WO2006065076A1

WO2006065076A1 - 5'-xmp aminase mutant

Info

Publication number: WO2006065076A1
Application number: PCT/KR2005/004308
Authority: WO
Inventors: Jae-Gu Pan; Heung-Chae Jung; Eui-Joong Kim; Han-Seung Lee; Young Hoon Park; Hyoung Suk Kim; Jong-Kwon Han; Jin Nam Lee; Ki Hoon Oh; Jeong Hwan Kim; Yoon Suk Oh; Jae Ick Sim; Kuk-Ki Hong; Kyung Oh Choi; Hyun Soo Kim; Min-Ji Baek; Tae Sun Kang
Original assignee: Cj Corporation
Priority date: 2004-12-14
Filing date: 2005-12-14
Publication date: 2006-06-22
Also published as: KR20060067894A; KR100751099B1; TWI323283B; TW200637912A

Abstract

The present invention relates to the development of 5'- xanthylic acid (XMP) aminase mutants having enhanced activity using artificial mutagenesis. The 5'-XMP aminase mutants were obtained by performing error-prone PCR to introduce mutations into the native 5'-XMP aminase gene, screening the resulting mutant library for highly active 5'-XMP aminase, performing molecular evolution using the obtained highly active 5'-XMP aminase mutant as a parent gene to obtain 5 ' -XMP aminase having much higher activity, and determining nucleotide sequences of the resulting mutants. The 5'-XMP aminase mutants effectively convert 5 ' -XMP to 5 ' -guanylic acid because they have higher activity than the native form.

Description

5'-XMP AMINASE MUTANT

Technical Field

The present invention relates to a 5'-xanthylic acid (XMP) aminase mutant, a nucleic acid molecule encoding the same, an expression vector comprising the nucleic acid molecule, a transformant transformed with the expression vector, a method of preparing a 5'-XMP aminase mutant by culturing the transformant, and a method of producing 5'-XMP aminase using the 5'-XMP aminase mutant.

Background Art

5'-guanylic acid (GMP), along with 5'-inosinic acid (IMP), is widely used as a chemical seasoning component for food. 5'-GMP, which by itself gives a rich mushroom flavor, is known to mainly enhance the flavor of monosodium glutamate (MSG) . 5'-GMP creates a synergistic flavor-enhancing effect in combination with 5'-IMP.

Some methods are known for producing 5'-GMP, which include (1) a method in which ribonucleic acid (RNA) isolated from yeast is enzymatically degraded; (2) a microbial fermentation method in which 5'-GMP is directly fermented; (3) a method in which guanosine produced by microbial fermentation is chemically phosphorylated; (4) a method in which guanosine produced by microbial fermentation is enzymatically phosphorylated; (5) a method in which 5'-xanthylic acid (XMP) produced by microbial fermentation is converted to 5'-GMP using coryneform bacteria; and (6) a method in which 5'-XMP produced by microbial fermentation is converted to 5'-GMP using Escherichia coli. Of these methods, Method (1) has problems in material supply and economical efficiency, and Method (2) has a disadvantage of having low yield because the cell membrane is not permeable to GMP. For these reasons, the other methods are mainly applied in the industrial field.

When 5'-xanthylic acid (XMP or xanthosine monophosphate) is converted to 5'-GMP as in Methods (5) and (6), 5'-XMP aminase participates as follows (Pantel et al. (1975), J. Biol. Sci., 250(7), 2609-2613) .

5'-XMP aminase is a member of the glutamine amidotransferase superfamily. Glutamine amidotransferases hydrolyze glutamine at the gamma-amide group to generate ammonia. The resulting free ammonia is assimilated into amino acids, nucleotides, sugars, coenzymes, and the like through polymerization reactions. Glutamine amidotransferases have very various target substances, but the method by which glutamine is hydrolyzed to form ammonia is well conserved during evolution. Glutamine amidotransferases have been divided into two subfamilies: class I and class II. The class I enzymes includes anthranilate synthase, carbamoyl phosphate synthetase, CTP synthetase, formylglycinamidine synthetase, 5'-XMP aminase, imidazole glycerol phosphate synthase, and aminodeoxychorismate synthasea. These enzymes use, in addition to glutamine, external ammonia as an anime donor (Cell MoI. Life Sci. 54, 205- 222, 1998) . Unlike how ammonia, released from glutamine, is transferred to a substrate, the external ammonia is considered to directly transfer to transferase. In the context of protein structure, glutamine amidotransferases are well separated between a domain having glutaminase activity responsible for catalytic hydrolysis of glutamine and another domain having transferase activity. The glutaminase activity is mainly achieved by a catalytic triad of cysteine, histidine and glutamate residues, which is similar to the catalytic mechanism of cysteine protease (Cell MoI. Life Sci. 54, 205-222, 1998) . Typically, since native enzymes have been evolved at degrees suitable for cells, they often exhibit improper results due to low activity in industrial applications. To overcome this problem, a method of cloning a gene encoding an enzyme of interest and overexpressing the gene is generally used. In the case of 5'-XMP aminase, a recent study revealed that 5'-GMP is produced from 5'-XMP by overexpressing a 5'-XMP aminase gene (guaA) , which is isolated from wild-type Escherichia coli and cloned into an inducible expression plasmid (Biosci. Biotech. Biochem. 61(5), 840-845, 1997).

Generally-used inducible expression vectors require expensive expression inducers such as IPTG, and are thus not suitable for industrial applications involving protein production in a large scale. This problem can be solved by employing a constitutive expression system. A great number of constitutive expression systems have been reported. In particular, a novel constitutive expression promoter was developed for Corynebacterium ammoniagenes known to be suited for fermentative production of nucleic acids (Korean Pat. Application No. 2004-107215) . The constitutive expression systems are useful because they sustain the expression of an introduced protein for a cultivation period of host cells with no use of an expression inducer. However, when the overexpression of an introduced protein affects the growth of host cells, the cells stop their growth, or a vector introduced into the cells is removed, resulting in low expression efficiency. The same results have been reported for 5'-XMP aminase (Biosci. Biotech. Biochem. 61(5), 840-845, 1997) .

Another method of increasing protein expression of wild- type bacteria is described in Korean Pat. Laid-open Publication No. 2000-0040840. In this publication, to increase the expression of a gene of interest using drug resistance, there is provided a mutant strain having enhanced activity of 5'-XMP aminase, which is prepared by imparting decoyinine resistance to a wild-type Escherichia coli strain.

In addition, the activity of a native enzyme can be improved by a method including introducing mutations into a native gene and screening the resulting mutant library to isolate an enzyme having a desired character. The mutagenesis is carried out either employing chemical mutagens, or by PCR using error-prone DNA polymerase in which mistakes are introduced into a target gene during DNA amplification. In particular, a DNA polymerase derived from Thermus aquatics is widely used because it is deficient in 3^I→5' exonuclease activity and thus generates high mutation rates during DNA synthesis (Leung et al., Technique, 1989, 1, 11-15) . Error- prone PCR using Taq DNA polymerase is useful because it is able to produce a great quantity of mutant libraries.

Leading to the present invention, intensive and thorough research into the production of 5'-XMP aminase having enhanced activity in culture fluid, conducted by the present inventors, resulted in a 5'-XMP aminase mutant, which is derived from Escherichia coli and has enhanced activity, being developed by molecular evolution error-prone PCR and molecular biological techniques.

Disclosure of the Invention

It is therefore a main object of the present invention to provide a 5'-xanthylic acid (XMP) aminase mutant which is derived from Escherichia coli and has enhanced activity.

It is another object of the present invention to provide a nucleic acid molecule encoding the 5'-XMP aminase mutant. It is a further object of the present invention to provide an expression vector comprising the nucleic acid molecule encoding the 5'-XMP aminase mutant.

It is yet another object of the present invention to provide a transformant transformed with the expression vector. It is still another object of the present invention to provide a method of converting 5'-XMP to 5'-guanylic acid (GMP) by using the 5 '-XMP aminase mutant.

Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

Fig. 1 schematically shows a process for producing a 5'- XMP aminase mutant, comprising constructing a 5'-XMP aminase mutant library by random mutagenesis and screening the mutant library to select for highly active 5'-XMP aminase mutants;

Fig. 2 shows an expression vector pG3 carrying a gene encoding a highly active 5'-XMP aminase mutant, G3;

Fig. 3 shows an expression vector pFl2 carrying a gene encoding a highly active 5'-XMP aminase mutant, F12;

Fig. 4 shows an expression vector pF63 carrying a gene encoding a highly active 5'-XMP aminase mutant, F63;

Fig. 5 shows an expression vector pCJ-G3-l carrying a gene encoding a highly active 5'-XMP aminase mutant, G3-1;

Fig. 6 shows an expression vector pCJ-F12-l carrying a gene encoding a highly active 5'-XMP aminase mutant, F12-1; and

Fig. 7 shows an expression vector pCJ-F63-l carrying a gene encoding a highly active 5'-XMP aminase mutant, F63-1.

Best Mode for Carrying Out the Invention

In one aspect, the present invention provides a 5'- xanthylic acid (XMP) aminase mutant which is derived from Escherichia coli and has enhanced activity.

In order to obtain a vector expressing 5'-XMP aminase derived from Escherichia coii, a vector containing a gene of 1,578 bp (SEQ ID No. 1) derived from Escherichia coli K12 was prepared, and error-prone PCR was carried out using the vector as a template. As a result, 5'-XMP aminase mutant DNA molecules, into which mutations were randomly introduced, were obtained. The mutant DNA molecules were inserted into an expression vector suitable for expressing 5'-XMP aminase mutants. The resulting vectors were transformed into an E. coli strain deficient in the 5'-XMP aminase gene to construct a mutant library. Since E. coli deficient in the 5'-XMP aminase gene is able to grow only when transformed with a vector expressing a mutant having 5'-XMP aminase activity, only active mutants of 5'-XMP aminase are obtained from the mutant library. To select E. coli clones transformed with a vector expressing a highly active mutant form of 5'-XMP aminase from grown E. coli colonies, the conversion of 5'-XMP into 5'- guanylic acid (GMP) was performed on 98-well microplates. After the reaction was terminated, E. coli clones producing 5'- XMP aminase having increased activity were selected by comparing absorbance values with a control.

The nucleotide sequences of 5'-XMP aminase mutants having enhanced activity were determined by a known method. When the nucleotide sequences of 5'-XMP aminase mutants were compared to the nucleotide sequence of native 5'-XMP aminase, six selected mutants of 5'-XMP aminase were found to have new amino acid sequences each altered in two, two, four, four, three and six amino acid residues, and were designated "5'-XMP aminase G3", "5'-XMP aminase F12", "5'-XMP aminase F63", "5'- XMP aminase G3-1", "5'-XMP aminase F12-1" and "5'-XMP aminase F63-1", respectively.

In detail, each mutant has an alteration in its amino acid sequence, as follows. The G3 mutant has an amino acid sequence (SEQ ID No. 4) in which amino acid residues at positions 52 and 91 are replaced by cysteine and threonine, respectively. The F12 mutant has an amino acid sequence (SEQ ID No. 6) in which amino acid residues at positions 93 and 152 are replaced by valine and proline, respectively. The F63 mutant has an amino acid sequence (SEQ ID No. 8) in which amino acid residues at positions 93, 113, 191 and 467 are replaced by valine, alanine, threonine and glycine, respectively. The G3-1 mutant has an amino acid sequence (SEQ ID No. 10) in which amino acid residues at positions 52, 191, 253 and 454 are replaced by cysteine, threonine, arginine and isoleucine, respectively. The F12-1 mutant has an amino acid sequence (SEQ ID No. 12) in which amino acid residues at positions 93, 152 and 454 are replaced by valine, praline and isoleucine, respectively. The F63-1 mutant has an amino acid sequence (SEQ ID No. 14) in which amino acid residues at positions 93, 100, 113, 191, 454 and 467 are replaced by valine, isoleucine, alanine, threonine, isoleucine and glycine, respectively.

Thus, in a detailed aspect, the present invention provides 5 '-XMP aminase mutants each having the amino acid sequence of SEQ ID No. 4, 6, 8, 10, 12 or 14.

The 5'-XMP aminase mutant of the present invention, in addition to proteins each having the amino acid sequence of of SEQ ID No. 4, 6, 8, 10, 12 or 14, includes a functional equivalent exerting the activity identical to the mutant proteins. The term "functional equivalent", as used herein, refers to a protein that has a s different sequence from an amino acid sequence of the 5'-XMP aminase mutant of the present invention, by a deletion, an insertion, a non-conservative or conservative substitution or combinations thereof in one more amino acid residues, and that exerts 5'-XMP aminase activity almost exactly as high as that of the 5'-XMP aminase mutant.

Amino acid exchanges in proteins and peptides which do not generally alter the activity of the proteins or peptides are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979) .

The 5' -XMP aminase mutants according to the present invention may be prepared by a chemical synthesis method (Merrifleld, J. Amer. chem. Soc. 85:2149-2156, 1963), or by a DNA sequence-based recombinant method (Sambrook et al. , Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2d Ed., 1989) . When a genetic recombination technique is used, a 5'-XMP aminase mutant may be obtained by inserting a nucleic acid encoding the 5'-XMP aminase mutant into a suitable expression vector, transforming a host cell with the recombinant expression vector, culturing the host cell to express the 5'-XMP aminase mutant, and recovering the produced 5'-XMP aminase mutant from the culture. When the 5'-XMP aminase mutants of the present invention were expressed using a constitutive expression system, they were found to have higher activity per reaction solution than the native form. In detail, the G3 5'-XMP aminase mutant had 1.8-fold higher activity, the F12 5'-XMP aminase mutant had 3.1-fold higher activity, the F63 5'-XMP aminase mutant had 2.3-fold higher activity, the G3-1 5'-XMP aminase mutant had 3- fold higher activity, the F12-1 5'-XMP aminase mutant had 3.8- fold higher activity, and the F63-1 5'-XMP aminase mutant had 3.07-fold higher activity. Thus, the 5'-XMP aminase mutants may be used for effectively producing 5'-GMP. In another aspect, the present invention provides a nucleic acid molecule encoding the 5'-XMP aminase mutant.

The 5'-XMP aminase mutants formed by random mutagenesis are preferably encoded by the following nucleic acid molecules. The G3 5'-XMP aminase mutant of SEQ ID No. 4 is encoded by the nucleic acid molecule of SEQ ID No. 3. The F12 5'-XMP aminase mutant of SEQ ID No. 6 is encoded by the nucleic acid molecule of SEQ ID No. 5. The F63 5'-XMP aminase mutant of SEQ ID No. 8 is encoded by the nucleic acid molecule of SEQ ID No. 7. The G3-1 5'-XMP aminase mutant of SEQ ID No. 10 is encoded by the nucleic acid molecule of SEQ ID No. 9. The F12-1 5'-XMP aminase mutant of SEQ ID No. 12 is encoded by the nucleic acid molecule of SEQ ID No. 11. The F63-1 5'-XMP aminase mutant of SEQ ID No. 14 is encoded by the nucleic acid molecule of SEQ ID No. 13. The sequences of the nucleic acid molecules may be single-stranded or double-stranded, and may be RNA (mRNA) formed by a substitution of uracil (U) for thymine (T) in a DNA molecule or sequence.

The nucleic acid sequence encoding the 5'-XMP aminase mutant of the present invention may be introduced into a vector for expressing the mutant so as to be expressed as a protein.

In a further aspect, the present invention provides an expression vector comprising the nucleic acid molecule encoding the 5'-XMP aminase mutant.

The term "expression vector", as used herein, which describes a vector capable of expressing a protein of interest in a suitable host cell, refers to a genetic construct that comprises essential regulatory elements to which a gene insert is operably linked in such a manner as to be expressed in a host cell.

The term "operably linked", as used herein, refers to a functional linkage between a nucleic acid expression control sequence and a second nucleic acid sequence coding for a target protein in such a manner as to allow general functions. For example, a promoter may be operably linked to a nucleic acid coding for a protein and affect the expression of the coding nucleic acid sequence. The operable linkage to a recombinant vector may be prepared using a genetic recombination technique well known in the art, and site-specific DNA cleavage and ligation may be easily achieved using enzymes generally known in the art. Examples of promoters useful in an expression vector are as follows. Promoters available in Escherichia species as hosts include trc promoter, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter and T7 promoter. Examples of promoters available in Bacillus species as hosts include SPOl promoter, SPO2 promoter and penP promoter. The initiation and stop codons are necessary to be functional in an individual to whom a genetic construct has been administered, and must be in frame with the coding sequence. An expression vector may also include a selectable marker that allows selection of host cells containing the vector. A replicable expression vector may include a replication origin.

In a detailed practice of the present invention, expression vectors each comprising a gene encoding a 5'-XMP aminase mutant, pG3, pF12, pF63, pCJ-G3-l, pCJ-F12-l and pCJ- F63-1, were constructed. The expression vectors were individually introduced into Escherichia coli JM105 to obtain transformed Escherichia coli. The transformants thus produced were designated "Escherichia coli JM105/pG3 (KCCM-10626) ", "Escherichia coli JM105/pF12 (KCCM-10627) ", "Escherichia coli JM105/pF63 (KCCM-10625)", "Escherichia coli JM105/pCJ-G3-l", "Escherichia coli JMlO5/pCJ-F12-l", and "Escherichia coli JM105/pCJ-F63-l". Of these, "Escherichia coli JM105/pG3 (KCCM- 10626)", "Escherichia coli JM105/pF12 (KCCM-10627)" and "Escherichia coli JM105/pF63 (KCCM-10625)" were deposited at the Korean Culture Center of Microorganisms (KCCM; 361-221, Yurim B/D, Hongjae 1-dong, Seodaemun-gu, Seoul, Republic of Korea) on November 30, 2004. The remaining transformants, "Escherichia coli JM105/pCJ-G3-l (KCCM-10716P) ", "Escherichia coli JM105/pCJ- F12-1 (KCCM-10718P)" and "Escherichia coli JM105/pCJ-F63-l (KCCM-10719P)", were deposited at the Korean Federation of Culture Collections (KFCC) on December 2, 2005 and assigned accession number KCCM-10716P, KCCM-10718P and KCCM-10719P, respectively.

Fig. 1 schematically shows a process for producing a 5'- XMP aminase mutant having enhanced activity according to the present invention.

In a yet another aspect, the present invention provides a transformant transformed with the expression vector.

"Transformation" includes any method by which nucleic acids can be introduced into organisms, cells, tissues or organs, and, as known in the art, may be performed by selecting suitable standard techniques according to host cells. These methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO₄) precipitation, calcium chloride (CaCl₂) precipitation, agitation with silicon carbide fiber, agrobacterium-mediated transformation, and PEG-, dextran sulfate- and lipofectamine-mediated transformation.

Host cells most suitable for objects may be selected and used because expression levels, modification, or the like of proteins vary depending on host cells into which an expression vector expressing the 5'-XMP aminase mutant of the present invention is transformed. Host cells include, but are not limited to, prokaryotic cells such as Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis or Staphylococcus. Also, eukaryotic cells useful as host cells include lower eukaryotic cells, such as fungi (e.g., Aspergillus species) and yeasts (e.g., Pichia pastoris,

Saccharomyces cerevisiae, Schizosaccharomyces, Neurospora crassa) .

In still another aspect, the present invention provides a method of preparing a 5'-XMP aminase mutant, comprising culturing the transformant and isolating the mutant protein of

5'-XMP aminase from the culture fluid.

The cultivation of host cells (transformants) transformed with an expression vector expressing the 5'-XMP aminase mutant of the present invention may be performed under culture conditions suitable for expressing a target protein,

5'-XMP aminase mutant, through a method generally known to those skilled in the art.

The 5' -XMP aminase mutant proteins of the present invention, expressed in host cells, may be purified by various common methods, which may be used separately or in combination, for example, salting out (e.g., ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation

(e.g., protein fraction precipitation using acetone, ethanol, etc.), dialysis, gel filtration, chromatographic methods such as ion exchange chromatography and reverse phase chromatography, and ultrafiltration.

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention. EXAMPLE 1: Construction of 5'-XMP aminase mutant library

To prepare multiple 5'-XMP aminase mutants, random mutations were introduced into a 5'-XMP aminase gene by error- prone PCR. First, a 5'-XMP aminase gene (SEQ ID No. 1) of 1,578 bp derived from Escherichia coli, guaA, was operably linked to an expression vector, pTrc99a, which includes a trc promoter and a replication origin functional in Escherichia coli, thus yielding a recombinant plasmid, pGl. Error-prone PCR was then carried out using the recombinant plasmid as a template with a pair of an N-terminal primer, represented by SEQ ID No. 15, and a C-terminal primer, represented by SEQ ID No. 16. The primers were synthesized based on the nucleotide sequence of the 5'-XMP aminase gene derived from E. coli.

SEQ ID No. 15: 5'-CGCGAATTCATGACGGAAAACATTCATAA-S' SEQ ID No. 16: 5'-TAGTCTAGATCATTCCCACTCAATGGT-S'

A PCR mixture was composed of 5 ng of recombinant plasmid pGl, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 7 mM MgCl₂,

0.1 inM MnCl₂, 0.2 mM dATP, 0.2 mM dGTP, 1 mM dCTP, 1 mM dTTP and 5 units of Taq polymerase, in a final volume of 50 μ&.

Error-prone PCR was carried out under conditions including 25 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min and elongation at 72°C for 1 min, followed by final elongation at 72°C for 10 min, thereby introducing mutations into the gene.

Amplified PCR products were electrophoresed on agarose gels and purified. The gel-purified DNA fragments were digested with EcoRI and HindIII and inserted into an expression vector, pTrc99a, useful for expressing 5'-XMP aminase, thus constructing recombinant plasmids. The recombinant vectors carrying the 5 '-XMP aminase gene mutants were introduced into Escherichia coli BW (guaA gene-knockout strain) , thus constructing a mutant library of 5'-XMP aminase. The guaA gene-deleted Escherichia coli BW strain was prepared by an ordinary molecular biological method (Datsenko KA, 2000, Proc. Natl. Acad. Sci., 97(12), 6640-6645) .

EXAMPLE 2: Screening for 5'-XMP aminase mutants

The 5'-XMP aminase mutant library prepared in Example 1 was smeared onto LB plates containing 0.5% bactotrypton, 1% yeast extract, 1% NaCl, 1.5% agar and 0.2 mM IPTG. The grown Escherichia coli colonies were cultured in LB medium in deep- well microplates. The culture was then diluted according to growth degree to give a final volume of 100 βl. 5 μl of xylene was added to each well of the plates, and the plates were incubated at 37°C for 30 min. 100 μl of a substrate solution preheated to 42°C was added to each well, and the plates were incubated at 42°C for 20 min. The substrate solution was prepared by dissolving 30 mM XMP, 13 itiM ATP, 16 itiM MgSO₄ ^• 7H₂O and 40 mM (NH₄)₂SO₄ in 16 mM Trizma HCl buffer (pH 8.6) . After the reaction was terminated by adding 800 μl of 3.5% perchloric acid to each well, 200 μJt of the reaction mixture was transferred into a 96-well UV-transparent microplate, and absorbance was measured at 290 ran. The yield of 5'-GMP was measured, and enzyme activity comparison was performed. As a result, an Escherichia coli JM105 transformant expressing a 5'- XMP aminase mutant with enhanced activity was selected.

The thus obtained pG3 plasmid carrying a 5' -XMP aminase mutant gene and the pGl plasmid carrying a native 5'-XMP aminase gene were digested with proper restriction enzymes, ligated, and subjected to error-prone PCR under the same conditions as in Example 1. The screening for enzyme activity and activity comparison resulted in the obtainment of plasmids pFl2, pF63, pCJ-G3-l, pCJ-F12-l and pCJ-F63-l, which expressed 5'-XMP aminase having enhanced activity relative to the parent enzyme. Fig. 1 schematically shows a process for producing a 5'- XMP aminase mutant having enhanced activity, described in Examples 1 and 2.

The scale of the 5'-XMP aminase mutant library and mutants, prepared in Example 2, are summarized in Table 1, below. TABLE 1 Molecular evolution of 5'-XMP aminase and selected mutants

EXAMPLE 3: Determination of nucleotide sequences of genes encoding 5'-XMP aminase mutants

Nucleotide sequences of 5'-XMP aminase mutants were analyzed using an automatic sequencer ABI3730 (Applied Biosystems) . The determined nucleotide sequences were represented by SEQ ID No. 3 for the G3 mutant, SEQ ID No. 5 for the F12 mutant, SEQ ID No. 7 for the F63 mutant, SEQ ID No. 9 for the G3-1 mutant, SEQ ID No. 11 for the F12-1 mutant, and SEQ ID No. 13 for the F63-1 mutant. Also, amino acid sequences deduced from the nucleotide sequences were represented, respectively, by SEQ ID No. 4 (G3 mutant), SEQ ID No. 6 (F12 mutant), SEQ ID No. 8 (F63 mutant), SEQ ID No. 10 (G3-1 mutant), SEQ ID No. 12 (F12-1 mutant), and SEQ ID No. 14 (F63-1 mutant) . Schematic maps of plasmids containing genes encoding the mutants, G3, F12, F63, G3-1, F12-1 and F63-1, are given in Figs. 2, 3, 4, 5, 6 and 7, respectively. In detail, the amino acid sequence of the G3 mutant was deduced from the nucleotide sequence of the highly active G3 5'-XMP aminase mutant gene contained in the pG3 plasmid, and was represented by SEQ ID No. 4. The amino acid sequence of the F12 mutant was deduced from the nucleotide sequence of the highly active F12 5'-XMP aminase mutant gene contained in the pF12 plasmid, and was represented by SEQ ID No. 6. The amino acid sequence of the F63 mutant was deduced from the nucleotide sequence of the highly active F63 5'-XMP aminase mutant gene contained in the pF63 plasmid, and was represented by SEQ ID No. 8. The amino acid sequence of the G3-1 mutant was deduced from the nucleotide sequence of the highly active G3-1 5'-XMP aminase mutant gene contained in the Pg3-1 plasmid, and was represented by SEQ ID No. 10. The amino acid sequence of the F12-1 mutant was deduced from the nucleotide sequence of the highly active F12-1 5'-XMP aminase mutant gene contained in the pCJ-F12-l plasmid, and was represented by SEQ ID No. 12. The amino acid sequence of the F63-1 mutant was deduced from the nucleotide sequence of the highly active F63-1 5'-XMP aminase mutant gene contained in the pCJ-F63-l plasmid, and was represented by SEQ ID No. 14.

When the amino acid sequences of the highly active 5'- XMP aminase mutants, G3, F12, F63, G3-1, F12-1 and F63-1, were compared with the amino acid sequence of native 5'-XMP aminase, represented by SEQ ID No. 2, they were found to be proteins which have novel amino acid sequences having amino acid substitutions for two, two, four, four, three and six amino acid residues, respectively.

In detail, the G3 mutant has an amino acid sequence in which amino acid residues at positions 52 and 91 are replaced by cysteine and threonine, respectively. The F12 mutant has an amino acid sequence in which amino acid residues at positions 93 and 152 are replaced by valine and proline, respectively. The F63 mutant has an amino acid sequence in which amino acid residues at positions 93, 113, 191 and 467 are replaced by valine, alanine, threonine and glycine, respectively. The G3-1 mutant has an amino acid sequence in which amino acid residues at positions 52, 191, 253 and 454 are replaced by cysteine, threonine, arginine and isoleucine, respectively. The F12-1 mutant has an amino acid sequence in which amino acid residues at positions 93, 152 and 454 are replaced by valine, praline and isoleucine, respectively. The F63-1 mutant has an amino acid sequence in which amino acid residues at positions 93, 100, 113, 191, 454 and 467 are replaced by valine, isoleucine, alanine, threonine, isoleucine and glycine, respectively. The results of amino acid sequence analysis and enzyme activity assay indicate that the G3, F12, F63, G3-1, F12-1 and F63-1 mutants are novel 5'-XMP aminase mutant forms, each of which has a different amino acid sequence from that of native E. coli 5'-XMP aminase and having high activity.

EXAMPLE 4: Evaluation of activity of the 5'-XMP aminase mutants The nonspecific activity of 5'-XMP aminase was assessed as follows. First, protein expression levels were measured on an SDS-PAGE gel using an analyzer for protein concentration. As a result, the 5'-XMP aminase mutants exhibited similar expression levels to each other, indicating that the enhanced activity of the 5'-XMP aminase mutants resulted from the increased nonspecific activity.

The activity of the 5'-XMP aminase mutants was compared with that of the native form, as follows. First, transformants expressing mutants were individually inoculated in 30 ml of a culture medium containing 16 g/L of bactotrypton, 10 g/L of yeast extract, 5 g/L of NaCl and 100 mg/L of ampicillin, cultured at 37°C. When the culture reached a desired optical density, 0.5 mM IPTG was added to the culture medium, and the cells were further cultured at 37°C for 1 hr. After the cultured cells were removed, 1 ml of each culture fluid was mixed with 20 ml of xylene and incubated at 37°C for 20 min with agitation at 250 rpm. The reaction mixture was diluted by 10 times. To measure the catalytic activity of the 5'-XMP aminase mutants with ammonia as a substrate, 200 /Λ of the 1:10 diluted enzyme solution was mixed with 800 βi of a substrate solution, which was prepared by dissolving 30 mM XMP, 13 mM ATP, 16 mM MgSO₄ • 7H₂O and 10 mM (NH₄)₂SO₄ in 200 mM Trizma HCl buffer (pH 8.6), and was incubated at 42°C for 15 min. 200 fd of the resulting reaction mixture was mixed with 3.8 ml of 0.175% TCA to terminate the reaction, and was subjected to HPLC to determine the amount of produced GMP. One unit of activity of 5'-XMP aminase was defined as the enzyme amount that forms one micromole of 5'-GMP per minute. HPLC was performed under the following conditions.

Eluent A: 0.02% tetrabutylammonium dihydrogen phosphate, 0.2% ammonium dihydrogen phosphate, pH 2.4 Eluent B: acetonitrile A:B = 97:3 Measurement wavelength: 254 nm Flow rate: 1.0 ml/min

The enzyme activity (per ml and OD) measured using ammonia as a substrate was as follows. The wild-type E. coli JM105/pGl strain exhibited an enzyme activity of 0.39 U/ml/OD. The E. coli JM105/pG3 transformant exhibited an enzyme activity of 0.712 U/ml/OD. The E. coli JM105/pF12 transformant exhibited an enzyme activity of 1.22 U/ml. The E. coli JM105/pF63 transformant exhibited an enzyme activity of 0.90 U/ml. The E. coli JM105/pCJ-G3-l transformant exhibited an enzyme activity of 1.17 U/ml/OD. The E. coli JM105/pCJ-F12-l transformant exhibited an enzyme activity of 1.47 U/ml. The E. coli JM105/pCJ-F63-ltransformant exhibited an enzyme activity of 1.20 U/ml.

When the activity of 5'-XMP aminase was compared between the native form and the mutant forms, the mutant forms of 5'- XMP aminase were found to have 1.8- to 3.8-fold higher activity than the native form. These results confirm that the 5'-XMP aminase mutants are novel 5'-XMP aminase mutant forms having enhanced activity.

Industrial Applicability

As described and demonstrated hereinbefore, in order to effectively produce 5'-GMP useful as a flavor-enhancing seasoning, the present invention provides 5'-XMP aminase mutants having enhanced activity, G3, F12, F63, G3-1, F12-1 and F63-1. Also, the present invention provides a gene encoding the mutant, a transformant transformed with an expression vector comprising the gene encoding the mutant, and a method of preparing a 5'-XMP aminase mutant using the transformant. The 5'-XMP aminase mutants of the present invention are useful in a biological process for producing 5'-GMP because they have enhanced activity relative to the native form.

Claims

1. A 5'-xanthylic acid aminase mutant having any one of amino acid sequences of SEQ ID Nos. 4, 6, 8, 10, 12 and 14.

2. A nucleic acid molecule encoding the 5'-xanthylic acid aminase mutant of claim 1.

3. An expression vector comprising the nucleic acid molecule of claim 2.

4. A prokaryotic transformant transformed with the expression vector of claim 3.

5. A method of converting 5'-xanthylic acid to 5'- guanylic acid, which is based on using the 5'-xanthylic acid aminase mutant of claim 1.