JP2008011710A - Glutamic acid aminopeptidase originating from aspergillus oryzae - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a seasoning with strong relish by using an Aspergillus oryzae obtained by specifying a gene encoding glutamic acid aminopeptidase originating from an Aspergillus oryzae and preparing an Aspergillus oryzae with a high glutamic acid aminopeptidase productivity through the use of the gene. <P>SOLUTION: The seasoning liquid with strong relish is prepared in the following processes: (1) specifying the gene encoding the glutamic acid aminopeptidase originated from the Aspergillus oryzae expressed by a specific sequence; (2) preparing a high expression vector of the glutamic acid aminopeptidase gene by connecting the gene on the downstream of a high expression promotor; (3) preparing a transformant by transforming the Aspergillus oryzae by using the expression vector; (4) preparing the Aspergillus oryzae producing a greater amount of glutamic acid aminopeptidase than the parent strain by culturing the transformant; and (5) acting an enzyme extract liquid of the Aspergillus oryzae to a partial hydrolysate of protein. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a glutamic acid aminopeptidase derived from Aspergillus, a gene encoding the enzyme, an Aspergillus oryzae highly producing glutamic acid aminopeptidase using the gene, and a seasoning using an enzyme extract of persimmon prepared using the Aspergillus It relates to the manufacturing method.

  Glutamyl aminopeptidase (EC 3.4.11.7) is an enzyme that acts on a partial hydrolyzate of protein to break peptide bonds at the amino terminus, and specifically releases glutamic acid, an amino acid that contributes to umami. Therefore, it is an important enzyme in the production of seasonings.

  On the other hand, Aspergillus oryzae is also certified by the US Food and Drug Administration (FDA) as GRAS (Generally Recognized As Safe), the enzyme it produces is used for the production of seasonings, etc. It is used a lot.

  Conventionally, as aminopeptidase derived from Aspergillus oryzae, leucine aminopeptidase, which is considered to be important in soy sauce production, has been reported mainly (Japanese translations of PCT publication No. 10-508475, Japanese Patent Laid-Open No. 11-346777, special table. No. 11-509082, Special Table 2000-502912, Special Table 2001-500022, JP 2002-355047, Special Table 2002-514920, Special Table 2002-511746, WO 97/04108, WO 2002/77223), There has been no report on glutamate aminopeptidase.

Special table hei 10-508475 JP-A-11-346777 Special table Hei 11-509082 Special table 2000-502912 Special table 2001-500022 JP 2002-355047 A Special table 2002-514920 Special table 2002-511746 WO97 / 04108 WO2002 / 77223 JP 2005-176602 A Nature 438, 1157-1161 (2005)

  Although the entire genome sequence of Aspergillus oryzae has been published (Japanese Patent Laid-Open No. 2005-176602, Nature 438, 1157-1161 (2005)), the glutamic acid aminopeptidase gene derived from the Aspergillus is unknown and has not yet been identified. . Moreover, in the conventional koji mold, the production amount of glutamic acid aminopeptidase is very low, and it is not practical to use this enzyme for the production of foods such as seasonings such as soy sauce.

  Therefore, the present invention specifies a gene encoding glutamic acid aminopeptidase derived from Aspergillus, prepares an Aspergillus oryzae with high productivity of glutamic acid aminopeptidase using this gene, and provides a seasoning with a strong umami taste using this Aspergillus The purpose is to do.

  As a result of intensive investigations to achieve the above object, the present inventors have succeeded in specifying (1) a gene encoding glutamic acid aminopeptidase derived from Aspergillus oryzae, and (2) A high expression vector of a glutamic acid aminopeptidase gene linked downstream of a high expression promoter was prepared. (3) Successful preparation of a transformant obtained by transforming koji mold using the expression vector. (4) The transformation By culturing the body, it is possible to prepare a cocoon that produced a larger amount of glutamate aminopeptidase than the parent strain, and (5) by causing the enzyme extract of the cocoon to act on the partial hydrolyzate of the protein, The present invention was completed. Therefore, the present invention is as follows.

[1] A glutamic acid aminopeptidase having the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted or added.
[2] The glutamic acid aminopeptidase according to [1] above, which is derived from Aspergillus oryzae.

[3] A glutamic acid aminopeptidase gene encoding the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted or added.
[4] The above-mentioned [3], wherein the glutamic acid aminopeptidase gene has the base sequence shown in SEQ ID NO: 2 or a base sequence in which one or several bases of the base sequence are deleted, substituted or added. gene.

[5] The gene according to [3] or [4] above, wherein the gene is derived from Aspergillus oryzae.
[6] A DNA fragment that hybridizes with the gene of [3] or [4] above under stringent conditions and encodes a polypeptide having glutamate aminopeptidase activity.

[7] A glutamic acid aminopeptidase high productivity plasmid containing the gene according to [3] or [4] or the DNA fragment according to [6].
[8] The plasmid according to [7] above, which has a promoter of an α-amylase gene (amyB).
[9] A koji mold transformed with the plasmid described in [7] above.
[10] A method for producing a seasoning, comprising preparing a koji using the koji mold described in [9] above, and hydrolyzing protein resources using the koji or an enzyme extract thereof.

  The present inventors identified for the first time a gene encoding glutamate aminopeptidase derived from Aspergillus oryzae, and introduced the cloned glutamate aminopeptidase gene in multiple copies under the control of a high-expression promoter, resulting in very efficient amino acid glutamate. Successful acquisition of koji mold producing peptidase. In particular, it is possible to efficiently produce glutamate aminopeptidase by using an amyB promoter as a high expression promoter.

  Producing a koji using such koji molds, and using this koji or its enzyme extract to decompose protein resources such as soybeans to produce a seasoning with a strong umami taste with a significantly increased amount of free glutamic acid The present invention is extremely useful in food production.

  In the amino acid sequence represented by SEQ ID NO: 1, one or several amino acids may be deleted, substituted, modified or added as long as the activity catalyzing the above reaction is maintained. Deletion, substitution, modification or addition of the amino acid sequences described above can be performed by site-directed mutagenesis (eg, Proc. Natl. Acad. Sci. USA, 81, 4662-5666 (1984), Nucleic Acid Res. 10, 6487-6500 (1982), Nature 316, 601-605 (1985), etc.).

  Further, the glutamic acid aminopeptidase of the present invention includes an enzyme having 90% or more homology with the amino acid sequence shown in SEQ ID NO: 1 as long as the activity of catalyzing the above reaction is maintained.

  Such a glutamic acid aminopeptidase is obtained by cloning a gene encoding an enzyme having the amino acid sequence shown in SEQ ID NO: 1 from Aspergillus oryzae, specifically, a glutamic acid aminopeptidase gene consisting of a base sequence shown in SEQ ID NO: 2, Prepare to use.

  In the present invention, as long as glutamate aminopeptidase can be produced, a gene in which one or more bases in the base sequence represented by SEQ ID NO: 2 have been deleted, substituted, inserted or added, or their A DNA fragment that hybridizes with a gene under stringent conditions, and a DNA fragment having 90% or more homology with the base sequence represented by SEQ ID NO: 2 can also be used.

  In addition, a gene in which one or more bases have been deleted, substituted, inserted or added is similar to the above-described amino acid sequence by deletion, substitution, substitution by a known method such as site-directed mutagenesis. It means that as many bases as can be modified or added are deleted, substituted, modified or added. The stringent conditions are 5 × SSC (1 × SSC is 8.76 g of sodium chloride, 4.41 g of sodium citrate dissolved in 1 liter of water), 0.1% (w / v) N-lauroyl monkey Perform a hybridization reaction at 60 ° C. for about 20 hours using a solution containing kosine sodium salt, 0.02% (w / v) SDS, and 0.5% (w / v) blocking reagent. Means.

  Cloning of such genes, preparation of expression vectors using cloned DNA fragments, preparation of glutamic acid aminopeptidase using expression vectors (or plasmids), etc. are well known to those skilled in the field of molecular biology. It is a technology and can be performed according to conventional methods.

  For example, a part of amino acid sequence such as N-terminal and C-terminal of glutamic acid aminopeptidase purified from Aspergillus oryzae is determined by a known method, and the corresponding oligonucleotide is synthesized, and the synthesized oligonucleotide is used as a probe. A DNA fragment containing a gene encoding glutamate aminopeptidase may be cloned from a cDNA library of Aspergillus oryzae RIB40 (ATCC 42149).

  In addition, a PCR primer was designed based on the DNA base sequence predicted to encode aminopeptidase in the entire genome sequence of Aspergillus oryzae that has already been published, and has already been cloned. The target gene can be cloned by screening from the cDNA library of Aspergillus oryzae RIB40 (ATCC 42149) based on the homology with the structure of the glutamate aminopeptidase gene derived from the isolated animal and the glutamate aminopeptidase activity. it can.

  The host used for cloning is not particularly limited, but it is appropriate to use a microorganism such as Escherichia coli as the host in terms of operability and convenience.

  Next, a high expression system of the cloned glutamic acid aminopeptidase gene is constructed. For example, the base sequence of the cloned DNA fragment is analyzed using the method of Maxam-Gilbert (Methods in Enzymology, 65,499 (1980)) or the dideoxy chain terminator method (Methods in Enzymology, 101, 20 (1983)). Recombinant expression in which the coding region of the gene is specified and expression control signals (transcription initiation and translation initiation signals) are linked upstream so that the gene can be self-expressed in the microorganism according to the host microorganism. Create a vector.

  The vector to be used is not particularly limited, and those usually used for breeding filamentous fungi can be used. For example, vectors used for Aspergillus oryzae include pUSA, pUNG (Appl. Microbiol. Biotechnol., 44, 425-431 (1995)), pMARG (Appl. Microbiol. Biotechnol., 40, 327-332 (1993)), pUSC ( Agric. Biol. Chem. 51, 2549-2555 (1987)).

  Among the above-mentioned vectors, pUSA has an α-amylase gene (amyB) promoter and terminator, and a glutamic acid aminopeptidase gene is inserted downstream of the promoter in a frame, whereby glutamic acid is controlled under the promoter. An aminopeptidase gene can be expressed efficiently.

  Transformation of Neisseria gonorrhoeae using such a vector can be performed by a known method. For example, a bacterial body (conidia) is inoculated in DPY medium (glucose 2%, peptone 1%, yeast extract 0.5%, pH 5.0), and cultured at 30 ° C. for about 24 hours. And the cells are collected.

  The collected cells are put into a test tube, protoplasts are added with an enzyme solution, and the obtained protoplasts are used to culture in a selective medium corresponding to the marker. Confirm that it is a converter.

  By culturing the obtained transformant under conditions suitable for the promoter to be used, the glutamate aminopeptidase gene is expressed, and glutamate aminopeptidase can be obtained. For example, when Aspergillus oryzae is used as the host and the α-amylase promoter is used as the promoter, the spores of transformed Asbergillus oryzae are suspended in a medium containing cracked wheat and cultured at about 30 ° C. Can produce glutamate aminopeptidase.

  An enzyme extract containing glutamic acid aminopeptidase can be obtained by placing the culture thus obtained in a container such as an Erlenmeyer flask and adding a suitable amount of distilled water or the like and stirring well. Further, if necessary, the glutamic acid aminopeptidase can be further purified from the enzyme extract by using gel filtration, various chromatography or the like.

  Preparation of seasonings and the like in the present invention is characterized by preparing koji using the koji mold of the transformant and hydrolyzing protein resources using the koji or enzyme extract thereof. That is, if the culture of the transformant or the enzyme extract thereof is mixed directly with a protein resource together with a proteolytic enzyme as necessary and an enzyme such as glutamate aminopeptidase is allowed to act on the protein, the free amino acid content is high. A strong protein hydrolyzate can also be obtained.

  Examples of protein resources to be acted on include soybeans, wheat, wheat gluten, and the like, and also defatted soybeans, various proteins processed such as expansion and solubilization, or separated proteins from these various raw materials. Good.

  As the reaction conditions for the enzyme, for example, a culture of a transformant or an enzyme extract thereof is mixed in a protein resource of 0.2 to 50% concentration in the presence of a proteolytic enzyme, and the reaction is performed at 5 to 60 ° C. for 4 hours to 20 What is necessary is just to react for one day.

  After completion of the reaction, undegraded protein raw materials and insoluble matters such as bacterial cells are removed using a conventional separation method such as centrifugation or filtration. In addition, if necessary, concentration may be performed by vacuum concentration, reverse osmosis, etc., and the resulting concentrate may be pulverized or granulated by a drying treatment such as freeze drying, vacuum drying, spray drying, etc., if necessary. it can.

  Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

Example 1
(1) Acquisition of glutamic acid aminopeptidase gene by PCR The following PCR primers (M13-Rv, AP-Rv3) were obtained with reference to the DNA base sequence predicted to be aspartyl aminopeptidase from the results of EST analysis of Aspergillus oryzae RIB40 (ATCC42149). Designed. Next, PCR using primers M13-Rv and AP-Rv3 was performed using plasmid JZ3909f containing koji mold cDNA as a template to amplify the gene encoding glutamate aminopeptidase.

M13-Rv: 5′-CAGGAAACAGCATTGAC-3 ′
AP-Rv3: 5′-TTCTAGGTACCCCCTATTCTACCAAGTAACCAACGC-3 ′

That is, in a 0.2 ml capacity PCR tube (manufactured by Eppendorf), distilled water 54 μl, 10 × buffer (attached to KOD-Plus- (manufactured by Toyobo)) 10 μl, primer DNA 10 pmol / μl solution 10 μl × 2 types , 1 μl of a 100 ng / μl solution of genomic DNA, 10 μl of 2 mM dNTP solution (Toyobo), ₄ μl of 25 mM MgSO ₄ and 1 μl of KOD-Plus- (Toyobo) were mixed, and then a thermal cycler (TaKaRa PCR Thermal Cycler Dice (Takara Bio) (1) 94 ° C. for 2 minutes, (2) 94 ° C. for 30 seconds, (3) 56 ° C. for 60 seconds, (4) 72 ° C. for 90 seconds, (2) to (4) 30 cycles, (5) A program of 7 minutes at 72 ° C. was combined to amplify the target gene.

  Next, 50 μl of the PCR product was cleaved with KpnI (Takara Bio Inc.), the reaction solution was electrophoresed with 0.9% agarose, a part of the gel containing the target gene sequence was excised, and Wizard SV Gel and PCR. DNA was eluted using a Clean-Up System (Promega).

On the other hand, pUSA which is a vector having an amyB promoter and terminator was cleaved with KpnI, and the cleaved vector was dephosphorylated with alkaline phosphatase (manufactured by Wako Pure Chemical Industries, Ltd.) and the glutamic acid obtained above. A DNA fragment considered to encode aminopeptidase was mixed with ligation high (Toyobo), and then incubated at 16 ° C. for 30 minutes to transform competent cells of E. coli DH5α.

  From the transformant thus obtained, a transformant containing a glutamic acid aminopeptidase gene is selected in a plasmid, the selected transformant is cultured in an LB medium containing ampicillin, and QIAprep is used for the culture. Using Spin Miniprep Kit (manufactured by QIAGEN), a plasmid was obtained according to the manual attached to the kit, and the resulting plasmid was named pUSA-AP. This pUSA-AP was constructed by introducing the glutamic acid aminopeptidase gene cloned between the amyB promoter and terminator in an expressible state, and was deposited with the Patent Organism Depositary Center on June 13, 2006. FERM AP-20934 is given as the receipt number.

(2) Acquisition of transformant introduced with glutamic acid aminopeptidase gene Aspergillus oryzae NS4 strain (Biosci. Biotech. Biochem., 61 (8), 1367-1369) was obtained using plasmid pUSA-AP thus obtained. (1997)) was transformed by a conventional method, several transformant colonies were obtained on a minimal medium, genomic DNA was prepared from this transformant, and then cleaved with restriction enzyme BamHI (manufactured by Takara Bio Inc.). After blotting with Hybond-N + (manufactured by GE Healthcare Bioscience), Southern hybridization analysis of the transformant was performed using the cloned glutamic acid aminopeptidase gene as a probe. As a result, it was confirmed that a gene that is supposed to encode glutamate aminopeptidase has not been introduced at all, that one copy has been introduced, and that a plurality of copies have been introduced. From these, a strain into which multiple copies were introduced was selected and named NAP10.

(3) Preparation of enzyme solution by transformant After preparing bran in a 500 ml Erlenmeyer flask, add spore (5 × 10 ⁶ ) of NAP10 strain and 4 ml of 4.5% maltose solution and stir well. The culture was performed in an incubator at 30 ° C. for 72 hours. After culturing, 70 ml of deionized water was added and shaken well, then allowed to stand at room temperature for 4 hours, and the filtrate obtained by filtration using No. 2 filter paper (ADVANTEC) was used as the enzyme extract.

As a result of measuring the glutamic acid aminopeptidase activity of this enzyme extract according to a conventional method using Glu-pNA as a substrate, as shown in Table 1, the cloned gene has a glutamic acid aminopeptidase activity. When the activity of NS4, which is the host strain, was 1, the relative activity of the NAP10 strain was 141113.

(4) Manufacture of seasoning liquid The obtained enzyme extract prepared in the same manner as described above was filtered twice with a 0.2 μm membrane to obtain a sterile enzyme liquid. Next, 7 ml of water was added to 5 g of defatted soybeans in a 500 ml Erlenmeyer flask, and after 30 minutes of water supply, autoclaved at 121 ° C. for 40 minutes to obtain steamed beans. 10 ml of sterile enzyme solution and 10 ml of sterilized water were added to the steamed beans, and the mixture was incubated at 50 ° C. for 20 hours. After the reaction, the filtrate obtained by filtering the filter paper was used as a soybean decomposition solution. Amino acids were analyzed. As a result, as shown in Table 2, it became clear that the amount of free glutamic acid was significantly increased compared to the parent strain, and it was possible to obtain a seasoning liquid having a strong umami taste.

<Receipt>

Claims (10)

A glutamic acid aminopeptidase having the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted or added. The glutamic acid aminopeptidase according to claim 1, which is derived from Aspergillus oryzae. A glutamic acid aminopeptidase gene encoding the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted or added. The gene according to claim 3, wherein the glutamate aminopeptidase gene has the base sequence shown in SEQ ID NO: 2 or a base sequence in which one or several bases of the base sequence are deleted, substituted or added. The gene according to claim 3 or 4, wherein the gene is derived from Aspergillus oryzae. A DNA fragment that hybridizes with the gene according to claim 3 or 4 under stringent conditions and encodes a polypeptide having glutamate aminopeptidase activity. A glutamic acid aminopeptidase high productivity plasmid containing the gene according to claim 3 or 4 or the DNA fragment according to claim 6. The plasmid according to claim 7, which has a promoter of an α-amylase gene (amyB). A koji mold transformed with the plasmid of claim 7. A method for producing a seasoning comprising preparing a koji using the koji mold of claim 9 and hydrolyzing a protein resource using the koji or an enzyme extract thereof.