Mutagenesis of the l-Amino Acid Ligase RizA Increased the Production of Bioactive Dipeptides

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 hav...

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Autores principales:	Sven Bordewick, Ralf G. Berger, Franziska Ersoy
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	<span style="font-variant: small-caps">l</span>-amino acid ligase biocatalysis mutagenesis coupled catalysis arginyl dipeptides salt taste Chemical technology TP1-1185 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/d96e8e8bbf8546eab0b76151de352822
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spelling	oai:doaj.org-article:d96e8e8bbf8546eab0b76151de3528222021-11-25T17:06:37ZMutagenesis of the <span style="font-variant: small-caps">l</span>-Amino Acid Ligase RizA Increased the Production of Bioactive Dipeptides10.3390/catal111113852073-4344https://doaj.org/article/d96e8e8bbf8546eab0b76151de3528222021-11-01T00:00:00Zhttps://www.mdpi.com/2073-4344/11/11/1385https://doaj.org/toc/2073-4344The <span style="font-variant: small-caps;">l</span>-amino acid ligase RizA from <i>B. subtilis</i> 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 <i>E. coli</i> 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.Sven BordewickRalf G. BergerFranziska ErsoyMDPI AGarticle<span style="font-variant: small-caps">l</span>-amino acid ligasebiocatalysismutagenesiscoupled catalysisarginyl dipeptidessalt tasteChemical technologyTP1-1185ChemistryQD1-999ENCatalysts, Vol 11, Iss 1385, p 1385 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	<span style="font-variant: small-caps">l</span>-amino acid ligase biocatalysis mutagenesis coupled catalysis arginyl dipeptides salt taste Chemical technology TP1-1185 Chemistry QD1-999
spellingShingle	<span style="font-variant: small-caps">l</span>-amino acid ligase biocatalysis mutagenesis coupled catalysis arginyl dipeptides salt taste Chemical technology TP1-1185 Chemistry QD1-999 Sven Bordewick Ralf G. Berger Franziska Ersoy Mutagenesis of the <span style="font-variant: small-caps">l</span>-Amino Acid Ligase RizA Increased the Production of Bioactive Dipeptides
description	The <span style="font-variant: small-caps;">l</span>-amino acid ligase RizA from <i>B. subtilis</i> 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 <i>E. coli</i> 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.
format	article
author	Sven Bordewick Ralf G. Berger Franziska Ersoy
author_facet	Sven Bordewick Ralf G. Berger Franziska Ersoy
author_sort	Sven Bordewick
title	Mutagenesis of the <span style="font-variant: small-caps">l</span>-Amino Acid Ligase RizA Increased the Production of Bioactive Dipeptides
title_short	Mutagenesis of the <span style="font-variant: small-caps">l</span>-Amino Acid Ligase RizA Increased the Production of Bioactive Dipeptides
title_full	Mutagenesis of the <span style="font-variant: small-caps">l</span>-Amino Acid Ligase RizA Increased the Production of Bioactive Dipeptides
title_fullStr	Mutagenesis of the <span style="font-variant: small-caps">l</span>-Amino Acid Ligase RizA Increased the Production of Bioactive Dipeptides
title_full_unstemmed	Mutagenesis of the <span style="font-variant: small-caps">l</span>-Amino Acid Ligase RizA Increased the Production of Bioactive Dipeptides
title_sort	mutagenesis of the <span style="font-variant: small-caps">l</span>-amino acid ligase riza increased the production of bioactive dipeptides
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/d96e8e8bbf8546eab0b76151de352822
work_keys_str_mv	AT svenbordewick mutagenesisofthespanstylefontvariantsmallcapslspanaminoacidligaserizaincreasedtheproductionofbioactivedipeptides AT ralfgberger mutagenesisofthespanstylefontvariantsmallcapslspanaminoacidligaserizaincreasedtheproductionofbioactivedipeptides AT franziskaersoy mutagenesisofthespanstylefontvariantsmallcapslspanaminoacidligaserizaincreasedtheproductionofbioactivedipeptides
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Mutagenesis of the <span style="font-variant: small-caps">l</span>-Amino Acid Ligase RizA Increased the Production of Bioactive Dipeptides

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