Details
| Original language | English |
|---|---|
| Article number | 4352 |
| Journal | MOLECULES |
| Volume | 27 |
| Issue number | 14 |
| Publication status | Published - 7 Jul 2022 |
Abstract
The biocatalytic system comprised of RizA and acetate kinase (AckA) combines the specific synthesis of bioactive arginyl dipeptides with efficient ATP regeneration. Immobilization of this coupled enzyme system was performed and characterized in terms of activity, specificity and reusability of the immobilisates. Co-immobilization of RizA and AckA into a single immobilisate conferred no disadvantage in comparison to immobilization of only RizA, and a small addition of AckA (20:1) was sufficient for ATP regeneration. New variants of RizA were constructed by combining mutations to yield variants with increased biocatalytic activity and specificity. A selection of RizA variants were co-immobilized with AckA and used for the production of the salt-taste enhancers Arg-Ser and Arg-Ala and the antihypertensive Arg-Phe. The best variants yielded final dipeptide concentrations of 11.3 mM Arg-Ser (T81F_A158S) and 11.8 mM Arg-Phe (K83F_S156A), the latter of which represents a five-fold increase in comparison to the wild-type enzyme. T81F_A158S retained more than 50% activity for over 96 h and K83F_S156A for over 72 h. This study provides the first example of the successful co-immobilization of an L-amino acid ligase with an ATP-regenerating enzyme and paves the way towards a bioprocess for the production of bioactive dipeptides.
Keywords
- ATP regeneration, L-amino acid ligase, acetate kinase, antihypertensive dipeptide, arginyl dipeptides, biocatalysis, co-immobilization, covalent immobilization, protein engineering, salt-taste enhancer
ASJC Scopus subject areas
- Chemistry(all)
- Analytical Chemistry
- Chemistry(all)
- Chemistry (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Medicine
- Pharmacology, Toxicology and Pharmaceutics(all)
- Pharmaceutical Science
- Pharmacology, Toxicology and Pharmaceutics(all)
- Drug Discovery
- Chemistry(all)
- Physical and Theoretical Chemistry
- Chemistry(all)
- Organic Chemistry
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In: MOLECULES, Vol. 27, No. 14, 4352, 07.07.2022.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Co-Immobilization of RizA Variants with Acetate Kinase for the Production of Bioactive Arginyl Dipeptides
AU - Bordewick, Sven
AU - Berger, Ralf G.
AU - Ersoy, Franziska
N1 - Funding Information: The project was supported by funds from 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 program. The publication of this article was funded by the Open Access Fund of Leibniz Universität Hannover.
PY - 2022/7/7
Y1 - 2022/7/7
N2 - The biocatalytic system comprised of RizA and acetate kinase (AckA) combines the specific synthesis of bioactive arginyl dipeptides with efficient ATP regeneration. Immobilization of this coupled enzyme system was performed and characterized in terms of activity, specificity and reusability of the immobilisates. Co-immobilization of RizA and AckA into a single immobilisate conferred no disadvantage in comparison to immobilization of only RizA, and a small addition of AckA (20:1) was sufficient for ATP regeneration. New variants of RizA were constructed by combining mutations to yield variants with increased biocatalytic activity and specificity. A selection of RizA variants were co-immobilized with AckA and used for the production of the salt-taste enhancers Arg-Ser and Arg-Ala and the antihypertensive Arg-Phe. The best variants yielded final dipeptide concentrations of 11.3 mM Arg-Ser (T81F_A158S) and 11.8 mM Arg-Phe (K83F_S156A), the latter of which represents a five-fold increase in comparison to the wild-type enzyme. T81F_A158S retained more than 50% activity for over 96 h and K83F_S156A for over 72 h. This study provides the first example of the successful co-immobilization of an L-amino acid ligase with an ATP-regenerating enzyme and paves the way towards a bioprocess for the production of bioactive dipeptides.
AB - The biocatalytic system comprised of RizA and acetate kinase (AckA) combines the specific synthesis of bioactive arginyl dipeptides with efficient ATP regeneration. Immobilization of this coupled enzyme system was performed and characterized in terms of activity, specificity and reusability of the immobilisates. Co-immobilization of RizA and AckA into a single immobilisate conferred no disadvantage in comparison to immobilization of only RizA, and a small addition of AckA (20:1) was sufficient for ATP regeneration. New variants of RizA were constructed by combining mutations to yield variants with increased biocatalytic activity and specificity. A selection of RizA variants were co-immobilized with AckA and used for the production of the salt-taste enhancers Arg-Ser and Arg-Ala and the antihypertensive Arg-Phe. The best variants yielded final dipeptide concentrations of 11.3 mM Arg-Ser (T81F_A158S) and 11.8 mM Arg-Phe (K83F_S156A), the latter of which represents a five-fold increase in comparison to the wild-type enzyme. T81F_A158S retained more than 50% activity for over 96 h and K83F_S156A for over 72 h. This study provides the first example of the successful co-immobilization of an L-amino acid ligase with an ATP-regenerating enzyme and paves the way towards a bioprocess for the production of bioactive dipeptides.
KW - ATP regeneration
KW - L-amino acid ligase
KW - acetate kinase
KW - antihypertensive dipeptide
KW - arginyl dipeptides
KW - biocatalysis
KW - co-immobilization
KW - covalent immobilization
KW - protein engineering
KW - salt-taste enhancer
UR - http://www.scopus.com/inward/record.url?scp=85133743618&partnerID=8YFLogxK
U2 - 10.3390/molecules27144352
DO - 10.3390/molecules27144352
M3 - Article
VL - 27
JO - MOLECULES
JF - MOLECULES
SN - 1420-3049
IS - 14
M1 - 4352
ER -