KR100757280B1 - 10859 New Hyperthermophilic KCTC 10589BP Strain and Hyperthermophilic Amylase Produced Therefrom - Google Patents

Abstract

The present invention relates to a highly thermophilic new strain KCTC 10859BP and a highly thermophilic amylase produced therefrom, Thermococcus sp. A highly thermophilic amylase isolated from NA1, a gene encoding the same, and a method of producing the same.

Hyperthermia, amylase, new strain KCTC 10859BP    

Description

New hyperthermophilic KCTC 10589BP Strain and Hyperthermophilic Amylase Produced Therefrom

1 is Thermococcus sp. Sequence comparisons of α-amylase from NA1 (TNA1), T. kodakarensis KOD1 189 (TkKOD1, gi: 57641390), T. hydrothermalis (PaGE5, gi: 14521082) and P. furiosus (PhOT3, gi14590819) are shown. The dash means a gap and the number on the right indicates the position of the last residue in the original sequence. The same residues between the four enzymes are indicated with *, and the conserved substitutions and the semiconserved chiwans, respectively: and. As shown. Residues that participate in Ca ²⁺ , which are important for thermostability, are indicated in bold and underlined. Revised or tentative signal peptides are underlined.

2 shows the expression of α-amylase in E. coli (A) and the expression of SDS-PAGE (B) of purified enzyme. A: Plate analysis of α-amylase activity of E. coli transformants. B: Coomassie blue staining of separated TNA1_amylm. The molecular weight standards of M lanes are phosphorylase b (103 kDa), bovine serum albumin (77 kDa), ovalbumin (50 kDa), carbonic anhydrase; 34.3 kDa), soybean trypsin inhibitor (28.8 kDa) and lysozyme (20.7 kDa). The bands corresponding to the enzymes of the invention are indicated by arrows.

3 shows the effect of temperature (A) and pH (B) on TNA1_amyl activity according to the invention. A: The sample temperature was increased from 30 to 95 ° C., and activity analysis was performed at standard conditions. B: Activity assays were performed at standard conditions using the following buffers (50 mM each), pH 4.0-6.0; Tris-HCl, pH 6.5-9.5

4 shows heat inactivation for α-amylase. A semilog plot of the remaining activity versus incubation time is shown. α-amylase (2nM) was incubated at 80 ° C. (original) or 90 ° C. (square) in 50 mM sodium acetate buffer (pH 6.0) in the presence or absence (0.5 figure) of 0.5 mM Ca ²⁺ . . At the indicated time, a portion was taken out and the activity was measured in the same buffer using soluble starch as substrate at 80 ° C. Straight lines were obtained through linear analysis of the data.

5 shows the effect of metal ions on the activity of α-amylase. The analysis was different for divalent metal ions, but the analysis was performed under standard conditions.

6 shows the measurement of the dynamic constants using a Lineweaver-Burke plot. Assays were performed under standard conditions with varying substrate concentrations.

7 shows thin layer chromatography of the products of the TNA_amyl reaction on various substrates. Reaction products from metallooligosaccharides (G2, G3, G4, G5, G6), starch (S), amylopectin (A), flulan (P), cyclodextrins (αCD and βCD) were analyzed. M represents glucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4), maltopentose (G5) and maltohexose (G6).

8 shows a cleavage map of a recombinant plasmid having a recombinant α-amylase enzyme according to the present invention.

The enzymes that degrade starch are distributed in many microorganisms and are used in the textile, food, fermentation, papermaking, casting and distillation industries, as well as in the glycosylation of sugars as well as in industrial applications in a wide range of fields such as clinical, pharmaceutical and analytical chemistry Pandey et al., 2000; Vihinen et al., 1989). There is a high demand for suitable enzymes for specific applications that make the process economical in terms of temperature, use of high starch, substrate specificity and pH. In the process of saccharification and liquefaction of starch at 80 to 90 ° C, the enzyme needs to be stable at high temperatures. Therefore, thermophilic and thermostable enzymes were required. In addition to thermal stability, highly thermophilic enzymes may be advantageous because of their resistance to denaturing agents, solvents, and proteolytic enzymes. Thermostable amylose degrading enzymes, including α-amylase, have been reported in mesophilic Bacillus sp., thermophilic archaea and sedative bacteria (Brown and Kelly 1993; Chung et al., 1995; Dong et al., 1997; Frillingos et. al. 2000; Morgan and Priest, 1981; Tachibana et al., 1996). Most of them belong to glycosyl hydrolase family 13 (Henrissat, 1991), based on amino acid homology. Although this population has low overall sequence similarity, it has four conserved catalytic sites and a common (α / β) ₈ -barrel structure (van der Maarel et al., 2002). In addition to the search for useful enzymes, manipulation of useful enzymes to meet important criteria for commercialization can improve enzyme stability.

In a combination of common genetic engineering and genome research methods, the gene sequence of highly thermophilic microorganisms is of biotechnological interest due to thermostable enzymes, and many highly thermostable enzymes have been developed for biotechnological purposes. Recently, the inventors have found that the thermophilic archaea Thermococcus sp. NA1 was isolated and the entire genome sequence was determined to find many useful extremely thermally stable enzymes. Analysis of genomic information showed that the strain had starch-degrading enzymes such as α-amylase, α-glucosidase, pullanase and cyclodextrinase. The present inventors cloned the α-amylase gene, expressed it in E. coli using a recombinant vector, and purified it to confirm enzymatic activity to complete the present invention.

The present invention provides a novel highly thermophilic Thermococcus sp. NA1 (KCTC 10859BP) is provided.

The present invention provides a novel highly thermophilic α-amylase.

The present invention provides a method for producing a novel highly thermophilic α-amylase.

The present invention is a new strain Thermococcus sp. NA1 (KCTC 10859BP) is provided.

The present invention provides α-amylases and methods of making them. The manufacturing method is not limited thereto, but is preferably a manufacturing method by a genetic engineering method.

The present invention provides an isolated DNA sequence encoding α-amylase and a recombinant vector containing them.

In a first aspect, the present invention provides a novel strain of Thermococcus sp. NA1 (KCTC 10859BP) is provided. Thermococcus sp. NA1 was isolated from deepwater hydrothermal vents in the area in the Park Manus region (3 ° 14 'S, 151 ° 42' E) in East Manus Basin. YPS medium was used for thermococcus sp. It was used to culture NA1, Thermococcus sp. Culture of NA1 and strain maintenance were done by standard methods. Thermococcus sp. To prepare NA1 seed cultures, YPS medium in 25 ml serum bottles was inoculated with a single colony formed on a physicalel plate and incubated at 90 ° C. for 20 hours. Seed culture was used to inoculate 700 ml of YPS medium in an anaerobic jar and incubated at 90 ° C. for 20 hours. The thermophilic Thermococcus sp. NA1 was deposited with the Korea Institute of Biotechnology and Biotechnology Center (KCTC) on October 7, 2005, and was assigned the accession number of KCTC 10859BP on October 20, 2005. DNA polymerase (Korean Patent Application No. 10-2005-0094644), DNA ligase (Korean Patent Application No. 10-2005-094693), and carboxypeptidase (Korean Patent Application No. 10-2005-0103489), which are highly thermophilic enzymes from the strain. Proyl oligopeptidase (Korean Patent Application No. 10-2005-0103487), Aminopeptidase P (Korean Patent Application No. 10-2005-0103488) and Methionyl Aminopeptidase (Korean Patent Application No. 10-2005-0103493) Filed.

In a second aspect, the present invention provides a nucleic acid sequence encoding α-amylase that is stable at high temperature and an equivalent nucleic acid sequence to the sequence. The nucleic acid sequences are more specifically SEQ ID NO: 1 or SEQ ID NO: 2.

"Equivalent nucleic acid sequence" includes codon degenerate sequences of the α-amylase sequence.

By "codon degenerate sequence" is meant a nucleic acid sequence that encodes a polypeptide that is different from the naturally occurring sequence but identical to the naturally occurring α-amylase enzyme disclosed herein.

In a third aspect, the present invention provides α-amylase. More specifically, α-amylase represented by SEQ ID NO: 3 and functional equivalents thereof are provided.

“Functional equivalents” of the invention include amino acid sequence variants in which some or all of the α-amylase enzyme of SEQ ID NO: 3 is substituted, or a portion of the amino acid is deleted or added. Substitutions of amino acids are preferably conservative substitutions. Examples of conservative substitutions of amino acids present in nature are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Deletion of amino acids is preferably located in portions not directly involved in the activity of α-amylase.

In a fourth aspect, the present invention provides a recombinant vector comprising an isolated DNA segment encoding the α-amylase.

The term vector, as used herein, refers to a nucleic acid molecule capable of binding and transferring another nucleic acid thereto. The term expression vector includes plasmids, cosmids or phages capable of synthesizing proteins encoded by each recombinant gene carried by the vector. Preferred vectors are vectors capable of self-replicating and expressing the nucleic acid to which they are bound.

In a fifth aspect of the invention, there is provided a cell transformed with a recombinant vector comprising an isolated DNA segment encoding the α-amylase.

The term 'transformation' used in the present invention means that foreign DNA or RNA is absorbed into a cell and the genotype of the cell is changed.

Suitable transformed cells include, but are not limited to, prokaryotes, fungi, plants, animal cells, and the like. Most preferably, E. coli is used.

A sixth aspect of the present invention provides a method for producing α-amylase using the transformed cells or the Thermococcus sp.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following Examples are intended to illustrate the present invention, but the content of the present invention is not limited by the Examples.

Example 1 Primary Structure of TNA1_amyl Gene and Expression of Recombinant Enzyme

(1) strains and growth conditions

Thermococcus sp. NA1 was isolated from deepwater hydrothermal vents in the area in the Park Manus region (3 ° 14 'S, 151 ° 42' E) in East Manus Basin. YPS medium was used for thermococcus sp. It was used to culture NA1, Thermococcus sp. Culture of NA1 and strain maintenance were done by standard methods. Thermococcus sp. To prepare NA1 seed cultures, YPS medium in 25 ml serum bottles was inoculated with a single colony formed on a pattagel plate and incubated at 90 ° C. for 20 hours. Seed culture was used to inoculate 700 ml of YPS medium in an anaerobic jar and incubated at 90 ° C. for 20 hours.

E. coli DH5α was used for plasmid propagation and nucleic acid sequencing. E. coli BL21-Codonplus (DE3) -RIL cells (stratazine, Lazola, Calif.) And plasmid pET-24a (+) (Novagen, Madison, Wisconsin) were used for gene expression. E. coli strains were incubated in Luria-Bertani medium at 37 ° C. and kanamycin was added to the medium to a final concentration of 50 μg / ml.

(2) DNA manipulation and sequencing

DNA manipulations were done in a standard manner as described by Samprock and Russell. Genomic DNA of Thermococcus sp. Was isolated by standard methods. Restriction enzymes and other modifying enzymes were purchased from Promega (Madison, Wisconsin). Small amounts of plasmid DNA from E. coli cells were made using the plasmid mini kit (Qiagen, Hilden, Germany). DNA sequencing was performed with an automated sequencer (AB3100) using the Big Die Terminator Kit (PE Applied Biosystems, Foster City, CA).

(3) Cloning and Expression of α-amylase Coding Gene

Thermococcus sp . Flanked by Nde I and Xho I. The full length of the gene of α-amylase of NA1 (TNA1_amyl) is determined by genomic DNA and two primers (sense [5′-CG ACC CGG CAT ATG GCC AGA AAA GCA GCC GTT GCA GTT TTG-3 ′; SEQ ID NO: 5] and antisense). [5′-CT CCA CAT CTC GAG GCC AAC ACC ACA GTA GCT CCA GAC-3 ′; SEQ ID NO: 6]; the sequence underlined in the sense primer is the NdeI site and the sequence underlined in the antisense primer is Xho I). The full-length length of the gene of α-amylase was determined by genomic DNA and two primers (sense [5′-CG ACC CGG CAT ATG GCG GAA ACA CTG GAA AAC GGC GGA GTC-3 ′). SEQ ID NO: 7 and antisense [5′-CT CCA CAT CTC GAG GCC AAC ACC ACA GTA GCT CCA GAC ′; SEQ ID NO: 8]; the underlined sequence in the sense primer is the NdeI site and the underlined sequence in the antisense primer Amplified by Xho I). The amplified sequence was digested with Nde I and Xho I and linked to Nde I / Xho I digested pET-24a (+). The conjugate was transformed into E. coli DH5α, and the resulting plasmids were pET-amylf (full length) and pET-amylm (mature form), and E. coli BL21-CodonPlus for sequencing and expression. (DE3) -RIL and E. coli Rosetta (DE3) pLysS, respectively. Overexpression of the gene was induced by adding isopropyl-β- _D -thiogalactopyranoside (IPTG) to the mid-grade water growing phase and incubating at 37 ° C. for 3 hours. Cells were obtained via centrifugation (6,000 × g for 20 min at 4 ° C.) and resuspended in 50 mM Tris-HCl buffer (pH 8.0) containing 0.1M KCl and 10% glycerol. Cells were pulverized by ultrasound and separated by centrifugation (20,000 xg for 30 minutes at 4 ° C). The resulting supernatant was treated on a column of TALON ^™ metal affinity resin (BD Bioscience Clontech, Palo Alto, Calif.) And 10 mM imidazole in 50 mM Tris-HCl buffer (pH 8.0) containing 0.1 M KCl and 10% glycerol. (Sigma, St. Louis, Missouri) and α-amylase eluted at 300 mM in buffer. The collected fractions were then buffer exchanged with 50 mM Tris-HCl (pH 8.0) buffer containing 10% glycerol using Centricon YM-10 (Millipore, Bedford, Mass.).

Protein concentration was measured by the method of Bradford (1976). The degree of purification of the protein was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis performed by standard methods (Laemmli, 1970).

(4) Primary structure of TNA1-amyl coding gene and expression of recombinant enzyme

Thermococcus sp. In genome sequencing of NA1, the inventors found an open reading frame (ORF) encoding a protein consisting of 1,377 bp encoding a protein consisting of 458 amino acids (SEQ ID NO: 4) and encoding a predicted molecular weight of 51,948 Da. .

Sequence comparisons were T. hydrothermalis (85% identity; Leveque et al., 2000a) and T. kodakarensis KOD1 (82% homology; Tachibana et al., 1996), and P. furiosus or P. woesei (82% Homology; high similarity with amylase from Dong et al., 1997).

The inferred amino acid sequence of the region encoding α-amylase included a putative signal peptide of 25 amino acids and a mature enzyme of 433 amino acids (expected molecular weight 50,531 Da) (FIG. 1). Sequencing revealed four active site consensus regions of glycosyl hydrolase population 13 (Nakajima et al., 1986) in TNA1_amyl. Data on amino acid composition showed that the amount of Asp and Gln was low in TNA amyl (31 compared to 47 B acillus licheniformis α-amylase), which would contribute to the stability of the protein at high temperatures. TNA_amyl has low Lys and Arg residues (32 similar to 35 P. furiosus α-amylase), which will account for the isoelectric point (4.61 isoelectric point) of the enzyme. The residues corresponding to amino acid residues that Linden et al. (2003) claimed to be Ca ²⁺ binding centers near the active site gap in P. woesei were preserved.

The full length of the TNA1_amyl gene was cloned into the pET-23a (+) vector by PCR (pET-amylf) and transformed into E. coli BL21-Codonplus (DE3) -RIL. When cultured on starch medium plates, E. coli transformed with an empty vector could not degrade this substrate after incubation at 60 ° C, whereas E. coli transformed with pET-amylf had a distinct halo indicating effective starch degradation of the TNA1_amyl gene. Formed (FIG. 2A). The activity of α-amylase was observed in the extracellular fraction of recombinant E. coli with pET-amylf. The inventors prepared the plasmid pET-amylm without signal peptide. The mature form of α-amylase was isolated from the soluble fraction of induced E. coli (pET-amylm), and the isolated protein remained soluble after repeated freezing and thawing. In SDS-PAGE, a band of isolated enzyme was observed at a size smaller than 51 kDa estimated from the amino acid sequence of mature form including His ₆ -tag (FIG. 2B).

Example 2 Biochemical properties of α- amylase enzyme

(1) Method for measuring α-amylase enzyme activity

Thermococcus sp . The activity of NA1 α-amylase (TNA1_amyl) was determined by measuring the amount of reducing sugar liberated during enzymatic degradation of 50 mM sodium acetate buffer (pH 6.0) dptj 1% soluble starch at 80 ° C. for 15 minutes. The amount of reducing sugar was determined by the modified dinitrosalicylic acid method (Bernfeld, 1955). One unit of α-amylase activity was defined as the amount of enzyme that liberates 1 μmole of reducing sugar equivalent to maltose per minute under the assay conditions.

(2) Screening of α-amylase activity in thin layer chromatography (TLC) and agar plates

Recombinant E. coli clones expressing thermostable α-amylase were grown on LB agar plates supplemented with 50 μg ml ⁻¹ kanamycin, 1% soluble starch and 1 mM IPTG and incubated continuously at 60 ° C. for 12-18 hours. Was identified. Positive colonies were sprinkled with Lugol's solution (I ₂ 5 gl ⁻¹ ; KI 10 gl ⁻¹ ) to create starch degradation regions that appeared as white halo against dark backgrounds.

Analysis of oligosaccharides of G1 to G7 was performed by modified thin layer chromatography (TLC) as a synergistic technique on silica gel coated TLC plates (type K6F; Whatman, UK; Dong et al., 1997). After developing with a solvent mixture (isopropyl alcohol: ethyl acetate: water = 3: 1: 1, v / v / v), the TLC plate was completely dried and the staining solution (N- (1-naphthyl) -ethylenediamine 3 g, 50 ml of sulfonic acid, adjusted to 1 L with methanol) was quickly immersed in color. The TLC plate was incubated at 110 ° C. for 10 minutes.

(3) Biochemical Characterization of TNA1_amyl

The specific activity of the separated TNA1_amyl was 7238 U mg ⁻¹ . The enzyme showed optimal activity at 80 ° C. and less than 20% of maximum activity at 40-50 ° C. (FIG. 3). The optimum pH for the enzyme was 5.5 to 6.0 (Figure 3B). The thermal stability of TNA1_amyl was determined by incubating the enzyme in 50 mM sodium acetate buffer (pH 6) at 80 and 90 ° C. TNA1_amyl was thermostable and had a half-life (t _1/2 ) of 30 minutes at 80 ° C. and 10 minutes at 90 ° C., respectively (see FIG. 4). The thermal stability of TNA1_amyl increased significantly significantly at 7,581 min at 80 ° C. and 153 min at 90 ° C., respectively, in the presence of 0.5 mM Ca ²⁺ (see FIG. 4), indicating that Ca ²⁺ bonds are necessary to increase thermal stability. Means that. In contrast to the requirement of Ca ²⁺ ions for improved thermal stability, the addition of 0.5 mM Ca ²⁺ did not increase the enzyme activity (FIG. 5). However TNA1_amyl are when treated with ethylenediaminetetraacetic acid (EDTA) treatment of 1mM, decreased activity is up to 4%, because been reduced activity was recovered by addition of Ca ²⁺ by EDTA, Ca ²⁺ on the enzyme activity Was dependent on its presence. Other divalent ions did not replace Ca ²⁺ for TNA1_amyl activity. Zn ²⁺ and Cu ²⁺ significantly inhibited TNA_amyl activity.

Kinetic analysis was performed using soluble starch, and K _m (4.57 mg ml ⁻¹ ) and V _{max (} 10,000 μmole min ⁻¹ mg ⁻¹ ) values were measured using a Lineweaver-Burk plot (FIG. 6). .

TNA1_amyl made soluble starch from maltose (G2) to maltoheptaose (G7) as the main product and hydrolyzed several substrates such as amylose, oligomaltosaccharides, amylopectin and soluble starch. No activity to degrade pullulan and cyclodextrin was observed. In conclusion, TNA1_amyl can be classified as a liquefiing enzyme such as P. furiosus (FIG. 7).

(4) Characteristic comparison analysis

The biochemical properties of TNA1_amyl were compared with thermococal α-amylase from other T. profundus and T. hydrothermalis at pH optimum, temperature and substrate profile, and the increased hydrolytic activity shown in Table 1 appears to be slightly increased. (Chung et al., 1995; Lee et al., 1996; Leveque et al., 2000b; Tachibana et al., 1996)

Table 1 Comparison of general biochemical properties of α-amylase from TNA1_amyl and Thermococcales

Ca ²⁺ has been reported to increase amylolytic activity and thermostability of α-amylase. However, most of these enzymes were active even in the absence of Ca ²⁺ . In P. furiosus α-amylase, Ca ²⁺ ions are very tightly attached to the enzyme, which does not allow EDTA to remove it, retained 95% α-amylase in 1 mM EDTA (11.5 ng) and even 86% activity in 5 mM EDTA. Was maintained (Linden et al., 2003). However, recent reports on the presence of calcium-binding residues in P. furiosus and treatment data of EDTA at high temperatures after much dialysis for metal-free buffers suggest that calcium binding is essential for the amylolytic activity of P. furiosus α-amylase. (Savchenko et al. 2002). In comparison with amylase of P. furiosus , inhibition of TNA1_amyl by EDTA was significant and reversible by the presence of Ca ²⁺ . In conclusion, calcium binding appears to be a major factor in the thermal stability of TNA1_amyl. It is reported that the amount of cysteine probably affects thermal stability (Tomazic and Kilbanov, 1988) and Savchenko et al. (2002) reported that five cysteines of α-amylase from P. furiosus (Cys 152, Cys 153, Cys165). , Cys387, alc Cys430) claimed that Cys165 plays a major role in thermal stability.

The optimum temperature of the extracellular α-amylase derived from Thermococcus was 80 ° C.

The new strain Thermoccus sp.NA1 (KCTC 10859BP) according to the present invention has a gene of novel highly thermophilic DNA polymerase, DNA ligase, carboxypeptidase, proryloligopeptidase, aminopeptidase P, methionylaminopeptidase and amylase. If these genes can be amplified by PCR, these genes can be inserted into an expression vector to express active enzymes.

Α-amylase enzymes according to the invention are novel proteases that are highly thermophilic. The optimum temperature of the extracellular α-amylase is 80 ° C., and is quite thermostable with a half-life of 7,581 minutes at 80 ° C. and 153 minutes at 90 ° C. in the presence of 0.5 mM Ca ²⁺ . Thus, it can be used in the textile, food, fermentation, papermaking, casting and distillation industries, sugars as well as industrial applications in a wide range of fields such as clinical, pharmaceutical and analytical chemistry.

references

Bernfeld, P. 1955. Amylases, α and β. Meth. Enzymology 1, 149-158.

Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

Brown, SH, Kelly, RM, 1993. Characterization of amylolytic enzymes, having both α-1,4 and α-1,6 hydrolytic activity, from the thermophilic archaea Pyrococcus furiosus and Thermococcus litoralis . Appl. Environ. Microbiol. 59, 2614-2621.

Chung, YC, Kobayashi, T., Kanai, H., Akiba, T., Kudo, T., 1995. Purification and properties of extracellular amylase from the hyperthermophilic archaeon Thermococcus profundus DT5432. Appl. Environ. Microbiol. 61, 1502-1506.

Dong, G .., Vieille, C., Savchenko, A., Zeikus, JG, 1997. Cloning, sequencing, and expression of the gene encoding extracellular α-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme. Appl. Environ. Microbiol. 63, 3569-3576.

Frillingos, S., Linden, A., Niehaus, F., Vargas, C., Nieto, JJ, Ventosa, A., Antranikian, G., Drainas, C., 2000. Cloning and expression of alpha-amylase from the hyperthermophilic archaeon Pyrococcus woesei in the moderately halophilic bacterium Halomonas elongata. J. Appl. Microbiol. 88, 495-503.

Henrissat, B. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 1991 280 (Pt 2), 309-316.

Jorgensen S., Vorgias, CE, Antranikian, G., 1997. Cloning, sequencing, characterization, and expression of an extracellular α-amylase from the hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli and Bacillus subtilis . J. Biol. Chem. 272, 16335-16342.

Koch, R., Spreinat, K., Lemke, K., Antranikian, G .., 1991. Purification and properties of a hyperthermoactive α-amylase from the archaeobacterium Pyrococcus woesei . Arch. Microbiol. 155, 572-578.

Laderman, KA, Davis, BR, Krutzsch, HC, Lewis, MS, Griko, YV, Privalov, PL, Anfinsen, CB, 1993.The purification and characterization of an extremely thermostable α-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus . J. Biol. Chem. 268, 24394-24401.

Laemmli, U. K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

Lee, JT, Kanai, H., Kobayashi, T., Akiba, T., Kudo, T., 1996. Cloning and nucleotide sequence and hyperexpression of α-amylase gene from an archaeon, Thermococcus profundus . J. Ferment. Bioeng. 82, 432-438.

Leveque, E., Janecek, S., Haye, B. and Belarbi, A., 2000a. Cloning and expression of an α-amylase encoding gene from the hyperthermophilic archaebacterium Thermococcus hydrothermalis and biochemical characterization of the recombinant enzyme. FEMS Microbiol. Lettes, 186, 67-71.

Leveque, E., Janecek, S., Haye, B., Belarbi, A., 2000b. Thermophilic archaeal amylolytic enzymes. Enzyme Microb. Technol. 26, 3-14.

Linden A., Mayans, O., Klaucke, W. M., Antanikian, G., Wilmanns, M., 2003. Differential regulation of a hyperthermophilic α-amylase with a novel (Ca, Zn) two-metal center by zinc. J. Biol. Chem. 278, 9875-9884.

Morgan, FJ, Priest, FG, 1981. Characterization of a thermostable α-amylase from Bacillus licheniformis NCIB6346. J. Appl. Bacteriol. 50, 107-114.

Nakajima R., Imanaka, T., Aiba, S., 1986. Comparison of amino acid sequences of eleven different α-amylases. Appl. Microbiol. Biotechnol. 23, 355-360.

Pandey, A., Nigam, P., Soccol, C. R., Soccol, V. T., Singh, D., Mohan, R., 2000. Advances in microbial amylases. Biotechnol. Appl. Biochem. 31, 135-152.

Sambrook, J., Russell, D. W., 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Savchenko, A., Vieille, C., Kang, S., Zeikus, JG., 2002. Pyrococcus furiosus α-amylase is stabilized by calcium and zinc. Biochemistry 41, 6193-6201.

Tachibana, Y., Leclere, MM, Fujiwara, S., Takagi, M., Imanaka, T., 1996. Cloning and Expression of the α-amylase gene from the hyperthermophilic archaeon Pyrococcus sp. KOD1, and characterization of the enzyme. J. Ferment. Bioeng. 82, 224-232.

Tomazic, S. J., Klibanov, A. M., 1988. Mechanisms of irreversible thermal inactivation of Bacillus alpha-amylases. J Biol. Chem. 263, 3086-3096.

van der Maarel, M. J., van der Veen, B., Uitdehaag, J. C., Leemhuis, H., Dijkhuizen, L., 2002. Properties and applications of starch-converting enzymes of the α-amylase family. J. Biotechnol. 94, 137-155.

Vihinen, M., Mantsala, P., 1989. Microbial amylolytic enzymes. Crit. Rev. Biochem. Mol. Biol. 24, 329-418.

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Claims (14)

New strain Thermococcus sp. NA1 (KCTC 10859BP).  A highly thermophilic α-amylase enzyme having the amino acid sequence of SEQ ID NO: 3.  A protein having the amino acid sequence of SEQ ID 3;  The gene of SEQ ID NO: 1 or 2.  A gene encoding the amino acid sequence of SEQ ID NO. The nucleic acid sequence encoding the highly thermophilic α-amylase enzyme according to claim 2. The DNA nucleic acid sequence of claim 6 which encodes said highly thermophilic α-amylase enzyme of SEQ ID NO: 1 or 2. 8. 7. The nucleic acid sequence according to claim 6, wherein the DNA nucleic acid sequence encoding the highly thermophilic α-amylase enzyme is equivalent to SEQ ID NO: 1 or 2 due to codon degeneracy. delete delete Recombinant vector comprising a nucleic acid sequence according to any one of claims 6 to 8. 12. The recombinant vector according to claim 11, wherein said recombinant vector has a cleavage map as described in FIG. A cell transformed with the recombinant vector according to claim 11. Strain Thermococcus sp. A method of producing a highly thermophilic α-amylase enzyme, comprising culturing NA1 (KCTC 10859BP) or the transformed cell according to claim 13, and separating the α-amylase enzyme of SEQ ID NO: 3 from the cultured cells.

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