★★★★★

Background of the invention

The present application refers to the field of bioremediation in particular of polluted water since it discloses the use of specific strain of genus Rhodococcus and Marinomonas for bioremediation of water from contaminants, in particular fuels, toxic metals and cocaine.

Prior art

Italian patent application n. 102019000014121 filed on 06/08/2019 of the same inventors discloses an in vitro process for the preparation of metabolites by Marinomonas efl or Rhodococcus efl, such as Ag, a process for the preparation of antiseptic textiles impregnated of Ag nanoparticles by coulturing Marinomonas efl or Rhodococcus efl, a process for the preparation of fluorescent dyes from Marinomonas efl or Rhodococcus efl.

The Rhodococcus strain from a bacterial consortium associated to the Antarctic ciliate Euplotes focardii named Rhodococcus efl has been isolated by the same inventor (Pucciarelli S, Devaraj RR, Mancini A, Ballarini P, Castelli M, Schrallhammer M, Petroni G, Miceli C. Microbial Consortium Associated with the Antarctic Marine Ciliate Euplotes focardii: An Investigation from Genomic Sequences. Microb Ecol. 2015 Aug;70(2):484-97.)

A 2-1 kbp DNA fragment from Rhodococcus strain ATCC 21145 that gave rise to the production of blue and pink pigments in Escherichia coli when cloned downstream of a strong promoter has been reported (Hart S, Kirby R, Woods DR. Structure of a Rhodococcus gene encoding pigment production in Escherichia coli. J Gen Microbiol. 1990 Jul;136(7):1357-63), that most probably metabolize a substrate of the E. coli, as also reported in (Hill R, Hart S, Tiling N, Kirby R, Woods DR. Cloning and expression of Rhodococcus genes encoding pigment production in Escherichia coli. J Gen Microbiol. 1989 Jun;135(6):1507-13) since no pigment was detected in Streptomyces griseus transformants containing the same encoding genes. The pigment has been characterized as an indigo-related pigment (Hart S, Koch KR, Woods DR. Identification of indigo-related pigments produced by Escherichia coli containing a cloned Rhodococcus gene. J Gen Microbiol. 1992 Jan;138(1):211-6).

Phenazines are heterocyclic compounds, with chemistry formula C6H4N2C6H4, that are produced naturally and substituted at different points around their rings by different bacterial species. They are pigments with acid-base properties, due to their redox ability, they show a characteristic Uv-Vis spectrum with an intense absorption band from 250nm to 360 nm, and a lower broad absorption band that can goes from 400nm to 600nm. Tipical Extinction Coefficient e of phenazine compounds of the main peak is around 3-104 cm-1 mol-1 L, this means that a low concentration of pigment shows an high intense colour [Olajire and Essien (2014) Aerobic degradation of petroleum components by microbial consortia Journal of Petroleum & Environmental Biotechnology, 5:5.) Small modifications of the core phenazine structure give rise to a wide range of colors, ranging from the deep red of 5-methyl-7-amino-l-carboxyphenazinium betaine, to the lemon yellow of phenazine-l-carboxylic acid (PCA), to the bright blue of l-hydroxy-5-methylphenazine (pyocyanin, PYO). Its activity is regulated by a gene, PhzH, that catalyzes the transamidation of the molecule. This secondary metabolite production prevents the excessive accumulation of primary metabolites at the end of the growth phase by synthesis and excretion of innocuous end products. Therefore, it is likely that the phenazine production, due to the apparent antibiotic activity, helps to protect the producing organism and its habitat against other microorganisms and microbial competitors, thus improving the living conditions for the host organism. Furthermore in oxygen lack, Rhodococcus Efl could use phenazine as energy source by the producing bacteria Costa KC, Bergkessel M, Saunders S, Korlach J, Newman DK. Enzymatic Degradation of Phenazines Can Generate Energy and Protect Sensitive Organisms from Toxicity. MBio. 2015 Oct 27;6(6):e01520-15. doi: 10.1128/mBio.01520-15.

Surfactants are surface active agents, that can be produced as secondary metabolites by a large number of microorganisms, as bacteria, yeasts or fungi including Rhodococcus species (Flavio Correa Bicca, Leonardo Colombo Fleck ; Marco Antonio Zachia Ayub. PRODUCTION OF BIOSURFACTANT BY HYDROCARBON DEGRADING RHODOCOCCUS RUBER AND RHODOCOCCUS ERYTHROPOLIS, Revista de Microbiologia (1999) 30:231-23. They have both hydrophilic and hydrophobic/lipophilic portion in the molecule, that confer to them a wide range of properties, including the ability to lower surface and interfacial tension of liquids and to form micelles and microemulsions between two different phases. These compounds can be divided into two main classes: low-molecular-weight compounds called biosurfactants, and high-molecular-weight polymers, called bioemulsans [Bioremediation Resource Guide,(1993) A bibliography of publications and other sources of information about bioremediation technologies. EPA 542-B-93- 004]. Some microorganisms are able to enhance oil displacement by producing and high-molecular-weight polymers that can reduce interfacial tension, making an emulsion with oil, easy to recover. These organic molecules have a complex structure with specific functional groups, that work with specific mechanisms, that can influence their bioremediation ability, besides pH, aeration status, nutrient and temperature. The biosurfactants are generally harmless and less toxic in the environment, and their production do not require high temperatures. Costs of microbial products are not affected by the crude oil price and can be produced using inexpensive raw substrates or even waste materials.

A review on bacterial pigments is in Hizbullahi M Usman, Nafi'u Abdulkadir, Mustapha Gani, Hauwa'u M Maiturare, Bacterial pigments and its significance, MOJ Bioequivalence & Bioavailability, 2017;4(3):285-288. Rhodococcus strains AC 74 Rhodococcus ruber, AC 87 Rhodococcus ruber,AC 239 Rhodococcus ruber, AC 265 Rhodococcus erythropolis, AC 272 Rhodococcus erythropolis, are able to grow on hydrocarbons as sole carbon sources and to produce biosurfactantst (Flavio Correa Bicca, Leonardo Colombo Fleck, Marco Antonio Zachia Ayub, Production of biosurfactant by hydrocarbon degrading rhodococcus ruber and rhodococcus erythropolis, Revista de Microbiologia, 1999, 30:231-236).

Chinese patent n. CN 102180334 discloses the rhodobacterium CGMCC No.3512 which can be used to reduce the Cr (VI) in wastewater to Cr (III), in particular is carried out a chemical removal of Chrome generating a precipitate of Cr(OH)3, thus reducing Cr(VI) to Cr(III), in the presence of the rhodobacterium strain.

Inserire stato dell'arte sulla degradazione batterica della cocaina.

Technical problem

In view of the findings of the prior art, the inventors of the present invention investigated if the strains Marinomonas efl and Rhodococcus efl can be used for bioremediation, in particular for removing from polluted water diesel fuel, chromium, cadmium, nichel and cocaine.

Budapest treaty

Marinomonas efl E Rhodococcus efl has been filed at Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia- Romagna "Bruno Ubertini", according to Budapest treaty.

Marinomonas efl Access .n. DPS RE RSCIC 4 of 08/05/2019

Rhodococus efl Access n. DPS RE RSCIC 17 of 08/05/2019

Object of the invention The above technical problem is solved by the use of bacterial strains Marinomonas efl and Rhodococcus efl for bioremediation of polluted water from contaminants.

In particular by the use of bacterial strains Marinomonas efl and Rhodococcus efl for bioremediation of polluted water from fuels, toxic post-transition metals and/or toxic transition metals, and cocaine.

Another object of the present invention is a method for removing diesel fuel from polluted water by adding bacterial strain Rhodococcus efl to the water polluted with diesel fuel for a time sufficient for the bacterial strains Rhodococcus efl to growth and naturally produce a phenazine-based dye and a surfactant wherein the phenazine-based dye acting as biosensor becoming coloured in the presence of diesel fuel has a peak of mass spectrum of m/z=274,3 uma and the surfactant traps and remove the diesel fuel.

A further object of the present invention a method for removing toxic post-transition metals and/or toxic transition metals from polluted water by adding Marinomonas efl and/or Rhodococcus efl to the water polluted with toxic post-transition metals and/or toxic transition metals in the presence of a precursor until nanoparticles containing toxic post-transition metals and/or toxic transition metals in a non-toxic chemical form are formed and optionally removing nanoparticles from bio-remediated water.

A final object of the present invention a method for removing cocaine from water by adding Rhodococcus efl to the water polluted with cocaine.

Brief description of drawings

Figure 1 shows The UV-vis spectrum of the extracted blue pigment in water solution at basic pH with main peak with high absorption at 300 nm, and a lower broadened peaks around 367 nm, 414 nm and 600 nm. Figure 2 shows the Uv-visible spectral change during titration with HC1.

Figure 3 shows absorbance trend as function of the pH at 300 nm.

Figure 4 shows first derivative graph and pKa values. Figure 5 shows HPLC chromatogram at 279 nm.

Figure 6 shows Molecular mass spectrum (M+H)+ peak.

Figure 7 shows a representation of the method (a) for Cr(III) test and Method (b) for Cr(VI) test.

Figure 8 shows a representation of the method confirming the formation of Cr nanoparticles.

Figure 9 shows a representation of the cocaine test.

Detailed description of the invention

Definitions

Within the meaning of the present invention Marinomonas sp. efl is a member of the bacterial consortium associated with the ciliate Antarctic psychrophile E. focardii endemic in Antarctic coastal waters. The genome of Marinomonas sp. efl is known and described in a uniprot. Access Number 2005043. It is deposited at Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna "Bruno Ubertini", according to Budapest treaty Access .n. DPS RE RSCIC 4 of 08/05/2019.

Within the meaning of the present invention Rhodococus efl belongs to the bacterial genus Rhodococcus, it is a Gram positive bacteria, member of the bacterial consortium associated with the antarctic ciliate Euplotes. It is deposited at Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia- Romagna "Bruno Ubertini", according to Budapest treaty Access .n DPS RE RSCIC 17 of 08/05/2019. Within the meaning of the present invention bioremediation means processes for treating contaminated environments, including polluted water and soil, by introducing in the environment growing microorganism able to alter the environmental conditions and degrade pollutants.

Within the meaning of the present invention pollutants are fuels, toxic post-transition metals,toxic transition metals and cocaine.

Within the meaning of the present invention fuels are diesel and biodiesel.

Within the meaning of the present invention toxic post transition metals and toxic transition metals are chromium, cadmium, nichel.

Within the meaning of the present invention a culture medium is a nourishing broth or lysogenic broth which contains ingredients such as yeast extract, peptides deriving from enzymatic digestion such as hydrolyzed casein, which consists of a mixture of different chemical components in unknown proportions.

Within the meaning of the present invention a minimal medium is a culture medium comprising just the ingredients to support growth, i.e. the minimum nutrients possible for colony growth, tipically comprising a carbon source, salts, essential elements such as magnesium, nitrogen, phosphorus, and sulfur and water.

Within the meaning of the present invention phenazine-based dyes means Phenazines or derivatives thereof produced by bacterial species which are pigments with acid-base properties, due to their redox ability, showing a Uv-Vis spectrum with an intense absorption band from 250nm to 360 nm, and a lower broad absorption band that can goes from 400nm to 600nm, Tipical Extinction Coefficient e is around 3-104 cm-1 mol-1 L, with colours deep red, lemon yellow, bright blue. Within the meaning of the present invention surfactants are surface active agents, that can be produced by bacteria which can be classified as low-molecular-weight compounds called biosurfactants, and high-molecular-weight polymers, called bioemulsans.

Within the meaning of the present invention LB Medium means Lysogeny broth comprising peptides and casein peptones, vitamins, trace elements and minerals, optionally comprising sodium chloride or tryptone.

The invention is the use of bacterial strains Marinomonas efl and Rhodococcus efl for bioremediation of polluted water from contaminants.

In particular bioremediation of polluted water from fuels, toxic post-transition metals and/or toxic transition metals and cocaine.

More preferably fuel is diesel fuel.

More preferably post-transition metals and/or toxic transition metals are selected from the group consisting of chromium, cadmium, nichel.

Another object of the present invention is a method for removing diesel fuel from polluted water by adding bacterial strain Rhodococcus efl to the water polluted with diesel fuel for a time sufficient for the bacterial strain Rhodococcus efl to growth and naturally produce a phenazine-based dye and a surfactant wherein the phenazine-based dye acting as biosensor becoming coloured in the presence of diesel fuel has a peak of mass spectrum of m/z=274,3 uma and the surfactant traps and remove the diesel fuel.

A further object of the present invention a method for removing toxic post-transition metals and/or toxic transition metals from polluted water by adding Marinomonas efl and/or Rhodococcus efl to the water polluted with toxic post-transition metals and/or toxic transition metals in the presence of a precursor until nanoparticles containing toxic post-transition metals and/or toxic transition metals in a non-toxic chemical form are formed and optionally removing nanoparticles from bio-remediated water.

In a preferred embodiment Marinomonas efl and/or Rhodococcus efl are added to polluted with CrIII and CrVI in the presence of CrS04 and K2Cr04 and wait until nanoparticles of reduced Cr are produced and Cr nanoparticles are optionally removed from the water with a filter.

Examples

Example 1: Production of pigment and surfactant by Rhodococcus efl

A Minimal Medium solution composed by: NaCl 0,5 g, MgS0 ₄ x 7 H20 1 g, KC10,70 g, KH2P042 g, NaHP043 g, NH4N031 g, In 1L of deionized water, was prepared. Diesel, diesel without additive, benzene, hexadecane, pentane, octane and hexane, to a final concentration of 1% was added. As a control, glucose was used as the carbon source. Therefore, in a flask of 500ml, having a right oxygenation, were mixed 100 ml of Minimal Medium, 1% in volume of organic compound and bacteria.Bacteria were prepared from a LB medium over night culture. 2 ml of the culture was centrifuged at 13.000 rpm for 2 minutes. The supernatant was removed and the bacterial pellet was washed with 1 ml of Minimal Medium. The washing step consisted in resuspension of the pellet in 1ml of MM, followed by a centrifugation at 13000 rpm for 3 min. The supernatant discarded again and the pellet suspended in lml of Minimal Medium. After 3-4 hours of incubation at 30°C under shacking, it appeared a pink layer, with many floes, on the surface of the solution. Said solution become white after additional 4/5 hours, making the solution turbid and opalescent. The experiment was repeated with 1% of the following organic substances: benzene, no additive diesel, biodiesel, hexadecane, pentane, octane, hexane, glucose (control). The incubation was performed with MM and at 30°C. Therefore, in a flask of 500ml, having a right oxygenation, were mixed:100 ml Minimal Medium, 1% in volume of organic compound and bacteria. Bacteria were prepared from a LB medium over night culture. 2 ml of the culture was centrifuged at 13.000 rpm for 2 minutes. The supernatant was removed and the bacterial pellet was washed with 1 ml of Minimal Medium. The washing step consisted in resuspension of the pellet in 1ml of MM, followed by a centrifugation at 13000 rpm for 3 min. The supernatant discarded again and the pellet is suspended in 1ml of MM. The pink pigment and white flocculate production was visible only in presence of common diesel, no additive diesel and hexadecane. With the other organic compounds, no production of pink pigment and white flocculate was observed. Different concentration of the organic substances: 0,5%, 2% and 4% final concentration, in MM at 30 °C, under shacking were also tested. The solution became pink faster respect the solution with lower amount of carbon source, after only 2 hours. No production of pink pigment and flocculate was observed with benzene, glucose, biodiesel, hexane, pentane and octane. The pink pigment was analysed by UV-Vis, High Pressure Liquid Chromatography (HPLC) and Mass Spectrometry (MS). The white floes was analysed in order to test the presence of surfactants. After solution decanting, the pink pigment concentrates on the top; 1,5 ml of the solution surface was sampled with a Pasteur pipette and transferred in Eppendorf. The solution was centrifuged at 13.000 rpm for 2 minutes at room temperature. Three layers with different density were observed: on the top was present the organic layer with the soluble pink pigment; then a lipid layer, and on the bottom the bacteria present in the aqueous layer. The soluble pink pigment can be easily collected. If the incubation solutions were left for other 4 hours in the water bath at 30 °C under shaking decolouration is observed with the formation of white floe. To purify the white floe 1,5 ml of the solution surface was sampled with a Pasteur pipette and transferred in Eppendorf. The solution was centrifuged at 13.000 rpm for 2 minutes at room temperature. Three layers were observed with different density: on the top was present the organic layer, then a lipid layer of white flock and, on the bottom, the bacteria present in the aqueous layer. The white floe can be easily collected from the solution with a small spoon. The organic layer with the soluble pink pigment was transferred from the Eppendorf's into a new tube. A water solution of NaOH 2 M was added to the pigment, that stratified on the bottom: in this way the dye was extracted from the organic solution at the top to the aqueous part in the bottom. The top organic layer became transparent whereas the aqueous layer became deep blue for the presence of the pink pigment that became blue for the presence of 2M of NaOH. The blue aqueous solution (500 mΐ) was transferred to a new tube. An equal volume of octane was added, to remove any possible organic substance contamination. This operation was repeated three times in order to extract the higher quantity of possible organic impurity. An equal volume of Chloroform (CHC1 ₃₎ with some drops of HC12M was added to the aqueous solution to re-extract the pigment onto the organic solvent in acidic conditions. The pigment became pink again. Therefore, the pigment is pink in acidic conditions and blue in basic condition, confirming that it can be used to check the acid/basic condition of a solution. The UV-vis spectrum of the extracted blue pigment in water solution at basic pH shows a main peak with high absorption at 300 nm, and a lower broadened peaks around 367 nm, 414 nm and 600 nm (Figure 1). This spectrum is typical of phenazine pigments, which present different colour intensity and range of wavelength, that depend on the source and the solvent conditions, with the main peak around 250-300 nm, and the lower one around 600 nm (Rajkumar Cheluvappa (2014), Standardized chemical synthesis of Pseudomonas aeruginosa pyocyanin, MethodsX 1 67-73). An acid-base study was performed by adding very low amount of HC1 at different concentrations starting from a pH= 12.33 up to pH 2,09 in water solution. The results show a spectral changes where is visible the decrease of the main band at 300 nm with the formation of blue-shifted band at 288 nm, while peak at 600 nm, is shifted at 540nm (Figure 2). The trend of the absorbance at 300 nm as a function of the pH (Figure 3) permitted, through the first derivative study, the calculation of the pKas (Figure 4), relative to the two inflection points of Figure 3. The two maximum peaks of Figure 5 are attributable of the pKal and pKa2 values. In this case is not possible to calculate the pKa3 relative to the formation of pigment neutral form since this is not soluble in water and aggregates at high acidity conditions. The obtained results indicate that this pigment present acidic groups in its molecule structure. In basic condition the molecule is completely deprotonated (Aº) and it is soluble in the aqueous part. During the titration, after the adding of HC1, these groups are protonated, passing through two intermediate forms: HA= and H2A . At the end of the titration, in very acidic state, the molecule is completely neutral (H3A), giving the aggregates formation in water solution. This particular condition prevents it to be detect by the Uv-visible spectrum. In the following table 1 the relative spectral data are resumed.

Table 1 The blue pigment in basic condition after the pre-treatment was analyzed by HPLC method by using a C18 column ( ALLTIMA C185 micron, 150 mmx4,6mm), at temperature of 23°C, and using an H20:Me0H 36:64 as eluent solution, flow lml/min. By this method, the dye was separated as function of its acidic-base character, obtaining three dye forms with retention time of 2.4, 5.3 and

8.9 min respectively (Figure 5); in this case the ionic form Aº is not possible to detect because not retained from the column. By this consideration the first compound that was eluted (2,4 min) was the most charged that is the less retained and has the lower affinity to the apolar stationary phase(C18); consequently the compound with retention time of 5,3 min was H2A-, and the last one was the compound with the higher affinity for the C18 stationary phase and it was the neutral form H3A. The acidic extracted pink pigment in Chloroform was dried and then solubilized in 1 drops of CH2C12 with Acetonitrile and successively injected in HPLC-MS showing the Mass spectrum reported in Figure 6 where the base peak is at m/z=274,3 uma, according to (M+H)+ peak, showing that the dye present a m/z of 273,3 uma.

Therefore, Rhodococcus efl can be used as a biosensor of diesel, no additive diesel and hexadecane by the formation of a colored pink pigment. In the presence of these compounds, this bacterium produces a phenazine type pigment of m/z 273,3 uma together with white floe of surfactant that, by the trapping of diesel molecules, facilitates their removal.

A drop-collapse method has been refined for use as both a qualitative assay to screen for surfactant-producing microbes, and as a quantitative assay to determine surfactant concentration. The assay is rapid, easy to perform, reproducible and requires little specialized equipment. The sample droplet is added to the centre of an oil or water drop and observed after 1 min. The droplet will either bead up, spread out slightly or collapse, depending on the amount of surfactant in the sample. The drop collapse indicate the presence of surfactant (positive result), or the drop remains beaded, indicating the absence of surfactant (negative response). The drop-collapse method has several advantages: a smaller volume is required (5 mΐ of sample), the effective range of measurement is greater and it does not require specialized equipment [Adria ABodour et al (1998), Journal of Microbiological Methods Volume 32, Issue 3, 1 May 1998, Pages 273-280, Application of a modified drop-collapse technique for surfactant quantitation and screening of biosurfactant-producing microorganisms]. In order to determine the presence or not of surfactants as products of the bacterium reaction incubated with diesel, two assay were set that exploit the capacity of these compounds to alter the surface tension of the paraffin and water drops. The samples were centrifuged for 5 min at 10,000 rpm, and the supernatant was used for assay. The first test was conducted using drops of water as support. The results revealed three different columns, formed by three drops of water. Each drops is made by 25 mΐ of water, as drop support. Then, in the A column was added 25 mΐ of water, as negative control, in the B 25 mΐ of Minimal Medium, also in this case as negative control, in the C 25 mΐ of supernatant white floe. Methylene blue was added to better visualize the drop in the picture. There is an evident collapse of the drops on C column, with the floe sample. The test was repeated in the same way also with the paraffinic drop as support: column A negative control (25 mΐ of paraffin† 25 mΐ of paraffin), B another negative control (25 mΐ of paraffin +25 mΐ of Minimal Medium), columns C and D there is the white floe. Column D revealed that there is the formation of a third face between the oily and aqueous parts. It could be possible that the oil became more soluble in the floe volume.

Example 2: Formation of Cr nanoparticles

The Marinomonas and Rhodococcus lawn (0.5 OD) was allowed to grow in LB Medium plus 1 mM of the CrS04 and K2Cr04, i.e. the salt precursors of the following chemical elements Cr(III) and Cr(VI), respectively. The cultures were incubated at 22°C under shaking conditions for 48h. Culture sample was centrifuged at 5000 rpm for 15 minutes. After centrifugation, supernatant and pellet was separated. The supernatant was purified using high speed centrifugation at 12000 rpm for 20 mins (4 degree C) followed by air drying of remaining contents from the petri- plate. Cr nanoparticles (CrNPs) were visualized with TEM. In order to evaluate the complete reduction of Cr(III) and Cr(VI) by bacteria, a colorimetric test was performed. First, two standard solutions containing Cr(III) and Cr(VI) respectively, were prepared. The method (a)-Cr(III) test is represented in figure 7: the method was optimized in order to prepare pink- violet Cr(III)-glycine complex [Budiasih et al. (2013), International Scholarly and Scientific Research & Innovation, Volume 7, Issue 6, pages 458-462]. Specifically, to Cr(III) solution, glycine in the molar ratio of 1:3, was added under stirring and heating at 80°C for five minutes. Method (b)-Cr(VI) test: the method was optimized in order to prepare pink-purple Cr(VI)-diphenyl carbazide complex. Specifically, to Cr(VI) solution, diphenyl carbazide and HC1 in the molar ratio of 1:3;:3, were added under stirring for five minutes [Lace et al., (2019), Int. J. Environ. Res. Public Health, Volume 16, Issue 10, 1803]. Similarly, to 1 ml of solution containing Cr(VI), pretreated with Bacteria for x hours, method (b) is applied, demonstrating the absence of Cr(VI). In order to confirm the formation of CrNPs, Marinomonas and Rhodococcus culture was treated with H202 (oxidant)in order to re-oxidize CrNPs and Methods (a) and (b) was applied demonstrating in both cases the formation of Cr(VI) solutions (Figure 8).From TEM image CrNPs have dimensions lower than lOnm. Therefore the bacteria can be used to remove the Cr(III) and Cr(VI) (highly toxic) from waste water as CrNPs. Bacteria can be therefore spread in water or dispersed into soil to allow the formation of nanoparticles that can be left in the environment (reduced Cr is not toxic), or eventually removed from water environment with a filter.

Example 3: Degradation of cocaine

From the genome annotation, we identified three predicted protein sequences that show similarity with the Rhodococcus CocE, an enzyme that can degrade cocaine. A strain of Rhodococcus designated MB1 was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. Rhodococcus strain MB1 is capable of utilizing cocaine as a sole source of carbon and nitrogen for growth. The cocaine esterase (cocE) from this Rhodococcal strain MB1 has been deeply characterized. The nucleotide sequence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids . The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocE is a serine esterase. CocE crystal structure was determined and showed that this enzyme is a serine carboxylesterase. CocE possesses a catalytic triad formed by S117, H287, and D259 within a hydrophobic active site, and an oxyanion hole formed by the backbone amide of Y118 and the Y44. The cocE coding sequence was subcloned into the pCFXl expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with an Mr of approximately 65,000. The apparent Km of the enzyme (mean y standard deviation) for cocaine was measured as 1.33 y 0.085 mM. CocE was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1 (Turner JM et al., 2002, Biochemical characterization and structural analysis of a highly proficient cocaine esterase 2002 Oct 15;41(41):12297-307).

SEQ.ID NO. 1

Rhodococcus sp. MB1 'Bresler 1999' (AAF42807.1)

MVDGNYSVASNVMVPMRDGVRLAVDLYRPDADGPVPVLLVRNPYDKFDVFAWSTQST NWLEFVR DGYAW IQDTRGLFASEGEFVPHVDDEADAEDTLSWILEQAWCDGNVGMFGVSYLGVTQWQAAV SGVGGLKAIAPSMASADLYRAPWYGPGGALSVEALLGWSALIGTGLITSRSDARPEDAAD FVQL AAILNDVAGAASVTPLAEQPLLGRLIPWVIDQW DHPDNDESWQSISLFERLGGLATPALITAG WYDGFVGESLRTFVAVKDNADARLVVGPWSHSNLTGRNADRKFGIAATYPIQEATTMHKA FFDR HLRGETDALAGVPKVRLFVMGIDEWRDETDWPLPDTAYTPFYLGGSGAANTSTGGGTLST SISG TESADTYLYDPADPVPSLGGTLLFHNGDNGPADQRPIHDRDDVLCYSTEVLTDPVEVTGT VSAR LFVSSSAVDTDFTAKLVDVFPDGRAIALCDGIVRMRYRETLVNPTLIEAGEIYEVAIDML ATSN VFLPGHRIMVQVSSSNFPKYDRNSNTGGVIAREQLEEMCTAVNRIHRGPEHPSHIVLPII KR

SEQ.ID NO. 2

Rhodococcus efl PROKKA_00430 Cocaine esterase

MGLTVPSAVASADPTGGSDGQAWLAATEAAPQYPGVSIEWDVPITMSDGTVLQANVY RPADASG

RAVESKTPSVLNITPYTKLLDTLVDSALSIPQVGETLMDVANSLDLAAPFDGVSELT GVIAGGG ARVLGVNRDLVQNGYTQWVDARGTGFSQGNWDVLGKREQQDSIEVIDWMSKQGWSDGKVG MAG ISYSAINSVQAASNNPPALKAIFPVEPGNDLLRDIVGTGGGLGVGFMPLWLTAVNGLKLI PNVQ DILQGNFDPLWLASRLEDPGTLIPELVQAMTAQRIEDVSPSTLQVAQDGQFYQDRSADVG NITA ATMVYGGWHDIFANSEPRIYNGIDLPPGQKQLIMGNGYHVTPGGGFGKDGAPPRLDVLER AWFD KWLKGIDNGIDRYGPVTMLQQGGGWITDDQFPRAGATYERMYLNSAPSGTAAHAAYDGSL TNDP SSAAARLTVAPGLRGFCSGDGTQGTAGISW LGASCSKDSRFQEAEGLTFTGEAVTEPTSLSGP INVHLETVLDATDGFWAATVNDVAPDGTSTPITNGALTASLRAVDDSKSTRSANGDYSEP HHYL TIDTRQPW PGEVTAVDINMLPTDAVLQPGHRLRVDVYAASVPRYLALGPMLADSQLKPQHIEL AADRPSFVNVPFVGGR

SEQ.ID NO. 3

Rhodococcus efl PROKKA_02245 Cocaine esterase

MIRTPYGRGWHLAEGIEWRSRGIAFLCQDVRGRHDSTGSWEPYVHEREDGAALAEWL KDQPWTP KCVIASGASYAAGTAWAFAAETNSADWSFRVDGVVSKVPTIGSDRVKRDPSGILLLAEHL AWWG EHGDSSSSREGFIPELMRSDSALLEHLPVQSMIEKGGLTSDSPGWVTPINRARERQPYGS DDLV

SADDLSELDVAGLHIGGWDDAMIRETLRHYASVERGPQSLIVGPWAHDLRPGMVGET SFGHLQV EWMESIFSARPVTINRVFDRGAGEWSTEPYVMGANRVRLEPDPGTAPFSFTHHADEGRND CVVW TFDMEVECVLSGTPSAELTVHSPEGEADWIVELSIVDSAGVSRSIARGAGVSLCSGTVTI DLDP VLHTLRPGDVLSLQIAGSDFPRLARNLGDGDRYRGTRCTALHQSFSGSLTLPILGGTGGE

SEQ.ID NO. 4

Rhodococcus efl PROKKA_05361 Cocaine esterase

MAHVYNSGVEIPMRDGVTLAALVFQPLEGNAPTLLLRTPYGLASHGAGPTEVLPFVD AGYAVVW VESRGTFKSEGEFRPKTNEPEDGYDTVEWIIARPWSDGQVGTYGPSYCGMTQWATASTGH PALK ASW KETAMNWYRGLWYHPGGALSQSFSTFWPTAMNASNERRKLAAGRGSLDRVLDLGGGLLNG RGLSLNDHTPISTHPLLPLEQWMSEILDHPDYDEFWKEQDFTTSVPEMTQPVLSIAGWYD IFIV EQLRDFERFRREAGSEEARTGSRLVVGPWIHELDYSSEFPDRDFGAIGAAAACDLTGAHL QHFN RWLRPGADAPAPSPAPVRLFVMGIDQWRDEQDWPLPDTTYVDYFLSGDAPNGRGELLLGG PGAA SESRYRYDPRDPVPTAGGPSMPARFGFNGPVDQKEVASRPDVLCFTGPALDEDTEVTGYV KATL FVTSDAVDTDFTAKLVDVFPDGKAINLCDGILRARYRNGLDRPEHLVPGAVYEVEIDMAA TSNV

FRAGHRIRVDISSSNFPHYDRNTNTGGFISRESIDDAWAHNTILTGPDHPSRLVLPR INR Rhodococcus Efl ability of to eat cocaine was evaluated. Rhodococcus Efl was grown in Minimal Medium containing 5%, 25%,

50% or 100% Luria-Bertani (LB) medium [10 g Tryptone, 5 g Yeast extract and 10 g f NaCl in 950 mL of deionaized water, pH 7]. A second set of culture was composed by Rhodococcus Efl in

Minimal Medium Containing 5%, 25%, 50% or 100% LB medium plus

ImM of cocaine. Turbidity at 600 nm at time 0, after 3, 18 and

20 hours of growing was evaluated and found that Rhodococcus Efl was growing more in the presence of 1 mM of cocaine (A'). To check the Rhodococcus ability to degrade cocaine, bacteria metabolites were sampled at different times from 0 to six hours and after one day of digestion. In order to identify cocaine a test was performed: to 750 mΐ of sample containing ImM of cocaine, 100 mΐ of Co(SCN)2 and 400 mΐ of CHC13 were added. Finally 50 mΐ of HCL 2M was added under stirring [da Silva et al. (2008) Analytica Chimica Acta, Volume 629, Pages 98-103].

The presence of cocaine is identified by the blue colour formation in organic layer, the blu colour of the organic layer decreases already after 2 hours, until it disappeared after one day (figure 10).

Applicant's or agent's International application file reference RBW16065-PCT PCT/EP2020/082865

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule 13 to)

Form PCI7RO 34 (July 1998; reprint Jai Applicant's or agent's International application file reference RBW16065-PCT PCT/EP2020/082865

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule 13 to)

Form PCI7RO 34 (July 1998; reprint Jai