluorescence units, an increase of almost six folds in fluorescence. FIG. 19B shows the scan results of -200,000 mutants. The fluorescence values of -32,000 of the clones are plotted in descending order. Three mutants that have a high activity compared to wild-type P450cam are indicated. These three clones with increased fluorescence were selected for growth and confirmation of increased activity towards naphthalene in a complete cell analysis. The fluorescence over time of these three mutants and wild type is shown in FIGS. 19C (wild type), 19D (Mutant M7-4H), 19E (M7-6H) and 19F (M7-8H). Clone M7-6H showed an increase of 1 1 times in activity compared to wild-type P450cam. Two other clones (M7-4H and M7-8H) identified by scanning digital images also showed enhanced activity on this substrate, with the longest increase of 3.2 times for M7-6H. For comparison, 96 randomly selected clones from a large library of mutants (-20,000 colonies) were analyzed in a 96-well fluorescence microplate reader (HTS 7000, Perkin Elmer). As shown in FIG. 20, approximately 80% of the clones in this library are mutants . inactive or less active, compared to wild-type P450cam. A percentage (-20%) of the randomly selected clones showed improved naphthalene hydroxylation activity. This result is similar to that obtained using 5 fluorescence image analysis (FIG 19). However, the image analysis is much faster and less expensive (estimated analysis time: approximately 3-5 seconds for analysis of 20,000 colonies). D. Kinetic Characterization of P450cam Mutants 10 Five positive P450cam variants (designated M7-4H, M7-6H, M7-8H, M7-H9 and M7-2R) were selected from -200,000 hills (Q-bot: 9 plates) which were selected by fluorescence image analysis. Three clones with fluorescence values close to the threshold (wild-type activity) were also selected for comparison (M7-1, M7-2, M7-3). These clones were grown and analyzed in a 96-well plate format for their activity for three different substrates, naphthalene, 3-phenylpropionate and coumarin. One clone, M7-2R, proved to be a false positive and was not analyzed further. The results of the kinetic analysis are summarized in Table 2. TABLE 2 Relative speeds for P450cam variants for 3-phenylpropionate, coumarin and naphthalene, as measured by time fluorescence generation in 96-well cavity plate analysis using whole cells íltá? li (iÉiÉifa? a & ^ ¿Ua ^ iáUJ ^ u For hydroxylation of naphthalene, the M7-6H variant showed a 9.4 fold increase in activity over the wild type. Four of the positive P450s showed highly improved activity for naphthalene and also for 3-phenylpropionate. In another series of experiments, M7-6H showed a 1-fold increase in Comparison to the wild type in naphthalene, and M7-4H and M4-8H showed at least a 5 to 8 fold increase in activity. These three clones also had increased activity in 3-phenylpropionate, with M7-6H showing a 3.2-fold increase. The activity for coumarin, as measured in this analysis, only increased slightly. For the microprincipal plate analysis, the cells (grown in 4 ml of TB / amp medium (100 ug / ml) were centrifuged for 10 min at 4 [deg.] C. After the supernatant solution was removed, the harvested cells were resuspended carefully in 1 ml of regulator solution (dibasic phosphate, 1 00 mM, pH 9.0). Then, 20 μl of aliquots were placed in a Nunc fluorescence microplate. The total reaction mixture of 1 80 μl was made up of 1 00 μl of dibasic sodium phosphate buffer (1 00 mM, pH 9.0), 20 μl of ethanol, 10 μl of substrate supply HWÉJÍÉÉßta (4.5 mM of coumarin in 1.0% ethanol, or 0 mM of 3-phenylpropionate in 1.0% ethanol, 2 mM of naphthalene in pure ethanol), and 10 μL of hydrogen peroxide provision (50 mM of H2O2 supply). The other reaction conditions are those described in EXAMPLE 2. The fluorescence was measured as a function of time, and the relative speeds presented in the Table are the inclinations of that measurement (RFU / min). E. Sequence characterization of P450cam mutants Sequence analysis of three mutant clones of P450cam of the invention, M7-4H, M7-6H and, 7-8H, revealed a mutation at position 331 of the amino acid sequence of FIG. 3B, in which glutamic acid (Glu or E) has been changed to lysine (Lys or K). In mutant M7-4H this was the only mutation. [I KNOW THAT. ID. DO NOT. eleven ]. The mutant M7-6H was found to have a second mutation at position 280 of the amino acid sequence of FIG. 3B, in which arginine (Arg or R) is changed to leucine (Leu or L). [I KNOW THAT. ID. DO NOT. 12]. It was found that the M7-8H mutant has a second mutation at position 242 of the amino acid sequence of FIG. 3B, in which the cysteine (Cys or C) was changed to phenylalanine (Phe or F). [I KNOW THAT. ID. DO NOT. 13]. F. Regiospecific P450 Enzymes The reaction products of the oxygenation reaction catalyzed by the P450 enzyme were reacted in the presence of HRP and hydrogen peroxide. The polymerization catalyzed by HRP In vitro of the different isomers of naphthol (alpha and beta) and different dihydric naphthalenes (1, 5-dihydroxy-, 2,3 dihydroxy- and 2,7-dihydroxy-) generated a variety of fluorescent products, ranging from dark blue (430-460 nm), blue-green (495 nm), yellow (580) fluorescence nm) to orange-red (620 nm). A combination of 1 - or 2-naphthol and 2,7-dihydroxy naphthalene produces a red fluorescent product (620 nm), while mixing 1,5-dihydroxy naphthalene with 2,7-dihydroxy naphthalene or 2-naphthol produces a pink fluorescence and yellow, respectively. The emission spectrum depends on the relative molar proportions of the reactants. Bacteria expressing wild-type P450cam generally only blue fluorescence (460 nm), which corresponds to the conversion of naphthalene to 1 - or 2-naphthol (and coupling by HRP). Bacteria expressing P450cam mutants, in contrast, generate a color palette, shown in Table 3 below, which reflects the altered regiospecificities of hydroxylations catalyzed by P450cam. In this way, the selection according to the invention is sensitive to regiospecificity of hydroxylation as well as total monooxygenase activity.

TABLE 3 Color reactions produced by Mutant Clones RD: red Fulas A-E of Column 1 correspond to the control strain, BL21 (DE3) E. coli. Column 2 (rows A-E) corresponds to the control strain expressing native P450cam. The remaining 10 columns show 50 different variants selected by fluorescence image scan in naphthalene as substrate. The hydroxylation activity of naphthalene was measured in 200 μL of reaction in the 96-well plate. Cells grown in 50 ml flasks were harvested by centrifugation (Beckman CS SR) at 3350 rpm and resuspended in 1 mL of 0.1 M sodium dibasic buffer (pH 9.0). An aliquot of 50 μL of this solution was added to the same buffer mixtures (total of 200 μL) containing 25% ethanol, naphthalene (6 mM) and hydrogen peroxide (10 mM). Fluorescence was measured using a 96-well microfluorimeter (Perkin Elmer HTS 7000) ^ | a ^ tt ^ Hhriti aj | ^^^^^ MÉ ^ Ulü The selection was also selective for one of the hydroxylated isomers of 3-phenylpropionate (3-PPA). Although an oxygenase can potentially hydroxylate different positions in the aromatic structure of 3-PPA, the hydroxylated product in the 4, 3- (4-hydroxyphenyl) propionate position, generates strong blue fluorescence (emission at 465 nm, 350 nm excitation) when it is coupled with HRP. In contrast, HRP does not generate any detectable fluorescence with 3-2 (-hydroxyphenyl) propionate as the substrate in an in vitro analysis. The genes encoding these and other improved P450 variants can be recombined by DNA redistribution methods or can be further mutated in additional cycles of directed evolution or error prone PCR in order to generate additional improved enzymes. P450s with improved thermostability, for example, can be obtained by measuring residual activity after incubation at elevated temperature. EXAMPLE 9 Expression of Horseradish Peroxidase in E. coli and Yeast A. Cloning of HRP The HRP gene was cloned from the pBBGI O plasmid (British Biotechnologies, Ltd, Oxford, UK) by PCR techniques to introduce an Msc I site into the initial codon and an EcoR I site immediately downstream from the final codon. This plasmid contains the synthetic horseradish peroxidase (HRP) gene described in Smith et al (26), whose DNA sequence is based on a published amino acid sequence for the HRP protein (38). pBBGI O AAyAA ^ A ^^^^^^ A ^ ^^^^ A ^^^^^ A ^ j ^ g ^ l, was done by inserting the HRP sequence between the Hindi and EcorRI sites of the polylinker in the PUC1 9 plasmid well known. The PCR product obtained from this plasmid was digested with Msc I and EcoR I and ligated into pET-22b (+) similarly digested (purchased from Novagen) to produce pETpe I BHRP. A map of this expression vector was shown in FIG. twenty-one . In this construct, the HRP gene was placed under the control of the T7 promoter and bound in structure to the signal sequence pe1 B (See [SEQ ID NO.14] and FIG.22), which theoretically directs protein transport in the periplasmic space, that is, to be delivered outside the cellular cytoplasm (25). The ligation product was transformed into BL21 (DE3) E. coli strain for the expression of the protein in cells with and without induction by 1 mM of IPTG. In the cells that were induced with IPTG, no previous peroxidase activity was detected, for BL21 (DE3) or BL21 (DE3) cells harboring pET-22b (+), even though the level of HRP polypeptides was considered up to 20% of total cellular proteins. This was consistent with the previous observations (26, 27, 28). In cells that were not induced with IPTG, it was found that the clones showed weak but measurable activity against azino-di- (ethylbenzthiazoline sulfonate (ABTS) .The T7 promoter in the vector pET-22b (+) is known to be punctured ( 29), in theory it is therefore possible that some of the HRP polypeptide chains produced at this basal level will be able to fold in the native form., the addition of IPTG leads to high-level HRP synthesis, which instead favors the aggregation of chains and prevents their proper folding. Subsequently, random mutagenesis and selection were used to identify mutations that can lead to elevated expression of HRP activity. fí. Selection and generation of random library One of the HRP clones showing detectable peroxidase activity was used in the first generation of error prone PCR mutagenesis. Random libraries were generated by a modification of the error prone PCR procedure previously described (15, 30), in which 0.15 mM of MnCl2 was used instead of 0.5 mM of MnCl2. This procedure incorporates both manganese ions and unbalanced nucleotides, and has been shown to generate both transitions and transversions and thus a wider spectrum of amino acid changes (31). Briefly, the PCR reaction solution contained 20 fmoles of tempering, 30 pmoles of each of two primaries, 7 mM of MgCl2, 50 mM of KCL, 10 mM of Tris-HCl (pH 8.3), 0.01% of gelatin, 0.2 mM of dGTP, 0.2 mM of dATP, 1 mM of dCTP, 1 mM of dTTP, 0.15 mM of MnCl2, and 5 units of Taq polymerase in a volume of 100 μL. The PCR reactions were run in a PTC-200 MJ cycle (MJ Research, MA) for 30 cycles with the following parameters: 94 ° C for 1 min, 50 ° C for 1 min, and 72 ° C for 1 min. The primary ones used were: a > _ ^ K £ B.

'-TTATTGCTCAGCGGTGGCAGCAGC [SEQ. ID. DO NOT. fifteen]; and 5'-AAGCGCTCATGAGCCCGAAGTGGC [SE.Q. ID. DO NOT. 16]. The PCR products were purified with a Promega Wizard PCR equipment, and digested with Nde I and Hind III. The digestion products were subjected to gel purification with a QUIAX II gel extraction kit, and the HRP fragments were ligated back into the pET-22b (+) vector gel-purified and similarly digested. The ligation mixtures are transformed into BL21 (DE3) cells by electroporation with a Gene Pulser II (Bio-Rad). The PCR products were ligated again to the vector pET-22b (+) which was transformed into BL21 (DE3) cells by electroporation. Cell growth and expression was carried out in either 384-cavity or 96-well microplates in LB medium at 30 ° C. The peroxidase activity tests were carried out with H2O2 and ABTS (32). For each generation, typically 12,000-15,000 colonies were selected and selected on 96-well plates. This number represents an exhaustive search of all accessible single mutants, with a 95% probability of any mutant to be sampled at least once (33). The colonies were chosen at least manually, or they used an automatic colony electrode in Caltech, Q-bot (Genetix, UK). Of the 12,000 colonies that were selected in the first generation (no IPTG was added), a mutant designated HRP1A6 showed 10-14 times more peroxidase activity than the clone of origin. This mutant clone also showed markedly reduced activity when as little as 5 μM of IPTG was added. The Sigma reports that 1 mg of HRP highly purified horseradish has a total activity of 1, 000 units, as determined by the ABTS analysis. Other works reported similar results (26). Based on these data, the concentration of active HRP was estimated to be -100 ug / L. HRP1A6 shows a total activity greater than 100 units / L. This compares favorably with the product obtained from re-folding HRP chains added in vitro (26). This level of expression for the mutant HRP is also similar to that of the bovine pancreatic trypsin inhibitor (BTP1) in E. coli (34), a non-glucosidated protein with three disulfide bonds. Again, more than 95% of the HRP activity was found in the LB culture medium as judged by the ABTS activity. The mutant HRP remained stable for up to one week at 4 ° C. IPTG was omitted in the HRP expression experiments, unless otherwise specified. Peroxidase activity tests on HRP were carried out with a classical analysis of peroxidase, ABTS and hydrogen peroxide (26). 15 μl of cell suspension was mixed with 140 μl of ABTS / H2O2 (2.9 mM ABTS, 0.5 mM H2O2, pH 4.5) in microplates and the activity was determined with a Spectra Max plate reader (Molecule Devices Sunnyval, CA) 25 ° C. One unit of HRP was defined as the amoof enzyme that oxidizes 1 μmol of ABTS per minute under chemiluminescent conditions. The sequencing of the mutant gene found a mutation at position 255, in which the AAC codon for amino acid asparagine (Asn or N) was changed to the GAC codon for the amino acid aspartic acid (Asp or D). This residue is a putative glycoxylation site and is located on the surface of the protein. The sequence of this mutant (HRP1 A6) is shown in Figure 23 [SED ID NO. 16]. A map of a pETpelBHRP1 A6 plasmid containing this mutant is shown in Figure 24. C. Functional Expression of HRP in Yeast The native HRP protein contains four disulfide linkages and E. coli has only a limited capacity to support disulfide formation. In theory, these disulfides conserved in cavities in HRP (and other plant peroxidases) are likely to be important for the structural integrity of the protein and may not be responsible for mutations anywhere.

Yeast has a much greater ability to support the formation of disulfide bonds. In this manner, the yeast can be used as a suitable expression host, rather than E. coli particularly if it is desired to remedy the apparent limitation in the fold of HRP imposed by any constraint on disulfide formation in E. coli. For example, S. can be used.

Cerevisiae as a host for the expression of mutant HRP and protein genes.

The HRP mutant (HRP1 A6) was cloned into the secretion vector pYEX-S1 obtained from Clontech (Palo Alto, CA) (1 9), yielding pYEXS1 -HRP (Figure 25). This vector uses the constitutive promoter of phosphoglycerate kinase and a secretion signal peptide of Kluveromyces lactis. The plasmid was first propagated in E. coli, and then transformed into S. cerevisiae strain BJ5464, obtained from the Yeast Genetic Deposit Center (YGSC), University of California, Berkeley, by using the LiAc method as described (twenty). BJ5464 is a deficient protease and has been found generally suitable for secretion. A first generation of error-prone PCR of HRP in the yeast was carried out. Among the first 7,400 mutants selected four variants showed an activity 400 greater than the HRP1A6 in the yeast.

EXAMPLE 10 Selection of Other Catalysts and Optimization of Reaction Conditions Empirical approaches are the only successful approaches tested in the development of novel catalysts. However, empirical approaches are often slow, costly and laborious. Parallel research of a large number of candidate catalysts can significantly reduce the time, cost and labor associated with catalyst discovery. In addition to the enzymatic catalysts, the methods of this invention can be applied to the selection of chemical libraries for oxidation catalysts. In addition to the discovery of the catalyst, it is also important to optimize the reaction conditions for a given catalyst. This requires simultaneous optimization of several parameters, each of which can have a significant effect on catalyst performance. Important parameters include the choice of solvent, the profile of the reagent, the presence of other compounds or contaminants in the reaction mixture, temperature, pressure, etc. Given the large number of potential variables, optimization is also preferably done in parallel tests, in which dozens or even hundreds of conditions are examined. Once an oxidation catalyst is at hand, the invention can be used to easily evaluate or optimize the conditions for that catalyst. The invention can be used with individual catalysts (eg, installed in individual cavities of a microtiter plate) or can be used with various grouping strategies in which multiple candidate catalysts are tested simultaneously. If a particular set of catalysts shows reactivity in a given reaction, members of that set can be tested individually to discover the catalyst of interest. Combinatorial Approaches for the Design and Discovery of the Catalyst. The invention can be used to select catalyst libraries without enzyme for their ability to oxygenate substrates (eg, hydroxylate), such as aromatic substrates. The catalysts identified in this way can in turn be used as "guides" for the discovery of catalysts that hydroxylate other substrates, catalyze other oxygen insertion reactions or which have more activity or stability or which can function under different conditions. For example, combinatorial chemistry techniques can be used to generate additional libraries of compounds for examination, once a guide compound is identified (43, 48, 49).

To use the invention in this application, the selection reaction would generally be carried out with the coupling enzyme after the oxygenation reaction has been completed. If necessary or appropriate, the reaction conditions can be adjusted after the oxygenation reaction in order to promote the coupling reaction. That is, conditions must be provided that are compatible with the maintenance of the activity of the coupling enzyme. Alternatively, the oxygenates could be extracted in a solvent (e.g., dichloromethane or a solvent in which the coupling enzyme, such as HRP, is known to work). HRP, a preferred coupling enzyme, is known to function as a coupling enzyme in an aqueous regulator and also in various organic solvents (including hexane, acetonitrile, t-butanol and others) and operates over a temperature range of about 4. up to 65 ° C, with better performance at approximately 20-50 ° C. The conditions of the coupling reaction are also preferably chosen to reduce the dilution of the oxygenated product. This mode also allows the measurement of a "end point" of the oxygenation reaction. For example, the oxygenation reaction would be allowed to proceed for a given amount of time. At this point, the conditions are changed to allow the coupling reaction (and a coupling enzyme and an oxygen donor would be added). The generation of colored or fluorescent products (or the absorption of UV light, chemiluminescence, etc.) indicates the total concentration of the oxygenated product produced during that time. If the oxygenation catalyst works under conditions that are also compatible with the coupling enzyme, both reactions are performed simultaneously or contemporaneously (oxygenation and coupling). Optimization of Reaction Conditions The invention can also be used to optimize the reaction conditions in any given catalyst. The hydroxylation of aromatic compounds can be difficult. The results may be poor due to the introduction of a hydroxyl group that activates the ring for a subsequent reaction and oxidation. In addition, the reaction conditions are often severe and potentially explosive (45). In this way, the evaluation and optimization of the reaction conditions for a given catalyst can be beneficial. There are several catalysts without an enzyme that are known to catalyze aromatic hydroxylations, similar to the enzymes of monooxygenase and dioxygenase. DeHaan et al. (46) describe the hydroxylation of various aromatics in high production, by the use of a bis (trimethylsilyl) peroxide / triflic acid system. The product was extracted in an organic solvent (dichloromethane) for analysis. The present invention can be used to determine the progress of the reaction by the addition of HRP (or other suitable coupling enzyme) and peroxide. Alternatively, a solvent that both extracts the product and supports HRP activity can be used. As another example, a large class of catalysts that can carry out hydroxylations are substituted porphyrins, which have been characterized as "imitations" without enzyme P450 enzymes (47). This invention can be used to select combinatorial libraries of porphyrin-based catalysts for the hydroxylation of aromatics under a variety of conditions. It can also be used to select libraries of di-iron compounds that mimic di-iron oxygenates (51). Having thus described the exemplary embodiments of the invention, it should be observed by those skilled in the art that the exhibits included are only exemplary. and that various other alternatives, adaptations and modifications may be made within the scope of the invention. For example, it will be understood by the practitioners that the steps of any method of the invention can generally be carried out in any order, including simultaneously or contemporaneously, unless a particular order is expressly required or is necessarily inherent or implicit in order to practice the invention. In accordance with the above, the invention is not limited to any modality or specific illustration herein. The invention is defined according to the appended claims and is limited only according to the claims. BILIOGRAPHY 1. Faber, K. Biotransformations in Organic Chemistry, Springer-Verlad, Berlin, p. 214, 21 7 (1 997). 2. Cook, D. L. and Atkins, W. M. Biochemistry, 36, 10801 (1 997). 3. Short, J. Nature Biotechnol. 15, 1322 (1997). 4. Sheldon, R. A. Catalysis: the key to waste minimization. J. Chem. Tech. Biotechnol. 68, 381 (1997). 5. 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Claims (8)

1. CLAIMS 1. A method for detecting an oxidation enzyme comprising the steps of: providing an organic substrate and an oxygen donor; supplying a test enzyme comprising a protein-containing material selected for evaluation with respect to its ability to influence the formation of an oxygen-containing product from the organic substrate and the oxygen donor; introduce the test enzyme into the organic substrate and the oxygen donor; provide an enzyme 10 coupling selected to promote the formation of a detectable composition of the oxygen-containing product; and examining the detectable composition, wherein the presence of the detectable composition indicates that the test enzyme is an oxidation enzyme.
2. 2. A method according to claim 1, characterized in that the test enzyme is introduced into the organic substrate and the oxygen donor under reaction conditions which are selected in order to evaluate the ability of the test enzyme to mediate the addition of oxygen to the substrate.
3. 3. A method according to claim 2, characterized in that the reaction conditions vary.
4. 4. A method according to claim 2, characterized in that the coupling enzyme is provided under selected coupling conditions to promote the formation of a Oxygenated polymeric composition comprising two or more of the , -aa »^ - ^ rÉ4aa». ^ same products that contain oxygen linked together.
5. 5. A method according to claim 1, characterized in that the organic substrate is an aromatic compound.
6. 6. A method according to claim 1, characterized in that the organic substrate is selected from the group consisting of naphthalene, 3-phenylpropionate, benzene, toluene, benzoic acid, anthracene, benzfetamine and coumarin. 7. A method according to claim 1, characterized in that the oxygen donor is molecular oxygen. 8. A method according to claim 1, characterized in that the oxygen donor is a peroxide. 9. A method according to claim 8, characterized in that the oxygen donor is selected from the group consisting of hydrogen peroxide and t-butyl peroxide. A method according to claim 1, characterized in that the coupling enzyme is used to drive together the molecules of the oxygen-containing product in order to form a poiimeric composition that is detectable by the use of at least one ultraviolet light, a change in color, fluorescence or luminescence. 1. A method according to claim 1, characterized in that the coupling enzyme is a peroxidase enzyme. 12. A method according to claim 6, characterized in that the coupling enzyme is a peroxidase enzyme. 13. A method according to claim 8, characterized in that the coupling enzyme is a peroxidase enzyme. A method according to claim 1, characterized in that the coupling enzyme is selected from the group consisting of horseradish peroxidase, cytochrome peroxidase c, tulip peroxidase, lignin peroxidase, carrot peroxidase, peanut peroxidase, Peroxidase of soybean and peroxidase Nobozyme® 502. 1 5. A method according to claim 1, characterized in that the coupling enzyme is a laccase enzyme. 16. A method according to claim 1, characterized in that the organic substrate is an aromatic compound, the oxygen donor is a peroxide and the coupling enzyme is a peroxidase enzyme.
7. 7. A method according to claim 16, characterized in that the organic substrate is selected from the group consisting of naphthalene, 3-phenylpropionate, benzene, toluene, benzoic acid, altracene, benzfetamine and coumarin.
8. 8. A method according to claim 16, characterized in that the coupling enzyme is selected from the group consisting of horseradish peroxidase., cytochrome peroxidase c, tulip peroxidase, lignin peroxidase, carrot peroxidase, peanut peroxidase, soybean peroxidase and Nobozyme® 502 peroxidase. A method according to claim 1, characterized in that it also comprises the step of providing a chemiluminescent agent. 20. A method for detecting an oxygenase enzyme, characterized in that it comprises the steps of: providing an aromatic organic substrate and a peroxide oxygen donor; supplying a test enzyme comprising a protein-containing material selected for evaluation with respect to its ability to influence the formation of an oxygen-containing product from the organic substrate and the oxygen donor; introduce the test enzyme into the organic substrate and the oxygen donor; providing a selected peroxide coupling enzyme to promote the formation of a detectable composition of the oxygen-containing product; and examining the detectable composition by evaluating at least one ultraviolet light, a color change, fluorescence and luminescence, wherein the presence of the detectable composition indicates that the test enzyme is an oxygenase enzyme. twenty-one . A method according to claim 20, characterized in that the organic substrate is selected from the group consisting of naphthalene, 3-phenylpropionate, benzene, toluene, benzoic acid, anthracene, benzfetamine, coumarin. 22. A method according to claim 20, characterized in that the oxygen donor is selected from the group consisting of hydrogen peroxide and t-butyl peroxide. 23. A method according to claim 20, characterized in that the coupling enzyme is selected from the group consisting of c 24. A method according to claim 20, characterized in that it further comprises the step of providing a chemiluminescent agent. 25. A method according to claim 23, characterized in that it further comprises the step of providing a chemiluminescent agent. 26. A method according to claim 20, characterized in that the chemiluminescent agent is luminol. 27. A method according to claim 23, characterized in that the chemiluminescent agent is luminol. 28. A method according to claim 20, characterized in that the oxygenase enzyme is a dioxygenase enzyme or a monooxygenase enzyme. 29. A method according to claim 20, characterized in that the reaction catalyzed by the oxygenase enzyme is a hydroxylation reaction, an epoxidation reaction or a sulfoxidation reaction. 30. A method according to claim 1, characterized in that the oxygenase enzyme is selected from the group consisting of chloroperoxidase, cytochrome P450, methane monooxygenase, toluene monooxygenase, toluene dioxygenase, biphenyl dioxygenase and naphthalene dioxygenase. 31 A method according to claim 20, characterized in that the oxygenase enzyme is selected from the group consisting of chloroperoxidase, cytochrome P450, methane monooxygenase, toluene monooxygenase, toluene dioxygenase, biphenyl dioxygenase and naphthalene dioxygenase. 32. A method according to claim 20, characterized in that the organic substrate is naphthalene. 33. A method according to claim 20, characterized in that the oxygen donor is hydrogen peroxide. 34. A method according to claim 20, characterized in that the coupling enzyme is horseradish peroxidase. 35. A method according to claim 1, characterized in that the introduction step comprises the introduction of the organic substrate and the oxygen donor into the host cells expressing at least one test enzyme or a coupling enzyme. 36. A method according to claim 20, characterized in that the introduction step comprises the introduction of the organic substrate and the oxygen donor into the host cells expressing at least one test enzyme or a coupling enzyme. 37. A method according to claim 1, characterized in that the test enzyme is a variant of an enzyme that is selected from the group consisting of chloroperoxidase, cytochrome P450, methane monooxygenase, toluene monooxygenase, toluene dioxygenase, dioxygenase biphenyl and naphthalene dioxygenase. 38. A method according to claim 20, characterized in that the test enzyme is a variant of an enzyme that is selected from the group consisting of chloroperoxidase, cytochrome P450, methane monooxygenase, toluene monooxygenase, toluene dioxygenase, biphenyl dioxygenase and naphthalene dioxygenase. 39. A method according to claim 35, characterized in that the host cells are bacterial cells. 40. A method according to claim 36, characterized in that the host cells are bacterial cells. 41 A method according to claim 39, characterized in that the host cells are EE cells. coli 42. A method according to claim 40, characterized in that the host cells are E. coli cells. 43. A method according to claim 45, characterized in that the host cells are yeast cells. 44. A method according to claim 36, characterized in that the host cells are yeast cells. 45. A method according to claim 43, characterized in that the host cells are S. cerevisiae cells. 46. A method according to claim 44, characterized in that the host cells are S cells. Cerevisiae 47. A method according to claim 20, characterized in that: the organic substrate is selected from the group consisting of naphthalene, 3-phenylpropionate, benzene, toluene, benzoic acid, anthracene, benzfetamine and coumarin; the oxygen donor is selected from the group consisting of hydrogen peroxide and t-butyl peroxide; and the coupling enzyme is selected from the group consisting of horseradish peroxidase, cytochrome peroxidase c, tulip peroxidase, lignin peroxidase, carrot peroxidase, peanut peroxidase, soybean peroxidase and peroxidase Nobozyme® 502 48. A method according to claim 47, characterized in that the test enzyme is a variant of an enzyme that is selected from the group consisting of chloroperoxidase, cytochrome P450, methane monooxygenase, toluene monooxygenase, toluene dioxygenase, dioxygenase. of biphenyl and naphthalene dioxygenase. 49. A method according to claim 48, characterized in that it further comprises the step of providing a chemiluminescent agent. 50. A method according to claim 1, characterized in that the organic substrate, the oxygen donor and the test enzyme are introduced in the absence of at least one coenzyme or auxiliary protein. 51. A method according to claim 20, characterized in that the organic substrate, the oxygen donor and the test enzyme are introduced in the absence of at least one of their coenzymes or auxiliary proteins. 52. A method according to claim 47, characterized in that the organic substrate, the oxygen donor and the test enzyme are introduced in the absence of at least one of their coenzymes or auxiliary proteins. 53. A method according to claim 50, characterized in that at least one coenzyme is selected from the group consisting of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH). 54. A method according to claim 50, characterized in that at least one auxiliary protein is selected from the group consisting of putidaredoxin and putidaredoxin reductase. 55. A method according to claim 51, characterized in that at least one coenzyme is selected from the group consisting of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH). 56. A method according to claim 51, characterized in that at least one auxiliary protein is selected from the group consisting of putidaredoxin and putidaredoxin reductase. 57. A method according to claim 1, characterized in that the organic substrate, the oxygen donor and the test enzyme are introduced in the presence of one or more cofactors. 58. A method for detecting an oxygenase enzyme, characterized in that it comprises the steps of: providing an aromatic organic substrate and a peroxide oxygen donor; 5 supplying a test enzyme comprising a protein-containing material selected for evaluation with respect to its ability to influence the formation of an oxygen-containing product from the organic substrate and the oxygen donor; introduce the test enzyme into the organic substrate and the donor Oxygen in the absence of at least one of its coenzymes or auxiliary proteins; providing a selected peroxide coupling enzyme to promote the formation of a detectable composition of the oxygen-containing product; and examine the detectable composition by evaluating the 15 minus an ultraviolet light, a color change, fluorescence and luminescence, wherein the presence of the detectable composition indicates that the test enzyme is an oxygenase enzyme. 59. A method according to claim 63, characterized in that at least one coenzyme is selected from 20 of the group consisting of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH). 60. A method according to claim 58, characterized in that at least one auxiliary protein is selected from the group consisting of putidaredoxin and reductase of 25 putidaredoxin. «Ii ^^ g ^^ 61. A method according to claim 58, characterized in that the organic substrate, the oxygen donor and the test enzyme are introduced in the presence of one or more cofactors. 62. A method according to claim 61, characterized in that at least one cofactor is selected from the group consisting of thiamine (vitamin B1), ferrous chloride (FeCl2) and delta-aminolevulinic acid (ALA). 63. A method according to claim 58, characterized in that the test enzyme is a variant of an enzyme that is selected from the group consisting of chloroperoxidase, cytochrome P450, methane monooxygenase, toluene monooxygenase, toluene dioxygenase, dioxygenase biphenyl and naphthalene dioxygenase. 64. A method according to claim 38, characterized in that at least one coenzyme is selected from the group consisting of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), or at least one auxiliary protein is select from the group consisting of putidaredoxin and putidaredoxin reductase. 65. A method for evaluating a test enzyme for its ability to mediate the production of an oxygenated product when it is introduced into an organic substrate in the presence of an oxygen donor, characterized in that it comprises the steps of: providing a host cell that is capable of expressing a test enzyme; provide a vector encoding the test enzyme; inserting the vector into the host cell to provide a transformed host cell expressing the test enzyme; provide an oxygen donor; introducing an organic substrate 5 to provide a reaction with the oxygen donor in the presence of the test enzyme in order to produce an oxygenated product; reacting the oxygenated product in the presence of a coupling enzyme to form a polymeric oxygenated product; and detect the oxygenated polymeric product when examining the 10 minus fluorescence, luminescence, ultraviolet light or color change. 66. A method according to claim 65, characterized in that the host cell also expresses the coupling enzyme. 67. A method according to claim 65, characterized in that it comprises the additional step of introducing a second vector expressing the coupling enzyme in the host cell. 68. A method according to claim 65, Characterized in that it comprises the additional step of reacting the oxygenated polymer product with a chemiluminescent agent to produce a chemiluminescent compound and the detection step comprises evaluating the luminescence of the chemiluminescent compound. 69. A method according to claim 65, iiijj iiMiraiii- characterized in that the test enzyme is a mutant oxygenase enzyme. 70. A method according to claim 69, characterized in that the mutant oxygenase enzyme is mutant of an enzyme that is selected from the group consisting of chloroperoxidase, cytochrome P450, methane monooxygenase, toluene monooxygenase, toluene dioxygenase, dioxygenase biphenyl and naphthalene dioxygenase enzymes. 71 A method according to claim 65, characterized in that the organic substrate is selected from the group consisting of naphthalene, 3-phenylpropionate, benzene, toluene, benzoic acid, anthracene, benzfetamine and coumarin. 72. A method according to claim 65, characterized in that the oxygen donor is a peroxide. 73. A method according to claim 65, characterized in that the coupling enzyme is a peroxidase. 74. A method according to claim 69, characterized in that the mutant oxygenase enzyme is a mutant of a P450 enzyme. 75. A method according to claim 74, characterized in that the oxygen donor is a peroxide. 76. A method according to claim 74, characterized in that the coupling enzyme is a peroxidase. 77. A method according to claim 74, characterized in that the organic substrate is selected from the • a- - "*" "- group consisting of naphthalene, 3-phenylpropionate, benzene, toluene, benzoic acid, anthracene, benzfetamine and coumarin, the oxygen donor is a peroxide and the coupling enzyme is a peroxidase. A method according to claim 75, characterized in that the coupling enzyme is horseradish peroxidase 79. A method according to claim 74, characterized in that the enzyme P450 is a P450cam enzyme 80. A method according to claim 1, characterized in that because the test enzyme is a mutant enzyme obtained by at least one of random mutagenesis, site-specific mutagenesis and intermixing of DNA.A method according to claim 20, characterized in that the test enzyme is a mutant enzyme obtained by less one of random mutagenesis, site-specific mutagenesis and intermixing of DNA 82. A method according to claim 58, characterized in that the enzyme ba is a mutant enzyme 20 obtained by at least one of random mutagenesis, site-specific mutagenesis and intermixing of DNA. 83. A method according to claim 65, characterized in that the test enzyme is a mutant enzyme obtained by at least one of random mutagenesis, Site-specific mutagenesis and intermixing of DNA. Aiεißlttii 84. A method according to claim 65, characterized in that the vector comprises a variant of the nucleotide sequence P450cam of [SEQ. ID. DO NOT. 1] of Figure 3A. 85. A method according to claim 65, characterized in that the vector encodes a mutation of the amino acid sequence P450cam of [SEQ. ID. DO NOT. 2] of Figure 3B. 86. A method according to claim 66, characterized in that the second vector comprises the horseradish peroxidase nucleotide sequence of [SEQ. ID. DO NOT. 16] 10 of Figure 23. 87. A method according to claim 66, characterized in that the second vector encodes the amino acid sequence of horseradish peroxidase of [SEQ. ID. DO NOT. 17] of Figure 23. 15 88. A method for selecting oxygenase enzymes, characterized in that it comprises the steps of: providing host cells; provide a plurality of vectors each of which encodes a test enzyme; insert each vector into one or more host cells to provide the transformed cells 20 corresponding ones that express each one, a corresponding test enzyme; introducing each test enzyme into an oxygen donor and an organic substrate under conditions that provide a reaction between the substrate and the donor in order to produce an oxygenated product; react the oxygenated product in the presence 25 of a coupling enzyme to form a polymeric product oxygenated iliiilii; and detecting the oxygenated polymer product by examining the presence or degree of at least one indicator selected from the group consisting of fluorescence, luminescence, ultraviolet light and a color change, wherein the detection of the oxygenated polymeric product indicates that a The corresponding test enzyme is an oxygenase. 89. A method according to claim 88, characterized in that the plurality of oxygenase enzymes is compared to each other when evaluating the corresponding degrees of the detected indicator. 90. A method according to claim 88, characterized in that it further comprises the step of reacting the oxygenated polymer product with a chemiluminescent agent in order to form a chemiluminescent composition and wherein the detection step comprises the examination of the luminescence of the chemiluminescent composition. 91 A method according to claim 88, characterized in that each vector encodes a test enzyme that is a variant of an oxygenase enzyme. 92. A method according to claim 88, characterized in that each test enzyme is a variant or an oxygenase enzyme selected from the group consisting of chloroperoxidase, cytochrome, P450, methane monooxygenase, toluene monooxygenase, toluene dioxygenase, dioxeganase of biphenyl and naphthalene dioxygenase. 93. A method according to claim 88, characterized in that each vector encodes a test enzyme which is a variant enzyme obtained by at least one of random mutagenesis, specific mutagenesis, directed evolution, DNA intermixing and error-prone polymerase chain reaction. 94. A method according to claim 88, characterized in that the oxygen donor is a peroxide. 95. A method according to claim 92, characterized in that the oxygen donor is a peroxide, the coupling enzyme is a peroxidase and the organic substrate is an aromatic compound selected from the group consisting of naphthalene, 3-phenylpropionate, benzene, toluene, benzoic acid, anthracene, benzfetamine and coumarin. 96. A method according to claim 58, characterized in that the detection step includes the ultraviolet light image analysis, color change, fluorescence or luminescence, detected. 97. A method according to claim 65, characterized in that the detection step includes the ultraviolet light image analysis, color change, fluorescence or luminescence, detected. 98. A method according to claim 88, characterized in that the detection step includes the ultraviolet light image analysis, color change, fl uorescence or luminescence, detected. 99. A method according to claim 88, characterized in that one or more steps are automated. 100. A method according to claim 88, characterized in that the steps of the method are carried out independently for each test enzyme and corresponding transformed cells. 101 A method according to claim 100, characterized in that one or more steps of the method are carried out contemporaneously for each test enzyme and corresponding transformed cells. 1 02. A method according to claim 88, characterized in that the steps of the method are repeated until at least one oxygenase enzyme is identified. 103. A method according to claim 102, characterized in that each test enzyme is a variant of at least one identified oxygenase enzyme. 104. A method according to claim 88, characterized in that each transformed cell expresses the coupling enzyme. 1 05. A method according to claim 88, characterized in that the step of introduction comprises the proportion of a supply of organic substrate and oxygen donor to the transformed cells. 1 06. A method according to claim 1, characterized in that the step of introduction comprises the proportion of a supply of organic substrate and donor of oxygen to the transformed cells. 107. A variant of oxygenase enzyme obtained by the method according to claim 1. 1 08. A variant of oxygenase enzyme obtained by the method according to claim 20. 109. A variant of oxygenase enzyme obtained by the method according to claim 47. 10. A variant of oxygenase enzyme obtained by the method according to Claim 58. 1 1 1. A variant of oxygenase enzyme obtained by the method according to claim 65. 12. A variant of oxygenase enzyme obtained by the method according to claim 88. 13. A variant of oxygenase enzyme obtained by the method according to the claim. 103. 1 14. An oxygenase enzyme variant obtained by the method according to claim 104. 1 1 5. A P450 oxygenase enzyme variant having at least one mutation at a position corresponding to position 331 of the amino acid sequence of a wild-type P450 enzyme. 16. An enzyme variant of P450 oxygenase according to claim 15, characterized in that the glutamic acid is changed to lysine. 1 1 7. A P450 oxygenase enzyme variant having at least one mutation at a position corresponding to position 280 of the amino acid sequence of a wild-type P450 enzyme. 1 1 8 A P450 oxygenase enzyme variant according to claim 1 17, characterized because arginine is changed to lysine. 1 1 9. A P450 oxygenase enzyme variant having at least one mutation at a position corresponding to position 242 of the amino acid sequence of a wild-type P450 enzyme. 120 A P450 oxygenase enzyme variant according to claim 1 1 9, characterized in that the cysteine is changed to phenylalanine. 1 21. A P450 oxygenase enzyme variant having at least one mutation at a position corresponding to any of positions 242, 280 and 331 of the amino acid sequence of a wild-type P450 enzyme. 122. A P40 oxygenase enzyme variant characterized in that it has at least one mutation in which: glutamic acid is changed to lysine at a position corresponding to position 331 of the amino acid sequence of a wild-type P450 enzyme; the arginine is changed to leucine in a position corresponding to position 331 of the amino acid sequence of a wild-type P450 enzyme; and the cysteine is changed to phenylalanine at a position corresponding to the position 331 e of the amino acid sequence of a wild-type P450 enzyme; 123. A conservative variant of the sequence of an enzyme according to claim 122. 124. A conservative variant of the binding of an enzyme according to claim 122. 125. A variant of oxygenase enzyme encoded by a first polynucleotide that hybridizes to a second polypeptide encoded by an enzyme according to claim 1 under high conditions of accuracy. 26. A method for developing an oxidation enzyme, characterized in that it comprises the steps of: supplying an organic substrate and an oxygen donor; providing at least one host cell that expresses a DNA sequence encoding a provided oxidation enzyme, capable of promoting the formation of an oxygen-containing product from the organic substrate and the oxygen donor; generating a library of test enzymes comprising a plurality of oxidation enzyme mutants, each of which is a variant of at least one DNA sequence provided; expressing a plurality of variants in host cells in order to produce a plurality of test enzymes from the library; introduce each test enzyme into the organic substrate and the oxygen donor; providing a selected coupling enzyme to promote the formation of a detectable composition of the oxygen-containing product; examining the detectable composition, wherein the presence of the detectable composition identifies the test enzyme as an oxidation enzyme; selecting at least one oxidation enzyme identified by comparison with at least one oxidation enzyme provided according to at least one property. 127. A method according to claim 126, characterized in that the method is repeated, by using at least one oxidation enzyme identified as a proportionate oxidation enzyme. 128. A method according to claim 126, characterized in that the generation step includes the use of error-prone PCR to provide mutants. 129. A method according to claim 126, characterized in that the oxygen donor is hydrogen peroxide, and the selection step includes a comparison of the activity of the enzyme. 130. A P450 enzyme developed according to claim 1 29 and characterized in that it has an improved enzyme activity or stability as compared to a provided P450 enzyme. 1 31. A P450 enzyme for use with hydrogen peroxide and obtained by a method according to claim 126. 32. A method for evaluating the reaction conditions for an oxidation catalyst, characterized in that it comprises the steps of: providing an oxidation catalyst; supply an organic substrate and an oxygen donor; introducing the catalyst into the organic substrate and the oxygen donor under each of the plurality of reaction conditions in order to form a plurality of test combinations; providing each test combination with a selected coupling enzyme to promote the formation of a detectable composition of the oxygen-containing product; examining the test combinations for the production of the detectable composition, wherein the reaction conditions are evaluated according to the differences in at least one of the relative rates and production amounts of the detectable composition. 1 33. A variant of developed P450 enzyme, characterized in that it has a catalytic activity at least twice as active as the corresponding wild type enzyme P450, by facilitating the oxidation of a substral or in the presence of hydrogen peroxide. 134. A variant of developed P450 enzyme, characterized in that it has a catalytic activity at least ten times as active as the corresponding wild-type enzyme P450, by facilitating the oxidation of a substrate in the presence of hydrogen peroxide. 1 35. A variant of developed P450 enzyme, characterized in that it has a catalytic activity at least twice as stable as a corresponding wild-type P450 enzyme. 136. A variant of developed P450 enzyme, characterized in that it has a catalytic activity at least ten times as stable as a corresponding wild-type P450 enzyme. 137. A method for evaluating the reaction conditions for an oxidation catalyst, characterized in that it comprises the steps of: providing an organic substrate and an oxygen donor; supplying a selected test catalyst for the evaluation of its ability to influence the formation of an oxygen-containing product from the organic substrate and the oxygen donor; introduce the test catalyst into the organic substrate and the oxygen donor; providing a selected coupling enzyme to promote the formation of a detectable composition of the oxygen-containing product, and examining the detectable composition, wherein the presence of the detectable composition indicates that the test catalyst is an oxidation catalyst. 138. A method according to claim 137, characterized in that the organic substrate is selected from the group consisting of naphthalene, 3-phenylpropionate, benzene, toluene, benzoic acid, anthracene, benzfetamine and coumarin. 139. A method according to claim 1, characterized in that the oxygen donor is a peroxide. 140. A method according to claim 137, characterized in that the coupling enzyme is used to drive together the molecules of the oxygen-containing product in order to form a polymeric composition that is detectable by using at least one ultraviolet light, a change of color, fluorescence and luminescence. 141. A method according to claim 137, characterized in that the coupling enzyme is at least one peroxidase enzyme or a laccase enzyme. 142. A method according to claim 137, characterized in that the coupling enzyme is selected from the group consisting of horseradish peroxidase, cytochrome peroxidase c, tulip peroxidase, lignin peroxidase, carrot peroxidase, peroxidase peanut, Novozyme® soybean peroxidase peroxidase 502. 143. A method according to claim 137, characterized in that the organic substrate is an aromatic compound, the oxygen donor is a peroxide and the coupling enzyme is a peroxidase enzyme. 144. A method according to claim 137, characterized in that it further comprises the step of providing a chemiluminescent agent. 145. A method according to claim 137, characterized in that a plurality of indicated oxidation catalysts is compared to each other by evaluation of the corresponding examination for the detectable composition.