Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Pyroglutamyl peptidase and its gene

a technology of pyroglutamyl peptidase and pyroglutamyl peptide, which is applied in the field of pyroglutamyl peptidase, can solve the problems of low amino acid release rate, inability to isolate pyroglutamyl peptidase, and inability to bind filamentous proteins, and achieve the effect of efficient cultur

Inactive Publication Date: 2006-10-19
KYOWA HAKKO FOOD SPECIALTIES +3
View PDF2 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a novel pyroglutamyl peptidase derived from filamentous fungi that can be used in the production of a protein hydrolysate with a higher releasing rate of amino acids, particularly glutamic acid, than conventional protein hydrolysates. The invention also provides a DNA encoding this pyroglutamyl peptidase and a method for its production using site-directed mutagenesis or gene homologous recombination. The pyroglutamyl peptidase activity of the invention can be determined using a substrate solution and a sample solution, and the DNA of the invention can be easily produced by combining methods known to a person skilled in the art. The invention provides a safer and more economical method for producing protein hydrolysates with improved functionality.

Problems solved by technology

However, there may be a case where a generated peptide has a bitter taste depending on properties of a protein and enzymes used in proteolysis and therefore it is not functionally preferred.
As shown in the fermentation of soy sauce and bean paste, in the case of using the conventional Aspergillus culture products, although the hydrolysis of proteins requires enormous manpower and time, it results in a low releasing rate of amino acid.
However, pyroglutamyl peptidase derived from filamentous fungi and a gene thereof have not been isolated yet.
It is considered that it results in high cost and deteriorated flavor.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

example 1

Method of Producing Whole Genome Shot Gun Library

1. Production of Insert DNA

(1) Obtainment f Chromosomal DNA

[0151] Spores of filamentous fungus Aspergillus oryzae RIB 40 (ATCC No. 42149) were inoculated into a YPD medium (0.5% yeast extract, 1% peptone, 2% glucose), and the mixture was subjected to a shaking culturing at 30° C. overnight. Thereafter, genomic DNA was extracted in accordance with the method of Iimura [Agric. Biol. Chem., 51, 323 (1987)]. Mitochondrial DNA that had existed with the genomic DNA was purified by cesium chloride ultracentrifugation in accordance with the method of Watson et al. [Methods Enzymol., 118, 57 (1986)], so as to obtain only chromosomal DNA.

(2) Fragmentation of Chromosomal DNA

[0152] Using a random DNA fragmentation device HydroShear (Tomy Seiko Co., Ltd.), the obtained pure chromosomal DNA was degraded into fragments with a size of approximately 1 to 2 kb.

(3) Treatment of Fragmented DNA Termini

[0153] The fragmented chromosomal DNA was t...

example 2

Identification of Gene

[0157] Identification of gene from the nucleotide sequence of the genomic DNA was carried out using, in combination, a gene finding system GeneDecoder based on the algorithm according to Kiyoshi Asai et al. [Pacific Symposium on Biocomputing, 98, 228 (1998)] and a gene finding system ALN based on the algorithm according to Osamu Goto [Bioinformatics, 16, 190-202 (2000)] to the contigs of the nucleotide sequence of the genomic DNA, while taking into consideration the EST sequence information that had already been obtained, and information regarding homology to the known protein amino acid sequence database, by applying the methods according to the following (1) to (7).

(1) Extraction of BLAST Homologous Gene Candidate Regions

[0158] A region having a high homology with the amino acid sequence of a known protein is extracted from the contigs of the nucleotide sequence of the genomic DNA. The homology of an amino acid sequence can be determined by the algorithm ...

example 3

Cloning of Pyroglutamyl Peptidase cDNA of Aspergillus Oryzae

(1) Preparation of cDNA of Aspergillus Oryzae RIB 40 Strain

[0169]Aspergillus oryzae RIB 40 strain was cultured under the following conditions. This is to say, this strain was inoculated into 60 ml of a DPY medium (2% dextrin, 2% polypeptone, 0.5% yeast extract, 0.5% monopotassium phosphate, 0.05% magnesium sulfate 7 hydrates), and the mixture was placed in a 300 ml Erlenmeyer flask with a baffle, followed by a shaking culturing at 30° C. at 150 rpm for 2 days. Thereafter, the culture was filtrated, and 1 g of the obtained wet cell bodies was transferred into a mortar containing liquid nitrogen, so that the cell bodies were frozen by the liquid nitrogen. Thereafter, the cell bodies were ground with a pestle into fine powders.

[0170] Total RNA was obtained from this fine powders, using an RNeasy Midi Kit manufactured by Qiagen.

[0171] From the obtained total RNA, 100 μL of 0.6 μg / ml mRNA solution was obtained, using an Oli...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
pHaaaaaaaaaa
pHaaaaaaaaaa
temperatureaaaaaaaaaa
Login to View More

Abstract

The present invention provides DNA encoding novel pyroglutamyl peptidase derived from Aspergillus oryzae, pyroglutamyl peptidase which is produced by using the DNA, and a method for producing a protein lysate with a good flavor at a high hydrolysis rate.

Description

TECHNICAL FIELD [0001] The present invention relates to a pyroglutamyl peptidase used in production of protein hydrolysates and DNA encoding the pyroglutamyl peptidase. BACKGROUND ART [0002] Among filamentous fungi, in particular, Koji molds including Aspergillus oryzae has been used for a long time in the field of fermented food production in our country, which produces sake, bean paste, soy sauce, Japanese sweet rice wine for cooking, etc., and thus, this fungus has been directly eaten. Koji molds are safe gene sources that have been listed up as GRAS (Generally Recognized as Safe) by the FDA (Food and Drug Administration) of USA. Thus, filamentous fungi, and especially, Koji molds are considered to be a treasure trove of genes with extremely high utility value in terms of safety. [0003] Protein hydrolysates can be produced by hydrolysis of a raw material comprising a protein with acid. From the viewpoint of the use of protein hydrolysates as natural seasonings, a production proce...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/569C07H21/04C12P21/06C12N9/50C12N15/74C12N1/16C12N1/15C12N1/19C12N1/21C12N9/06C12N9/48C12N9/62C12N9/80C12N15/52C12N15/53C12N15/57
CPCC12N9/0022C12P21/06C12N9/80C12N9/48
Inventor MACHIDA, MASAYUKIABE, KEIETSUGOMI, KATSUYAASAI, KIYOSHISANO, MOTOAKIKIN, TAISHINNAGASAKI, HIDEKIHOSOYAMA, AKIRAAKITA, OSAMUOGASAWARA, NAOTAKEKUHARA, SATORUTOKUNAGA, CHIKARATODA, ITARUSAITOH, CHIAKISENOH, AKIHIRO
Owner KYOWA HAKKO FOOD SPECIALTIES
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com