WO2018085906A1

WO2018085906A1 - Vector, recombinant micro-organism, method for obtaining a recombinant epoxide-hydrolase enzyme, recombinant epoxide-hydrolase enzyme and use thereof

Info

Publication number: WO2018085906A1
Application number: PCT/BR2017/000056
Authority: WO
Inventors: Gabriela Desiree Tormet GONZALEZ; Luciana Gonzaga De Oliveira
Original assignee: Universidade Estadual De Campinas - Unicamp
Priority date: 2016-11-10
Filing date: 2017-05-30
Publication date: 2018-05-17
Also published as: BR102016026341A2

Abstract

The present invention relates to a method for obtaining a recombinant epoxide-hydrolase enzyme with a gene taken from the total genome of Streptomyces sp. bacteria B1 and subsequently expressed in E. coli genetically modified by the pGTEH2 vector comprising the gene sequence of interest. Protection is also claimed in the application for said vector and for the genetically modified E. coli bacteria. Said enzyme has functional activity on a large range of substrates, besides promoting the enantioselective hydrolysis of racemic epoxides, and can promote the production of vicinal diol and enantiopure epoxide compounds of great commercial interest, since they are used as intermediates or final products in the fine chemical, pharmaceutical and food industries.

Description

VECTOR, RECOMBINANT MICRO-ORGANISM, METHOD OF OBTAINING RECOMBINANT EPOXIDE-HYDROLASE ENZYME, ENZYME. RECOMBINANT EPOXIDE HYDROLASE AND USE

Field of the invention:

This invention is part of the biotechnology group and describes the method for obtaining the recombinant epoxide hydrolase enzyme obtained from Stzeptomyces sp. RI, an endophytic actinobacterium of the plant Citrus reticulata., And expressed in a bacterium Escherichia coli (E. coli.) Genetically modified by the vector pET29b (+) containing the gene sequence of interest. and the genetically modified E. coli microorganism.

[2] The recombinant enzyme acts enantioselectively in the hydrolysis of racemic epoxides, making it possible to obtain enantiomerically pure vicinal epoxides and diols, which are compounds of great commercial interest as they are used as intermediates or end products in the fine chemical, pharmaceutical and pharmaceutical industries. of food.

Background of the invention:

[33 Epoxide hydrolases (EH) enzymes are present in a wide variety of organisms from prokaryotes to eukaryotes and act as catalysts for the epoxide conversion reaction to the corresponding vicinal diols resulting from the apparent addition of water molecules (as shown in Scheme 1 below). ! .

[4] Enantiomerically pure epoxides and diols are important function blocks present in numerous compounds of economic importance for the food and pharmaceutical industries. As an example, tryptolide is a medicine used in traditional Chinese medicine as anticancer and anti-inflammatory. and more recently in the treatment of polycystic kidney disease.

[5] Epoxides and diols are also used as intermediates in organic synthesis reactions for the formation of other compounds of major interest such as N1999A2, an enediin class antitumor agent that can be synthesized by following a synthetic route using epoxide (R). -glycidol as a starting material.

{6} Historically, the introduction of drugs in the form of pure enantiomers has been particularly relevant with the case of racemic thalidomide, a drug widely used by women in the 1950s in the first weeks of pregnancy to ease nausea, causing congenital anomalies in numerous children. .

[7] Analysis indicated that (R) -enantiomer had the desired anti-nausea pharmacological action, while (S) -thalidomide had the tsratogenic effect responsible for the observed malformations.

[8] From this case, it was evidenced the need to develop appropriate methods to obtain enantiomerically pure (non-racemic) compounds, which demanded the effective development of asymmetric synthesis methodologies.

[9] Strategies for obtaining enantiomerically pure epoxides and diols include chemical methodologies such as the Sharpless or Jacobsen-Katsuki reactions, which include the use of titanium or manganese as catalysts.

[10] Among the disadvantages of these two protocols are the restricted use of. allylic alcohols in the Sharpless reaction and the low enantioselectivity for trans isomers in the Jacobsen-Katsuki reaction, which limits the broad application of these reactions in the organic synthesis of enantiopure epoxides.

[11] Alternatively to conventional organic synthesis strategies, biocatalytic processes employ whole cells or enzymes. Monooxygenases, for example, are used in the stereospecific epoxidation of alkanes. The use of these enzymes generally results in high enantiomeric excesses, however, there are limitations such as low yields and the requirement for cofactors.

[12] Among the various enzymes studied as catalysts in epoxide hydrolysis to the corresponding vicinal diols, epoxide hydrolases stand out, which can be divided into two. main groups: from higher organisms (eukaryotes) and from microorganisms (prokaryotes and eukaryotes).

[13] Eukaryotic enzymes generally result in greater enantioselectivity and acceptance by bulky substrates, but are difficult to produce. In this sense, prokaryote enzymes are more versatile, since they can be applied in industry due to the lower difficulty in cultivating these organisms for large-scale enzyme production. However, the disadvantages include low enantioselectivity and limited acceptance by bulky substrates,

[14] Distinguishing the potential of microorganisms in the production of vicinal epoxides and diols, the US5672504 describes a procedure capable of catalyzing the enrichment of one of [R, S) -1,2-epoxide enantiomers by bacterial strains. In this document, a brief mention is made of a strain of Streptomyces albosporeus, promoter of oxirane ring opening reaction and vicinal diol formation and tested against racemic mixtures of monosubstituted epoxides leading to the obtainment of the respective [R] -epoxide. However, this document does not detail the type (s) of enzyme (s) that catalyze the reported hydrolysis,

[15] US6823115 specifically describes the use of epoxide hydrolase from Streptomyces sp. to the process of separating racemic epoxides by enantioselective hydrolysis to form the respective vicinal diols. The invention also details a rapid screening process for identifying epoxide hydrolases. This document is the first study that reports the use of epoxide hydrolases enzymes from Streptomyces microorganisms, however, in the proposed process whole cells are used.

[16] The present invention details the cloning of a Streptomyces sp BI gene whose function has been attributed to the production of an epoxide hydrolase and relates to the method of obtaining the recombinant enzyme in Bscheríchia coli genetically modified by pET29b-derived vector pGTE.H2 (+).

[17] The described process has the advantage of obtaining purified and interference-free enzyme from other enzymes present in the reaction medium when compared to the use of whole cells.

[18] US 6828115 details the process of epoxide hydrolysis promoted by Streptomyces epoxide hydrolases for the specific cases of styrene oxide and / or its mono- or polysubstituted epoxide derivatives in the oxirane ring or aromatic ring as the ethyl-3-phenylglycidate, indene oxide compounds, 1,2 -epoxyhexane, 1,2-epoxidecane.

[19] The patent is however specific for the conversion of styrene oxide with an E> 2 enantioselectivity factor to the formation of (S) -phenyl-1,2-ethanol using intact S. grlseus, S. thermovulgarls cells. , S. antibioticus TU4, S. azenae Til and S. fradiaa TU27 in the presence of DMSO.

t.20] In order to elucidate the enantio- and regioselectivity governing epoxide hydrolase-catalyzed reactions from Streptomyces sp, a comparative study was conducted between the SgçF, NcsF2 and KedF SHs involved in the biosynthesis of the enedi antitumor antibiotics C-1027, neocarzinostatin and kedarcidine,

[21] Styrene (S) -oxide substrate was used for. to study the enantioselectivity in the hydrolysis reaction promoted by the 3 enzymes. The results showed the formation of (i¾) -diol by SgcF action, while NcsF2 and KedF led to (Sj-diol), indicating different regioselectivities.

[22] The observed results were attributed to a point mutation in a tyrosine residue that directly participates in the catalytic activity of NcsF2 and KedF which in the case of SgcF is replaced by a tryptophan residue. A second mutation corresponds to the presence of a methionine residue. which replaces a glutamate near the predicted active site cavity and acts directly to promote inversion of the configuration in the product formed by SgcF-mediated hydrolysis.

[23] This paper presents a proposal to explain different regioselectivities observed in epoxide hydrolysis and opens new perspectives to studies of directed evolution of structurally similar epoxide-hydrolases enzymes.

[24] WO2014127438 describes a process for obtaining enantiomerically pure vicinal diols starting from racemic epoxides by a reaction catalyzed by a recombinant epoxide hydroiasis from the fungus Aspergillus brasiliensis in aqueous medium or biphasic systems. This enzyme is considered the first recombinant epoxide hydrolase produced in Brazil.

[25] In view of the foregoing, no prior art document or disclosed knowledge provides for the obtention of a recombinant epoxide hydroxyase enzyme from a genetically modified E. coli bacterium by the pGTEH2 vector comprising the gene sequence of interest.

Brief Description of the Invention:

[26] This invention relates to the method of obtaining a recombinant epoxide hydroxyase enzyme whose gene was annotated from the total genome of the bacterium Streptomyces sp BI and later expressed in E. coli genetically modified by the pGTEH2 vector derived from pET29bi +). Also protected by this application are said vector and genetically modified E. coli bacteria (DSM 32387).

[27] This enzyme shows functional activity against a diverse scope of substrates, in addition to promoting enantioselective hydrolysis of racemic epoxides. It is capable of promoting the production of vicinal diols and enanti-enriched epoxides, compounds of great commercial interest for use as intermediates or end products in the fine chemicals, pharmaceutical and food industries.

Brief description of the figures:

[28] For a complete view of the object of this invention, reference figures are given as follows.

[29] Figures 1A and 1B correspond to the photograph of the plate with the recombinant epoxide hydrclase adrenaline test and corresponding gray scale, respectively.

[30] Figure 2 graphically represents the preference for epoxide hydrolase enzyme substrates (evaluation after 30 minutes of substrate enzyme incubation at a concentration of 200 µg rnL ^-1 enzyme and 15μ9 mlr ¹ substrate).

[31] Figure 3 graphically measures the DC spectrum measurements of the epoxide hydrolase enzyme.

[32] Figure 4 graphically represents the relative activity (%) of the epoxide hydrolase enzyme as a function of pH and temperature variation in DMSO.

[33] Figure 5 graphically shows the relative activity (%) of the epoxide hydrolase enzyme as a function of pH and temperature variation in acetonitrile (ACN).

[34] Figure 6 graphically compares the relative activities (%) of the epoxide hydrolase enzyme in ACN and DMSO.

[35] Figure 7 graphically shows the relative activity (%) of the epoxide hydrolase enzyme in DMSO at the different temperatures tested. Detailed Description of the Invention:

[363 This invention relates to the vector, recombinant microorganism, method of obtaining the recombinant epoxide hydrolase enzyme, the gene encoding for which was noted from the total genome of the bacterium Streptomyces sp BI, a plant endophytic actinobacterium Citrus reticulata and then the expression of the gene mentioned in E. coli is genetically modified by the pGTEH2 vector derived from pET29b (+).

(37} The method for obtaining the recombinant epoxide hydrolase enzyme consists of the steps of r

a) Amplification of the annotated bleph2 gene from the Streptomyces sp genome and cloning of the amplified fragment into pET29b (+) yielding plasmid pGTEH2;

b) Obtaining and confirming the nucleotide sequence and translation thereof;

ç) Obtaining recombinant Escherichia coli BL21 (DE3) strain transformed with plasmid pGTEH2;

d) Expression of the recombinant E. coJi-pGTEH2 strain for production of the epoxide hydrolase enzyme;

e) Purification of the enzyme.

[38] To start the method, the bleph gene ?. Related to the epoxide hydrolase enzyme of interest is amplified using the genomic DNA of Streptomyces sp. B1 as template and cloned into pET29b (+) digested between Ndel and EcoRI sites. To this end, the oligonucleotides (primers) EPH2_NdeI_F (5'-GACCATATC5ACGGACACACCCACCACC-3 ') and

F.PH2_EcoRI_R (5'-GACGAATTCCCGCAGCCCGTCCAGCC-3 ') as indicated in SEQ. ID 1 and 2 respectively. [39] Others promote the amplification of a 1032 nucleotide sequence as indicated in SEQ. ID No. 3, which translates to the recombinant 344 amino acid ar? / 8-epoxide hydrolase protein according to SEQ. ID No. 4.

[40] Expression of the epoxide hydrolase enzyme occurs in chemically competent cells, preferably from the E lineage. col i BL2KDE3) (UniProt Identifier EC03D,

with the vector defined in SEQ ID NO: 2 and resulting in the microorganism DSM 32387 containing the target gene sequence (SEQ ID NO: 1). .For this, the pET29b (+) vector is previously digested with the restriction enzymes Ndel and EcoRI and then the coding gene is ligated to the digested vector to maintain a histidine-caudaβ tail at the C-terminal portion. to facilitate the purification of the enzyme epoxide hydrolase enzyme obtained.

[41] As a result of the expression experiments, a pel let containing a mixture of proteins produced by the microorganism E was obtained. recombinant coli. Disruption of the bacterial wall to extract the enzyme epoxide hydrolase is therefore a fundamental procedure in the purification process.

[42] Thus, the purification process consists in resuspending the pellet at the ratio of 50 to 100 mg 20 ml buffer ^_1 Skip ¹ mmol sodium phosphate, 10 to 20 mirto1. ¹ go of imidazole and 500 mmol of ⁱ br sodium chloride pR 7.4. Subsequently, the solution is sonicated 7 times with a power of 0.02 Wrras, ice bath for 30 seconds and rest intervals of 1.5 minutes between each pulse. Optimal conditions were observed in the resuspended pellet at a rate of ^one 100 mg bm buffer 20 mmol L ^_i sodium phosphate, 10 mmol ^-J L and 500 mmol of imidazole ^L-l sodium chloride at pH 7, 4. The suspension is then centrifuged and the supernatant containing the soluble fraction (bacterial lysate) is transferred to another centrifuge tube.

[43] The purification of enzymes is performed on affinity columns, wherein the elution is carried out with the lysate buffer containing 20-500 mmol of imidazole Skip ^1, 2Q mmol sodium phosphate and 500 U ^-i ¹ mmol Go chloride desalination and concentration of the epoxide hydrolase enzyme are performed in millipore filter tubes. All affinity column purification steps were performed at room temperature (25 ° C).

[44] Once purified, the enzyme is used to promote epoxide hydrolysis, which occurs in aqueous media in the pH range 6 to 8, mainly in sodium phosphate / sodium chloride buffer, in the presence of 10% DMSO as co-solvent to aid in dissolving the substrates in the reaction medium. The reaction is kept under stirring and gentle heating at 30 ° C.

[45] The substrates employed in the hydrolytic process may be epoxides of formula (I) where Rx, Rs, R. and R-. they may be different, same or distinct substituents.

[46] Ri, _R2, R_t, substituents R4 can be hydrogen, aryl, alkyl, cycloalkyl, heterocycles, arylalkyl, alkoxides, aryloxides, acyl and acyl derivatives, among others.

Tests:

- Measurement of activity as epoxide hydrola3e:

[41] For the functional characterization of the enzyme epoxide hydrolysis, the epinephrine test was performed, which allows quantitative measurement of the enzyme's ability to promote hydrolysis of epoxide functionality to the corresponding 1,2-diol.

[48] This reaction is based on a back-titration process, whereby the biocatalytic reaction between the enzyme and the substrates dissolved in acetonitrile (ACN) is added a known amount of sodium periodate, a reagent that oxidizes 1,2-diodes. .is to form the corresponding aldehydes according to the protocol of Fluxa et al (Fluxa, VS, Wahler, D. & Reymond, J.-L. Enzyrae assay and fingerprinting of hydrolases with the red-chromogenic adrenaline test. Nat Protoc 3, 1270-1277 (2008)). ¹

[49] Quantification of this reaction is possible with the addition, after 30 minutes of incubation, of the L-adrenaline compound, which forms a red-colored adrenochrome with the remaining periodate in solution, which is readily detectable at 490 nm.

[50] Thus, if the enzyme promotes hydrolysis, the periodate in solution will be consumed in the oxidation of diols and the formation of adrenochrome will not be possible, leaving the solution colorless. In the opposite case the solution will turn red.

[51] The activity of the epoxide hydrosis enzyme was tested against 12 substrates (Table 1}. Selected compounds have different structural characteristics, which also allows to verify the comprehensiveness, the replacement patterns of the hydrolyzed substrates by the enzyme.

[52] Negative control corresponds to reaction of sodium periodate with L-adrenaline in the absence of enzyme and substrates (0% activity) and positive control corresponds to reaction in the presence of diol (1,2-cyclohexanediol, 100 % activity) are added to the assay plate (Figure IA) to allow quantification of enzyme activity (activity percentage).

[53] Spontaneous hydrolysis of all substrates was also measured as part of the test and subtracted from the enzyme-promoted hydrolysis data,

Table 1. Substrates used in the adrenaline test.

[54] For the enzyme epoxide hydrolase, concentrations 1 were tested; 2.5; 5; 10 ^' ; 15 and 20 μg ml ^-1 - of enzyme against the 12 selected substrates and the minimum concentration from which hydrolytic activity was observed was 2.5 μς ml ^-1 .

[55] With the data resulting from the coorimetric test, a grayscale map was constructed in which white is assigned to 0% activity (negative control), and black to 100% activity (positive control, as can be observed in Figure 1B). The gray scale revealed that an increase in enzyme concentration promotes a direct increase in catalytic activity (higher product formation) against the 12 tested substrates.

[56] The graph generated with the adrenaline test measurements shows high enzyme coverage once hydrolysis of the 12 substrates tested is observed (Figure 2). The enzyme epoxide hydrolase, however, has a greater preference for the substrates butyl glycidyl ether, phenyl glycidyl ether and 1,2-epoxyoctane.

[57] This higher enzyme efficiency can be attributed to the lower stereo impedance these substrates present which allows better positioning in the active site cavity to result in a more efficient hydrolysis process.

- Measurement of circular dichroism spectrum:

[58] Measurements of the circular dichroism spectrum predict the secondary structure of the epoxide hydrolase enzyme (Figure 3). These measurements were performed at wavelengths between 250 nm and 195 nm using a 125 µg mL ^{-1 solution} of the enzyme in 20 mmol Lr ¹ sodium phosphate buffer, pH 7 in 1 cm x 1 cm quartz cuvettes.

[59] The CDNN program (Btíhm., G., Muhr, R. & Jaenícke, R. Quantitative analysis of circular UV protein dichroism spectra by neural networks. "Protei ;: Eng Des Sei 5, 191-195 (1992 )) was used for data deconvolution, allowing to generate the percentages of each conformation type in the total secondary structure of the epoxide hydrolase enzyme (as Table 2 below).

Table 2. Contributions participating in the structure

secondary to the epoxide hydrolase enzyme

- Measurement of enzyme activity dependence on pH and temperature:

[60] During the adrenaline test, the absorbance values obtained from the measurements were converted to relative activity percentage, generating graphs representing the relative activity of the enzyme at different pHs. as a function of temperature (Figures 4 and 5).

[61] When DMSO was used as a co-solvent, the epoxide hydrolase enzyme reached maximum activity a. 45 ° C and pH 6.0. The pH value 6.0 was the best (relative activity over 70%) at temperatures from 25 to 45 ° C. When the co-solvent used was acetonitrile, the relative activity trend is very similar between all temperatures and pH tested, and it is only possible to give a slight preference for pH 5.0 and 9.0.

(62] Figure 6 shows the comparison between the enzymatic activities of the enzyme epoxide hydrolase when DMSO and ACN are used as co-solvents.These curves show that the biocatalytic reaction in the presence of DMSO occurs more efficiently, achieving a better performance. relative catalytic range 70-100% These data suggest that the ACN solvent acts as a possible inhibitor of the catalytic activity of the epoxide hydrolase enzyme.

[63] Thermostability, a measure of the enzyme's residual activity when subjected to elevated temperatures, was also defined by determining Tso (temperature required to reduce initial enzyme activity by 50% over a given period of time). epoxide hydrolase enzyme

is 50 ° C (Figure 7).

[64] Those skilled in the art will appreciate the knowledge presented herein and may reproduce the invention in the embodiments disclosed and in other embodiments within the scope of the appended claims.

Claims

1. Recombinant microorganism characterized in that it is DSM 32337.

Recombinant microorganism comprising at least SEQ ID NOil.

Microorganism according to Claim 2, characterized in that it is preferably E. coli.

4. A vector comprising SEQ ID NO: 1.

5. A vector comprising a DNA construct consisting of the sequence defined by SEQ ID NO: 2 operably linked to the transcriptional promoter and terminator sequences.

6. A vector comprising the vector pET29b (+), the gene sequence as defined in SEQ ^' ID NO 1 and a histidine tail.

Epoxide hydrolases enzyme purification method characterized in that it is from the microorganism defined in claims 1 to 3 and comprises the following steps:

-a) resuspension of the microorganism at a rate of 50 to ICO mg κ mL sodium phosphate 2Q iranol buffer ¹ , 10 to 20 runol L ^-1 imidazole and 500 mmol L ^-1 sodium chloride at pH 7 4;

-b) sonication of the solution obtained in (a) with a power of 0.02 Wrms, bathing at 4 ° C, for at least 7 30 second cycles and 1.5 minute rest intervals between each pulse;

-c) Centrifugation of the solution obtained in (b);

-d) Transfer supernatant obtained in (c). containing the. bacterial lysate for further purification on columns of affinity,

- e) such enzyme classification and concentration.

Epoxide hydrolases enzyme characterized in that it is obtained as defined in claim 7, is a recombinant Cf, β-epoxide hydrolase protein (SEQ ID NO 3) and promotes enantioselective hydrolysis of racemic epoxides.

Epoxide hydrolases enzyme characterized in that it is obtained from the microorganisms as described in claims 1 to 3, is a recombinant α, β-epoxide hydrolase protein (SEQ ID NO 3) and promotes enantioselective hydrolysis of epoxides. racemic.

9. Use of the enzyme epoxide hydrolase characterized by its application in obtaining enantiomerically pure vicinal epoxides and diols, which are compounds of great commercial interest as they are used as intermediates or end products in the fine chemical, pharmaceutical and food industries.