WO2022060246A1

WO2022060246A1 - Method for producing l-(+)-tartaric acid

Info

Publication number: WO2022060246A1
Application number: PCT/RU2021/000387
Authority: WO
Inventors: Алексей Сергеевич РОЗАНОВ; Сергей Евгеньевич ПЕЛЬТЕК; Антон Владимирович КОРЖУК; Константин Викторович СТАРОСТИН; Валерия Николаевна ШЛЯХТУН
Original assignee: Публичное Акционерное Общество "Сибур Холдинг" (Пао "Сибур Холдинг")
Priority date: 2020-09-16
Filing date: 2021-09-10
Publication date: 2022-03-24
Also published as: RU2756179C1

Abstract

The invention relates to the field of producing L-(+)-tartaric acid from cis-epoxysuccinates of alkali metals or alkaline earth metals. More particularly, the invention relates to: an enzyme with an amino acid sequence having at least 70% identity with SEQ ID NO:1 for the hydrolysis of cis-epoxysuccinates of alkali metals or alkaline earth metals; a polynucleotide that codes for said enzyme, a plasmid construct containing said polynucleotide, and a host cell containing said polynucleotide or said plasmid construct; and a method for producing L-(+)-tartaric acid by the enzymatic hydrolysis of cis-epoxysuccinates of alkali metals or alkaline earth metals using said enzyme.

Description

METHOD FOR PRODUCING L-(+)-Tartaric Acid

The field of technology to which the invention belongs

The invention relates to a method for producing L-(+)-tartaric acid, which includes the step of enzymatic hydrolysis of alkali or alkaline earth metal cisepoxysuccinate.

State of the art

Carboxylic and dicarboxylic acids, diols and alcohols are increasingly used in various industries. In particular, this applies to L- (+) -tartaric acid. L-(+)-tartaric acid is used in the food, cosmetic, textile, pharmaceutical, chemical and construction industries as an acidulant, flavor enhancer, antioxidant and chemical solvent. Traditionally, L-(+)-tartaric acid is obtained from winemaking waste.

In addition, L-(+)-tartaric acid can be obtained by chemical synthesis (see document US5087746 A (publ. 11.02.1992, MONSANTO CO [US] ) or fermentation (see Bhat H. K. et al. PRODUCTION OF TARTARIC ACID BY IMPROVED RESISTANT STRAIN OF GLUCONOBACTER-SUBOXYDANS Research and Industry 1986 vol 31 no 2 pp 148-152) of maleic anhydride and 5-oxogluconic acid or glucose as starting material.

Currently, the most efficient method is the enzymatic production of L-(+)-tartaric acid from inexpensive and readily available cis-epoxysuccinate (CES) using cisepoxysuccinate hydrolases (CESH), as described, for example, in US 4010072 A (publ. 01.03 .1977, TOKUYAMA SODA [JP] ) .

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

The 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 epoxy lytus, Ale. margaritae, Corynebacterium sp. , Nocardia tartaricans , Pseudomonas sp. , Rhizobium validum , Rhodococcus rhodochrous , Klebsiella sp. and Labrys sp.

Despite the rather wide distribution of CESH among bacteria, only 2 amino acid sequences of CESH are presented in the databases, for which the conversion reaction of CESH to L-tartrate is reliably described.

Document US6379938 B1 (publ. 02.30.2002 PURATOS NV [BE]) determined the first complete sequence of the CESH protein from the bacterium Rhodococcus rhodochrous LMGP-18079 and demonstrated that the gene protein has CESH hydrolase activity. An identical sequence was also found in the bacterium Rhodococcus 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, V. 74, No. 1, C. 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 - V. 97. - No. 6. - C. 2433-2441) The enzyme sequence is currently listed in the NCBI database (GenBank ID: JQ267565) .

The second known sequence is the CESH gene sequence 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. - C. 2325-2330) .

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

Thus, at present, there is a need to identify new sequences that exhibit CESG activity and allow optimizing 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 SEQ ID N0 : 1 in the production of tartrates from cis-epoxysuccinates (CES) of alkali and alkaline earth metals.

This enzyme is obtained from a host cell culture carrying a plasmid construct containing the nucleotide sequence of SEQ ID N0:2.

The invention also relates to a polynucleotide encoding the sequence SEQ ID N0 : 1 , a plasmid construct containing a polynucleotide encoding an enzyme containing the amino acid sequence SEQ I D N0 : 1 .

In addition, the invention relates to a host cell containing a polynucleotide or plasmid construct encoding an enzyme containing the amino acid sequence of SEQ ID N0 : 1 .

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

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 plasmid pQE30-PrCo expressing the CESH gene of Prauserella coralliicola.

FIG. 2 1H NMR spectrum ^of the resulting tartaric acid in D2O. FIG.3 Profiles of enzyme activity depending on the temperature of the incubation 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 discloses an enzyme having the amino acid sequence SEQ IN N0:1, useful for the hydrolysis of alkali and alkaline earth metal cis-epoxy succinates.

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 according to the composition of codons (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. Destruction of cells to obtain a lysate containing the specified sequence.

The described method for obtaining a sequence is given by way of example only and does not limit the present invention. Examples of specific steps for obtaining a sequence are the steps for obtaining, 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. - C. 99-106, EP 2840135 Al (published 03/25/2015 HANGZHOU BIOKING BIOCHEMICAL ENGINEERING CO LTD [CN] ) , US 6379938 Bl (published 04/30/2002 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. - C. 1859- 1863.

In another embodiment of the invention, the invention relates to a polynucleotide encoding an enzyme having the amino acid sequence of SEQ ID N0:1. In some embodiments, the polynucleotide is represented by the nucleotide sequence of SEQ ID N0:2.

In yet another embodiment, the invention relates to a plasmid construct for expression of an enzyme having the amino acid sequence of SEQ ID N0:1 into which the nucleotide sequence of SEQ ID N0:2 has been cloned.

In another embodiment, the invention relates to a host cell for expression of an enzyme having the amino acid sequence of SEQ ID N0:1 into which the nucleotide sequence of SEQ ID N0:2 has been cloned, or a plasmid construct containing the nucleotide sequence of SEQ ID N0:2.

Any cell known in the art, such as bacteria, fungi or yeast, can be used as the host cell. Preferably, due to technological convenience and availability, 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 containing the amino acid sequence of SEQ ID N0:1 in the production of L-(+)-tartaric acid.

In another embodiment of the invention, the invention relates to a method for producing L-(+)-tartaric acid by enzymatic hydrolysis of cis-epoxysuccinates of alkali and alkaline earth metals, where as the enzyme is used the sequence of SEQ ID N0:1. 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% is also used. % homologous to the above.

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

Enzymatic hydrolysis is carried out by any method known from the prior art. Examples of such methods are, but are not limited to: EP 2840135 A1 (publ. 25.03.2015 HANGZHOU BIOKING BIOCHEMICAL ENGINEERING CO LTD [CN] ) , US 6379938 Bl (publ. 30.04.2002 PURATOS NV [BE] ) , GB 1534195 A ( 11/29/1978, Takeda Chemical Industries [JP] ) .

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

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

Implementation of the invention

Methods of determination

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

For protein electrophoresis, 40 μl of sodium dodecyl sulfate (SDS) lysis buffer (0.05 M Tris-HC1 pH 6.8; 2% SDS; 0.002% bromophenol blue) was added to the cell precipitate after washing from the medium. ; 10% glycerol; 5% mercaptoethanol), resuspended and sonicated in an ultrasonic bath for 15 min. The resulting lysate was boiled for 5 min at 95°C, centrifuged for 15 min at 16100 d. After that, the supernatant was applied to a polyacrylamide SDS gel for electrophoresis according to the Laemmli method (34) in a mini-gel (8><10 cm, 1 mm thick) .

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

For protein electrophoresis, after chromatographic purification, the samples were mixed in a 2:1 ratio with loading 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, M.M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding // Anal. Biochem. -1976. -Vol. 2. - P.248 - 254).

Obtaining a gene sequence

To create a molecular biological construct SEQ ID N0:2 expressing the CES hydrolase PrCo protein, a chemical synthesis of a gene sequence with a codon composition optimized for expression in E. coli was carried out.

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 gene of Prauserella coralliicola is shown in FIG. 1.

Obtaining transformed cells E. coli

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

The minimum salt medium containing 2.0 g/l Na2SO ₄ , 6.12 g/l (NH ₄ ) ₂ SO ₄ , 0.50 g/l NH ₄ C1, 14.60 g/l K ₂ was used as a basis. HPO ₄ , 3.60 g/l NaH ₂ PO ₄ 'H ₂ O, 1.00 g/l dibasic sodium citrate, 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 0.20, 1 g / l Pa ₂ -EDTA, 16.70 g / l GeS1 ₃ '6H ₂ O, 0.16 g / l CuSO ₄ -5H ₂ O, 0.18 g / l CoC1 ₂ -6H ₂ 0. pH level nutrient medium was adjusted to a value of 7.5. Glycerol and glucose, 7.5 g/l and 2.5 g/l, respectively, were used as the carbon source.

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

Further incubation of cultures was carried out at a temperature of 32°C for 7 hours. The optical density OD600 of the culture at the end of cultivation was ~6.2. The cell culture was precipitated 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 recombinant protein using one-dimensional gel electrophoresis in PAAG.

Getting a lysate

Cell culture 50 ml of recombinant E. coli XL blue (with plasmid pQE30-PrCo) after induction was besieged by centrifugation at 3000g for ten minutes. Cells were resuspended in 10 ml of mineral medium and destroyed by ultrasound. 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°C. After that, the presence of tartrate in the solution was determined by the metavanadate method. The test lysate demonstrates the presence of activity in comparison with the analogous lysate obtained from the E. coli XL blue strain (with pQE30 plasmid without inserting the target gene).

Cultivation of E. coli

The overnight culture was grown in 500 ml conical flasks, in which 100 ml of LB medium (1% peptone, 1% NaCl and 0.5% yeast extract, medium pH 8.0) was placed with the addition of ampicillin at a concentration of 100 μg/ml. The total overnight culture volume was 400 ml. Cultivation was carried out at 37°C, 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 C1, 14.60 g/ l K ₂ HPO ₄ , 3.60 g / l NaH ₂ PO ₄ -H ₂ O, 1.00 g / l dibasic sodium citrate, 0.36 g / l MgSO ₄ and 0.1 g / l thiamine hydrochloride with the addition to the prepared mixture of 1/1000 solution of microelements 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 FeCl3 -6H ₂ O, 0.16 g/l CuSO ₄ -5H ₂ O, 0.18 g/l CoCl ₂ -6H ₂ 0. The mineral base was autoclaved , trace elements were sterilized by filtration through 0.22 µm filters.

400 ml of overnight culture was added to a five liter Biostat B bioreactor (Sartorius) containing 4 liters of the mineral medium described above supplemented with 2.5 g/l glucose and 7.5 g/l glycerol. 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 an optimum level for the growth of E. coli equal to 7.6. Before adding the overnight culture, the medium was heated to 37°C, 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 cultivation. Upon reaching the optical density OD600 = 8.0, induction was performed. 50 ml of glycerol and 16 ml of a solution (Trypton 100 g/l and yeast extract 50 g/l) were added to the medium. IPTG was added as an inducer to concentration 1 µM/ml. Cultivated at 32°C for 6.5 hours.

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

The cis-epoxysuccinate hydrolase activity was determined by the ammonium metavanadate method. The activity of the resulting enzyme was ~ 40,000 U/l of culture fluid.

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

5.35 g of SaCES was suspended in 100 ml of distilled water.

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

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

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

According to XRD analysis, the obtained powder contains L-CaC4H ₄ O6 * 4H ₂ O and does not contain SaCES, which indicates the completeness of the reaction.

Example 2 . Comparison of enzymatic activity depending on temperatures.

Conducted a qualitative determination of CESG activity in the obtained solutions of proteins. Activity was demonstrated for clarified lysates of E. coli cell cultures carrying constructs expressing the following CESH: SEQ ID N0 : 3 (RhOp from Rhodococcus opacus); SEQ ID N0:4 (KlOx from Klebsiella oxytoca) ; SEQ ID N0:1 (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 is more stable than known sequences over the optimum temperature range for enzymatic hydrolysis (45-60°C). However, the sequence found in Prauserella coralliicola shows high selectivity in the hydrolysis of calcium cis-epoxysuccinate.

Claims

CLAIM

1. An enzyme having an amino acid sequence at least 70% identical to SEQ ID N0:1 for the hydrolysis of alkali and alkaline earth metal cis-epoxysuccinates.

2. Polynucleotide encoding the enzyme according to claim 1.

3. A polynucleotide according to claim 2, characterized in that it has the nucleotide sequence of SEQ ID N0:2.

4. Plasmid construction for the expression of the enzyme according to claim 1, containing a polynucleotide according to claim 2 or 3.

5. A host cell for expression of an enzyme according to claim 1, containing a polynucleoid according to claim 2 or 3 or a plasmid construct according to claim 4.

6. The host cell of claim 5, wherein the cell is a bacterium

E . coli.

7. The host cell according to claim b, where the E. coli cell is deposited in the All-Russian Collection of Industrial Organisms under the number B-13524.

8. The method of obtaining the enzyme according to claim 1, including the following stages: a) chemical synthesis of a polynucleotide encoding the enzyme; b) cloning the polynucleotide into a plasmid construct or host cell; c) in the case of cloning the polynucleotide into a plasmid construct, introducing the plasmid construct into host cells; d) culturing the host cells; e) destruction of host cells to obtain a lysate containing the enzyme according to claim 1.

9. The method according to claim 8, in which, after step a), the nucleotide sequence is additionally optimized for the composition of codons.

10. The method according to claim 8, in which the polynucleotide in step a) is a polynucleotide according to claims. 2-3.

11. The method of claim 8 wherein the host cell is an E. coli cell.

SUBSTITUTE SHEET (RULE 26)

12. A method for producing L- (+) -tartaric acid by enzymatic hydrolysis of cis-epoxysuccinates of alkali and alkaline earth metals, characterized in that the enzyme according to claim 1 is used as the enzyme.

13. The method according to claim 12, in which the enzymatic hydrolysis is carried out at a temperature of more than 40°C.

14. The method according to claim 13, in which the enzymatic hydrolysis is carried out at temperatures up to 60°C.

15. Use of the enzyme according to claim 1 or the enzyme obtained by the method according to claim 8 for the production of L-(+)-tartaric acid.

SUBSTITUTE SHEET (RULE 26)