ES2678022T3 - Variant of L-arabinose isomerase to produce D-tagatose - Google Patents

Abstract

A variant of arabinose isomerase having an increased activity of converting D-galactose into Dtagatose, the variant of arabinose isomerase having a substitution of an amino acid other than phenylalanine for an amino acid at position 275 and a replacement of proline for an amino acid in position 469 of an amino acid sequence shown in SEQ ID NO: 1.

Description

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DESCRIPTION

Variant of L-arabinose isomerase to produce D-tagatose Technical field

The present invention relates to a variant of the L-arabinose isomerase of Thermotoga neapolitana DMS 5068, which is produced by protein engineering and has an increased activity of conversion of D-galactose into D-tagatose, a microorganism that expresses the variant of L-arabinose isomerase, and a method of producing D-tagatose from D-galactose using the microorganism. The variant is characterized by substitutions at positions 275 and 769 of the isomerase.

Background of the technique

D-tagatose is a monosaccharide that has sweetness equal to approximately 90% of that of sugar while it has properties, including low calones and non-caries properties. It can be used as a healthy sweetener without causing various adult diseases, unlike conventional sweeteners.

Due to such properties, D-tagatose is received as a sugar substitute and is known to have high commercial potential in the food market. However, tagatose is a rare sugar that is not abundantly present in nature, but is contained in dairy products or some plants in very small quantities. For this reason, in order for tagatose to be used as a functional low-calorie sweetener, the technology capable of producing tagatose must be developed.

D-tagatose was produced by a chemical isomerization process from D-galactose using Ca (OH) 2 as a catalyst by Arla Foods Ingredients Inc. in 2003, and has been marketed under the trade name "Gaiotagatose." However, it is known that the process of chemical isomerization is excellent in terms of isomerization conversion performance, but it has defects in the sense that recovery and purification are difficult and the process is complex and, therefore, the yield The total process is less than that of an enzymatic isomerization process.

L-arabinose isomerase (EC 5.3.1.5) is an enzyme that catalyzes a conversion isomerization reaction of L-arabinose into L-ribulose. Furthermore, it is known that L-arabinose isomerase converts not only L-arabinose (ie its natural substrate) into L-ribulose, but also D-galactose (ie a substrate structurally similar to L-arabinose) into D-tagatose.

The most important factor capable of contributing to an increase in the productivity of a production process of D-tagatose from D-galactose using L-arabinose isomerase is to develop an enzyme, which has good reactivity and can be applied successfully to the production process, through the modification of the isomerase. Because an increase in productivity plays a crucial role in maximizing profit by a decrease in production cost and business success, there has been a continuing need to modify arabinose isomerase.

Thermotoga neapolitana DSM 5068 arabinose isomerase which is a thermophilic microorganism has a very high thermal stability, but needs to improve further to ensure the economic productivity of arabinose isomerase, which is comparable to that of glucose isomerase.

WO 2014/137148 (September 2014) describes a variant of L-arabinose isomerase derived from Thermotoga neapolitana and a method for producing D-tagatose from D-galactose using the enzyme or a Corynebacterium microorganism that expresses the same.

Generally, the methods of producing varying enzymes to increase the activities of enzymes or to produce active enzymes for new substrates are largely divided into a random mutagenesis method and a rational design method. The random mutagenesis method is widely used, because they can be used without requiring special information about a target enzyme. However, a screening system capable of processing a large number of variant enzymes is required. On the other hand, the modification of enzymes by rational design does not require a special screening system, because it produces only a limited number of variant enzymes. However, in the case of rational design, the factors that determine the catalytic mechanism, substrate binding property or substrate specificity of a target enzyme should be investigated in detail.

Divulgation

Technical problem

Therefore, the applicant has attempted to increase the substrate specificity of the Thermotoga neapolitan DSM 5068 L-arabinose isomerase for D-galactose by changing the three-dimensional structure of the L-arabinose

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isomerase based on protein engineering, molecular modeling and analysis of the enzymatic reaction mechanism so that arabinose isomerase that has the potential to produce tagatose can have the ability to produce industrially tagatose.

It is an objective of the present invention to provide a variant of arabinose isomerase from Thermotoga neapolitana DSM 5068, which has increased conversion activity, and a gene nucleotide sequence encoding the variant.

Another objective of the present invention is to provide a recombinant vector comprising the gene nucleotide sequence, and a microorganism of the Corynebacterium genus transformed with the recombinant vector.

A further objective of the present invention is to provide a method of producing D-tagatose from D-galactose using either the arabinose isomerase variant or the transformed microorganism or a culture of the transformed microorganism.

Technical solution

The present invention provides variants of arabinose isomerase, polynucleotides, vectors, microorganisms and methods as defined in the appended claims.

To achieve the above objectives, the present invention provides a variant of arabinose isomerase that has an increased activity of converting D-galactose to D-tagatose, the arabinose variant isomerase having a proline substitution for leucine at position 469 and a substitution of an amino acid other than phenylalanine for an amino acid at position 275 of the thermotoga neapolitan ismrase Arabometose DSM 5068, and a gene nucleotide sequence encoding the arabinose isomerase variant.

The present invention also provides a recombinant vector comprising the gene nucleotide sequence, and a microorganism of the Corynebacterium genus transformed with the recombinant vector.

The present invention also provides a method of producing D-tagatose from D-galactose using the arabinose isomerase variant.

The present invention also provides a method for producing D-tagatose, the method comprising culturing the transformed microorganism of the invention.

Advantageous effects

According to the present invention, the production of D-tagatose can be increased using a microorganism of the genus Corynebacterium transformed with the new variant of arabinose isomerase or the gene nucleotide sequence encoding the variant of arabinose isomerase, thereby reducing the cost of Production and infrastructure investment.

Description of the drawings

Figure 1 shows a composite structure of the active site and manganese ion cofactor of the L-arabinose isomerase of Thermotoga neapolitana.

Figure 2 shows the sites of L-arabinose and D-galactose, which bind to the active site of L-arabinose isomerase, and the functional groups of L-arabinose and D-galactose, which interact with the active site of L-arabinose isomerase. It is shown that carbon 6 of D-galactose causes steric hindrance with the rest of phenylalanine at position 275 of L-arabinose isomerase.

Figure 3 shows the results obtained by replacing the 275 moiety of L-arabinose isomerase with saturated mutagenesis and comparing the relative activity of the variants, selected by the cystem-carbazole method, through a color development reaction. A considerable number of variants were found that showed an activity greater than that of a control.

Figure 4 shows the structures of selected variants (F275V / L469P, F275M / L469P, and F275I / L469P), provided by a molecular modeling technique.

Figure 5 shows the results of the SDS-PAGE analysis of isolated and purified variants of L-arabinose isomerase from Thermotoga neapolitana.

Figure 6 shows the results of the evaluation of the relative activity of selected variants at varying temperatures to determine the optimum temperatures of the variants.

Figure 7 shows the measurement results of the thermal stability of selected variants at 95 ° C as a function of time.

Figure 8 shows the results of the evaluation of the relative activity of selected variants to determine the dependence of the variants on manganese.

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Mode for the invention

A variant of the arabinose isomerase is described from Thermotoga neapolitana DSM 5068, which has increased conversion activity, and a gene nucleotide sequence encoding the variant.

The present invention provides a variant of arabinose isomerase that has an increased activity of converting D-galactose to D-tagatose, the variant of arabinose isomerase having a proline substitution for leucine at position 469 and a substitution of an amino acid other than phenylalanine. for an amino acid at position 275 of the Arabinose isomerase of Thermotoga neapolitana DSM 5068, and a gene nucleotide sequence encoding the arabinose isomerase variant.

As used herein, the expression "arabinose isomerase that converts D-galactose to D-tagatose" means an enzyme that catalyzes an isomerization reaction that uses D-galactose as a substrate to produce D-tagatose.

The arabinose isomerase variant according to the present invention preferably has a substitution of an amino acid having a non-polar aliphatic side chain for an amino acid at position 275 of the arabinose isomerase.

As used herein, the term "amino acid having a non-polar aliphatic side chain" means alanine, valine, isoleucine, leucine, methionine or proline.

Preferably, the amino acid at position 275 of the arabinose isomerase is substituted with any amino acid selected from the group consisting of valine, methionine and isoleucine.

Preferably, the arabinose isomerase variant according to the present invention also has a proline substitution for leucine at position 469 of the arabinose isomerase.

As used herein, the term "substitution" means substitution of an amino acid in a specific position with another amino acid to produce a mutation. A suitable mutagenesis method can be any method that can be used by those skilled in the art for this purpose. Particularly, the mutagenesis method may be a saturated mutagenesis method, a random mutagenesis method or a directed mutagenesis method ("Evolutionary molecular engineering based on RNA replication", Pure Appl. Chem. 1984, 56: 967-978; "Promoters selected from random DNA-sequences", Proc. Natl. Acad. Sci. USA, 1986, 83: 7.405-7.409; "Mutants generated by the insertion of random oligonucleotides into the active-site of the beta-lactamase gene", Biochemistry 1989, 28: 5,703-5,707).

Preferably, the arabinose isomerase variant according to the present invention has an amino acid at position 275, substituted using the saturated mutagenesis method, and leucine at position 469, substituted using the directed mutagenesis method.

In one embodiment of the present invention, random mutagenesis was performed using wild-type arabinose isomerase (which has an amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence of SEQ ID NO:

6) Thermotoga neapolitana DSM 5068 as a mold, thereby obtaining variants that have improved enzymatic characteristics and genetic information about the variants. The variants were analyzed together, and as a result, it was found that variation in the amino acid sequence of the C-terminal region of the arabinose isomerase causes an increase in enzymatic activity.

In addition, the amino acids in the C-terminal region that constitute the active site of each of the wild-type arabinose isomerase and the arabinose isomerase variant were analyzed by molecular modeling. As a result, the variant was found to be a substitution of proline for leucine at position 469 of the wild-type arabinose isomerase, and thus the beta sheet disappears at position 18 of the protema, and the angle and the main chain inclined when the three-dimensional structure of the alpha helix at position 17 moved towards the protein body, indicating that the structure of the protein was changed.

Based on the results described above, leucine at position 469 of wild-type arabinose isomerase was substituted with proline using directed mutagenesis, thereby preparing a variant (L469P) (which feeds an amino acid sequence of SEQ ID NO: 2 and a nucleotide sequence of SEQ ID NO:

7). The variant was incubated and then its activity was measured, and as a result, it was found that the variant showed increased activity for the galactose substrate compared to the wild-type arabinose isomerase.

In one embodiment of the present invention, to obtain an enzyme that has increased conversion activity from the arabinose isomerase variant (L469P), the main residues of the substrate binding region and the active region of the enzyme were selected, and the reaction mechanism was estimated, thereby selecting the amino acid at position 275. Mutations were introduced at position 275 using the method of

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saturated mutagenesis, and the variants were screened, thereby selecting variants that fear increased conversion activity.

The selected variants were sequenced, and as a result, the variants were found to substitute valine (L469P / F275V), methionine (L469P / F275M) and isoleucine (L469P / F275I), respectively, for the amino acid at position 275. Each one of the three variants were transformed into microorganisms of the genus Corynebacterium, and an isomerization reaction was carried out by culturing the microorganisms. As a result, it was found that all three variants showed increased activity compared to the L469P variant that had a proline replacement for leucine at position 469.

To examine the characteristics of the three variants, the arabinose isomerases expressed were isolated from microorganisms of the Corynebacterium genus cultured under the conditions described above. The purified proteins were found to show arabinose isomerase activity for D-galactose. In addition, it was found by SDS PAGE that purified proteins fear molecular weights consistent with that of arabinose isomerase.

Using the purified variant enzymes, the optimum temperature, thermal stability, the use of metal ion and the enzymatic activity of the variant enzymes were measured. As a result, it was found that the three variants showed the highest activity at 75 ° C, but the thermal stability somewhat decreased, the use of the metal ion did not differ significantly, and the specific activities of the arabinose isomerase variants were approximately 5 , 5 times greater (F275V / L469P), 5 times greater (F275M / L469P) and 3.9 times greater (F275I / L469P), respectively, than those of the L469P variant.

In one embodiment, the present invention provides a genetic nucleotide sequence encoding the arabinose isomerase variant.

The gene nucleotide sequence may be one selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

Arabinose isomerase variants are described which may have gene nucleotide sequences encoding proteins that have a homology of at least 80%, preferably at least 90%, more preferably at least 95%, and particularly preferably at least 97%, at sequences of amino acids of SEQ ID NO: 3 to 5, provided that the arabinose isomerase activities of the arabinose isomerase variants of the present invention can be maintained or increased. Most preferably, arabinose isomerase variants according to the present invention have gene nucleotide sequences shown in SEQ ID NO: 8 to 10.

As used herein, the term "homology" refers to the identity between two amino acid sequences. Homology can be determined using methods well known to those skilled in the art, for example, BLAST 2.0 which calculates parameters such as punctuation, identity or similarity.

Variants encoding arabinose isomerase variants that can hybridize to polynucleotides shown in SEQ ID NO: 8 to 10 or polynucleotide probes under strict and normally functioning conditions are described.

As used herein, the term "strict conditions" means the conditions that allow specific hybridization between polynucleotides. For example, hybridization is performed in a hybridization buffer (3.5> SSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH2PO4 (pH 7), 0.5% SDS, 2 mM EDTA) at 65 ° C ("Molecular Cloning", A Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989) or "Current Protocols in Molecular Biology" (FM Ausubel et al., Editors, John Wiley & Sons, Inc., New York). Here, SSC is 0.15 M sodium chloride / 0.15 M sodium citrate (pH 7). After hybridization, the membrane that has DNA transferred to it is washed with 2> SSC at room temperature and then washed with 0.1 to 0.5> SSC / 0.1xSDS at a temperature of 68 ° C.

In one embodiment, the present invention provides an isomerase variant having any amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. Specifically, the variant is an arabinose mutant isomerase that has a substitution of any amino acid selected from the group consisting of valine, methionine and isoleucine for an amino acid at position 275 and that also has a proline substitution for an amino acid at position 469.

A variant is described which may be an artificial mutant or mutant encoding a polypeptide having an amino acid sequence comprising a substitution, deletion, insertion or addition of one or more amino acids in one or more positions other than positions 275 and 469 of the amino acid sequence of any one of SEQ ID NO: 3 to 5, provided that the activity of arabinose isomerase can be maintained or increased.

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As used herein, the term "various amino acids" means 2 to 20 amino acids, preferably 2 to 10 amino acids, and more preferably 2 to 5 amino acids, depending on the type or positions of the amino acid residues in the three-dimensional structure of the Protect

In addition, the described substitutions, deletions, insertions, additions or inversions of amino acids can include naturally occurring mutants or artificial variants, based on individual differences and / or species differences of the microorganism expressing arabinose isomerase.

In one embodiment, the present invention also provides a recombinant vector comprising a polynucleotide operably linked thereto.

As used herein, the term "vector" refers to a DNA construct that contains the nucleotide sequence of a target protein coding gene operably linked to a suitable regulatory sequence to be able to express the target gene. in a suitable host cell. The regulatory sequence includes a promoter capable of initiating transcription, any operator to regulate this transcription, a sequence encoding a mRNA ribosome binding site, and a sequence to regulate the termination of transcription and translation. Once transformed into a suitable host, the vector can replicate or function independently of the host genome, or it can be integrated into the genome itself.

The vector used in the present invention is not specifically limited and can be any vector known in the art, as long as it can be replicated in a host. Examples of commonly used vectors may include natural or recombinant plasmids, cosmetics, viruses and bacteriophages.

In addition, the vector used in the present invention is a vector capable of transforming host cells, to insert the polynucleotide encoding the target protein into the host cell chromosome. Specific examples of the vector include, but are not limited to, a shuttle vector pECCG112 that can self-replicate in both directions in E. coli and Coryne bacteria (Kap-Soo, Noh, Kor. Jour. Microbiol. July 1991, p 149-154).

Also, the polynucleotide encoding the endogenous target protein on the chromosome can be replaced with a new polynucleotide with a vector for insertion into the bacterial chromosome. The insertion of the polynucleotide into the chromosome can be performed by any method known in the art, for example, homologous recombination.

Because the vector of the present invention can be inserted into the chromosome by homologous recombination, it can also comprise a selection marker to confirm its insertion into the chromosome. The selection marker is used to select a cell transformed with the vector, that is, to confirm the insertion of the target polynucleotide. The selection marker that is used in the present invention can be selected from markers that provide selectable phenotypes, such as drug resistance, auxotrophic, resistance to cytotoxic agents, or surface protection expression. Only cells expressing the selection marker are capable of surviving or displaying different phenotypes under the environment treated with the selective agent, and thus transformed cells can be selected.

In one embodiment, the present invention also provides a microorganism of the genus Corynebacterium transformed with the recombinant vector.

As used herein, the term "transformation" means introducing a vector comprising a polynucleotide encoding a target protein in a host cell to be able to express a protein encoded by the polynucleotide in the host cell. Transformed polynucleotides include all genes inserted into the host cell chromosome or located outside the chromosome, provided they can be expressed in the host cell. In addition, polynucleotides include DNA and RNA, which encode the target protein. As long as the polynucleotide can be introduced into the host cell and expressed in it, the gene can be introduced in any way. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette that is a polynucleotide construct that includes all the elements for gene expression. The expression cassette includes a promoter that operably binds to the gene, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self replication. The polynucleotide can also be introduced into the host cell itself, and operably linked to the sequence necessary for expression in the host cell.

The microorganism of the present invention includes any of the prokaryotic microorganisms and eukaryotic microorganisms, provided that the isomerase variant can be expressed. For example, a microorganism belonging to the Escherichia genus, the Erwinia genus, the Serratia genus, the Providencia genus, the Corynebacterium genus or the Brevibacterium genus can be included. Preferably, the microorganism of the present invention is a microorganism belonging to the genus Corynebacterium. More preferably, it is Corynebacterium glutamicum.

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In one example of the present invention, Corynebacterium glutamicum that has the ability to produce L-amino acid was transformed with a vector that feeds a nucleotide sequence of each of SEQ ID NO: 8, 9 and 10, and the strains constructed are they named Corynebacterium glutamicum pFIS-1-TNAI-2, pFIS-1-TNAI-3 and pFIS-1- tNaI-4, respectively, and deposited in the "Korean Culture Center of Microorganisms" (361-221, Honje 1-dong , Seodaemun-gu, Seoul, South Korea), an international deposit authority, on February 14, 2013 under accession numbers KCCM11378P, KCCM11379P and KCCM11380P, respectively. The present description also relates to a culture of a microorganism of the genus Corynebacterium transformed with a recombinant vector of the invention.

The culture may be an undiluted culture comprising the cells of the microorganism or it may be a microbial cell obtained by removing the culture supernatant or concentrating the culture. A composition of the medium for culturing the microorganism can comprise not only conventional components required for the cultivation of microorganisms of the Corynebacterium genus, but also components that have a synergistic effect on the growth of microorganisms of the Corynebacterium genus, and can be readily selected by experts. in the technique In addition, the culture may be in a liquid or dry state, and methods for drying the culture include, but are not limited to, air drying, natural drying, spray drying and lyophilization.

The microorganism of the genus Corynebacterium according to the present invention can be cultured by any conventional method. Specifically, the microorganism can be cultured by inoculating it in a medium that contains totally or partially sucrose or glucose as a carbon source. The cultivation process can be performed under suitable culture media and conditions known in the art. This culture process can be easily modified by any person skilled in the art depending on the type of strain selected. Examples of the cultivation process include, but are not limited to, discontinuous culture, continuous culture, and discontinuous feed culture. The medium that is used in the cultivation of the microorganism of the present invention should properly meet the requirements of the microorganism of the present invention.

Specifically, the medium used in the present invention contains sucrose or glucose as the main source of carbon. In addition, molasses that contain a high concentration of sucrose can also be used as a carbon source. In addition, suitable amounts of various carbon sources can be used. Preferably, purified glucose is used. Examples of nitrogen sources that can be used in the present invention include organic nitrogen sources such as peptone, yeast extract, meat extract, malt extract, maceration liquor, and soybean meal, and inorganic nitrogen sources. such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Preferably, peptone is used. These nitrogen sources can be used alone or in combination. The medium may contain monobasic potassium phosphate, dibasic potassium phosphate and the corresponding sodium-containing salts, as phosphorus sources. In addition, the medium may contain a metal salt such as magnesium sulfate or ferrous sulfate. In addition, the medium may contain suitable amino acids, vitamins and precursors. These means or precursors can be added to the medium in a discontinuous or continuous manner.

The culture medium is generally maintained at a temperature ranging from 27 ° C to 37 ° C, and preferably from 30 ° C to 37 ° C. The culture of the microorganism can be continued until the desired level of the protein is obtained. Preferably the cultivation period is 10 to 100 hours.

The present invention provides a method for producing D-tagatose, the method comprising reacting a solution containing D-galactose with a metal ion source selected from the group consisting of manganese ions, magnesium ions and zinc ions in the presence of the arabinose isomerase variant that has a conversion activity of D-galactose into D-tagatose, the transformed microorganism of the genus Corynebacterium, or a culture of the transformed microorganism of the genus Corynebacterium, thereby producing D-tagatose.

To allow a substrate to be introduced into the transformed microorganism of the Corynebacterium genus, or a culture of the transformed microorganism of the Corynebacterium genus, the microbial cells obtained by centrifuging the transformed microorganism or the culture can be treated with a surfactant, lysozyme or xylene.

Preferably, microbial cells can be treated with 0.1% POESA.

The solution containing D-galactose, which is used in the present invention, can be selected from the group consisting of purified D-galactose, D-galactose derived from biomass, and D-galactose obtained by lactose hydrolysis, but not Limit them.

Arabinose isomerase is a metalloenzyme that uses a metal ion as a cofactor. The metal ion can be selected from the group consisting of manganese ions, magnesium ions and zinc ions, but it is not limited, and it can be any metal ion that can bind to the isomerase to perform an isomerization reaction. Specifically, the manganese ion source includes manganese chloride; the ion source of

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Magnesium includes magnesium chloride; and the zinc ion source includes zinc chloride; however, the scope of the present invention is not limited to these sources of metal ion.

A reaction solution to produce D-tagatose contains a buffer system to maintain the pH, such as Tris buffer or phosphate buffer. Preferably, it contains Tris buffer (pH 6.5 to 7.5). Manganese chloride, magnesium chloride or zinc chloride is contained at a concentration of 0.1 mM to 10 mM, and preferably 1 mM to 5 mM. The D-galactose substrate is added in an amount of 1 to 300 g / l, and preferably 18 to 300 g / l, and the isomerization reaction is induced at a temperature of 60 ° C to 95 ° C, preferably 70 ° C at 80 ° C, and more preferably 72 ° C to 78 ° C, thereby producing D-tagatose.

Later in this document, the present invention will be described in more detail in reference to the examples.

Examples

Example 1: Modeling of the Arabinose protein isomerase of Thermotoga neapolitana for the construction of the enzyme that has increased conversion activity and selection of the main amino acid mutations

Because the three-dimensional structure of the wild-type Arabinose isomerase of Thermotoga neapolitana DSM 5068 has not yet been found, the prediction of the three-dimensional structure was made using a molecular modeling technique that uses, as a model, the arabinose isomerase of Escherichia coli whose Three-dimensional structure has already been found and it has a high sequence homology. For the prediction of the three-dimensional structure, a comparative modeling technique was used, and a structural model was obtained using an APM module (Tripos, USA) in a molecular modeling package.

The comparative modeling technique is a method that is the most frequently used to predict the three-dimensional structures of the proteins. If the amino acid sequence of a desired protein is very similar to the sequence of another protein whose three-dimensional structure has been known, the three-dimensional structure of the desired protein can be easily predicted using the comparative modeling technique, and in this case, the precision The prediction is very high.

The structure of the E. coli arabinose isomerase used in this Example was a type-like structure registered as 2HXG.pdb in the "Protein Data Bank" (PDB).

From the modeling results, it was predicted that wild-type arabinose isomerase (which has an amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence of SEQ ID NO: 6) of Thermotoga neapolitana DSM 5068 is very similar to E. coli arabinose isomerase in terms of not only the sequence of

nucleotides, but also bi- and three-dimensional structures. Several studies on E. arabinose isomerase.

J 2+

coli gave information about the substrate binding site, the cofactor manganese ion (Mn) and the main amino acids (Manjasetty & Chance, J. Mol. Biol., 2006. 360: 297-309). Based on this information, the main isomerase remains of Thermotoga neapolitana DSM 5068 were selected.

For the analysis of the main amino acid residues, sequence alignment, molecular coupling simulation, and reaction mechanism analysis were performed. Sequence alignment was performed using the clustalW algorithm (//
www.ebi.ac.uk/Tools/msa/clustalw2/) based on the information on the sequences of 10 different L-arabinose isomerases and the arabinose isomerase of Thermotoga neapolitana.

The results of the sequence alignment suggest that the sequences of the metal binding site and the active site of the Arabinose isomerase of Thermotoga neapolitana DSM 5068 were very well preserved, like those of other isomerases. The thermotoga neapolitan isomerase DSM 5068 has an active site comprising amino acid residues E302 (glutamic acid at position 302), E329 (glutamic acid at position 329), H346 (histidine at position 346) and H445 (histidine at position 445) and manganese ions (Figure 1), and amino acid residues E302, E329, H346 and H445 have effects on the binding of the manganese ion. In particular, it was predicted that the remains of E302 and E329 were the most important factors that promote an isomerization reaction (Manjasetty & Chance, J. Mol. Biol., 2006. 360: 297-309).

It was assumed that the substrate specificity of the Arabinose isomerase of Thermotoga neapolitana was determined according to the size and morphology of the active site and the characteristics of the amino acid residues of the active site. Through the molecular coupling simulation (surflexDock; Tripos, USA) performed using the original L-arabinose substrate, the important residues in the substrate recognition were selected. In addition, through the analysis of the reaction mechanism (Adrian J. Mulholland, Drug Discov. Today. 2005. 10 (20): 1393-402), the most suitable binding position of D-galactose was selected. Based on this selection, an amino acid residue was selected that had a high possibility of interfering with the binding between the active site of arabinose isomerase and D-galactose.

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The amino acid at position 275 consisted of phenylalanine which has an aromatic side chain and is relatively large in size and less flexible, and it was predicted that the amino acid at position 275 caused steric hindrance with carbon 6 of D-galactose (Figure 2 ). Due to L-arabinose, a pentose, it basically shows the same structure as D-galactose, except that the number of carbon atoms is smaller than that of D-galactose by one, it was anticipated that the reactivity of arabinose D-galactose isomerase can be significantly increased even by only the substitution of the amino acid at position 275 with another amino acid.

As amino acids capable of replacing phenylalanine, valine, methionine and isoleucine were selected, which have polarity to that of phenylalanine, are relatively small in size and highly flexible, and thus are predicted to have less effect on the overall structure of arabinose isomerase while that the repulsion with carbon 6 of D-galactose is minimized.

Because these selected amino acids are composed of a non-polar aliphatic chain, unlike phenylalanine which comprises an aromatic chain, it was anticipated that these amino acids could substitute for phenylalanine while structurally free.

Variant structures (which have amino acid sequences of SEQ ID NO: 3 to 5 and nucleotide sequences of SEQ ID NO: 8 to 10) that have point mutations were predicted by a molecular modeling technique. As a result, it was predicted that the effect of mutations on the overall structure of arabinose isomerase will be negligible.

In addition, through random mutagenesis using wild-type arabinose isomerase from Thermotoga neapolitana DSM 5068 using a template, variants were obtained that showed improved enzymatic characteristics and genetic information about the variants. The variants were analyzed together, and as a result, it was found that the variation in the amino acid sequence of the C-terminal region of the arabinose isomerase is subject to an increase in enzymatic activity.

The phenomenon that the chain structure of the C-terminal region of arabinose isomerase is very changed was analyzed by a molecular prediction technique. As a result, it was anticipated that the reactivity of arabinose isomerase with D-galactose can be significantly increased even by only the replacement of proline for leucine at position 469 of wild-type arabinose isomerase.

Example 2: Preparation of the variants designed for arabinose isomerase

(1) Replacement of proline for leucine at position 469

Leucine at position 469 of the wild-type Arabinose isomerase of Thermotoga neapolitana DSM 5068 was replaced with proline by a method of directed mutagenesis using specific primers.

As primers, the N-terminal primer (SEQ ID NO: 13) and a C-terminal primer (SEQ ID NO: 14) were used, which are oligonucleotides having nucleotide sequences complementary to a mutation. When using a plasmid DNA as a template, a plasmid that feeds a new mutation in a test tube was amplified and synthesized and then the wild-type DNA was separated by cleavage with a restriction enzyme Dpn I. In other words, the Wild-type DNA used as a template was DNA isolated from E. coli and was digested with Dpn I that recognizes and cleaves Gm6ATC, but the DNA synthesized in the test tube was not cleaved.

The DNA was transformed into E. coli DH5 alpha to obtain a variant gene, and then the nucleotide sequence of the variant gene was analyzed to confirm that the mutation occurred properly. The variant gene was transformed into Corynebacterium glutamicum ATCC 13032 to produce a recombinant strain that was then called L469P. The recombinant strain was used as a control.

(2) Substitution of amino acids other than phenylalanine for the amino acid at position 275

To induce an additional mutation in the L469P constructed variant, an isomerase cloned vector was subjected to saturated mutagenesis using a pair of primers that contain a mutation.

The designed primer pair was designed so that the amino acid codon at position 275 is replaced with NNS (N: A, T, G or C; and S: G or C). The variants obtained by this method may comprise 20 types of amino acids (SEQ ID NO: 11 and SEQ ID NO: 12). Such variants provided simple colonies by transformation, and changes in activity by the 20 amino acid mutations in the corresponding positions can be confirmed without loss by screening simple colonies and analyzing their activities.

Specifically, to produce a library comprising 20 types of amino acids, the polymerase chain reaction (PCR) was carried out using 100 pl / ml of a plasmid pECCG117-CJ1-TNAI_L469P (publication open to public inspection of Korean patent No. 10 -2010-0016948) as a mold and direct and reverse primers. The PCR reaction was performed under the conditions shown in Tables 1 and 2 below. The library obtained

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by PCR reaction, it was transformed into a K12 DH5a strain of E. coli to produce colonies. The plasmid used was expressed in the E. coli strain and its activity was tested in the E. coli strain, because it could be replicated and expressed in both E. coli and Coryne type bacteria.

Isomerase activities expressed from 110 colonies obtained as described were analyzed by a cystem-carbazole method (Dische, Z., and E. Borenfreund., “A New Spectrophotometric Method for the Detection and Determination of Keto Sugars and Trioses ”, J. Biol. Chem., 192: 583-587, 1951), and as a result, a number of clones can be found that have an activity greater than that of the control (L469P) (Figure 3).

Among them, 10 colonies measured to have the highest activity were selected and sequenced. As a result, as expected, it could be seen that the activity was higher for valine, methionine and isoleucine (amino acid sequences of SEQ ID NO: 3 to 5 and nucleotide sequences of SEQ ID NO: 8 to 10). In addition, no mutations were found in amino acids other than amino acid at position 275, suggesting that the rest of phenylalanine at position 275 works to inhibit the reactivity of D-galactose.

To examine whether the mutation has any effect on isomerase, the structures of the variants were predicted by a molecular modeling technique (Figure 4). The results of the prediction of the structures indicated that all three amino acids substituted for phenylalanine at position 275 without causing significant changes in the two- and three-dimensional structures. Therefore, it can be anticipated that the reactivity of isomerase with incremental D-galactose without significant increases in thermal stability and other production indices.

Variants were transformed into Corynebacterium glutamicum ATCC 13032 to produce recombinant strains. The recombinant strains were named "Corynebacterium glutamicum pFlS-1-TNAI-2, pFlS-1- TNAI-3 and pFIS-1-TNAI-4", respectively, and were deposited in the "Korean Culture Center of Microorganisms" (361221, Honje 1-dong, Seodaemun-gu, Seoul, South Korea), an international deposit authority, on February 14, 2013 under accession numbers KCCM11378P, KCCM11379P and KCCM11380P, respectively.

Table 1: PCR of saturated mutagenesis

 Composition of the reaction solution

 Quantity (pl) added

 10x PCR buffer (pfu-ultra)

 5

 dNTP (2.5 mM)

 5

 pCJ1-TNAI L469P

 one

 TNAI275 F primer

 one

 TNAI275 R primer

 one

 pfu-ultra

 one

 doubly distilled water

 Up to 50 pl

Table 2: PCR reaction conditions

 Stage

 Temperature Time Cycles

 Initial Denaturation

 95 ° C 5 min 1

 Denaturation

 95 ° C 45 s 18

 Alignment

 60 ° C 45 s

 Extension

 68 ° C 18 min

 Final extension

 72 ° C 10 min 1

Example 3: Expression of arabinose isomerase variants in microorganisms of the genus Corynebacterium

To measure the degree of increases in the activities of the selected variants and the applicability of the variants to the current production of D-tagatose, the three variants were expressed in microorganisms of the genus Corynebacterium, and studies were conducted on indices related to productivity and the production.

The three variants selected in Example 2 were transformed into Corynebacterium glutamicum ATCC 13032 to produce recombinant strains. These recombinant strains were cultured in media (20 g / l of glucose, 10 g / l of poly peptone, 10 g / l of yeast extract, 10 g / l of ammonium sulfate, 5.2 g / l of KH2PO4, 10.7 g / l of K2HPO4, 0.5 g / l of MgSO4, 1.5 g / l of urea, 1.8 mg / l of D-biotin, 9 mg / l of thiamine, 9 mg / l of Ca-pantothenic, 60 mg / l of niacinamide) containing 50 pg / ml of kanamycin at 30 ° C for 20 hours to induce the expression of recombinant arabinose isomerase mutants.

To measure the activity of the expressed arabinose isomerases, the cultures were centrifuged at 8,000 g force for 10 minutes to collect the bacterial cells, which were then suspended again in 50 mM Tris-HCl buffer (pH 7.5). Suspended cells were treated with 0.1% POESA at room temperature for 1 hour to weaken the cell wall. Then, centrifugation was performed again under the conditions described above to collect the cells which were then suspended again in a mixed solution of 300 g / l of D-galactose, 5 mM manganese chloride and Tris-HCl 50 mM (pH 7.5) for a concentration of 4% (w / v). The suspension was allowed to react at 75 ° C for 1 hour, and HPLC analysis was performed (WATERS

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HPLC, EMPOWER system, 300 mm column of 6.5 l SugarPak ID of WATERS, refractive index detector 2414) for the quantification of D-galactose and D-tagatose.

The results of one hour of the reaction indicated that the L469P control produced 35 g / l of D-galactose, while the F275V / L469P variant showed a productivity of 121 g / lh, the F275M / L469P variant showed a productivity of 117 g / lh, and the F275I / L469P variant showed 101 g / lh.

Example 4: Isolation of arabinose isomerase variants expressed in Coryne bacteria

200 ml of the recombinant strain comprising each of the three variants (F275V / L469P, F275M / L469P, and TNAI-F275I / L469P) whose activities were confirmed, and the L469P control, were seeded in a 2L flask under the same conditions as those described in Example 3. To examine whether arabinose isomerases were expressed, isomerase activities were measured using a portion of each of the cultures under the same conditions described in Example 3.

Bacterial cells obtained from each of the cultures were resuspended in 20 mM Tris-HCl buffer (pH 7.5) containing 0.1 mM manganese chloride, and lysed using a high pressure cell T series homogenizer (4.0 kW: Constant systems, GB). To separate endogenous proteins other than isomerases, the cells were heat treated at 75 ° C for 20 minutes. The residues of the heat treated cells were separated by centrifugation at 8,000 g force for 10 minutes and then the residues and cell lipids were further separated by ultracentrifugation (Optima L-80 XP ultra-centrifugal from BECKMAN COULTER) at 60,000 g force .

The resulting cell extract was purified by anion exchange chromatography (Mono QTM 10 / 100GL, GE Healthcare). The purified cell extract was previously calibrated with a binding solution (50 mM NaCl, 0.1 mM manganese chloride, 20 mM Tris-HCl (pH 7.5) and then an excess amount of the cell extract was bound and fractionating while increasing the ratio of an eluent solution [1 M NaCl, 0.1 mM manganese chloride, 20 mM Tris-HCl (pH 7.5). Fractions showing activity for the D-galactose substrate were selected through the cystem-carbazole-sulfuric acid method and then analyzed by SDS-PAGE.

As a result, it can be seen that the purified protein has a molecular weight of approximately 56 kDa, which is consistent with the known molecular weight of Thermotoga neapolitana arabinose isomerase. A fraction having the highest degree of purification was selected by SDS-PAGE, and a high concentration of NaCl was separated therefrom using a PD-10 desalination column (GE Healthcare). The resulting purified enzyme was analyzed by SDS-PAGE (Figure 5). The isolated and purified protein was quantified using a Bradford assay, and BSA (bovine serum albumin) was used as a standard protein.

Example 5: studies on the characterization of arabinose isomerase variants

It was found that the three variants of arabinose isomerase prepared in the previous example showed significantly increased activities compared to the variant that had a substitution of proline for leucine at position 469. Based on this discovery, experiments were conducted on the parameters related to reaction conditions that have effects on the current production of D-tagatose.

5-1: Study on the optimum temperature

The arabinose isomerase of Thermotoga neapolitana, a thermophilic enzyme, has relatively high thermal stability and optimum temperature. The suitable temperature to produce D-tagatose from D-galactose is between 55 ° C and 75 ° C. At a temperature below 55 ° C, problems resulting from contamination with heterologous strains will occur, and at a temperature greater than 75 ° C, the problems will arise in terms of the stability of the D-tagatose produced. The wild type arabinose isomerase or the L469P control, the optimal reaction temperature of which is 85 ° C, is a problem in that it shows relatively low activity at a temperature at which the production process is applicable.

To examine the optimal temperatures of the arabinose isomerase variants purified in Example 4, each of the purified enzymes was added to 100 mM D-galactose substrate, and its activity was measured in 50 mM Tris-HCl buffer (pH 7.5 ) containing 1 mM manganese chloride (MnCh) at a temperature ranging from 60 ° C to 90 ° C at intervals of 5 ° C.

The measurement of the activity was done by a method of cystem-carbazole. As shown in Figure 6, the results of the measurement of enzymatic activity as a function of temperature indicated that the optimal reaction temperature of the three variants of arabinose isomerase was 75 ° C, which was 10 ° C lower than that of L469P. Therefore, it was found that the activity of the isomerases in the temperature range in which the production process is applicable will generally be high and that the application temperature range of the production process will be broader.

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In addition, the three variants showed similar temperature patterns, suggesting that the characteristics of the rest of phenylalanine at position 275 have effects on the optimum reaction temperature. Therefore, it can be seen that sufficient flexibility must be ensured so that the steric phenylalanine impediment at position 275 with D-galactose during the reaction of the isomerase with D-galactose can be minimized. As the temperature increases, the molecular mobility of the phenylalanine phenyl moiety and the surrounding moieties may be more active and, therefore, the steric hindrance with D-galactose carbon 6 can be reduced. At the same time, it can be anticipated that the optimum temperature can be formed within a range that does not significantly influence the three- and four-dimensional structures of the protein. Therefore, there may be a change in the optimum temperature of the variants whose steric impairment has been reduced basically by the mutation at position 275. However, additional analysis and experiments are required to scientifically approve this fact.

5-2: Study on thermal stability

To examine the thermal stability of arabinose isomerase variants, each of the enzymes was added to a solution of 50 mM Tris-HCl (pH 7.5) and 1 mM manganese chloride at a concentration of 20 pg / ml and incubated in a constant temperature water bath at 95 ° C for 180 minutes. An enzymatic reaction was performed using an enzymatic solution sampled at varying times to measure the residual activity of the enzyme.

As shown in Figure 7, the results of the thermal stability test indicated that arabinose isomerase variants showed a decrease in residual activity over time at 95 ° C, but the difference in thermal stability between the variants was not significant. To obtain more quantitative data, the asset half-life of each variant was measured. As a result, L469P was found to have a half-life of approximately 3 hours, and variants that fear the mutation at position 275 fear a half-life of approximately 2 hours (Table 3). The thermal stabilities of the variants that fear the mutation at position 275 were measured to be relatively low, but it is believed that the difference in thermal stability is not significant and can be sufficiently compensated by the increase in activity and the decrease in process temperature

Table 3: Half-lives of isomerase arabinoses, measured at 95 ° C

 Variants

 Half-life (minutes)

 L469P

 185

 F275V / L469P

 122

 F275M / L469P

 126

 F275I / L469P

 134

5-3: Study on the change in activity caused by the effect of metal ions

Many enzymes require metal ions for catalysis. For this reason, to examine the dependence of the thermal stability of the arabinose isomerase variants of the present invention on metal ions, the change in the activity of the variants by metal ions was examined using each of the purified enzymes.

To examine the change in enzyme activity as a function of the enzyme concentration, each of the purified enzymes was added to 100 mM D-galactose substrate, and the activity of each enzyme was measured in a Tris- buffer solution. 50 mM HCl (pH 7.5) containing 1 to 5 mM manganese chloride (MnCh) at 75 ° C for 10 minutes (Figure 8). As a result, it can be seen that all variants showed similar activities within the concentration range of manganese chloride used in the experiment and that the effect of manganese ions on the activity of isomerases to the concentration of manganese chloride essential for Enzyme activity was not significant.

5.4: Study on enzyme activity

To examine the reaction rates of the enzymes, the specific activities of arabinose isomerase variants were measured at a reaction temperature of 75 ° C. Specifically, each of the enzymes was added to 1 mM manganese chloride and 100 mM D-galactose, and the reactivity of 1 mg of the enzyme was measured at pH 7.5 and 75 ° C. The specific activity of each enzyme during the reaction was measured under the above conditions for 10 minutes, and as a result, the specific activities of the variants that fear the mutation at position 275 were shown to be approximately 5.5 times (F275V), 5 times (F275M) and 3.9 times (F275I), respectively, greater than those of L469P (Table 4).

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Table 4: Specific activities of the variants

 L469P F275V / L469P F275M / L469P F275I / L469P

 Specific activity (U / mg)

 2.4 13.1 12.1 9.3

Access numbers

Deposit Authority: Korean Culture Center of Microorganisms;

Access number: KCCM11378P;

Deposit date: February 14, 2013.

Deposit Authority: Korean Culture Center of Microorganisms;

Access number: KCCM11379P;

Deposit date: February 14, 2013.

Deposit Authority: Korean Culture Center of Microorganisms;

Access number: KCCM11380P;

Deposit date: February 14, 2013.

<110> CJ Cheiljedang Corporation

<120> variants of L-arabinose isomerase with improved conversion activity and method for the production of D-tagatose using them

<130> PP14-0114

<160> 14

<170> KopatentIn 2.0

<210> 1 <211> 496 <212> PRT

<212> Neapolitan Thermotoga <400> 1

Claims (13)

5 10 fifteen twenty 25 30 35 40 1. A variant of arabinose isomerase having an increased activity of conversion of D-galactose into D-tagatose, the variant of arabinose isomerase having a substitution of an amino acid other than phenylalanine for an amino acid at position 275 and a substitution of proline for an amino acid at position 469 of an amino acid sequence shown in SEQ ID NO: 1. 2. The arabinose isomerase variant of claim 1, wherein the amino acid substitution for the amino acid at position 275 is an amino acid selected from the group consisting of valine, methionine and isoleucine. 3. The arabinose isomerase variant of claim 1, which has an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. 4. A polynucleotide encoding the arabinose isomerase variant of any one of claims 1 to 3. 5. The polynucleotide of claim 4, which has a nucleotide sequence of SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10. 6. A recombinant vector comprising the polynucleotide of claim 4 or claim 5. 7. A microorganism of the genus Corynebacterium transformed with the recombinant vector of claim 6. 8. The microorganism of claim 7, which is Corynebacterium glutamicum. 9. The microorganism of claim 8, which is Corynebacterium glutamicum pFIS-1-TNAI-2 (KCCM 11378P), pFIS-1-TNAI-3 (KCCM 11379P) or pFIS-1-TNAI-4 (KCCM 11380P). 10. A method for producing D-tagatose, the method comprising converting D-galactose to D-tagatose using the arabinose isomerase variant according to claim 1. 11. A method for producing D-tagatose, the method comprising culturing the microorganism of any one of claims 7 to 9. 12. The method of claim 10 or claim 11, which comprises reacting a solution containing D-galactose with a metal ion source selected from the group consisting of manganese chloride, magnesium chloride and zinc chloride in the presence of the arabinose isomerase variant. 13. The method of claim 12, wherein the manganese chloride is added at a concentration of 0.1 to 10 mM. image 1 image2 image3 CO cn Relative attitude image4 Temperature (-C) H- IQ <1 image5 CO CD image6 m £ ^ o oo oo K) ^ n to