HEXURONATE C4-EPIMERASE VARIANT HAVING IMPROVED D-TAGATOSE CONVERSION ACTIVITY, AND D-TAGATOSE PRODUCTION METHOD USING SAME

Abstract

Provided are a hexuronate C4-epimerase variant with improved activity in converting D-fructose by D-tagatose of hexuronate C4-epimerase and a method for production of D-tagatose using them.

Claims

1. A hexuronate C4-epimerase variant in which threonine (T) as an amino acid residue at position 272 from the N-terminus of a hexuronate C4-epimerase consisting of the amino acid sequence set forth in SEQ ID NO: 1 is mutated.

2. The hexuronate C4-epimerase variant according to claim 1, wherein the threonine (T) residue at position 272 is replaced by alanine (A), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), valine (V) or tyrosine (Y).

3. The hexuronate C4-epimerase variant according to claim 1, wherein the serine (S) residue at position 125 from the N-terminus of the hexuronate C4-epimerase is further mutated.

4. The hexuronate C4-epimerase variant according to claim 3, wherein the serine (S) residue at position 125 is replaced by cysteine (C), tyrosine (Y), glutamine (Q), glutamic acid (E), threonine (T), asparagine (N) or aspartic acid (D).

5. The hexuronate C4-epimerase variant according to claim 3, wherein (i) the serine (S) residue at position 185 or (ii) the valine (V) residue at position 267, the serine (S) residue at position 268 or a combination of the valine (V) residue at position 267 and the serine (S) residue at position 268 from the N-terminus of the hexuronate C4-epimerase is further mutated.

6. The hexuronate C4-epimerase variant according to claim 5, wherein the serine (S) residue at position 185 is replaced by alanine (A), glycine (G), histidine (H), lysine (K), glutamine (Q) or arginine (R).

7. The hexuronate C4-epimerase variant according to claim 5, wherein one or more amino acid residues selected from the group consisting of the valine (V) residue at position 267, the serine (S) residue at position 268, and the tryptophan (W) residue at position 306 from the N-terminus of the hexuronate C4-epimerase in addition to the serine (S) residue at position 185 are further mutated.

8. The hexuronate C4-epimerase variant according to claim 7, wherein the valine (V) residue at position 267 is replaced by methionine (M), the serine (S) residue at position 268 is replaced by cysteine (C) or threonine (T), the tryptophan (W) residue at position 306 is replaced by phenylalanine (F), histidine (H), methionine (M) or valine (V).

9. The hexuronate C4-epimerase variant according to claim 5, wherein (i) the aspartic acid (D) residue at position 231, the arginine (R) residue at position 386 or a combination of the aspartic acid (D) residue at position 231 and the arginine (R) residue at position 386 from the N-terminus of the hexuronate C4-epimerase or (ii) one or more amino acid residues selected from the group consisting of the lysine (K) residue at position 164, the aspartic acid (D) residue at position 168, and the glutamic acid (E) residue at position 175 from the N-terminus of the hexuronate C4-epimerase in addition to the valine (V) residue at position 267, the serine (S) residue at position 268 or a combination of the valine (V) residue at position 267 and the serine (S) residue at position 268 are further mutated.

10. The hexuronate C4-epimerase variant according to claim 9, wherein the aspartic acid (D) residue at position 231 is replaced by arginine (R) and the arginine (R) residue at position 386 is replaced by proline (P) or valine (V).

11. The hexuronate C4-epimerase variant according to claim 9, wherein one or more amino acid residues selected from the group consisting of the threonine (T) residue at position 97, the glutamine (Q) residue at position 149, the proline (P) residue at position 166, and the proline (P) residue at position 351 from the N-terminus of the hexuronate C4-epimerase in addition to the valine (V) residue at position 267 and the arginine (R) residue at position 386 are further mutated.

12. The hexuronate C4-epimerase variant according to claim 11, wherein the threonine (T) residue at position 97 is replaced by alanine (A) or leucine (L), the glutamine (Q) residue at position 149 is replaced by arginine (R), the proline (P) residue at position 166 is replaced by arginine (R), and the proline (P) residue at position 351 is replaced by serine (S).

13. The hexuronate C4-epimerase variant according to claim 9, wherein the lysine (K) residue at position 164 is replaced by methionine (M), the aspartic acid (D) residue at position 168 is replaced by glutamic acid (E), and the glutamic acid (E) residue at position 175 is replaced by glycine (G).

14. A nucleic acid encoding the hexuronate C4-epimerase variant according to claim 1.

15. A method for D-tagatose production comprising bringing the hexuronate C4-epimerase variant according to claim 1, a microorganism or a culture thereof expressing the variant into contact with D-fructose.

Description

BEST MODE

[0103] Hereinafter, the present invention is described in more detail with reference to the following Examples. However, the following Examples are merely examples of the present invention, and the contents of the present invention should not be construed as being limited thereto.

EXAMPLE

Example 1. Improved Target Site Design and Analysis

[0104] Amino acids predicted to be functionally important were firstly selected based on analysis of the tertiary structure model of the active site of the ortholog (a homologous gene predicted to have the same function in different microbial species) which has identity with an amino acid of a hexuronate C4-epimerase derived from Thermotoga neapolitana (hereinafter referred to as wild-type). Then, based on analysis results of the docking model between D-fructose and the refined active site structure after the alanine scanning mutagenesis, a modified target site was designed for improvement of the unit activity of the conversion reaction of D-fructose by C4-epimerization. The details thereof are described as follows.

[0105] 1-1. Ortholog Analysis

[0106] The homologous genes (ortholog) having identity with the wild-type amino acid sequence (SEQ ID NO: 1) [about 60 homologous genes with 80% sequence coverage and 50% or more homology] were screened using GenBank gene database. Through multiple sequence alignment analysis among amino acid sequences of the selected homologous genes, conserved amino acid residues predicted to be functionally important in the wild-type amino acid sequence were identified.

[0107] 1-2. Analysis of Enzyme Tertiary Structure Model

[0108] There was no protein structure that appears to have 30% or more amino acid sequence identity with the homologous genes of the wild-type in Protein Data Bank database, and thus it was expected that accuracy in the prediction of the tertiary structure model of the wild-type by a homology modeling method would be low. Accordingly, the active sites among the tertiary structure models obtained from various modeling servers (RaptorX, Robetta, ModWeb, M4T, HHpred, PHYRE2, ITASSER and SWISS-MODEL) were compared and analyzed to obtain information about the structure sites that were predicted as the same.

[0109] 1-3. Alanine Scanning Mutagenesis and Docking Binding Analysis

[0110] The amino acids that were selected based on the amino acid sequence analysis and the analysis of the tertiary structural model of the active site among the homologous genes as described above were substituted and mutated with alanine, and these recombinant mutation enzymes were produced in Escherichia coli. Then, characteristics of each mutation site were analyzed. Amino acids predicted to be functionally important were selected through the docking simulation between D-fructose and the refined active site structure after the alanine scanning mutagenesis was analyzed. Then, the modified target site was designed for the improvement of the unit activity of the conversion reaction of D-fructose by C4-epimerization. The amino acid sites of which activity is completely lost through the alanine scanning mutagenesis [assuming catalytic metal ion binding residues and deprotonation/protonation involved catalytic residues] were excluded from the target site for activity improvement.

Example 2. Production of Mutation Enzyme and Selection of Activity-Modified Mutation Enzyme

[0111] Single-site saturation mutagenesis libraries of 54 target sites designed in Example 1 (amino acid residues at position Nos: 9, 21, 60, 62, 68, 77, 91, 97, 125, 126, 140, 141, 145, 149, 157, 158, 160, 163, 164, 166, 167, 168, 175, 176, 177, 185, 202, 218, 221, 231, 241, 242, 267, 268, 272, 276, 284, 295, 297, 302, 306, 316, 337, 351, 361, 366, 386, 388, 402, 403, 415, 429, 440, and 441 from the N-terminal of the wild-type hexuronate C4-epimerase) were constructed, and mutation sites of which the unit activity was improved and amino acids were screened. The multiple mutation enzyme was made by integrating the information of the screened modified sites to develop a mutation enzyme having improved unit activity of the conversion reaction of D-fructose by C4-epimerization.

[0112] 2-1. Saturation Mutagenesis

[0113] The recombinant expression vector constructed for expression of wild-type enzyme gene, wild-type Escherichia coli BL21 (DE3) (which expresses the recombinant enzyme in which the wild-type was introduced into the NdeI and XhoI restriction enzyme sites of pET21a and 6His-tag is bound at the C-terminal of the wild-type) was used as a template for saturation mutagenesis for producing a variant library. Inverse PCR-based saturation mutagenesis was used in consideration of diversity of mutation distribution and yield of variants (2014. Anal. Biochem. 449: 90-98), NDT, VMA, ATG and TGG mixed primers in which termination codon was excluded and rare codons of E. coli were minimized in order to minimize the screening scale of the constructed variant library (i.e., to minimize the number of codons introduced during saturation mutagenesis) were designed and used (2012. Biotechniques 52:149-158). Specifically, a mixed primer including 15 bp for the front base, 3 bp (NDT, VMA, ATG and TGG, respectively) for substituting the displaced site, and 15 bp for the back base of the respective mutated sites, i.e., 33 bp in total length was constructed and used. The PCR was repeated 30 times under conditions of denaturation at 94 C. for 2 minutes, denaturation at 94 C. for 30 seconds, annealing at 60 C. for 30 seconds, extension at 72 C. for 10 minutes, and extension at 72 C. for 60 minutes. After constructing the saturation mutagenesis libraries for each mutation site, variants for each library were randomly selected (<mutation 11), and base sequences were analyzed to evaluate amino acid mutation distribution. Based on the analysis results, the screening scale of 90% or more of the sequence coverage for each library was determined (2003. Nucleic Acids Res. 15; 31:e30).

[0114] 2-2. Screening of Activity-Modified Mutation Enzyme and Construction of Multiple Mutation Enzyme

[0115] A chromogenic assay was used to specifically quantify D-fructose in order to rapidly screen large quantities of activity-modified mutation enzymes in the produced saturation mutagenesis libraries. Specifically, a 70% folin-ciocalteu reagent (SIGMA-ALDRICH) and a substrate reaction solution were mixed at a ratio of 15:1 and reacted at 80 C. for 5 minutes. The OD values measured at 900 nm were compared and analyzed.

[0116] 54 variants in the mutation site with increased activity (D-tagatose production by conversion of D-fructose) as compared to the relative activity of the wild-type enzyme (SEQ ID NO: 1) were firstly selected. The base sequences of the corresponding genes were analyzed and the amino acid mutation information was analyzed (Tables 2 to 10).

[0117] The firstly selected mutation enzymes were reacted with D-fructose using a purified enzyme solution (His-tag affinity chromatography), and the reaction products were used to finally select 236 variants with the increased activity in producing D-tagatose by conversion from D-fructose as compared to the wild-type enzyme by using HPLC (column Shodex SUGAR SP-G, column analysis temperature of 80 C., mobile phase H.sub.2O, flow rate of 0.6 ml/min, Refractive Index Detector).

Example 3. Comparative Evaluation of Activity-Modified Mutation Enzyme Characteristics

[0118] In order to evaluate the relative activity of the D-fructose C4-epimerization on the mutation enzyme for a single site with improved unit activity and on the mutation enzyme for a multiple site in combination thereof, each enzyme was expressed in E. coli BL21 (DE3) by a conventional method (see Sambrook et al. 1989) and purified (by His-tag affinity chromatography). Then, in the presence of NiSO.sub.4, each enzyme at a concentration of 10 units/ml was added to 25% (w/v) D-fructose substrate and reacted at pH 8.0 [50 mM potassium phosphate buffer] and at 65 C. for 2 hours, and the relative activity of D-fructose C4-epimerization as compared to the wild-type recombinase (wild-type, SEQ ID NO: 1) derived from Thermotoga neapolitana was measured.

[0119] From the above results, it could be confirmed that the C4-epimerase variants of the present invention had the increased D-fructose C4-epimerization activity as compared to that of the wild-type enzyme, and in particular, the enzyme variant of M199 was analyzed as having increased the unit activity about 20 times, and thus, it could be confirmed that the activity of producing tagatose of the present invention was remarkably increased as compared to the wild-type enzyme.