ES2320505B1 - PROCEDURE FOR OBTAINING L-DOPA FROM L-THYROSINE USING THE THYROSINASE ENZYME NP 518458 OF THE BACTERIA RALSTONIA SOLANACEARUM. - Google Patents

Abstract

Procedure for obtaining L-dopa from L-tyrosine using the tyrosinase enzyme NP_518458 from the bacterium Ralstonia solanacearum .

The present invention relates to a process for producing 3,4-dihydroxy-L-phenylalanine (hereinafter L-dopa) from L-tyrosine, using as a catalyst a tyrosinase enzyme obtained from a microorganism, namely the tyrosinase NP_518458 obtained from the bacterium Ralstonia solanacearum . The process proposed by the present invention allows the accumulation of all the L-dopa formed, avoiding the disadvantage of the unwanted oxidation of the L-dopa that is formed and, therefore, the loss of product that occurs with the use of other tyrosinases described in the prior art.

Description

Procedure for obtaining L-dopa from L-tyrosine using the tyrosinase enzyme NP_518458 from the bacterium Ralstonia solanacearum .

Technical sector

The present invention relates to a process for producing 3,4-dihydroxy-L-phenylalanine (hereinafter L-dopa) from L-tyrosine, using as a catalyst a tyrosinase enzyme obtained from a microorganism, namely the tyrosinase NP_518458 obtained from the bacterium Ralstonia solanacearum .

State of the art

3,4-Dihydroxy-L-phenylalanine (hereafter L-dopa) is used for the treatment of Parkinson's disease. It is known that L-dopa is present in the shell and in the seeds of the Vicia faba plant, bean from where it can be obtained by extraction. L-dopa can be obtained by synthesis from intermediates such as vanillin and tyrosine.

L-dopa is also produced biochemically using L-tyrosine and tyrosinase, or with other substrates and β-tyrosinase produced by a microorganism as described below. The L-tyrosine is one of the essential amino acids and it produces on a commercial scale by extraction of natural substances and enzymatically

Katsuji Haneda, Shiro Watanabe, and Isao Takeda are the authors of "Synthesis of L-3,4-Dihydroxyphenylalanine from L-Tyrosine by Microorganisms" published in Applied Microbiology, 22, 721-722 (1971) in which they describe that L- Dopa can be efficiently synthesized from L-tyrosine in acidic conditions (pH 2 to 5) from Aspergillus fungi species, specifically A. chevalier and A. oryzae without the need for cofactors, while at a neutral pH or alkaline, L-tyrosine breaks down into other metabolites and L-dopa does not accumulate.

In GB 1268721 (Ajinomoto Co., Inc.), published on March 29, 1972, a process for producing L-dopa or its derivatives is described, comprising the catechol reaction and one or more amino acids selected from cysteine, S-alkyl p-cysteine (where alkyl p - means a small alkyl group, with one or two carbon atoms, usually methyl or ethyl), cystine, serine and alanine, and / or a salt of said amino acids, in an aqueous medium in the presence of β-tyrosinase as a catalyst; said beta-tyrosinase was produced by culture of one of the microorganisms selected from the following genus: Escherichia, Aerobacter, Erwinia, Citrobacter, Pseudomonas, Bacillus, Xanthomonas and Proteus . The same authors, in US Patent 3791924 (Ajinomoto Co., Inc.), published on February 12, 1974, describe a method for obtaining L-dopa by means of the action of a β-tyrosinase produced by the culture of microorganisms selected from the genus Citrobacter, Proteus, Pseudomonas, Aerobacter, Salmonella, Erwinia, Paracolobactrum or Alcaligenes , with a compound that can be: catechol, phenol and resorcinol, with a) one or more amino acids selected from cysteine, S-alkyl_ {p} -cysteine (small alkyl group), cystine, serine and alanine, or with b) ammonium and a second compound of the group consisting of pyruvic, oxalacetic, malico, fumarole, glyoxylic and lactic acid, in an aqueous medium to a pH between 4 and 11.

In EP 0618299 Al and EP 0636695 Al patents (both applied for by Ajinomoto Co., Inc) published respectively on 01.04.1993 and 21.07.1994, methods for the production of L-dopa from catechol, pyruvic acid and ions are described ammonium, or catechol and L-serine, in the presence of 13-tyrosinase produced by Erwinia herbicola . The method of EP 0618299 Al is characterized in that it uses the culture obtained after an additional incubation for 6 to 24 hours after the cell culture reaches the stationary phase, while the pH is maintained between 7 and 8.3. And the method of EP 0636695 Al is characterized in that the last part of the reaction period is carried out at a temperature less than 25 ° C with the condition that there are anhydrous crystals of L-dopa in the reaction mixture, while the L-dopa is formed and precipitated in the form of anhydrous crystals, which are obtained from the mixture.

In US 5,837,504 (Xun et al .), Published on November 17, 1998, a method for producing L-dopa from L-tyrosine is claimed, comprising a) the oxidation of L-tyrosine by adding said tyrosine and a substrate to a cell suspension to generate NADH, forming a reaction mixture; and b) incubating said reaction mixture for a time with the consumption of molecular oxygen and NADH to produce the L-dopa. The cell suspension is prepared from one of the bacteria in the group consisting of E. coli W, E. coli S17 λ and E. coli DH1 expressing the 4-hydroxyphenylacetate 3-hydroxylase activity. The substrate consists of small molecules of organic acids or alcohols and their combinations.

In WO 2004/015094 A1 (DSM IP ASSETS BV and DMS BIOTECH GMBH), published on February 19, 2004, they mention that in the process of US 5,837,504 when fermentation takes place in aqueous medium, L-dopa it oxidizes forming brown or black polymerization products, and its objective is to provide a fermentative production process in which the L-dopa obtained is stable. L-dopa is produced by aerobic fermentation of a recombinant microorganism that has L-tyrosine-3-hydroxy-mono-oxygenase activity and the metabolic pathway: glycolysis, pentose phosphate pathway, aromatic amino acid pathway, or its derivatives, comprising: a) growth phase and production phase, in which L-dopa is produced in the fermentation medium, and b) final phase
zation, in which L-dopa is produced from a carbon source and the pH is from 1 to 7 at least in this last phase.

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The authors of the present application, during the study of genes that putatively encode oxidizing polyphenol (PPOs) in bacteria interacting with plants in general and with pathogenic microbes in particular, thought that there is a connection between the ability of microorganisms to express the activities of PPOs and infection. To examine this possibility, they chose Ralstonia solanacearum as an appropriate model to see if these activities are really expressed in microorganisms, since this organism whose genome has been sequenced is a pathogenic bacterium that causes plants such as potatoes and tomatoes to wilt and die These authors found that at least three genes are expressed and the proteins show PPO activity ( Applied and Enviromental Microbiology , 71, 6808-6815; 2005). Continuing with studies of the activities of PPOs produced by R. solanacearum (FEBS Journal, 273, 257-270; 2006), the authors of the present application have surprisingly found that of the two PPOs that act as tyrosinase, with access numbers NP_519622 and NP_518458, its activity is different. NP_519622 is an enzyme with a clear preference for oxidation of non-carboxylated o- diphenols and only has a residual monophenolase activity, behaving like a catechol oxidase. In contrast to all the tyrosinases studied so far, and unexpectedly it has been found that NP_518458 has a greater activity as monophenolase than o-diphenolase, that is, it has a tyrosine hydroxylase (TH) activity greater than dopa oxidase ( DO), and also its initial activity does not depend just on the presence of an L-dopa cofactor.

The present invention, therefore, relates to a process for obtaining L-dopa from L-tyrosine using the tyrosinase enzyme NP_518458 (hereinafter referred to by its accession number) from Ralstonia solanacearum , an improved process in relation to the existing ones since the tyrosinase NP_518458 of R. solanacearum has a very effective hydroxylating activity and allows the accumulation of all the L-dopa formed, which does not occur in the tyrosinases described in the prior art. The main disadvantage of the use of other tyrosinases is precisely the unwanted oxidation of the dopa that accumulates, with the consequent loss of the desired product and therefore economic.

Description of the invention

For an efficient expression of the enzyme, and taking into account that it is a bacterial pathogen, the gene that encodes this enzyme will be amplified using Ralstonia solanacearum GMI1000 genomic DNA as a template and using appropriate primers with restriction sites that allow donation in a stable expression plasmid vector in E. coli .

There are several vectors available, in any of them it is interesting that the expression of the donated protein is inducible, for example by IPTG, in order to differentiate the growth phase from the expression phase of the enzyme tyrosinase. In this way, the recombinant E. coli strain will be cultured in aerobic conditions and in a complex medium until it reaches a high optical density and is then induced by the compound of choice.

The bioconversion of L-tyrosine in L-dopa it will be performed by the enzyme in optimal conditions and for this several are possible terms:

A- Adding L-Tyrosine directly to bacterial culture.

B- Preparation of cell extracts containing the enzyme, and

C- Isolation and purification of tyrosinase. In this case to monetize the cost associated with the process of isolation and purification the enzyme will be immobilized on a support suitable, being possible borosilicate or nylon.

Those skilled in the art know different supports for the production of L-Dopa using immobilized tyrosinase so that the reaction is carried out on a suitable support that favors the process of isolation and purification of the product. For example, it carried out the production of L-DOPA with tyrosinase immobilized on nylon membranes (Pialis, P;. Hamann, J MC and Saville, BA, L-DOPA production from tyrosinase immobilized on nylon 6,6 Biotechnology and bioengineering 51, 141-147 (1996) Also in CA 2 277 371 Al (National Silicates LTD), published on 08.01.2001, a method of immobilization of enzymes on silica supports is described, using glutaraldehyde as a polyfunctional agent. It is mentioned that the enzyme is much better stabilized on this support than other known ones and less enzyme is needed for the production of chemical and pharmaceutical products, such as L-dopa, and it has also been described that mushroom tyrosinase has been immobilized on polyamino-styrene (PSNH) and polymethyl-styrene chloride (PSCL), giving the latter support better results (Ho, P. Y; Chiou, MS and Chao, AC, Production of L-DOPA from tyrosinase immobilized on p modified oliestirene. Applied Biochemistry and Biotechnology-Part A Enzyme Engineering and Biotechnology , 111, 139-152 (2003).

Of the processes mentioned, the C presents advantages regarding the purity of the L-dopa obtained without enzyme losses, thereby facilitating processes subsequent product collection.

The person skilled in the art knows the different conditions of the transformation that will be conditioned by the protocol followed according to the enzyme used. In this case that the enzyme NP_518458 of R. solanacearum has been used, the protocol must meet the following requirements:

- pH lower than 7 to avoid self-oxidation of the L-dopa formed, considering pH 5 as close to optimal. The buffer of choice is phosphate or acetate buffer.

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- Tyrosine concentration of approximately 1 mg / ml (5.5 mM) that is as high as possible, taking into account its limited solubility and that an excess of this can cause a reaction inhibition.

- Temperature not exceeding 37ºC, preferably in the range 25-30 ° C.

- Addition of the SDS detergent at concentrations below the critical micellar concentration (0.1%), preferably at the 0.05% concentration. This compound is an agent that stimulates the tyrosinase activity of Ralstonia solanacearum .

- Adding EDTA traces to concentrations less than 0.1 mM. The goal is to eliminate metal cations free for its possible ability to mediate oxidation of L-dopa formed and destabilize the accumulation of East.

- The characteristics of the enzyme NP_518458 of R. solanacearum mean that it is not necessary to add L-dopa as a cofactor, a compound that should be used in the case of other tyrosinases to avoid a lag phase in the detection of enzymatic activity.

- Addition of reducing agents. There are several usable compounds. Ascorbic acid is one of the preferably usable. The concentration of this one, in the range from 10 to 1 mM, it will be adjusted to the highest possible without reaching produce tyrosinase inhibition.

- The bioconversion will be carried out in a Bioreactor operating in aerobic conditions. The bioreactor can operate in batch (Batch) but you can also do it in fed batch conditions (Feed-batch), in this way tyrosine and the reducer can be supplied appropriate in small quantities avoiding the problem of inhibition and increasing the production yield of L-dopa.

After the L-dopa has been formed it is necessary to collect and purify it. On this aspect it have described different protocols, known to experts in the matter, for example crystallization of the compound under conditions of Low aeration and slightly acidic pH.

Description of the figures

Figure 1. Figure 1 shows the dismutation reaction experienced by dopaquinone to generate dopa and dopachrome (DC), catalyzed by the enzyme NP_518458 from Ralstonia solanacearum .

Figure 2. Formation of DC and dopa by the enzyme NP_518458 of Ralstonia solanacearum . The absorbance graph shows that all the dopa formed by the dopaquinone dismutation accumulates when using the enzyme NP_518458 from Ralstonia solanacearum .

Figure 3. Formation of DC and dopa by commercial mushroom tyrosinase. The absorbance graph shows how when using commercial mushroom tyrosinase you don't know produces a stoichiometric accumulation of the dopa with the DC, due to to the use of the dopa by the enzyme.

Embodiment of the invention: Example Cell culture

R. solanacearum was grown in a basal saline medium (BSM) containing: 15 mM (NH 4) 2 SO 4, 0.8 mM MgCl 2, 2 µM FeSO 4 , 0.2 mM CaCl 2, 8 µM Na 2 MoO 4, 5 µM MnCl 2, 0.5% glycerol and 0.01% yeast extract in sodium phosphate buffer 50 mM, pH 7. E. coli was grown in the usual manner in Luria-Bertani medium (LB). When needed, the media was supplemented with kanamycin, ampicillin or rifampin 50 µg / ml, as necessary depending on the plasmid used and the necessary selection.

To obtain usual bacterial extracts, cells were grown in BSM (basal saline medium) for 48 h until an optical density of approximately 1.2 was obtained and centrifuged at 5000 x g for 10 min. The sediment was washed with 0.9% NaCl solution, resuspended in 1 ml of phosphate 0.1 M sodium, pH 7.0 containing phenylmethylsulfonyl fluoride 0.1 mM plus a 1/500 dilution of "Protease inhibitor cocktail" ® ("protease inhibitor cocktail") and was broken by discontinuous sonication using a Braun Labsonic sonicator for about 4 min. The homogenate was centrifuged at 12000 x g for 4 min, and the supernatant was used to purification and / or determinations of enzymatic activity. Be they obtained the reagents for the cell culture media and the Enzymatic determinations of Sigma Co. (St. Louis, Missouri, USA.).

Protein determination

Protein concentration was determined with the bicinconinic acid test. Alternatively, the absorbance at 280 nm to follow the elution profile of the proteins in chromatography purification columns.

Enzymatic determinations

TH and DO activities were determined respectively monitoring the oxidation of L-tyrosine or 2 mM L-dopa a L-dopachrome at 475 nm (Figure 1C, ε = 3700 M <-1> cm <-1>), in 0.1 M sodium phosphate buffer. The pH it was 5.0 or 7.0 depending on the activity and the enzyme PPO (polyphenoloxidase) tested, due to the different optimal conditions shown by the activities of this microorganism. In addition, 0.05% SDS was added for the TH assay pattern, while 0.02% SDS was added to determine the DO activity. For dopa titration, 50 ml of 10 mM sodium periodate and the increase was immediately determined of absorbance at 475 nm. Occasionally a small one was added L-dopa concentration as cofactor for determine the TH activity when appropriate. Be monitored the tyramine hydroxylase and dopamine oxidase of the same form, but dopaminechrome (ε = 3100 M <-1> cm <-1>). In all cases, a unit was defined as the amount of enzyme that catalyzes the appearance of 1 µmol of dopachrome / dopaminechrome per minute at 37 ° C.

Enzyme Purification

Extracts were concentrated using tubes 15 ml Amicon Millipore ultra centrifuge (cut-off point 10 kDa). Preparations were loaded in a column CM-Sephadex (Amersham Pharmacia Biotech, Amersham, RU) (3 cm in diameter x 18 cm in length). The column was eluted with 0.05 M sodium phosphate buffer, pH 7, up to one volume approximately equal to the total volume of the column. Once unbound proteins elute from the column, others are eluted proteins with a linear gradient of sodium chloride up to 1.5 M In the same buffer. The fractions were tested for determine PPO activities.

SDS-PAGE

The purity of the enzymes was confirmed by SDS-PAGE [24]. Dissociation was carried out analytical with SDS-PAGE using 9% acrylamide for the separation gel and 3% for the stacking gel. He Resolution buffer was Tris-HC1 (pH 8.8) and the Deposit buffer was Tris-glycine (pH 8.3), containing both 0.1% SDS. The samples were mixed in one 2: 1 ratio (vol / vol) with sample buffer (Tris-HC1 0.18 M, pH 6.8, 15% glycerol, 0.075% bromophenol blue, 7.5% 2-mercaptoethanol and 9% SDS) and heated at 95 ° C for 5 min before application. Ran electrophoresis at 20 ° C and a constant current of 15 mA during 20 min and 30 mA for approximately 90 min. The bands of proteins were visualized by bright blue staining of Coomassie Reagents for SDS-PAGE of Biorad Laboratories (Richmond, CA, USA).

Finally the bioconversion of L-tyrosine in L-dopa according to the procedures detailed above.

Comparative test of L-dopa obtained using the enzyme NP_518458 of R. solanacearum and the tyrosinase of mushroom A. bisporus

In TH activity tests you can record both the dopachrome (DC) formed and the dopa accumulated in reaction time. The first can be followed by registration I continue in spectrophotometer of its absorbance at 475 nm (AbS_ {475 [nm]), while the second can be determined by stopping the reaction at different times and adding excess periodate to which oxidizes instantaneously and stoichiometrically to DC, increasing the absorbance at 475 nm proportionally (Graham and Jeff, 1977).

Methodology

The test consisted of carrying out a measurement of the TH activity of R. solanacearum under normal conditions:

2 mM tyrosine (0.36 mg / ml) (in phosphate buffer pH 5).

0.05% SDS.

100 µl enzyme cell extract expressing the enzyme NP_518458 (usually contain between 0.6 and 1.5 mg protein / ml).

100 µL of mushroom tyrosinase 0.1 mg / ml (alternatively to the previous one).

37 ° C

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The reaction is allowed to proceed, recording the appearance of dopachrome (DC) as Abs 475 nm. To both reaction minutes 25 µl of sodium periodate are added (IO4 Na) 10 mM and the reaction is allowed to continue until the line of the absorbance measurement register is parallel to the previous one (prior to the addition of periodate). The amount of dopa, formed in those initial two minutes of reaction will correspond to the increase almost instantaneous of Abs_ {475 [nm]] due to chemical oxidation for the period of the dopa formed up to DC.

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The procedure must be repeated adding the periodate at different reaction times (4 ', 6', 8 ', 10' ...) with the objective of obtaining the increments of A_ {475 [nm]] along the entire reaction curve and finally compose the line of dopa accumulation compared to that of DC formation obtained with a measure of routine TH activity.

Results

In the case of tyrosinase NP_518458 of R. solanacearum , the dopa accumulates approximately equal to DC, which does not occur if mushroom is used. The product of the enzyme in both cases is dopaquinone, which undergoes a dismutation reaction (see figure 1) to generate dopa and dopachrome (DC):

2 Dopaquinone Dopa + DC

The released dopa can be used as a substrate for tyrosinase, competing with tyrosine. This competition depends on the amount of accumulated dopa and the affinity of the enzyme for o- diphenol dopa compared to monophenol tyrosine.

As can be seen in Figure 2, the data obtained indicate that the tyrosinase OD activity of R. solanacearum does not compete with the TH activity under the measurement conditions used, and therefore all the dopa formed by the dopaquinone dismutation it accumulates. When compared with the behavior of mushroom tyrosinase (see figure 3), the differences are clear, observing how that enzyme does use the dopa, not allowing stoichiometric accumulation with DC.

The accumulation of all the dopa formed is somewhat unusual in the tyrosinases described, and makes the enzyme NP_518458 of R. solanacearum a PPO different from the rest and with excellent properties for use in processes where a very effective hydroxylating activity is needed. The main disadvantage of the use of other tyrosinases is precisely the unwanted oxidation of the dopa that accumulates, with the consequent economic loss, a problem that is greatly attenuated in the tyrosinase of R. solanacearum , since the figure shows that the dopa in these conditions and concentration does not enter the catalytic cycle.

Claims (12)

1. Production process of 3,4-dihydroxyphenyl-L-alanine from L-tyrosine, characterized in that it comprises the following steps:

to)

use of the enzyme NP_518458, obtained from the microorganism Ralstonia solanacearum as a tyrosinase;

b)

cloning and expression of the enzyme NP_518458 in a strain of E. coli ; Y

C)

bioconversion of L-tyrosine in 3,4-dihydroxyphenyl-L-alanine by:

(i)

L-tyrosine addition directly to bacterial culture; or

(ii)

preparation of cell extracts containing the NP_518458 enzyme; or

(iii)

isolation, purification and fixing to a support inert of the enzyme NP_518458 that will act as a catalyst in the oxidation of L-tyrosine to form the 3,4-dihydroxyphenyl-L-alanine.

2. Method according to claim 1, in which stage c) still takes place pH between 2 and 7. 3. Method according to claims 1 and 2, in which step c) takes place at a pH between 4 and 6. 4. Method according to claims 1 to 3, in which step c) takes place at a pH 5. 5. Method according to claims 1 to 4, in which step c) takes place at a temperature not exceeding 37 ° C 6. Method according to claims 1 to 5, in which step c) takes place from 25 ° C to 30 ° C. 7. Method according to claims 1 to 6 in which EDTA is added as a chelating agent in concentrations less than 0.1 mM. 8. Method according to claims 1 to 7 in which a reducing agent is present. 9. Method according to claims 1 to 7 in which the reducing agent is ascorbic acid. 10. Method according to claims 1 to 9 in which SDS is added as a tyrosinase stimulating agent to a concentration between 0.01% and 0.1%. 11. Method according to claims 1 to 10 in which SDS is added as a tyrosinase stimulating agent at a concentration of 0.05%. 12. Procedure according to any of the previous claims wherein the addition of L-Dopa as cofactor.