WO2008020659A1

WO2008020659A1 - Thermophilic l-ribose isomerase and use thereof

Info

Publication number: WO2008020659A1
Application number: PCT/KR2006/003230
Authority: WO
Inventors: Yu-Ryang Pyun; Eun-Ah Cho; Myung-Ji Seo; Yun-Hwan Cha; Jeong-Nyeo Kim; Sang-Jae Lee; Yong-Ho Hong; Jae-Gu Pan; Heung-Chae Jung
Original assignee: Yu-Ryang Pyun; Eun-Ah Cho; Myung-Ji Seo; Yun-Hwan Cha; Jeong-Nyeo Kim; Sang-Jae Lee; Yong-Ho Hong; Jae-Gu Pan; Heung-Chae Jung
Priority date: 2006-08-17
Filing date: 2006-08-17
Publication date: 2008-02-21

Abstract

The present invention relates to a thermophilic L-ribose isomerase which is specifically reactive to L-ribose as substrates, a nucleic acid molecule encoding the same and a method for preparing an aldose or ketose using the same. In recent, L-ribose has been mostly prepared by chemical synthesis methods; however these methods have disadvantages: complexity of purification steps, and production of harmful chemical contaminants and by-products. However, because L-ribose isomerase of this invention has significant thermophilicity as well as substrate specificity to L-ribose, it has plausible advantages over chemical synthesis methods. For example, L-ribose isomerase enables to utilize substrates in higher concentrations since enzymatic reactions using L-ribose isomerase can be carried out with minimizing generation of byproducts in higher temperature conditions. Furthermore, where reactions are performed using the L-ribose isomerizing enzyme of this invention, their viscosity is low, their susceptibility to contamination by microorganisms is very low and their rate is very likely to be high. In addition to this, the L-ribose isomerizing enzyme of this invention contributes to minimization of byproducts generation.

Description

THERMOPHILIC L-RIBOSE ISOMERASE AND USE THEREOF

FIELD OF THE INVENTION

The present invention relates to a novel thermophilic L-ribose isomerase, more particularly, to the thermophilic L-ribose isomerase, a nucleic acid encoding the same and a method for preparing an aldose or ketose using the same.

DESCRIPTION OF THE RELATED ART

In the past decades, L-carbohydrates and their nucleoside derivatives have been increasingly used in the pharmaceutical field. Particularly, some modified nucleosides have been suggested to have a significant potential as an antiviral agent

(Ma, T., et 5/(1997) J. Med. Chem., 40, 2750-2754). L-ribose is an important key pentose that constitutes a backbone in the synthesis of L-ribonucleoside, L- oligoribonucleoside and many other therapeutic drugs. Compared with D-nucleoside, L-nucleoside has higher stability from nucleases attack in vivo conditions, so that it would be considered candidate materials with high potential that may be used as therapeutics.

L-ribose is one of rare sugars whose preparation processes has not yet been established. L-ribose has been suggested to be significantly useful as a basic raw material of drugs such as antiviral agents and anticancer agents, drawing attention of many researchers to establish scaled-up industrial schemes for preparing L-ribose.

Despite of the importance and usefulness of L-ribose as described above, it is likely to be naturally found in a very small amount, unlike D-ribose. Furthermore, the conventional methods for preparing L-ribose are generally accompanied with a relatively higher cost. Accordingly, the development of commercial methods for preparing L-ribose in lower cost remains to be needed.

Several methods converting L-arabinose, D-glucose, L-xylose, D-galactose and

D-ribose into L-ribose derivatives have been reported (Austin, W. C. and Humoller, F. L.

(1934) J. Am. Chem. Soc, 56, 1152-1153; Humoller, F. L: L-Ribose, p. 83-88. In Whistler, R. L. and Wolfram, M. L. (ed.) (1962) Methods in carbohydrate chemistry, vol. 1. Academic Press, London.; Matteson, D. S. and Peterson, M. L. (1987) J. Org. Chem., 52(23), 5116-5121; Yamaguchi, M. and Mukaiyama, T. (1981) Chem. Lett., 7, 1005- 1008). In addition, Ikegmi and co-researchers reported an improved method to synthesize L-sugars from D-sugar lactones. However these methods have demerits including a low yield, requirement for high cost starting materials, a lot of reaction steps and difficulty in mass production. Moreover, these methods are composed of a serial of chemical reactions, which require prolonged reaction time, relatively high cost materials and plenty of labor force and generate by-product. Therefore, biochemical methods for preparing L-ribose from L-ribulose have been suggested using microorganisms and their enzymes (Shimonishi, T., Izumori, K., J. (1996) J. Ferment Bioeng. 81, 493-497), being expected to overcome afore-mentioned shortcomings.

L-ribose isormerase (L-RI) as enzymes in cells catalyzes a reversible isomerization between L-ribose and L-ribulose which are metabolized into a pentos phosphate pathway (Lehninger, A. L., Nelson, D. L., and Cox, M. M. (2000) Principles of Biochemistry, 3rd edn., 1088 pp. Worth publishers.) or a phosphoketolase pathway (Doelle, H. W. (1965) phosphoketolase pathway, 2nd ed., 244-250 pp. Academic Press, New York.). L-ribose can be used as a carbon source in several microorganisms, and is sequentially converted to L-ribulose, L-ribuIose-5-phosphate and D-xylose-5-phosphate (intermediates in a pentose phosphate pathway) (Lehninger, A. L. et al. 2000). The conversion from L-ribose to L-ribulose as the first step in the metabolic pathway may be catalyzed by L-RI (Shimonishi, T. et al. 1996). It was proposed that L-RI derived from microorganisms catalyzes the isomerization reaction of L-ribose to L-ribulose and its reverse reaction in vitro (Shimonishi, T. et al. 1996). Recently, it was reported to isolate L-RI and its gene from Acinetobacter sp. DL-

28 and identify its sequence firstly (Mizanur, R. M., Takata, G., Izumori, K. (2001) Biochimica et Biophysica Acta 1521, 141-145). However, the enzyme has a serious problem not to maintain its isomerization activity for more than 10 min at even room temperature (Shimonishi, T. et al. 1996). In the industrial utilization of biocatalysts, a heat-induced enzyme inactivation should be addressed because industrial processes are often run under significantly severe conditions, e.g. high temperatures. In light of these, unstable enzymes are not suitable in industrial processes under severe conditions. Recently, thermophilic enzymes have attracted much attention in industrial applications. Thermophilic enzymes, stable and active under high temperatures, provide considerable biotechnological advantages compared to mesophilic enzymes. Firstly, their thermal stability is associated with resistance properties to chemical denaturants such as guanidine hydrochloride. Secondly, the possibility to perform enzymatic reactions in high temperatures demonstrates that substrates in higher concentrations may be utilized, contamination caused by microorganisms hardly occurs and higher reaction rate may be often obtained. Accordingly, it could be recognized that the researches on thermophilic enzymes are promising and contribute greatly to the development of protein engineering technologies and biotechnological application technologies for proteins with excellent efficiency.

Throughout this application, several patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications is incorporated into this application in order to more fully describe this invention and the state of the art to which this invention pertains.

DETAILED DESCRIPTION OF THIS INVENTION

The present inventors have made intensive researches to satisfy the needs of one skilled in the art as described hereinabove. As a result, we have successfully cloned and sequenced a novel L-ribose isomerase capable of retaining its stability and activity even at higher temperatures and substrate-specificity to L-ribose. Accordingly, it is an object of this invention to provide L-ribose isomerase having thermophilicity.

It is another object of this invention to provide a nucleic acid molecule encoding L-ribose isomerase.

It is still another object of this invention to provide a vector comprising the nucleic acid molecule encoding L-ribose isomerase. It is further object of this invention to provide a transformant transformed by the vector comprising the nucleic acid molecule encoding L-ribose isomerase.

It is still further object of this invention to provide a method for preparing an aldose or a ketose using the thermophilic L-ribose isomerase with a substrate specificity to L-ribose.

Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings.

In one aspect of the present invention, there is provided a L-ribose isomerase comprising the amino acid sequence of SEQ ID NO:2.

The present inventors have isolated Paenibacillus sp. RI-39 (KCCM 10653P) strain from soil of hot spring regions under conditions in which L-ribose was solely used as a carbon source. The microorganism has been characterized to be thermophilic and to be able to grow by use of L-ribose metabolized through pentose phosphate pathway and phosphoketolrase pathway. Furthermore, we have discovered that the novel L-ribose isomerase isolated from the bacterial strain has thermophilicity and very high substrate specificity to L-ribose. L-ribose isomerase according to the present invention shows thermophilicity.

The term "thermophilicity" as used herein refers to a feature that enzyme activities are stably maintained at high temperatures, having the same meaning as a thermal stability and a thermal resistance. In particular, the term thermophilicity means characteristics to exhibit at least half of the original activity of enzymes for at least 1-2 hr at 60-75⁰C.

L-ribose isomerase of this invention has the following features: (i) the optimum temperature of 60-80⁰C; (ii) the optimum pH of 6.0-7.0; (iii) a molecular weights of about 39-50 kDa; (iv) the activity increase in the presence of Co²⁺ or Ni²⁺; and (v) the activity decrease in the presence of Zn²⁺, Cu²⁺ or Mg²⁺. Preferably, L-ribose isomerase of the present invention has the optimum temperature of 65-78⁰C and the optimum pH of 6.3-6.8, more preferably 70-78⁰C and 6.3-6.7, and most preferably about 75⁰C and about 6.5.

According to a preferred embodiment, L-ribose isomerase of this invention has a molecular weight of about 39-50 kDa, more preferably about 40-58 kDa, most preferably about 42 kDa. L-ribose isomerase of the present invention has a homodimer structure.

L-ribose isomerase of the present invention is capable of catalyzing the conversion of L-ribose to L-ribulose and its reverse reaction, from L-ribulose to L- ribose.

According to a specific example, in the presence of 1 mM Co²⁺, Ni²⁺ or Mn²⁺, the activity of L-ribose isomerase increases dramatically by 907%, 1125% or 442%, respectively; in the presence of 1 mM Cu²⁺, Mg²⁺, Zn²⁺, Ca²⁺, Na⁺ or K⁺, the activity of L-ribose isomerase decreases by 0-39%. These characteristics address that the enzyme of the present invention requires a specific metal ion species to exhibit its maximal activity; however, its activity decreases by some other metal species. The enzyme kinetic parameters of L-ribose isomerase shows the K_m value of 4.76 mM and the V_max value of about 8.35 U/mg.

L-ribose isomerase of this invention is capable of catalyzing other aldoses and ketoses as well as L-ribose, preferably the conversion of D-lyxose to D-xylulose and its reverse reaction as well. The preferable substrate of this invention, L-ribose or L- ribulose is not D-type but L-type isomers and is likely to be naturally found in a very small amount. In light of this, the enzyme of this invention is considered very unique due to its activity to such a rare substrate. Preferably, L-ribose isomerase of this invention is derived from a thermophilic microorganism, most preferably Paen/bac/7/ussp. RI-39 (KCCM 10653P).

The term "protein" as used herein with referring to L-ribose isomrase, includes not only proteins isolated/purified from microorganisms but also recombinant proteins prepared by use of a gene encoding L-ribose isomerase. In another aspect of the present invention, there is provided a nucleic acid molecule encoding L-ribose isomerase as described above.

Most preferably, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:1.

The term "nucleic acid molecule" as used herein is intended to encompass DNA (gDNA and cDNA) and RNA molecules, including known analogs of natural nucleotides unless otherwise indicated (Scheit, Nucleotide Analogs, John Wiley, New York(1980); Uhlman and Peyman, Chemical Reviews, 90:543-584(1990)). It could be obvious to those skilled in the art that the present enzyme, L-ribose isomerase, is not limited by the amino acid sequence and nucleotide sequence set forth in the appended Sequence Listing, so long as the variants retain the most striking feature of L-ribose isomerase, i.e. the abilities to exhibit the optimum temperature at 60-80⁰C and to convert L-ribose to L-ribulose. The variations in nucleotide sequences may not cause changes in proteins.

Such a variation includes nucleic acid molecules comprising codons encoding functionally equivalent amino acids or identical amino acids {e.g., as a result of the degeneracy of genetic codes), or biologically equivalent amino acids.

In contrast, some variations in nucleotide sequences may cause changes in the L-ribose isomerizing enzyme. In such case, variants with substantially unimpaired activity may be obtained.

It would be obvious to one skilled in the art that the biologically functional equivalents falling into the scope of L-ribose isomerase of this invention are restricted to variations of amino acid sequences retaining the biological activities equivalent to L- ribose isomerase of this invention.

Such amino acid variations may be provided on the basis of a relative similarity of amino acid side chains, e.g., hydrophobicity, hydrophilicity, charge and size. By the analysis for size, shape and type of the amino acid side chains, it could be clear that all of arginine, lysine and histidine residues are those having positive charge; alanine, glysine and serine have a similar size; phenylalanine, tryptophan and tylosin have a similar shape. Accordingly, based on these considerable factors, arginine, lysine and histidine; alanine, glysine and serine; and phenylalanine, tryptophane and tylosin may be considered to be biologically functional equivalents.

For introducing mutation, a hydropathic index of amino acids may be considered. Based on the hydrophobicity and the charge, the hydropathic index is given to each amino acid: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glysine (- 0.4); threonine (-0.7); serine (-0.8); tryptophane (-0.9); tylosin (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagin (-3.5); lysine (-3.9); and arginine (-4.5).

For providing an interactive biological function of proteins, the hydropathic index of the amino acid is very important. It is well known to one of skill in the art that variations can possess a similar biological activity only where proteins are replaced with amino acids having similar hydropathic index. Where variations are intended to introduce based on the hydropathic index, the substitution is preferably performed between amino acid residues having no more than ±2 difference in hydropathic index values more preferably within ±1, much more preferably within ±0.5. It would be also obvious to those of skill in the art that substitutions of amino acids with other amino acids having similar hydrophilicity values may result in the generation of variants having biologically equivalent activities. As disclosed in U.S. Pat. No. 4,554,101, each amino acid residue is assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartate (+3.0±l); glutamate (+3.0±l); serine (+0.3); asparagin (+0.2); glutamine (+0.2); glysine (0); threonine (-0.4); proline (- 0.5±l); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (- 1.5); leucine (-1.8); isoleucine (-1.8); tylosin (-2.3); phenylalanine (-2.5); tryptophane (-3.4). Where variations are intended to introduce based on the hydrophilicity values, the substitution is preferably performed between amino acid residues having no more than ±2 difference in hydropathic index values more preferably within ±1, much more preferably within ±0.5. The alteration of amino acid residues not to substantially impair protein activity is well known to one skilled in the art (H.Neurath, R.LHill, The Proteins, Academic Press, New York, 1979). Such amino acid alteration includes Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, but not limited to.

Considering the afore-mentioned variations having biologically equivalent activities, it could be understood that either L-ribose isomerase of this invention or the nucleic acid encoding the same includes substantially identical sequences to the sequences set forth in the appended Sequence Listing. The substantially identical sequences refers to those showing preferably at least 61%, more preferably at least 70%, still more preferably at least 80%, most preferably at least 90% nucleotide similarity to the sequences of the appended Sequence Listing, as measured using one of the sequence comparison algorithms. Methods of alignment of sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482(1981); Needleman and Wunsch, J. MoI. Bio. 48:443(1970); Pearson and Lipman, Methods in MoI. Biol. 24: 307-31(1988); Higgins and Sharp, Gene 73:237-44(1988); Higgins and Sharp, CABIOS 5:151-3(1989); Corpet et al., Nuc. Acids Res. 16:10881-90(1988); Huang et al., Comp. Appl. BioSci. 8:155-65(1992); and Pearson et al., Meth. MoI. Biol. 24:307-31(1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. MoI. Biol. 215:403-10(1990)) is available from several sources, including the National Center for Biological Information (NBCI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blasm, blastx, tblastn and tblastx. It can be accessed at http://www.ncbi.nlm.nih.qov/BLAST/. A description of how to determine sequence identity using this program is available at http://www.ncbi.nlm.nih.gov/BI-AST/blast help.html.

In still another aspect of this invention, there is provided a vector comprising the nucleic acid encoding L-ribose isomerase as described above.

The vector system of this invention may be constructed according to the known methods in the art as described in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press(2001), which is incorporated herein by reference.

Typically, the vector may be constructed for cloning or expression. In addition, the vector may be constructed for use in prokaryotic or eukaryotic host cells.

In considering the facts that the nucleic acid molecule of this invention is originated from prokaryotic cells and prokaryotic cells are feasible in culturing, it is preferable that prokaryotic host cells are used.

For example, where the vector is constructed for expression in prokaryotic cells, it generally carries a strong promoter to initiate transcription {e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, p_L ^λ promoter, p_R ^λ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter), a ribosome binding site or translation initiation and a transcription/translation termination sequence. In particular, where E co/f ls used as a host cell, a promoter and operator in operon for tryptophan biosynthesis in E. coll

(Yanofsky, C, J. Bacteriol., 158:1018-1024(1984)) and a leftward promoter of phage λ (p_L ^λ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet, 14:399-445(1980)) may be employed as a control sequence.

Numerous conventional vectors used for prokaryotic cells are known to those of skill in the art, and the selection of an appropriate vector is a matter of choice.

Conventional vector used in this invention includes plasmids {e.g., pSClOl, CoIEl, pBR322, pUC8/9, pHC79, pUC19 and pET series), phages {e.g., λgt4λB, λ-Charon, λ Δzl and M13) and viruses {e.g., SV40).

For example, where the expression vector is constructed for eukaryotic host cell, inter alia, animal cell, a promoter derived the genome of mammalian cells (e.g., metallothionein promoter) or mammalian virus {e.g., adenovirus late promoter; vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) may be used. The vector generally contains a polyadenylation site of the transcript.

In addition, the vector of this invention further comprises a nucleotide sequence to conveniently purify the L-ribose isomerase protein expressed, which includes but not limited to, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6 x His (hexahistidine; Quiagen, USA), most preferably, 6 x His. Due to the additional sequence, the protein expressed can be purified with affinity chromatography in a rapid and feasible manner.

According to a preferred embodiment of this invention, the protein is purified by affinity chromatography. For example, in case of using glutathione S-transferase, the elution buffer containing glutathione is employed and in case of using 6 x His, Ni- NTA His-binding resin is generally employed to purify the protein of interest in a rapid and feasible manner.

It is preferable that the vector of this invention carries one or more markers which make it possible to select the transformed host, for example, genes conferring the resistance to antibiotics such as ampicillin, gentamycine, carbenicllin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

In further aspect of the present invention, there is provided a transformant comprising the vector as described above. The hosts useful in preparing the transformant may include any host cell well known to those skilled in the art. For example, as prokaryotic host, £ coli JM109, E CO// BL21 (DE3), E coli RRl, £ coli LE392, £ coli B, £ coli X 1776, £ α?// W3110, Bacillus sp. strains such as Bacillus subtillis and Bacillus thuringiensis, Bifidobacterium longum, Salmonella typhimurium, Serratia marcescens and various Pseudomonas sp. may be employed.

In addition, where the vector of this invention is transformed into eukaryotic cells, yeast (Saccharomyce cerevisiae), insect and human cells {e.g. CHO cell lines (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) may be used as host cells.

Where the host cell is the prokaryotic cell, the vector of this invention may be delivered into the host cell by CaCI₂ method (Cohen, S.N. et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114(1973)), Hanahan method (Cohen, S.N. et al., Proc. Natl. Acac. Sci. USA₁ 9:2110-2114(1973); and Hanahan, D., J. MoI. Biol., 166:557-580(1983)) and electroporation (Dower, WJ. et al., Nucleic. Acids Res., 16:6127-6145(1988)) etc. In addition, microinjection (Capecchi, M. R., Cell, 22:479(1980)), calcium phosphate precipitation (Graham, F.L. et al., Virology, 52:456(1973)), electroporation (Neumann, E. et al., EMBO J., 1:841(1982)), liposome-mediated transfection (Wong, T.K. et al., Gene, 10:87(1980)), DEAE-dextran treatment (Gopal, MoI. Cell Biol., 5:1188- 1190(1985)), and particle bombardment (Yang et al., Proc. Natl. Acad. Sci, 87:9568- 9572(1990)) can be used for introduction the vector into eukaryotic host cells.

The vector introduced into the host cell may express the protein, thereby producing a high level of L-ribose isomerase. For instance, where the expression vector carries the lac promoter, the induction of expression can be performed by treatment of IPTG to host cells.

In another aspect of the present invention, there is provided a method for preparing a ketose or aldose, which comprises contacting the L-ribose isomerase to an aldose or ketose as a substrate.

The enzyme of this invention may show the activity to various aldoses; preferably the enzyme is contacted to L-ribose or D-lyxose for preparing its counterpart isomer, L-ribulose or D-xylulose, respectively.

Moreover, the enzyme of this invention may show the activity to various ketoses; preferably the enzyme is contacted to L-ribulose or D-xylulose for preparing its counterpart isomer, L-ribose or D-lyxose, respectively.

More preferably, the suitable substrate in the method of this invention is L- ribose or L-ribulose.

The method for preparing ketoses or aldoses using L-ribose isomerase is preferably performed under conditions at temperature of 60-80⁰C and pH 6-7, most preferably approximately 75⁰C and pH 6.5.

According to a preferred example of this invention, the L-ribose isomerase- catalyzing reaction is performed in the presence of Co²⁺ or Ni²⁺.

Alternatively, for preparing aldoses or ketoses by L-ribose isomerase of this invention, cell extracts of Paenibacillus sp. RI-39 (KCCM 10653P) containing L-ribose isomerase may be used instead of the isolated L-ribose isomerase.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. Ia represents a SDS-PAGE gel photograph of L-ribose isormerase of this invention isolated and purified from Paenibacillus sp. RI-39.

Fig. Ib is a non-denaturing PAGE (Native-PAGE) gel photograph of L-ribose isormerase of this invention isolated and purified from Paenibacillus sp. RI-39.

Fig. 2 represents a graph showing a thermal stability of L-ribose isomerase of this invention isolated and purified from Paenibacillus sp. RI-39.

Fig. 3 is a graph demonstrating effects of reaction temperatures on the activity of L-ribose isomerase of this invention isolated and purified from Paenibacillus sp. RI- 39.

Fig. 4 is a graph showing the optimum pH of L-ribose isomerase of this invention isolated and purified from Paenibacillus sp. RI-39.

Fig. 5 is a graph showing effects of various metal ions on the activity of L- ribose isomerase of this invention isolated and purified from Paenibacillus sp. RI-39.

Fig. 6 is a graph showing substrate specificity to various pentoses of L-ribose isomerase of this invention isolated and purified from Paenibacillus sp. RI-39. Fig. 7 is a Lineweaver-Burk plot of L-ribose isomerase of this invention isolated and purified from Paenibacillus sp. RI-39.

Fig. 8 is a graph showing the production of L-ribulose from L-ribose by L-ribose isomerase of this invention isolated and purified from Paenibacillus sp. RI-39 (analyzed by GC-MC method). Fig. 9 is a graph showing the production of L-ribulose from L-ribose by L-ribose isomerase of this invention isolated and purified from Paenibacillus sp. RI-39 (analyzed by HPIC method).

Fig. 10 represents alignment results between well-known amino acid sequences (BSRI, BLRI and BCRI) and the amino acid sequence (RI39) of L-ribose isomerase of this invention.

EXAMPLES Example 1: Isolation of L-Ribose Isomerase

L-ribose isomerase was isolated from Paenibacillus sp. RI-39(KCCM 10653P) having been isolated and characterized by us from soil of hot spring region in

Indonesia

Paenibacillus sp. RI-39 was cultured for 50 hr at 5O⁰C, 220 rpm under aerobic conditions in 250 ml pleated flask and centrifuged (8000 x g, 20 min, 4⁰C) at an initial stable growth phase to harvest cells. After washing the harvested cells twice with 50 mM sodium phosphate buffer solution (pH 6.5), cells were suspended in the same buffer solution (200 ml) and ultrasonicated for cell lysis. The cell debris was removed by centrifugation (10,000 x g, 30 min, 4⁰C) and supernatant formed thus was used as enzyme crude extracts. Because the activity of L-ribose isomerase was stable at 4⁰C for several weeks, all purification procedures were performed at 4⁰C. First, the enzyme crude extracts were purified by DEAE-separose column chromatograpy using Hiprep 16/10 DEAE column (Pharmacia), and then by Phenyl column chromatograpy using Resource phenyl column (Pharmacia).

Example 2: Electrophoresis and Analysis of the N -Terminal and Internal

Amino Acid Sequences

Electrophoresis was performed using the protein samples purified in Example 1.

SDS-PAGE on 12% acrylamide gel slap was performed according to Laemmli (Laemmli, U. K. 1970) method, and non-denaturing electrophoresis (Native-PAGE) was carried out on 8.0% acrylamide gel slap (thickness: 0.75 mm). The protein bands were stained with Coomassie brilliant blue R-250 (Bio-Rad).

The results of SDS-PAGE showed a single protein band, demonstrating that the purified enzyme is homogeneous. The molecule weight was revealed to be about 21 kDa by SDS-PAGE (Rg. Ia: lane 1, SDS-PAGE standard maker; lane 2, finally purified

L-ribose isomerase). In the Native-Page, the protein band was detected at approximately 42 kDa (Fig. Ib: lane 1, standard maker; lane 2, finally purified L-ribose isomerase). The results of electrophoresis address that L-ribose isomerase of the present invention exists as a homodimer structure.

The analysis of N-terminal and internal amino acid sequences was carried out using the finally purified sample in Example 1. The sequence analysis was executed in the Analytical Core Facility of Tufts university (Boston, MA), thereby revealing the N- terminal and internal sequences as follows: MRGTEWREARDRVA (N-terminal sequence), QLVTYLNTDR (internal sequence 1), LVSEFSST (internal sequence 2) and GLALTPSELE (internal sequence 3).

Example 3: Characterization of L-Ribose Isomerase The physiochemical characteristics of L-ribose isomerase isolated in Example 1 were analyzed. The enzyme activities were measured using L-ribose as substrates, and the contents of L-ribulose produced were analyzed in accordance with cysteine- carbazole-sulfuric acid method (Dische, Z., and E. Borefreund. (1951) J. Biol. Chem. 192, 583-587).

Example 3-1: Thermal Stability and Optimum Temperature

To measure the thermal stability for isomerization activity of L-ribose isomerase, the enzyme was incubated in 50 mM sodium phosphate solution (pH 6.5) for 0-2 hr at 6O⁰C, 70⁰C or 8O⁰C in the presence of or the absence of 1 mM Co²⁺, and its aliquots were then sampled at constant time intervals, followed by measuring residual activities (Fig. 2). As shown in Fig. 2, L-ribose isomerase of this invention obtained from Paenibacillus sp. RI-39 exhibited the stability in the presence of metal ion (Co²⁺) at more than 6O⁰C. L-ribose isomerase of this invention showed the stability up to 7O⁰C in 50 mM sodium phosphate buffer solution (pH 6.5). However, where the metal ion was absent, the enzyme exhibited little or no activity. In contrast, L-ribose isomerase was evaluated to show higher optimum temperature of 70 to 75⁰C (Fig. 3).

Example 3-2: Optimum pH The optimum pH of enzymes was measured using the following buffer solution (100 mM): sodium acetate/acetic acid buffer (pH 4-6), potassium phosphate buffer (pH 6-7.5) and Tris-HCI buffer (pH 7-8.5). Aliquots were sampled to measure enzyme activities according to procedures of Example 2 (Fig. 4). As shown in Fig. 4, the optimum pH of L-RI was analyzed to be 6.5 at 75⁰C, and the activities corresponding to more than 50% of the highest activity were found between pH 5.5 and 8.0.

Example 3-3: Effect of Metal Ion Following treatment of the purified enzyme with 10 mM (final cone.) of EDTA, the enzyme was kept to stand over 12 hr and subjected to dialysis against 50 mM sodium phosphate buffer for further experiments. The enzyme was pre-incubated for 10 min in 50 mM sodium phosphate buffer containing 1 mM (final cone.) CoCI₂, MnCI₂, FeCI₃, KCI, MgCI₂, CaCI₂, ZnCI₂ or CuCI₂, and its residual activity was measured. Fig. 5 showed effects of 1 mM metals on L-RI activities. The addition of Co²⁺ and Ni²⁺ was responsible for increasing the enzyme activity up to 907% and 1125%, respectively compared to the control. In contrast, the enzyme activities significantly decreased by the addition of 1 mM Zn²⁺(8%), Cu²⁺(0%) and Mg²⁺(0%).

Example 3-4: Examination of Substrates

Substrates for L-ribose isomerase were examined using various aldoses as substrates. The reactions were conducted for 10 min at 75⁰C by use of 125 μl reaction mixtures (pH 6.5 at room temperature) containing 40 mM L-substrate, 1 mM CoCI₂ and 50 mM sodium phosphate. L-ribose and D-lyxose as substrates produced their ketose counterparts, L-ribulose and D-xylose (Fig. 6). In contrast, other aldoses were analyzed to produce little or no ketoses.

Example 3-5: Measurement of K_g Values The enzyme was incubated in 50 mM sodium phosphate buffer solution (pH

6.5) at 75⁰C for 5 min in the presence of 1 mM Co²⁺ with using various concentrations of L-ribose (5-1000 mM) for measuring several kinetic parameters. Lineweaver-Burk plots were shown in Fig. 4 (L-ribose). K_m value against L-ribose of L-RI derived from Paen/bac/llus sp. RI-39 was calculated 4.76 mM, and V_max value 8.35 U/mg.

Example 3-6: Detection of Final Products

The enzymatic reactions were induced using the finally purified enzyme in 50 mM sodium phosphate buffer (pH 6.5) over 6 hr at 6O⁰C in the presence of 1 mM Co²⁺ using L-ribose as substrates. Following the enzymatic reactions, the final products were identified using GC (Gas chromatography) and HPIC (High performance ionic chromatography) (Figs. 8 and 9). It was found that the enzymatic reactions using L- ribose as substrates resulted in the production of L-ribulose as final products.

Example 4: Construction of Genomic DNA Library of Paen/bac/7/us sp. RI-39

Following the isolation of gDNAs from Paen/bac/llus sp. RI-39 (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press(2001)), they were digested with a restriction enzyme Sau3M to produce about 2-5 kb fragments. The pBluescript II KS(+) vectors (Stratagen) were digested by a restriction enzyme BamHl, the 2-5 kb fragment and the digested vector were ligated by T4 DNA ligase. The resultant ligates were transformed into into E col/ TOPlO (Invitrogen) to construct the genomic DNA library of Paenibacillus sp. RI-39.

Example 5: Sequencing and Analysis of L-Ribose Isomerase Gene Based on the partial amino acid sequences of L-ribose isomerase described in

Example 2, we designed PCR primers as follows: NF-I: 5'-GGATGMGIGGNACNGA-3' (forward primer); IR-I: S'-GGTCIGTRTTIADRTANGTNAC-S' (backward primer 1), IR-2: 5'-GGGNGTIADNGCNADICC-S' (backward primer 2), IR-3: 5'- GGGYTTIACrrCIADYTCNC-3' (backward primer 3).

Next, the gDNA library constructed in Example 4 was used as templates, and PCR amplification was performed using a set of NF-I and IR-I primers. A reaction mixture was prepared to contain 10 x buffer, 1.5 mM MgCI₂, 1 mM dNTPs, 25 pM each primer, 2.5 unit Ex- Taq polymerase (Takara Biomedical) and 300 ng genomic DNA. The PCR amplifications were carried out on GeneAmp PCR system 9700 (Perkin-Elmer Cetus) under the following thermal conditions: 30 cycles of 50 sec at 94⁰C (denaturation), 40 sec at 5O⁰C (annealing) and 20 sec at 72⁰C (extension), followed by a 2 min final extension at 72⁰C. The amplified products were resolved by electrophoresis on a 0.8% agarose gel. We could reveal a partial nucleotide sequence (about 180 bp) of the L-ribose isomerase gene based on results of the PCR amplifications.

Based on the identified partial sequence of the L-ribose isomerase gene, the primers were designed to amplify ORF of the L-ribose isomerase gene as follows: AF: 5'-GGTGGCGGAGATGTTTC-S' (forward primer 1), BF: 5'-TAATACGACTCACTATAGGG-3' (forward primer T)₁ CF: 5'-GAACTGGAAAAGGTGGAGGTCG-S' (forward primer 3); AR: 5'-AATTAACCCTCACTAAAG-S' (backward primer 1), BR: 5'-GCCAAGTTCCCCAATCC-3' (backward primer 2), CR: 5'-CGATGGCGTCAGGGCGATG-S' (backward primer 3).

Afterwards, PCR amplifications were performed using the gDNA library constructed in Example 4 as template, and a set of AF and AR primers, a set of BF and BR primers or a set of CF and CR primers. A reaction mixture was prepared to contain 10 x buffer, 1.5 mM MgCI₂, 1 mM dNTPs, 25 pM each primer, 2.5 unit Ex- Tag polymerase (Takara Biomedical) and 500 ng genomic DNA. The PCR amplifications were carried out on GeneAmp PCR system 9700 (Perkin-Elmer Cetus) under the following thermal conditions: 30 cycles of 50 sec at 94⁰C (denaturation), 40 sec at 45- 55⁰C (annealing) and 90-300 sec at 72⁰C (extension), followed by a 2 min final extension at 72⁰C. The amplified products were resolved by electrophoresis on a 0.8% agarose gel. We could reveal a full sequence (about 0.55 kb) comprising the 180 bp- partial sequence of the L-ribose isomerase gene based on results of the PCR amplifications.

These results demonstrate that the ORF of the L-ribose isomerase gene is 549 bp in length (SEQ ID:1), coding for the amino acid sequence containing 182 amino acid residues (SEQ ID:2). The amino acid sequence containing 182 amino acid residues have a molecular weight of about 20 kDa which is similar to the molecular weight measured by SDS-PAGE in Example 2.

The nucleotide sequence of the L-ribose isomerase gene was subjected to similarity search using BLAST. As a result, the L-ribose isomerase gene was elucidated to show 60% similarity to three genes (Protein sequence database accession No. YP_077744, CAB12227 and BAD62991), of which functions have not yet been identified. The amino acid sequence alignment for L-ribose isomerase and proteins encoded by three genes was carried out, as represented in Fig. 10.

In recent, L-ribose has been mostly prepared by chemical synthesis methods; however these methods have disadvantages: complexity of purification steps, and production of harmful chemical contaminants and by-products. However, because L- ribose isomerase of this invention has significant thermophilicity as well as substrate specificity to L-ribose, it has plausible advantages over chemical synthesis methods. For example, L-ribose isomerase enables to utilize substrates in higher concentrations since enzymatic reactions using L-ribose isomerase can be carried out with minimizing generation of byproducts in higher temperature conditions. Furthermore, where reactions are performed using the L-ribose isomerizing enzyme of this invention, their viscosity is low, their susceptibility to contamination by microorganisms is very low and their rate is very likely to be high. In addition to this, the L-ribose isomerizing enzyme of this invention contributes to minimization of byproducts generation. Interestingly, L- ribose isomerase of this invention enables utilization of D-lyxose as well as L-ribose as substrates to be available. Moreover, L-ribose isomerase of this invention is able to catalyze the reverse reaction, so that L-ribulose and D-xylulose as substrates can be used to produce their corresponding aldoses. In particular, one of rare sugars, L- ribose is effectively obtained as L-ribulose as substrates is used.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is:

1. A L-ribose isomerase comprising the amino acid sequence of SEQ ID NO: 2.

2. A nucleic acid molecule encoding the L-ribose isomerase of claim 1.

3. The nucleic acid molecule according to claim 2, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO:2.

4. A vector comprising the nucleic acid molecule of claim 2 or 3 encoding the L- ribose isomerase.

5. A transformant comprising the vector of claim 4.

6. A method for preparing a ketose or aldose, which comprises contacting the L- ribose isomerase of claim 1 to an aldose or ketose as a substrate.

7. The method according to claim 6, wherein the aldose as the substrate is L-ribose or D-lyxose, and the ketose as the substrate is L-ribulose or D-xylulose.

8. The method according to claim 7, wherein the substrate is L-ribose or L-ribulose.

9. The method according to claim 6, wherein the method is performed at 60-80 ⁰C and pH 6-7.

10. The method according to claim 6, wherein the method is performed in the presence of Co²⁺ or Ni²⁺.