Peptidylarginine deiminase and uses thereof in the production of citrullinated proteins and peptides

A technology of arginine deiminase and citrulline, applied in the field of proteins and peptides, which can solve problems such as serious and taste problems

Inactive Publication Date: 2009-07-08
DSM IP ASSETS BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the use of free amino acids in foods is expected to lead to serious taste problems, which must be the case if the recommended amino acid dosages are considered

Method used

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  • Peptidylarginine deiminase and uses thereof in the production of citrullinated proteins and peptides
  • Peptidylarginine deiminase and uses thereof in the production of citrullinated proteins and peptides
  • Peptidylarginine deiminase and uses thereof in the production of citrullinated proteins and peptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0202] Example 1 Fusarium strain can secrete PAD-like activity

[0203] A large collection of molds was screened to identify secreted PAD-like activity. For this purpose, the strains were pre-cultured on Potato Dextrose Broth (PDB; Difco) at 30°C for 4-5 days. Then, the culture was harvested by centrifugation, washed with distilled water, and transferred to minimal medium enriched with rice protein hydrolyzate. Rice protein is relatively rich in arginine residues, and to increase its water solubility, rice protein (Remy Industries, Leuven, Belgium) was preincubated with Alcalase (NOVO, Bagsvaerd, Denmark) at pH 7.5 to obtain a DH of ca. The hydrolyzate of 15. The minimal medium used contained 0.52 g KCl, 1.52 g KH per liter 2 PO 4 , 1.3ml 4M KOH, 0.52g MgSO 4· 7H 2 O, 22mg ZnSO 4· 7H 2 O, 11 mg H 3 BO 3 , 5mg FeSO 4· 7H 2 O, 1.7mg CoCl 2· 6H 2 O, 1.6mgCuSO 4· 5H 2 O, 5mg MnCl 2· 4H 2 O, 1.5 mg Na 2 MoO 4· 2HO, 50mg EDTA, 40g glucose and 5g hydrolyzed rice...

Embodiment 2

[0204] Example 2 Identification of the PAD-encoding gene in the Fusarium genome sequence

[0205] Now that some Fusarium strains are known to secrete PAD-like activity, the genomic sequences of the genes encoding these PADs were isolated and analyzed. For this purpose, Fusarium graminearum strains CBS166.57, CBS316.73, CBS11063, CBS18432 and CBS792.70 were cultured in PDB (Potato Dextrose Medium, Difco) at 30 °C for 3 days and grown according to the supplier's Chromosomal DNA was isolated using the Q-Biogene kit (Cat. No. 6540-600; Omnilabo International BV, Breda, the Netherlands) according to the instructions. This chromosomal DNA was used to amplify the coding sequence of the PAD gene using PCR.

[0206]Two PCR primers were designed to specifically amplify the PAD gene from chromosomal DNA of Fusarium graminearum strains CBS166.57, CBS316.73, CBS11063, CBS18432 and CBS792.70. The primer sequence was obtained in part from a sequence found in the genomic DNA of Gibberella...

Embodiment 3

[0214] Example 3 Overexpression of possible Fusarium graminearum PAD by Aspergillus niger

[0215] From the pGBPAD plasmid containing the genomic PAD gene from Fusarium graminearum CBS166.57, the PacI / AscI fragment containing the PAD coding sequence was isolated and exchanged with the PacI / AscI phyA fragment in pGBFIN-5 (WO 99 / 32617). The resulting plasmid is a PAD expression vector called pGBFINPAD (see image 3 ). The expression vector pGBFINPAD was linearized by digestion with NotI, which removed all E. coli derived sequences from the expression vector. Digested DNA was purified using phenol:chloroform:isoamyl alcohol (24:23:1 ) extraction and ethanol precipitation. These vectors were used to transform Aspergillus niger CBS513.88. The Aspergillusniger transformation procedure is extensively described in WO 98 / 46772. It also describes how to select for transformants on agar plates containing acetamide and how to select for targeted multi-copy integrants. Preferably, A...

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Abstract

The present application concern the isolation of a peptidylarginine deiminase enzyme (EC 3.5.3.15) from Gibberella zeae (also called Fusarium graminearum). The application also discloses citrullinated proteins and peptides produced enzymatically using said arginine deiminase and uses of such citrullinated proteins and peptides in foodstuff. The present invention relates to a modified protein, peptide or protein hydrolysate wherein at least 15%, preferably at least 30%, more preferably at least 45%, still more preferably at least 60%, and most preferably at least 80% of the arginine residues which are originally present in the protein, peptide or protein hydrolysate is transformed into a citrulline residue.

Description

field of invention [0001] The present invention relates to proteins and peptides comprising citrulline residues. Background of the invention [0002] Arginine is a conditionally essential amino acid that plays a key role in mammals. The metabolic pathway of arginine has been well described. In its dietary intake, arginine is absorbed by the liver from the hepatic portal vein and rapidly converted to ornithine by arginase. In the latter process, urea is formed. Ornithine produced from arginine is then converted to citrulline, or can be metabolized to glutamate and alanine. Alternatively, the formed ornithine is incorporated into a polyamine compound, eg, putrescine. Dietary arginine that is not metabolized to ornithine can be processed, for example, to nitric oxide or arginyl-tRNA for protein synthesis purposes. Furthermore, there is an endogenous synthetic pathway leading to arginine. The latter process occurs mainly in the kidney, where arginine is synthesized from or...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/78A23L1/29A23L1/30C12R1/77A23J3/34C12N15/80
CPCA23J3/34A23L1/3051C12N9/78A23L33/175
Inventor 路泊·伊登斯尼古拉斯·埃米尔·保卢斯·迪乌特茨彼得吕斯·雅各布斯·西奥多瑞斯·蒂克尔
Owner DSM IP ASSETS BV
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