Details
| Original language | English |
|---|---|
| Article number | 1385 |
| Journal | CATALYSTS |
| Volume | 11 |
| Issue number | 11 |
| Publication status | Published - 17 Nov 2021 |
Abstract
The L-amino acid ligase RizA from B. subtilis selectively synthesizes dipeptides containing an N-terminal arginine. Many arginyl dipeptides have salt-taste enhancing properties while Arg-Phe has been found to have an antihypertensive effect. A total of 21 RizA variants were created by site-directed mutagenesis of eight amino acids in the substrate binding pocket. The variants were recombinantly produced in E. coli and purified by affinity chromatography. Biocatalytic reactions were set up with arginine and four amino acids differing in size and polarity (aspartic acid, serine, alanine, and phenylalanine) and were analyzed by RP-HPLC with fluorescence detection. Variant T81F significantly improved the yield in comparison to wild type RizA for aspartic acid (7 to 17%), serine (33 to 47%) and alanine (12 to 17%). S84F increased product yield similarly for aspartic acid (7 to 17%) and serine (33 to 42%). D376E increased the yield with alanine (12 to 19%) and phenylalanine (11 to 26%). The largest change was observed for S156A, which showed a yield for Arg-Phe of 40% corresponding to a 270% increase in product concentration. This study expands the knowledge about positions governing the substrate specificity of RizA and may help to inform future protein engineering endeavors.
Keywords
- Arginyl dipeptides, Biocatalysis, Coupled catalysis, L-amino acid ligase, Mutagenesis, Protein engineering, Salt taste, Substrate specificity
ASJC Scopus subject areas
- Chemical Engineering(all)
- Catalysis
- Chemistry(all)
- Physical and Theoretical Chemistry
Cite this
- Standard
- Harvard
- Apa
- Vancouver
- BibTeX
- RIS
In: CATALYSTS, Vol. 11, No. 11, 1385, 17.11.2021.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Mutagenesis of the l-Amino Acid Ligase RizA Increased the Production of Bioactive Dipeptides
AU - Bordewick, Sven
AU - Berger, Ralf G.
AU - Ersoy, Franziska
N1 - Funding Information: The project was supported by funds of the Federal Ministry of Food and Agriculture (BMEL) based on a decision of the Parliament of the Federal Republic of Germany via the Federal Office for Agriculture and Food (BLE) under the innovation support programme. The publication of this article was funded by the Open Access Fund of Leibniz Universität Hannover.
PY - 2021/11/17
Y1 - 2021/11/17
N2 - The L-amino acid ligase RizA from B. subtilis selectively synthesizes dipeptides containing an N-terminal arginine. Many arginyl dipeptides have salt-taste enhancing properties while Arg-Phe has been found to have an antihypertensive effect. A total of 21 RizA variants were created by site-directed mutagenesis of eight amino acids in the substrate binding pocket. The variants were recombinantly produced in E. coli and purified by affinity chromatography. Biocatalytic reactions were set up with arginine and four amino acids differing in size and polarity (aspartic acid, serine, alanine, and phenylalanine) and were analyzed by RP-HPLC with fluorescence detection. Variant T81F significantly improved the yield in comparison to wild type RizA for aspartic acid (7 to 17%), serine (33 to 47%) and alanine (12 to 17%). S84F increased product yield similarly for aspartic acid (7 to 17%) and serine (33 to 42%). D376E increased the yield with alanine (12 to 19%) and phenylalanine (11 to 26%). The largest change was observed for S156A, which showed a yield for Arg-Phe of 40% corresponding to a 270% increase in product concentration. This study expands the knowledge about positions governing the substrate specificity of RizA and may help to inform future protein engineering endeavors.
AB - The L-amino acid ligase RizA from B. subtilis selectively synthesizes dipeptides containing an N-terminal arginine. Many arginyl dipeptides have salt-taste enhancing properties while Arg-Phe has been found to have an antihypertensive effect. A total of 21 RizA variants were created by site-directed mutagenesis of eight amino acids in the substrate binding pocket. The variants were recombinantly produced in E. coli and purified by affinity chromatography. Biocatalytic reactions were set up with arginine and four amino acids differing in size and polarity (aspartic acid, serine, alanine, and phenylalanine) and were analyzed by RP-HPLC with fluorescence detection. Variant T81F significantly improved the yield in comparison to wild type RizA for aspartic acid (7 to 17%), serine (33 to 47%) and alanine (12 to 17%). S84F increased product yield similarly for aspartic acid (7 to 17%) and serine (33 to 42%). D376E increased the yield with alanine (12 to 19%) and phenylalanine (11 to 26%). The largest change was observed for S156A, which showed a yield for Arg-Phe of 40% corresponding to a 270% increase in product concentration. This study expands the knowledge about positions governing the substrate specificity of RizA and may help to inform future protein engineering endeavors.
KW - Arginyl dipeptides
KW - Biocatalysis
KW - Coupled catalysis
KW - L-amino acid ligase
KW - Mutagenesis
KW - Protein engineering
KW - Salt taste
KW - Substrate specificity
UR - http://www.scopus.com/inward/record.url?scp=85119117004&partnerID=8YFLogxK
U2 - 10.3390/catal11111385
DO - 10.3390/catal11111385
M3 - Article
VL - 11
JO - CATALYSTS
JF - CATALYSTS
SN - 2073-4344
IS - 11
M1 - 1385
ER -