Koji mold-origin phospholipase a2

a technology of phospholipase and koji mold, which is applied in the field of phospholipase a2, can solve the problem of about 1 billion yen cost and achieve the effect of higher homology

Inactive Publication Date: 2007-01-04
NATIONAL INSTITUTE OF TECHNOLOGY AND EVALUATION +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010] The present inventors have earnestly investigated, and as a result, succeeded in finding two kinds of sequences (hereinafter, referred to as “spaA gene” and “spaB gene”) which have higher homology to phospholipase A2 genes derived from the previously reported microorganism in genome of Aspergillus. When expressing two kinds of proteins encoded by the sequences (hereinafter, referred to as “phospholipase A2-spaA” and “phospholipase A2-spaB”) by using Escherichia coil and Aspergillus as a host, both prot

Problems solved by technology

Therefore, in the safety test such as a chronic toxicity test required when genes derived from general fungi are used for foods, etc., in the case of genes derived from gen

Method used

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  • Koji mold-origin phospholipase a2
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  • Koji mold-origin phospholipase a2

Examples

Experimental program
Comparison scheme
Effect test

example 1

Production Method of Whole-Genome Shotgun Library

1. Preparation of Insert Side

[0058] (1) Obtaining of Chromosomal DNA

[0059] Spores of filamentous fungi, Aspergillus oryzae RIB-40 strain (ATCC 42149) were inoculated in a YPD culture medium (0.5% yeast extract, 1% peptone and 2% glucose) and cultured with shaking ovemight at 30° C. Thereafter, a genome DNA was extracted in accordance with a method by Iimura (Argric. Biol. Chem. 323-328, 51 (1987)). In order to exclude a mitochondrial DNA mixed in the genome DNA, purification by cesium chloride ultracentrifugation was carried out so as to obtain only a chromosomal DNA in accordance with the method by Watson et al. (Methods Enzymol. 57-75 118 (1986)).

[0060] (2) Fragmentation of Chromosomal DNA

[0061] The obtained pure chromosomal DNA was placed in a DNA fragmentation device HydroShear (TOMY SEIKO Co., Ltd.) so as to form the chromosomal DNA into fragments of about 1-2 kb.

[0062] (3) End Treatment of Fragmented DNA

[0063] The fragme...

example 2

Identification of Gene

[0068] Identification of gene from a genome DNA base sequence was conducted by the following technique. In the technique of identifying genes, with respect to the contig sequence of the genome DNA base sequence, the combination of a gene region prediction system GeneDecoder based on algorithm by Kiyoshi Asai et al. (Pacific Symposium on Biocomputing 98, 228-239) and a gene region prediction system ALN based on algorithm (Bioinformatics 2000 16: 190-202) by Osamu Goto was used while considering the homology between sequence information on the previously obtained EST and the amino acid sequence database of the well-known protein. Furthermore, for predicting a tRNA gene, tRNA-scan was used.

“Extraction of BLAST Homologous Gene Candidate Region”

[0069] A region having a high homology to the amino acid sequence of the known protein was extracted from the contig sequence of the genome DNA base sequence. The homology of amino acid sequence can be determined by algorit...

example 3

Retrieving for Sequence Encoding Phospholipase A2

[0079] Based on DNA sequence of phospholipase A2 gene derived from filamentous fungi of Helicosporium sp., BLAST search (Standard protein-protein BLAST: blastp) provided by NCBI was carried out with respect to all DNA sequence of Aspergillus genome DNA. As a result, the present inventors succeeded in finding two regions having high homology to phospholipase A2 gene derived from filamentous fungi of Helicosporium sp. One of the found regions was a region that had been expected to be a sequence having certain functions in identifying the above-mentioned gene in which the function had not been estimated. Note here that a coding region of the gene (putative phospholipase A2-spaA coding region) is shown in SEQ ID NO: 3. Furthermore, an amino acid sequence encoded by the region is shown in SEQ ID NO: 1.

[0080] On the other hand, another region (sequence) was firstly expected to have a translation function and existed in the sequence whose ...

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Abstract

It is intended to provide koji mold-origin phospholipase A2 and a DNA encoding it. Namely, phospholipase A2 comprising the following protein (a) or (b): (a) a protein having an amino acid sequence represented by SEQ ID NO: 1 or 2; and (b) a protein having an amino acid sequence derived from an amino acid sequence represented by SEQ ID NO: 1 or 2 by a partial modification and serving as phospholipase A2.

Description

TECHNICAL FIELD [0001] The present invention relates to phospholipase A2 derived from Aspergillus and a DNA encoding the same, and uses thereof. BACKGROUND ART [0002] Among filamentous fungi, in particular, Aspergillus including Aspergillus oryzae (yellow Aspergillus) etc. has been traditionally used in brewing industry in Japan for producing sake, bean paste, soy sauce, mirin, and the like, and directly eaten. Aspergillus is a safe source of genes listed up in GRAS (Generally Recognized as Safe) by US FDA (Food and drug Administration). [0003] Therefore, in the safety test such as a chronic toxicity test required when genes derived from general fungi are used for foods, etc., in the case of genes derived from general fungi, the cost is about 1 billion yen. On the other hand, in the case of genes that are the above-mentioned GRAS genes, it is advantageous that the cost can be reduced to about one-third of the cost and further that it takes a shorter time to conduct the test as compa...

Claims

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Application Information

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IPC IPC(8): C12P21/06C12N9/16C07H21/04C12N1/16C12N15/74C12N1/15C12N9/20
CPCC12Y301/01004C12N9/20
Inventor KITAMOTO, KATSUHIKOARIOKA, MANABUYAMAGUCHI, SHOTAROMACHIDA, MASAYUKIABE, KELETSUGOMI, KATSUYAASAI, KIYOSHISANO, MOTOAKIKIN, TAISHINNAGASAKI, HIDEKIHOSOYAMA, AKIRAAKITA, OSAMUOGASAWARA, NAOTAKEKUHARA, SATORU
Owner NATIONAL INSTITUTE OF TECHNOLOGY AND EVALUATION
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