RU2756179C1 - Method for producing l-(+)-tartaric acid - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology, in particular, to the field of producing L-(+)-tartaric acid from cis-epoxysuccinates of alkaline or alkaline earth metals. In particular, the invention relates to the application of an enzyme with an amino acid sequence at least 70% identical to SEQ ID NO:1 for hydrolysis of cis-epoxysuccinates of alkaline and alkaline earth metals.EFFECT: invention provides a possibility of effectively producing L-(+)-tartaric acid.5 cl, 3 dwg, 2 ex

Description

The technical field to which the invention relates

The invention relates to a method for producing L - (+) - tartaric acid, comprising the step of enzymatic hydrolysis of an alkali or alkaline earth metal cis-epoxysuccinate.

State of the art

Carboxylic and dicarboxylic acids, diols and alcohols are increasingly used in various industries. This also applies to L - (+) - tartaric acid. L - (+) - tartaric acid is used in food, cosmetic, textile, pharmaceutical, chemical and construction industries as an acidifier, flavor enhancer, antioxidant and chemical solvent. Traditionally, L - (+) - tartaric acid is obtained from wine-making waste.

In addition, L - (+) - tartaric acid can be obtained by chemical synthesis (see document US5087746 A (published 02/11/1992, MONSANTO CO [US])) or by fermentation (see Bhat HK et al., PRODUCTION OF TARTARIC ACID BY IMPROVED RESISTANT STRAIN OF GLUCONOBACTER-SUBOXYDANS // Research and Industry. - 1986. - T. 31. - No. 2. - P. 148-152) maleic anhydride and 5-oxogluconic acid or glucose as a raw material.

Currently, the most effective method is the enzymatic production of L - (+) - tartaric acid from inexpensive and readily available cis-epoxysuccinate (CES) using cis-epoxysuccinate hydrolases (CESH), as described, for example, in document US 4010072 A (publ. . 03/01/1977, TOKUYAMA SODA [JP]).

Epoxy hydrolases are biocatalysts for the asymmetric hydrolysis of epoxides, which require neither cofactors nor metal ions.

Cis-epoxysuccinate hydrolase activity has been described for a wide group of microorganisms: Acetobacter curtus , Achromobacter tartarogenes, Ach. acinus and Ach. sericatus , Acinetobacter tartarogenes, Agrobacterium aureum, Agr. viscosum, Alcaligenes epoxylytus, Alc. margaritae, Corynebacterium sp., Nocardia tartaricans, Pseudomonas sp., Rhizobium validum, Rhodococcus rhodochrous, Klebsiella sp. and Labrys sp.

Despite the widespread distribution of CESG among bacteria, the databases contain only 2 amino acid sequences of CESG, for which the reaction of the conversion of CES into L-tartrate has been reliably described.

In document US6379938 B1 (publ. 30.02.2002 PURATOS NV [BE], the first complete sequence of the CESG protein from the bacteriumRhodococcus rhodochrousLMGP-18079 and it was demonstrated that the protein of the gene has CES hydrolase activity. Identical sequence also found in bacteriaRhodococcus opacus (document Liu Z. et al., Cloning, sequencing, and expression of a novel epoxide hydrolase gene from Rhodococcus opacus in Escherichia coli and characterization of enzyme // Applied microbiology and biotechnology. - 2007. - T. 74. - No. 1 . - S. 99-106),Nocardia tartaricans CAS-52 (Wang Z., Wang Y., Su Z. Purification and characterization of a cisepoxysuccinic acid hydrolase from Nocardia tartaricans CAS-52, and expression in Escherichia coli // Applied microbiology and biotechnology. - 2013. - T. 97. - No. 6. - P. 2433-2441). The enzyme sequence is currently listed in the NCBI database (GenBank ID: JQ267565).

The second known sequence is the sequence of the CESG gene from Klebsiella oxytoca (Cheng Y. et al., Purification and characterization of a novel cisepoxysuccinate hydrolase from Klebsiella sp. That produces L (+) - tartaric acid // Biotechnology letters. - 2014. - T. 36. - No. 11. - S. 2325-2330).

Despite the fact that these sequences have CESG activity, the process of enzymatic epoxide opening must be carried out at a temperature of no more than 37 ° due to insufficient enzyme stability. Using higher temperatures will improve the efficiency of the process and reduce the likelihood of microbial contamination.

Thus, there is currently a need to identify new sequences exhibiting CESH activity and allowing to optimize the process of obtaining tartrates from the corresponding CES with subsequent selective production of L - (+) - tartaric acid.

The essence of the invention

The invention consists in the use of an enzyme having the amino acid sequence of SEQ ID NO: 1 in the production of tartrates from cis-epoxysuccinates (CES) of alkali and alkaline earth metals.

This enzyme is obtained from a culture of a host cell carrying a plasmid construct containing the nucleotide sequence of SEQ ID NO: 2.

The invention also relates to a polynucleotide encoding the sequence of SEQ ID NO: 1, a plasmid construct comprising a polynucleotide encoding an enzyme comprising the amino acid sequence of SEQ ID NO: 1.

In addition, the invention relates to a host cell containing a polynucleotide or plasmid construct encoding an enzyme comprising the amino acid sequence of SEQ ID NO: 1.

Ultimately, the invention relates to a method for producing L - (+) - tartaric acid by hydrolysis of cis-epoxysuccinates of alkali and alkaline earth metals.

The technical result of the invention is to ensure the stability of the enzyme in the process of obtaining tartrate from the CES of alkali and alkaline earth metals at temperatures up to 60 ° C.

Description of figures

FIG. 1. Map of the plasmid pQE30-PrCo expressing the CESG Prauserella coralliicola gene.

FIG. 2. ¹ H NMR tartaric acid obtained in D ₂ O.

FIG. 3. Profiles of enzyme activity as a function of the incubation temperature of the enzyme solution before adding the substrate.

Detailed description of the invention

The following is a description of various aspects of the implementation of the present invention.

In one embodiment, the invention provides an enzyme having the amino acid sequence SEQ IN NO: 1 used for hydrolysis of cis-epoxysuccinates of alkali and alkaline earth metals.

The specified sequence can be obtained as follows.

1. Chemical synthesis of a nucleotide encoding the specified sequence.

2. Optional optimization of the nucleotide sequence in terms of codon composition (to increase the level of expression of the enzyme by the host cell).

3. Cloning of a nucleotide into a plasmid construct.

4. Introduction of the plasmid construct into the cells of the microorganism.

5. Cultivation of cells.

6. Disruption of cells to obtain a lysate containing the specified sequence.

The described method of obtaining a sequence is provided by way of example only and does not limit the present invention. Examples of specific steps for obtaining a sequence are those described in: Liu Z. et al., Cloning, sequencing, and expression of a novel epoxide hydrolase gene from Rhodococcus opacus in Escherichia coli and characterization of enzyme // Applied microbiology and biotechnology. - 2007. - T. 74. - No. 1. - P. 99-106, EP 2840135 A1 (published 25.03.2015 HANGZHOU BIOKING BIOCHEMICAL ENGINEERING CO LTD [CN]), US 6379938 B1 (published 30.04.2002 PURATOS NV [BE]).

The resulting sequence can be immobilized as described, for example, in Wang Z. et al. Production of tartaric acid using immobilized recominant cisepoxysuccinate hydrolase // Biotechnology letters. - 2017. - T. 39. - No. 12. - S. 1859-1863.

In another embodiment, the invention relates to a polynucleotide encoding an enzyme having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polynucletide is represented by the nucleotide sequence of SEQ ID NO: 2.

In another embodiment, the invention provides a plasmid construct for expressing an enzyme having the amino acid sequence of SEQ ID NO: 1 into which the nucleotide sequence of SEQ ID NO: 2 has been cloned.

In another embodiment, the invention provides a host cell for expressing an enzyme having the amino acid sequence of SEQ ID NO: 1 into which the nucleotide sequence of SEQ ID NO: 2 has been cloned, or a plasmid construct comprising the nucleotide sequence of SEQ ID NO: 2.

Any cell known in the art, for example bacteria, fungi or yeast, can be used as a host cell. Preferably, due to the technological convenience and accessibility, E. coli bacteria are used, in the most preferred embodiment of the invention, the E. coli strain deposited in the All-Russian collection of industrial organisms (VKPM) under the number B-13524 is used.

In one embodiment, the invention relates to the use of an enzyme comprising the amino acid sequence of SEQ ID NO: 1 in the production of L - (+) - tartaric acid.

In yet another embodiment, the invention relates to a method for producing L - (+) - tartaric acid by enzymatic hydrolysis of cis-epoxysuccinates of alkali and alkaline earth metals, wherein SEQ ID NO: 1 is used as the enzyme. As a sequence in enzymatic hydrolysis, a sequence of more than 70%, preferably more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 99 % homologous to the above.

As cis-epoxysuccinates of alkali and alkaline-earth metals, cis-epoxysuccinates of sodium, calcium, barium, etc. are used. Preferably, cis-epoxysuccinate calcium is used.

Enzymatic hydrolysis is carried out by any method known in the art. Examples of such methods are, but are not limited to: EP 2840135 A1 (published 03/25/2015 HANGZHOU BIOKING BIOCHEMICAL ENGINEERING CO LTD [CN]), US 6379938 B1 (published 04/30/2002 PURATOS NV [BE]), GB 1534195 A ( publ. 11/29/1978, Takeda Chemical Industries [JP]).

A distinctive feature of the method for producing L - (+) - tartaric acid according to the present method is the ability to carry out enzymatic hydrolysis at temperatures above 40 ° and up to 60 °.

L - (+) - tartaric acid is used both in the food industry as a flavor enhancer and sweetener, and in construction as part of dry building mixtures, etc.

Implementation of the invention

Determination methods

Polyacrylamide gel electrophoresis (PAGE) technique (one-dimensional protein gel electrophoresis)

To carry out electrophoresis of proteins, 40 μL of sodium dodecyl sulfate lysis buffer (SDS buffer) was added to the cell sediment after washing from the medium for application (0.05 M Tris-HCl pH 6.8; 2% SDS; 0.002% bromophenol blue ; 10% glycerol; 5% mercaptoethanol), resuspended and exposed to ultrasonic waves in an ultrasonic bath for 15 min. The resulting lysate was boiled for 5 min at 95 ° ^C, centrifuged 15 min at 16100 g. Then the supernatant was applied to a polyacrylamide SDS gel for electrophoresis by the Laemmli method (34) in a mini-gel (8 × 10 cm, 1 mm thick).

To carry out electrophoresis of proteins, supernatants after ultrasonic destruction of cells in saline buffer were mixed in a 1: 1 ratio with a loading buffer (0.05 M Tris-HCl pH 6.8; 2% SDS; 0.002% bromophenol blue; 10% glycerin; 5% mercaptoethanol), then boiled for 5 minutes.

To carry out electrophoresis of proteins after chromatographic purification, the samples were mixed in a 2: 1 ratio with an application buffer (0.05 M Tris-HCl pH 6.8; 2% SDS; 0.002% bromophenol blue; 10% glycerol; 5% mercaptoethanol), then boiled for 5 minutes.

Protein concentration was determined by the Bradford method (see Bradford, MM A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding // Anal. Biochem. - 1976. - Vol. 72. - P. 248-254).

Gene Sequence Obtaining

To create a molecular biological construct SEQ ID NO: 2 expressing the protein CES hydrolase PrCo , a chemical synthesis of a gene sequence with a codon composition optimized for expression in E. coli was performed.

The resulting gene was cloned into the pQE30 plasmid construct under the control of a powerful T5 promoter under the regulation of the lactose operon operator. A map of the plasmid pQE30-PrCo expressing the CESG Prauserella coralliicola gene is shown in FIG . 1.

Getting transformed cells E.coli

The molecular genetic construct pQE30-PrCo was introduced into E. coli Xl-blue cells by electroporation. The cultivation of the transformed E. coli cells was carried out as follows.

As a basis, we used a minimal saline medium containing 2.0 g / l Na ₂ SO ₄ , 6.12 g / l (NH ₄ ) ₂ SO ₄ , 0.50 g / l NH ₄ Cl, 14.60 g / l K ₂ HPO ₄ , 3.60 g / l NaH ₂ PO ₄ · H ₂ O, 1.00 g / l sodium citrate dibasic, 0.36 g / l MgSO ₄ and 0.1 g / l thiamine hydrochloride. To the prepared mixture was added 250 μL of a solution of trace elements containing 0.50 g / L CaCl ₂ 2H ₂ O, 0.18 g / L ZnSO ₄ 7H ₂ O, 0.10 g / L MnSO ₄ H ₂ O, 20 , 1 g / l Na ₂ -EDTA, 16.70 g / l FeCl ₃ 6H ₂ O, 0.16 g / l CuSO ₄ 5H ₂ O, 0.18 g / l CoCl ₂ 6H ₂ O. Level The pH of the nutrient medium was adjusted to 7.5. Glycerin and glucose were used as carbon sources, 7.5 g / L and 2.5 g / L, respectively.

To induce protein expression after 24 hours of incubation, 150 μl of a 1M aqueous solution of IPTG (isopropyl-β-D-1-thiogalactopyranoside) and ampicillin were added to the medium at a rate of 100 μg / ml. Simultaneously with the beginning of the induction of protein expression, additional carbon sources - glycerol and a solution containing 100 g / L of tryptone and 50 g / L of yeast extract - were added to the bacterial culture.

Further incubation of cultures was carried out at a temperature of 32 ° for 7 hours. The optical density OD600 of the culture at the end of cultivation was ~ 6.2. The cell culture was besieged at 3000-4000 rpm for 10 minutes, washed with minimal saline medium, frozen in liquid nitrogen and stored at -70 ° C.

An aliquot of cells from the sample was analyzed for the presence of the recombinant protein using one-dimensional gel electrophoresis in PAGE.

Obtaining a lysate

Cell culture of 50 ml of recombinant E. coli XL blue (with plasmid pQE30-PrCo) after induction was precipitated by centrifugation at 3000g for ten minutes. The cells were resuspended in 10 ml of mineral medium and sonicated. The lysate was centrifuged at 16100g for ten minutes.

To determine the presence of an enzymatic reaction, 10 μl of the lysate after centrifugation was added to 90 μl of a solution containing 25 g / l of sodium cis-epoxysuccinate, and vortexed. The mixture was incubated for 1 hour at 32 °. Then the presence of tartrate in the solution was determined by the metavanadate method.

The investigated lysate demonstrates the presence of activity in comparison with the analogous lysate obtained from the E. coli XL blue strain (with plasmid pQE30 without insertion of the target gene).

Cultivation E.coli

The overnight culture was grown in 500 ml conical flasks, into which were placed 100 ml of LB medium (1% peptone, 1% NaCl and 0.5% yeast extract, pH 8.0) with the addition of ampicillin at a concentration of 100 μg / ml. The total volume of the overnight culture was 400 ml. The cultivation was carried out at 37 °, at 250 rpm on an ES-20/60 shaker-incubator (Biosan) for 16 hours.

For cultivation in a bioreactor, a mineral medium of the following composition was used: 2.0 g / l Na ₂ SO ₄ , 6.12 g / l (NH ₄ ) ₂ SO ₄ , 0.50 g / l NH ₄ Cl, 14.60 g / l K ₂ HPO ₄ , 3.60 g / l NaH ₂ PO ₄ · H ₂ O, 1.00 g / l sodium citrate dibasic, 0.36 g / l MgSO ₄ and 0.1 g / l thiamine hydrochloride with the addition of to the prepared mixture of 1/1000 of a solution of trace elements containing 0.50 g / l CaCl ₂ 2H ₂ O, 0.18 g / l ZnSO ₄ 7H ₂ O, 0.10 g / l MnSO ₄ H ₂ O, 20 , 1 g / l Na2-EDTA, 16.70 g / l FeCl ₃ 6H ₂ O, 0.16 g / l CuSO ₄ 5H ₂ O, 0.18 g / l CoCl ₂ 6H ₂ O. Mineral base autoclaved, microelements were sterilized by filtration through 0.22 µm filters.

400 ml of the overnight culture was added to a five-liter Biostat B bioreactor (Sartorius) containing 4 liters of the above-described mineral medium supplemented with 2.5 g / L glucose and 7.5 g / L glycerol. The cultivation was carried out with air supply: 2 liters per liter of medium per minute. The stirrer rotation speed was 800 rpm. The pH of the medium was maintained at the optimum level for E. coli growth, equal to 7.6. Before adding the overnight culture, the medium was warmed up to 37 °, then the same temperature was maintained throughout the experiment.

The optical density of the culture was measured every 30 minutes 8 hours after the start of the culture. Upon reaching the optical density OD600 = 8.0, induction was performed. 50 ml of glycerol and 16 ml of solution (Trypton 100 g / L and yeast extract 50 g / L) were added to the medium. IPTG was added as an inducer to a concentration of 1 μM / ml. Cultivated at 32 ° for 6.5 hours.

The cell culture was concentrated using tangential ultrafiltration on filters with a pore size of 0.2 μm. 56 g of cells were obtained from a total of four liters of culture. Six grams of the obtained cell pellet was suspended in 40 ml of distilled water, the test tube was placed in an ice bath. The cells were disrupted by ultrasound. The lysate was clarified by centrifugation at 12000 g for 30 minutes.

Cis-epoxysuccinate hydrolase activity was determined by the method using ammonium metavanadate. The activity of the resulting enzyme was ~ 40,000 U / L of the culture liquid.

Example 1. Enzymatic hydrolysis of calcium cis-epoxysuccinate (SACES)

In 100 ml of distilled water, 5.35 g of CaCES was suspended.

To the resulting suspension, 5 ml of clarified lysate of the preparatively produced enzyme CES hydrolase PrCo was added and incubated for 20 hours at 28 ° C with constant stirring at 150 rpm on an ES-20/60 incubator shaker (Biosan).

At the end of the cultivation, the precipitate was filtered and dried to an air-dry state.

Inspection showed that the resulting powder contained 90 ± 5% calcium L-tartrate tetrahydrate hydrate.

According to the XRD analysis data, the resulting powder contains L-CaC ₄ H ₄ O ₆ • 4H ₂ O and does not contain CaCES, which indicates the completeness of the reaction.

Example 2. Comparison of enzymatic activity depending on temperatures.

A qualitative determination of the CESG activity in the obtained protein solutions was carried out. Activity has been demonstrated for clarified lysates of E. coli cell cultures carrying constructs expressing the following CSEG: SEQ ID NO: 3 (RhOp from Rhodococcus opacus) ; SEQ ID NO: 4 (KlOx from Klebsiella oxytoca) ; SEQ ID NO: 1 (PrCo from Prauserella coralliicola) .

The result of the determination is shown in FIG. 3.

As illustrated in FIG. 3, the sequence found in Prauserella coralliicola, compared to the known sequences, is more stable in the optimal temperature range for enzymatic hydrolysis (45-60 ° C). At the same time, the sequence found in Prauserella coralliicola is highly selective in the hydrolysis of calcium cis-epoxysuccinate.

Claims (5)

1. The use of an enzyme having an amino acid sequence identical at least 70% to SEQ ID NO: 1 for the hydrolysis of cis-epoxysuccinates of alkali and alkaline earth metals. 2. Use according to claim 1 for the hydrolysis of cis-epoxysuccinates of alkali and alkaline earth metals to obtain L - (+) - tartaric acid. 3. A method for producing L - (+) - tartaric acid by enzymatic hydrolysis of cis-epoxysuccinates of alkali and alkaline earth metals, characterized in that an enzyme is used as an enzyme having an amino acid sequence identical by at least 70% to SEQ ID NO: 1, for hydrolysis of cis-epoxysuccinates of alkali and alkaline earth metals. 4. The method according to claim 3, wherein the enzymatic hydrolysis is carried out at a temperature of more than 40 ° C. 5. The method according to claim 3, wherein the enzymatic hydrolysis is carried out at temperatures up to 60 ° C.

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