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Glutaminase, glutaminase gene, novel recombinant DNA, and process for manufacturing glutaminase


No:

6919195 -

Application no:

10175002 -

Filed date:

2002-06-20 -

Issue date:

2005-07-19

Kind:

B2


Claims:

12

Abstract:


Glutaminase which comprises (a) a protein comprising an amino acid sequence shown in SEQ ID NO: 2, or (b) a protein which comprises an amino acid sequence having deletion, substitution, or addition of one or plurality of amino acids relative to the amino acid sequence (a), and has glutaminase activity. A glutaminase gene which encodes (a) a protein comprising an amino acid sequence shown in SEQ ID NO: 2, or (b) a protein which comprises an amino acid sequence having deletion, substitution, or addition of one or a plurality of amino acids relative to the amino acid sequence (a), and has glutaminase activity. The present invention enables glutaminase to be produced efficiently and thus greatly contributes to the relevant industries.

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Inventors:



Agents:


Assignees:


Claims:


What is claimed is:

1. An isolated nucleic acid comprising a polynucleotide which hybridizes to the complement of SEQ ID NO: 1 under stringent conditions, and which encodes a protein having glutaminase activity; wherein stringent conditions comprise hybridization in 150 mM sodium at a temperature of 65.degree. C.

2. The nucleic acid of claim 1, which encodes the polypeptide of SEQ ID NO: 2 or a fragment of SEQ ID NO: 2 having glutaminase activity.

3. The nucleic acid of claim 1 which comprises SEQ ID NO: 1.

4. An isolated vector comprising the nucleic acid of claim 1.

5. An isolated host cell comprising the nucleic acid of claim 1.

6. The isolated host cell of claim 5 which is selected from the group consisting of a fungi, yeast and bacterium.

7. An isolated polypeptide which has glutaminase activity, which is encoded by a polynucleotide which hybridizes to the complement of SEQ ID NO: 1 under stringent conditions, wherein stringent conditions comprise hybridization in 150 mM sodium at a temperature of 65.degree. C.

8. The polypeptide of claim 7 which comprises SEQ ID NO: 2 or a fragment of SEQ ID NO: 2.

9. The polypeptide of claim 7 which is encoded by SEQ ID NO: 1.

10. A method for producing an isolated polypeptide which has glutaminase activity comprising: expressing the isolated nucleic acid of claim 1 and recovering the polypeptide expressed by said nucleic acid.

11. The method of claim 10 further comprising purifying the polypeptide having glutaminase activity.

12. A method for producing L-glutamic acid comprising: contacting a substrate containing L-glutamine with the polypeptide of claim 7 for a time and under conditions suitable for hydrolysis of L-glutainine into L-glutamic acid.

Text:


BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a glutaminase, a glutaminase gene, a novel recombinant DNA, and a process for manufacturing a glutaminase.

2. Description of the Relevant Art

Glutaminase is an enzyme which hydrolizes L-glutamine to ammonia and L-glutamic acid, which is a umami (sweetener) flavor-enhancing component. Glutaminase plays an important role in food processing industry. It is useful, for example, in the manufacture of soy sauce or those seasonings which are produced by enzymically hydrolysing proteins. Glutaminase has been isolated from various species, and its enzymological properties and genes have been reported (e.g., in Japanese Patent Publication (Kokoku) No. 6-38748).

In the manufacture of soy sauce, and the manufacture of seasonings with the use of koji malt, in order to improve glutaminase by genetic engineering techniques and abundantly produce the enzyme, it is important to obtain the enzyme from the koji malt.

In this manner, hydrolysis products of proteins (such as soy sauce) can be easily improved in quality and provided at low prices.

SUMMARY OF THE INVENTION

It is therefore the object of the present invention to provide a koji malt-derived glutaminase, a glutaminase gene, a novel recombinant DNA, and a process for manufacturing a glutaminase.

After extensive research and analysis, the present inventors succeeded in isolating a glutaminase gene derived from Aspergillus sojae and determining its structure, and arrived at the present invention.

Namely, a first invention is a glutaminase according to the following (a) or (b):

(a) a protein comprising an amino acid sequence shown in SEQ ID NO: 2;

(b) a protein which comprises an amino acid sequence having deletion, substitution or addition of one or a plurality of amino acids relative to the amino acid sequence (a), and has glutaminase activity.

A second invention is a glutaminase gene which encodes a protein according to the following (a) or (b):

(a) a protein comprising an amino acid sequence shown in SEQ ID NO: 2;

(b) a protein which comprises an amino acid sequence having deletion, substitution or addition of one or a plurality of amino acids relative to the amino acid sequence (a), and has glutaminase activity.

A third invention is a glutaminase gene comprising a DNA according to the following (a) or (b):

(a) a DNA comprising a base sequence shown in SEQ ID NO: 1;

(b) a DNA which hybridizes with the DNA comprising the base sequence of (a) under stringent conditions, and which encodes a protein having glutaminase activity.

A fourth invention is a novel recombinant DNA characterized in that the above-mentioned glutaminase gene is inserted into vector DNA.

A fifth invention is a transformant or transductant containing the above-mentioned recombinant DNA.

A sixth invention is a process of manufacturing a glutaminase which comprises culturing the above-mentioned transformant or transductant on a culture medium, and collecting glutaminase from the culture product.

DESCRIPTION OF EMBODIMENTS

The present invention will be hereafter described in detail.

1. A Glutaminase and a Gene which Encodes It

The glutaminase according to the present invention is a glutaminase according to the following (a) or (b):

(a) a protein comprising an amino acid sequence shown in SEQ ID NO: 2;

(b) a protein which comprises an amino acid sequence having deletion, substitution, or addition of one or a plurality of amino acids relative to the amino acid sequence (a), and has glutaminase activity.

The protein (a) can be obtained by cloning a naturally occurring glutaminase gene derived from a chromosome DNA or cDNA of a filamentous fungi of the genus Aspergillus, and then introducing the clone into an appropriate vector-host system to express.

As indicated in (b), this protein may comprise an amino acid sequence having deletion, substitution, or addition of one or a plurality of amino acids relative to the amino acid sequence (a), as long as the amino acid sequence has glutaminase activity. In the present invention, "a plurality of" means usually 2 to 300, preferably 2 to 170, more preferably 2 to 50, and most preferably 2 to 10, depending on the position of the amino acid residues in the three-dimensional structure of the glutaminase protein or kind of the amino acids.

Such a mutated glutaminase, i.e., the protein (b), can be obtained by producing a mutated glutaminase gene by introducing into the base sequence of a naturally occuring glutaminase gene a mutation such as substitution, deletion, insertion, addition, or inversion, and introducing it into an appropriate vector-host system to express.

The methods of introducing the mutation into the gene include, for example, a site-specific mutagenesis, a random mutation inducing method by PCR, and a method whereby the gene is selectively cleaved and then rejoined after removal or addition of selected nucleotides.

The glutaminase gene according to the present invention is a gene containing a DNA which encodes the protein (a) or (b). The glutaminase gene according to the present invention may be a gene which hybridizes with a DNA encoding the protein (a) or (b) under stringent conditions, and which encodes a protein having glutaminase activity. In the present invention, "stringent conditions" means such conditions where the sodium concentration is 50 to 300 mM, preferably 150 mM, and the temperature is 42 to 68.degree. C., preferably 65.degree. C.

An example of the gene containing the DNA which encodes the protein (a) is a DNA containing the base sequence indicated by SEQ ID NO: 1. This DNA is a naturally occuring glutaminase gene.

The naturally occuring glutaminase gene can be obtained by cloning a naturally occuring gene derived from chromosome DNA or cDNA of a filamentous fungi of the genus Aspergillus. Gene cloning methods include, for example, one in which an appropriate probe DNA is synthesized after purifying glutaminase and determining the partial amino acid sequence, and then Aspergillus sojae chromosome DNA is screened using the probe DNA. Another method involves the production of an appropriate primer DNA based on a partial amino acid sequence, followed by amplification of a DNA containing fragments of the above-mentioned gene by an appropriate polymerase chain reaction (PCR) such as the 5'-RACE method or 3'-RACE method, so that the fragments are joined to produce a DNA containing the full-length gene.

Specifically, the naturally occuring glutaminase gene can be obtained as follows. Initially, Aspergillus sojae FERM BP-6820 is cultured, and a resultant bacterial body is frozen in liquid nitrogen, then physically milled or ground by using a mortar or the like, thereby obtaining bacterial body fragments in a fine powder form, from which an entire RNA fraction is extracted in a usual manner. In the extraction procedure, commercially available RNA extraction kits can be used.

Alternatively, an RNA may be collected from the resultant RNA liquid extract by ethanol precipitation, and then an RNA with a poly A chain may be fractionated in a usual manner. In this fractionation procedure, a commercially available Oligo dT column can be used.

Primers for use in PCR are synthesized by referring to the DNA sequence in SEQ ID NO: 2. Using this primer DNA and the RNA obtained in the above-described manner, a DNA containing fragments of the gene is amplified by an appropriate RT-PCR reaction such as the 5'-RACE method and the 3'-RACE method, and a DNA containing the full-length gene is obtained by joining these fragments. In the partial cDNA synthesizing procedure by the 5'-RACE method and 3'-RACE method, commercially available kits may be used.

Using the above cDNA as a template, PCR is performed with the synthesized primer complementary to the 5'-end sequence and the 3'-end sequence, thereby amplifying the DNA. The amplified DNA can be cloned according to conventional methods.

The amplified DNA is inserted into an appropriate vector to obtain a recombinant DNA. For cloning, a commercially available kit such as TA Cloning Kit (Invitrogen Corporation), commercially available plasmid vector DNAs such as pUC119 (Takara Shuzo Co., Ltd.), pBR322 (Takara Shuzo Co., Ltd.), and pBluescript SK+ (Stratagene), and a commercially available bacteriophage vector DNA such as .lambda. EMBL3 (Stratagene), or the like can be used.

By using the resultant recombinant DNA, a transformant or a transductant is obtained by transforming or transducing Escherichia coli K-12, preferably Escherichia coli JM109 (Takara Shuzo Co., Ltd.), or XL-Blue (Stratagene), for example. The transformation can be performed by D. M. Morrison's method (Methods in Enzymology, 68, 326-331, 1979), for example. The transduction, on the other hand, can be performed by B. Hohn's method (Methods in Enzymology, 68, 299-309, 1979), for example. As a host cell, in addition to Escherichia coli, other microorganisms such as bacteria, yeasts, filamentous fungus and actinomycete, and animal cells may be used.

The entire base sequence (see SEQ ID NO: 1) of the thus amplified DNA can be analyzed by, for example, Li-COR MODEL 4200L Sequencer (purchased from ALOKA CO., LTD.), 370DNA Sequence System (PerkinElmer Inc.), or CEQ2000XL DNA Analysis System (Beck man Coulter). By comparing the base sequence with information about the partial amino acid sequence, it can be determined whether or not naturally occuring glutaminase gene was obtained.

And by analyzing the naturally occuring glutaminase gene, the amino acid sequence of the translated polypeptide, i.e., the protein (a), can be determined.

2. Process of Manufacturing Glutaminase

When manufacturing the glutaminase according to the present invention, initially a recombinant DNA containing the glutaminase gene is produced. A transformant or transductant containing that recombinant DNA is then produced and cultured, and glutaminase can be collected from the cultured product.

In order to manufacture the protein having glutaminase activity by using the glutaminase gene according to the present invention, it is necessary to select an appropriate vector-host system. As such a system, there can be mentioned, e.g., a system of pST14 (Unkles et al., 1989, Mol. Gen. Genet., 218, 99-104) and a filamentous fungi (Aspergillus sojae, Aspergillus oryzae, Aspergillus nidulans, Aspergillus niger, Penicillium chrysogenum, etc.), a system of a yeast expression vector pYES2 (Invitrogen) and a yeast Saccharomyces cerevisiae, and a system of an Escherichia coli expression vector pTE (Stratagene) and Escherichia coli. It is preferable to use a filamentous fungi or yeast system in which there occurs sugar chain addition to the protein.

The recombinant DNA can be obtained by inserting the glutaminase gene into an appropriate vector. As the vector, commercially available products can be used, such as yeast expression vectors pYES2, pYD1 (Invitrogen), pUR123 (Takara Shuzo Co., Ltd.), pYEX-BX, pYEX-S1, pYEX-4T (CLONTECH), Escherichia coli expression vector pSET (Invitrogen), and pTE (Stratagene), for example.

Thereafter, the recombinant DNA is transformed or transducted into a host cell. The transduction into the yeast can be performed by, for example, the method of Becker D M. et al. (Methods in Enzymology, 194, 182-187, 1991). As the host cell, in addition to the Escherichia coli and yeast, there can be used microorganisms such as other bacteria, filamentous fungus, and actinomycetes, or animal cells.

There is thus obtained a transformant or transductant with a glutaminase production capacity. Though the transformant or transductant may be cultured by the conventional solid culture method, it is preferable to adopt the liquid culture method as much as possible.

When a yeast is used as a host, general eutrophic media such as YPD media and YM media may be used. When a selective medium is used in view of the genetic properties of the host, an SD medium being a minimal medium can be used. When using the selective medium, since the selection pressure differs depending on the host-vector system used, amino acids or nucleic acids other than the selection pressure, for example, are added to the minimal medium, in accordance with the host's genetic requirement as needed.

In addition, inorganic salts, saccharide materials, vitamins, etc., may be added to the medium as necessary. The initial pH of the medium is appropriately adjusted to pH 6 to 9. Depending on the vector used, the expression of the protein can be controlled. When using such a vector, glutaminase can be induced by adding an inducer appropriate for the vector, such as galactose or copper ions.

When the yeast is cultured, there should preferably be used such methods as an aeration-agitation submerged culture, shaking culture, static culture, etc., at temperatures of 25 to 35.degree. C., preferably at more or less 30.degree. C., for 24 to 48 hours.

The glutaminase that expressed can be purified by a method partly modified from the method described in Japanese Patent Application Laid-Open (Kokai) No. 11-332553.

In the case of the yeast, after the transformed yeast is cultured by the above-mentioned appropriate method, the culture solution is centrifuged to obtain a yeast body. After the cell wall is sufficiently lysed by the addition of a cell-wall lysing enzyme to the yeast body, a supernatant liquid is obtained by centrifugation. Ammonium sulfate is then added to the supernatant liquid to thereby salt out, and the liquid is further centrifuged to remove insoluble proteins, thereby obtaining a crude enzyme solution containing glutaminase.

From the crude enzyme solution is purified a glutaminase active fraction by means of the Phenyl Sepharose column, DEAE-Sepharose column, a gel filtration column, and HPLC, thereby obtaining a purified glutaminase.

The genetic engineering method according to the present invention can be performed according to the descriptions in "Molecular Cloning: A Laboratory Manual 2.sup.nd edition". (1989), Cold Spring Harbor Laboratory Press, ISBN 0-8769-309-6, and "Current Protocols in Molecular Biology" (1989), John Wiley & Sons, Inc., ISBN 0-471-50338-X, for example.

EXAMPLES

The present invention will be hereafter described in greater detail by way of examples.

Example 1

Acquisition of a Glutaminase cDNA

(1) Retrieval of a Glutaminase Homologous Gene in a Bacterium of the Genus Aspergillus

After searching the koji mold EST library of the International Patent Organism Depositary at the National Institute of Advanced Science and Technology for a gene which is highly homologous to a known glutaminase gene derived from Cryptococcus (Japanese Patent Application No. 2000-270371), an EST clone Contig Mix0010110003775.sub.-- 1 comprising 711 bases was obtained. The clone was presumed to be a fragment of the glutaminase gene, and a cDNA of the gene was cloned.

Extraction of the Entire RNA from Aspergillus sojae

Spores of the Aspergillus sojae FERM BP-6820 were inoculated into 50 ml of a soybean power medium (3% soybean powder, 1% KH.sub.2 PO.sub.4, pH6.0) to a density of 3.times.10.sup.5 /ml, and the spores were shake-cultured in a 150 ml Erlenmeyer flask at 30.degree. C. for 48 hours, at 150 r.p.m.

A resultant culture solution was filtered by Miracloth (Calbiochem) to thereby collect a bacterial body. After washing the collected bacteria with sterilized water, it was frozen in liquid nitride and then physically ground in a mortar, thereby obtaining fine powdery bacterial fragments. From the bacterial fragments were extracted an entire RNA by means of ISOGEN (Nippon Gene Co., Ltd.). The entire procedure was performed in accordance with the attached protocol.

(2) Acquisition of a Glutaminase cDNA by the RACE Method

From about 200 .mu.g of the entire RNA thus obtained, 4 .mu.g of mRNA was obtained by using the Oligotex-dT30<Super> mRNA Purification Kit (Takara Shuzo Co., Ltd.). Of the mRNA, 1 .mu.g was subjected to 5'-RACE and 3'-RACE performed by using the Marathon cDNA Amplification Kit (Clontech) and Advantage cDNA PCR kit (Clontech). As primers for the RACE method, oligo DNAs respectively expressed by SEQ ID NOS: 3-6 were synthesized, namely antisense primers (SEQ ID NOS: 3 and 4) for the EST clone CONTIG MIX0010110003775.sub.-- 1 for the 5'-RACE and sense primers (SEQ ID NOS: 5 and 6) for performing the 3'-RACE. The entire procedure was performed in accordance with the attached protocol. As PCR apparatus, there was used the GeneAmp5700 DNA detection system (PerkinElmer). As a result, amplification of about 1.7 kb of a DNA fragment corresponding to the glutaminase cDNA 5'-region, and about 0.8 kb of a DNA fragment corresponding to the 3'-region were confirmed.

The amplified DNA fragments were separated on a 0.7% agarose gel, and extracted by using the QIAquick Gel Extraction Kit (QIAGEN). The procedure was in accordance with the attached protocol. The extracted DNA fragment was incorporated into a pCR2.1-TOPO vector by using the TOPO TA Cloning Kit (Invitrogen). The resultant recombinant plasmid was subjected to sequence reaction by using the Thermo Sequence Cycle Sequencing Kit (Amersham Pharmacia Biotech), and the base sequence was determined by the LI-COR MODEL4200L sequencer (purchased from Aloka). As a result, the DNA sequence of approximately 1.9-kb open reading frame (ORF) indicated by SEQ ID NO: 1 was determined, and it became clear that the EST clone CONTIG MIX0010110003775.sub.-- 1 was a partial fragment thereof.

This DNA encoded a protein comprising 643 amino acids. This amino acid sequence is described in SEQ ID NO: 2. Furthermore, a homology search was performed on a known amino acid sequence database with regard to this amino acid sequence. For the homology search, NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) was used. As a result, there was no known protein matching the above-mentioned ORF.

However, when a homology search was performed with respect to a glutaminase derived from Cryptococcus albidus and Cryptococcus nodaensis, homology was recognized in a region which is expected to be an active center, which suggested that the glutaminase was encoded by the ORF.

PCR was performed by using as a template the cDNA which was produced during a 5'-RACE, whereby the full-length cDNA of glutaminase was cloned. As a primer, oligo DNA indicated by SEQ ID NOS: 7 and 8 were used. The resultant amplified DNA fragment of about 2.1 kb was extracted by the above-mentioned method and incorporated into the pCR2.1-TOPO vector by using the TOPO TA Cloning Kit (Invitrogen), thereby obtaining a recombinant plasmid pASgln containing the full-length cDNA of glutaminase.

The base sequence of the recombinant plasmid pASgln was again analyzed to determine the base sequence of the glutaminase cDNA (SEQ ID NO: 1).

The full-length glutaminase cDNA, i.e., the plasmid pASgln containing the base sequence indicated by base Nos. 1 to 1932 of SEQ ID NO: 1, is deposited with the International Patent Organism Depositary at National Institute of Advanced Science and Technology, as FERM BP-7634.

Example 2

Expression of Glutaminase cDNA

The above-mentioned plasmid pASgln was subjected to an enzyme treatment with restriction enzymes Bam HI and Sph I (both by Takara Shuzo Co., Ltd.). Then, it was subjected to 0.7% agarose gel electrophoresis. Thereafter, DNA fragments of a desired size (about 2.0 kbp) were sliced and purified.

These DNA fragments were incorporated into a yeast expression vector pYES2 (Invitrogen) which has been subjected to an enzyme treatment by the above-mentioned restriction enzyme, to thereby produce a recombinant plasmid pYES-ASgln. This recombinant plasmid is capable of inducing a target protein glutaminase to express. As a host, the attached INVSc1 (Genotype: MATa, his3.DELTA.1, leu2, trp1-289, ura3-52/MAT.alpha., his3.DELTA.1, leu2, trp1-289, ura3-52) was used, and the host yeast was transformed by the above-mentioned plasmid pYES-ASgln by the lithium acetate method. As a selective medium, there was used 0.67% Yeast Nitrogenbase without amino acids (Difco), 2% raffinose (Wako Pure Chemicals Industries, Ltd.), and 0.192% Yeast Synthetic Dropout Medium Supplement without uracil (SIGMA). The lithium acetate method was performed in accordance with the description in "Tanpakushitsu Jikken Purotokoru--Kino Kaiseki-hen--" ("Protein Experiment Protocol--Function Analysis--") (A supplement to a magazine Saibo Kogaku (Cell Technology; Shujun-sha).

Thereafter, the resultant transformant was used to express a protein according to the protocol attached to the pYES2 vector (Invitrogen). The transformant was transplanted from a colony into a 20 ml of the selective medium by using a 200-ml Erlenmeyer flask with bumps, and shake-cultured at 30.degree. C., 140 r.p.m., for about 14 hours, thereby obtaining a seed culture.

The turbidity (OD.sub.600) of the seed culture was then measured, and the seed culture was inoculated into a protein expression-inducing medium such that the initial turbidity is OD.sub.600 =0.4. For culturing in the protein expression-inducing medium, a 500 ml Sakaguchi flask was used, and a shaking culture was performed in 50 ml of the medium at 30.degree. C., 140 r.p.m. As a protein expression-inducing medium, there were used a 1% Yeast Extract (Difco), a 2% Poly Peptone (Nippon Seiyaku K.K.), a 1% raffinose and a 2% galactose (both SIGMA). A centrifuged and collected yeast body was suspended in distilled water and supplied as an enzyme solution.

Glutaminase activity was measured by a method partly modified from the method described in Japanese Patent Application Laid-Open (Kokai) No. 11-332553. Specifically, 500 .mu.l of 0.2 M phosphoric acid buffer solution (pH 6.5) and 500 .mu.l of the enzyme solution were added to 250 .mu.l of 2% (W/V) L-glutamine solution and reacted at 37.degree. C. for 30 minutes. The reaction was then terminated by adding 250 .mu.l of 0.75 N perchloric acid solution, and the reaction solution was neutralized by adding 125 .mu.l of 1.5 N sodium hydroxide solution. This reaction solution was centrifuged (10000 r.p.m., 10 min), and to 100 .mu.l of the supernatant liquid were added 1.0 ml of 0.1 M hydroxylamine hydrochloride buffer solution (pH 8.0), 1.0 ml of 20 mM NAD+ solution (Oriental Yeast Co., Ltd.), and 50 .mu.l of 500 units/ml L-glutamic acid dehydrogenase solution (SHIGMA). The supernatant solution was then reacted at 37.degree. C. for 30 minutes, and the absorbance at 340 nm was measured by a spectrophotometer. Under these conditions, a single unit (U) of glutaminase activity was defined as the amount of enzyme which generates 1 .mu.mol of glutamic acid per minute.

The result of measuring glutaminase activity of the transformant is shown in Table 1. The values in the table indicate glutaminase activity per 1 ml of the culture liquid (mU/ml) 24 hours after the start of culturing (OD.sub.600 =15). The designation "pYES2" indicates the transformant by plasmid pYES2, and the designation "pYES-ASgln" indicates the transformant by plasmid pYES-ASgln. The signs "-" and "+" indicate induction by a protein expression non-inducing medium containing no galactose and by a protein expression-inducing medium containing galactose, respectively.

TABLE 1 Plasmid/galactose - + PYES2 0.33 2.50 PYES-Asgln 4.64 32.87

When cultured on the protein expression-inducing medium containing galactose, the transformant by the plasmid pYES-ASgln was higher in glutaminase activity than the transformant by the plasmid pYES2. On the other hand, when cultured in the protein expression non-inducing medium containing no galactose, the transformant by the plasmid pYES-ASgln showed no increase in glutaminase activity. This indicated that the glutaminase activity of the transformant by the plasmid pYES-ASgln derived from the introduced glutaminase gene (Table 1). When the INVSc1 was used as the host to express glutaminase, too, the glutaminase activity expressed on the surface of the yeast body, as in the case of the bacterium of the genus Aspergillus.

Thus, in accordance with the present invention, glutaminase can be efficiently manufactured. The present invention therefore has great industrial applicability.

SEQUENCE LISTING <100> GENERAL INFORMATION: <160> NUMBER OF SEQ ID NOS: 8 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 1 <211> LENGTH: 1932 <212> TYPE: DNA <213> ORGANISM: Aspergillus sojae <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1932) <223> OTHER INFORMATION: <400> SEQUENCE: 1 atg ttt ctt agt aca ctc ctc tca ctg gcg gcg gtc gtt gcc ggc gct 48 Met Phe Leu Ser Thr Leu Leu Ser Leu Ala Ala Val Val Ala Gly Ala 1 5 10 15 gcc atc ccc aat ggc cag acg ctt tct ctc aat gac att cct tac tat 96 Ala Ile Pro Asn Gly Gln Thr Leu Ser Leu Asn Asp Ile Pro Tyr Tyr 20 25 30 gtg agc ggc att cct gtg tca act ttg caa ggg tac aat gcc tct gca 144 Val Ser Gly Ile Pro Val Ser Thr Leu Gln Gly Tyr Asn Ala Ser Ala 35 40 45 tat gct gct ttg aca aag gga ata gat ttg gtg cca tta act gtc att 192 Tyr Ala Ala Leu Thr Lys Gly Ile Asp Leu Val Pro Leu Thr Val Ile 50 55 60 cct gta act cct acc acg aac ttg gag tcg ctg cta tcg gac tat gtt 240 Pro Val Thr Pro Thr Thr Asn Leu Glu Ser Leu Leu Ser Asp Tyr Val 65 70 75 80 gaa cgc gat gat gtc ttc cag ccg gct ttt ctg cgt gca gtc tat ctc 288 Glu Arg Asp Asp Val Phe Gln Pro Ala Phe Leu Arg Ala Val Tyr Leu 85 90 95 aca gct tcc act gct gat gac att gac tcc caa ctg agc aat tat gcg 336 Thr Ala Ser Thr Ala Asp Asp Ile Asp Ser Gln Leu Ser Asn Tyr Ala 100 105 110 tca att ctc aag tct tcc ggc acc gac gtg ctg ctg gtt gat tca gaa 384 Ser Ile Leu Lys Ser Ser Gly Thr Asp Val Leu Leu Val Asp Ser Glu 115 120 125 gta cac acc gct tcg tca gat tcc aca atc aca gcg cag ttg acc aaa 432 Val His Thr Ala Ser Ser Asp Ser Thr Ile Thr Ala Gln Leu Thr Lys 130 135 140 gag ctg ccg agt ggg cct tat ttt gtc tcc ttg tat act gga gag gtg 480 Glu Leu Pro Ser Gly Pro Tyr Phe Val Ser Leu Tyr Thr Gly Glu Val 145 150 155 160 ttt aga gcg tac cgg ttg tac cct gac gac aac cta gct ttc att caa 528 Phe Arg Ala Tyr Arg Leu Tyr Pro Asp Asp Asn Leu Ala Phe Ile Gln 165 170 175 gca gga atc agt gac gag aag gga ggt gtc ctg ccc cta cca gcc gtg 576 Ala Gly Ile Ser Asp Glu Lys Gly Gly Val Leu Pro Leu Pro Ala Val 180 185 190 aca gaa aac gcg atg acc aaa gac gtt gcc gtg cct tca cgt ctc tat 624 Thr Glu Asn Ala Met Thr Lys Asp Val Ala Val Pro Ser Arg Leu Tyr 195 200 205 tat aca ccg acc gca gaa aag cca tta gcc ggt ctg agg tta ggt gtc 672 Tyr Thr Pro Thr Ala Glu Lys Pro Leu Ala Gly Leu Arg Leu Gly Val 210 215 220 aag gat atc tac cac gtt aaa ggt ctc aag acg agt ggc ggc agt cgc 720 Lys Asp Ile Tyr His Val Lys Gly Leu Lys Thr Ser Gly Gly Ser Arg 225 230 235 240 tcc tat tat tat tta tac gga act cag aat gtc act gcc cca tct att 768 Ser Tyr Tyr Tyr Leu Tyr Gly Thr Gln Asn Val Thr Ala Pro Ser Ile 245 250 255 cag aga ctg ttg gac tta ggc gcg gtc ttt gtc ggt aaa act ggg acc 816 Gln Arg Leu Leu Asp Leu Gly Ala Val Phe Val Gly Lys Thr Gly Thr 260 265 270 gtt cag ttt gct aac ggt gat cga cct act gcc gac tgg gtg gat ttc 864 Val Gln Phe Ala Asn Gly Asp Arg Pro Thr Ala Asp Trp Val Asp Phe 275 280 285 cac tgt cca ttc aac caa cgc gga gaa gga tat cag gca cct agc ggt 912 His Cys Pro Phe Asn Gln Arg Gly Glu Gly Tyr Gln Ala Pro Ser Gly 290 295 300 tcc tcc tcc ggc tca ggt gtg gct att gca gcc tac gac tgg ttg gac 960 Ser Ser Ser Gly Ser Gly Val Ala Ile Ala Ala Tyr Asp Trp Leu Asp 305 310 315 320 ctt gct gtc ggt agt gac act ggc ggt tca atg cgt tcc cca gct gca 1008 Leu Ala Val Gly Ser Asp Thr Gly Gly Ser Met Arg Ser Pro Ala Ala 325 330 335 gtt caa ggg ata tat ggc aac agg cca tct act ggc gct atc tct cta 1056 Val Gln Gly Ile Tyr Gly Asn Arg Pro Ser Thr Gly Ala Ile Ser Leu 340 345 350 gat cat gtc tta cct ctc tcg ccg gct ctg gat aca gcg ggc gtc ttt 1104 Asp His Val Leu Pro Leu Ser Pro Ala Leu Asp Thr Ala Gly Val Phe 355 360 365 gcc cga agt gcc tca cta tgg tcc cat act gtg caa gct tgg tat cct 1152 Ala Arg Ser Ala Ser Leu Trp Ser His Thr Val Gln Ala Trp Tyr Pro 370 375 380 cat ctc cag cac aat ttt acg tcc ttc cct cgg cag ctg ctc cta gcc 1200 His Leu Gln His Asn Phe Thr Ser Phe Pro Arg Gln Leu Leu Leu Ala 385 390 395 400 ggt ggt gga tgg gat ggt aaa ggc atc agt ccc gag gcc cat cag agt 1248 Gly Gly Gly Trp Asp Gly Lys Gly Ile Ser Pro Glu Ala His Gln Ser 405 410 415 ctt acc aca ttc aca cgt ggg ctt gag gca ttc ctc gga aca aac cat 1296 Leu Thr Thr Phe Thr Arg Gly Leu Glu Ala Phe Leu Gly Thr Asn His 420 425 430 acc aat gtc gac gtg tcg cag cga tgg ctt gac aca cac tct ccc acc 1344 Thr Asn Val Asp Val Ser Gln Arg Trp Leu Asp Thr His Ser Pro Thr 435 440 445 aca cca agc ctg gaa gag atg ctc aac ctg acc tat gcc aca ctt act 1392 Thr Pro Ser Leu Glu Glu Met Leu Asn Leu Thr Tyr Ala Thr Leu Thr 450 455 460 tct gtg gat cag ttc aac cac cta gcc gtc cct ctc ttt gct gac tat 1440 Ser Val Asp Gln Phe Asn His Leu Ala Val Pro Leu Phe Ala Asp Tyr 465 470 475 480 aaa gcc gtc cac cgc ggt cgt cag cct ttc att aac ccc ggc cca tta 1488 Lys Ala Val His Arg Gly Arg Gln Pro Phe Ile Asn Pro Gly Pro Leu 485 490 495 gcg cgt tgg cag tgg ggc cag gcg aat ggc gga aac acc tcg tac gat 1536 Ala Arg Trp Gln Trp Gly Gln Ala Asn Gly Gly Asn Thr Ser Tyr Asp 500 505 510 gca gct ctg cgc aac atg act act ttc cga aac tgg tgg gag aag tcc 1584 Ala Ala Leu Arg Asn Met Thr Thr Phe Arg Asn Trp Trp Glu Lys Ser 515 520 525 ggg tat ggt cag tcc gat aat gcc tct tgc tcc agg tcg ctt ttc gtc 1632 Gly Tyr Gly Gln Ser Asp Asn Ala Ser Cys Ser Arg Ser Leu Phe Val 530 535 540 agt gtg tat tcg gtc ggc acc act gac tac cgt aac caa tat tat gag 1680 Ser Val Tyr Ser Val Gly Thr Thr Asp Tyr Arg Asn Gln Tyr Tyr Glu 545 550 555 560 gcg ccc act aca ccc cca ctg gga ttc tcg atc gga cgc atc gcg gta 1728 Ala Pro Thr Thr Pro Pro Leu Gly Phe Ser Ile Gly Arg Ile Ala Val 565 570 575 tta ggt gga gca cct gag gtt gtt gtt cct gtg gga gag tcc cca tac 1776 Leu Gly Gly Ala Pro Glu Val Val Val Pro Val Gly Glu Ser Pro Tyr 580 585 590 aat agt act atc tct ttg cag acc gag tat ttg ccg gtc agt gtt gcg 1824 Asn Ser Thr Ile Ser Leu Gln Thr Glu Tyr Leu Pro Val Ser Val Ala 595 600 605 ctg cag atg gcg cga gga tgt gac cat gtt ctg gct tcc ttg gtc gct 1872 Leu Gln Met Ala Arg Gly Cys Asp His Val Leu Ala Ser Leu Val Ala 610 615 620 ggc ctt gag aag aag ggc gtc ctc cga cct gtc agt acc ggc tct cgc 1920 Gly Leu Glu Lys Lys Gly Val Leu Arg Pro Val Ser Thr Gly Ser Arg 625 630 635 640 cta tac tcc taa 1932 Leu Tyr Ser <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 2 <211> LENGTH: 643 <212> TYPE: PRT <213> ORGANISM: Aspergillus sojae <400> SEQUENCE: 2 Met Phe Leu Ser Thr Leu Leu Ser Leu Ala Ala Val Val Ala Gly Ala 1 5 10 15 Ala Ile Pro Asn Gly Gln Thr Leu Ser Leu Asn Asp Ile Pro Tyr Tyr 20 25 30 Val Ser Gly Ile Pro Val Ser Thr Leu Gln Gly Tyr Asn Ala Ser Ala 35 40 45 Tyr Ala Ala Leu Thr Lys Gly Ile Asp Leu Val Pro Leu Thr Val Ile 50 55 60 Pro Val Thr Pro Thr Thr Asn Leu Glu Ser Leu Leu Ser Asp Tyr Val 65 70 75 80 Glu Arg Asp Asp Val Phe Gln Pro Ala Phe Leu Arg Ala Val Tyr Leu 85 90 95 Thr Ala Ser Thr Ala Asp Asp Ile Asp Ser Gln Leu Ser Asn Tyr Ala 100 105 110 Ser Ile Leu Lys Ser Ser Gly Thr Asp Val Leu Leu Val Asp Ser Glu 115 120 125 Val His Thr Ala Ser Ser Asp Ser Thr Ile Thr Ala Gln Leu Thr Lys 130 135 140 Glu Leu Pro Ser Gly Pro Tyr Phe Val Ser Leu Tyr Thr Gly Glu Val 145 150 155 160 Phe Arg Ala Tyr Arg Leu Tyr Pro Asp Asp Asn Leu Ala Phe Ile Gln 165 170 175 Ala Gly Ile Ser Asp Glu Lys Gly Gly Val Leu Pro Leu Pro Ala Val 180 185 190 Thr Glu Asn Ala Met Thr Lys Asp Val Ala Val Pro Ser Arg Leu Tyr 195 200 205 Tyr Thr Pro Thr Ala Glu Lys Pro Leu Ala Gly Leu Arg Leu Gly Val 210 215 220 Lys Asp Ile Tyr His Val Lys Gly Leu Lys Thr Ser Gly Gly Ser Arg 225 230 235 240 Ser Tyr Tyr Tyr Leu Tyr Gly Thr Gln Asn Val Thr Ala Pro Ser Ile 245 250 255 Gln Arg Leu Leu Asp Leu Gly Ala Val Phe Val Gly Lys Thr Gly Thr 260 265 270 Val Gln Phe Ala Asn Gly Asp Arg Pro Thr Ala Asp Trp Val Asp Phe 275 280 285 His Cys Pro Phe Asn Gln Arg Gly Glu Gly Tyr Gln Ala Pro Ser Gly 290 295 300 Ser Ser Ser Gly Ser Gly Val Ala Ile Ala Ala Tyr Asp Trp Leu Asp 305 310 315 320 Leu Ala Val Gly Ser Asp Thr Gly Gly Ser Met Arg Ser Pro Ala Ala 325 330 335 Val Gln Gly Ile Tyr Gly Asn Arg Pro Ser Thr Gly Ala Ile Ser Leu 340 345 350 Asp His Val Leu Pro Leu Ser Pro Ala Leu Asp Thr Ala Gly Val Phe 355 360 365 Ala Arg Ser Ala Ser Leu Trp Ser His Thr Val Gln Ala Trp Tyr Pro 370 375 380 His Leu Gln His Asn Phe Thr Ser Phe Pro Arg Gln Leu Leu Leu Ala 385 390 395 400 Gly Gly Gly Trp Asp Gly Lys Gly Ile Ser Pro Glu Ala His Gln Ser 405 410 415 Leu Thr Thr Phe Thr Arg Gly Leu Glu Ala Phe Leu Gly Thr Asn His 420 425 430 Thr Asn Val Asp Val Ser Gln Arg Trp Leu Asp Thr His Ser Pro Thr 435 440 445 Thr Pro Ser Leu Glu Glu Met Leu Asn Leu Thr Tyr Ala Thr Leu Thr 450 455 460 Ser Val Asp Gln Phe Asn His Leu Ala Val Pro Leu Phe Ala Asp Tyr 465 470 475 480 Lys Ala Val His Arg Gly Arg Gln Pro Phe Ile Asn Pro Gly Pro Leu 485 490 495 Ala Arg Trp Gln Trp Gly Gln Ala Asn Gly Gly Asn Thr Ser Tyr Asp 500 505 510 Ala Ala Leu Arg Asn Met Thr Thr Phe Arg Asn Trp Trp Glu Lys Ser 515 520 525 Gly Tyr Gly Gln Ser Asp Asn Ala Ser Cys Ser Arg Ser Leu Phe Val 530 535 540 Ser Val Tyr Ser Val Gly Thr Thr Asp Tyr Arg Asn Gln Tyr Tyr Glu 545 550 555 560 Ala Pro Thr Thr Pro Pro Leu Gly Phe Ser Ile Gly Arg Ile Ala Val 565 570 575 Leu Gly Gly Ala Pro Glu Val Val Val Pro Val Gly Glu Ser Pro Tyr 580 585 590 Asn Ser Thr Ile Ser Leu Gln Thr Glu Tyr Leu Pro Val Ser Val Ala 595 600 605 Leu Gln Met Ala Arg Gly Cys Asp His Val Leu Ala Ser Leu Val Ala 610 615 620 Gly Leu Glu Lys Lys Gly Val Leu Arg Pro Val Ser Thr Gly Ser Arg 625 630 635 640 Leu Tyr Ser <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 3 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 3 tagctatggt cccgtactgt gcaagcttgg 30 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 4 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 4 atggcttgac acacaatctc ccaccacacc 30 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 5 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 5 gcagcgcaac actgaccggc aaatactcgg 30 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 6

<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 6 aagagcgact tggagcagga ggcacatcgg 30 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 7 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 7 ggtgacagac tggatccatc atgtttctta 30 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 8 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 8 ttgtttgaac cggcatgctc tactttgtac 30

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