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14 pages, 2508 KiB

Open AccessArticle

Co-Immobilization of RizA Variants with Acetate Kinase for the Production of Bioactive Arginyl Dipeptides

by Sven Bordewick, Ralf G. Berger and Franziska Ersoy

Molecules 2022, 27(14), 4352; https://doi.org/10.3390/molecules27144352 - 7 Jul 2022

Cited by 2 | Viewed by 2127

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. Full article

(This article belongs to the Special Issue The Latest Trends in Catalyst Immobilization)

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14 pages, 3646 KiB

Open AccessArticle

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

by Sven Bordewick, Ralf G. Berger and Franziska Ersoy

Catalysts 2021, 11(11), 1385; https://doi.org/10.3390/catal11111385 - 17 Nov 2021

Cited by 2 | Viewed by 2694

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. Full article

(This article belongs to the Special Issue Enzyme Catalysis, Biotransformation and Bioeconomy)

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11 pages, 3883 KiB

Open AccessArticle

Boosted Prediction of Antihypertensive Peptides Using Deep Learning

by Anum Rauf, Aqsa Kiran, Malik Tahir Hassan, Sajid Mahmood, Ghulam Mustafa and Moongu Jeon

Appl. Sci. 2021, 11(5), 2316; https://doi.org/10.3390/app11052316 - 5 Mar 2021

Cited by 13 | Viewed by 3227

Abstract

Heart attack and other heart-related diseases are among the main causes of fatalities in the world. These diseases and some other severe problems like kidney failure and paralysis are mainly caused by hypertension. Since bioactive peptides extracted from naturally existing food substances possess antihypertensive activity, these antihypertensive peptides (AHTP) can function as prospective replacements for existing pharmacological drugs with no or fewer side effects. Such naturally existing peptides can be identified using in-silico approaches. The in-silico methods have been proven to save huge amounts of time and money in the identification of effective peptides. The proposed methodology is a deep learning-based in-silico approach for the identification of antihypertensive peptides (AHTPs). An ensemble method is proposed that combines convolutional neural network (CNN) and support vector machine (SVM) classifiers. Amino acid composition (AAC) and g-gap dipeptide composition (DPC) techniques are used for feature extraction. The proposed methodology has been evaluated on two standard antihypertensive peptide sequence datasets. The model yields 95% accuracy on the benchmarking dataset and 88.9% accuracy on the independent dataset. Comparative analysis is provided to demonstrate that the proposed method outperforms existing state-of-the-art methods on both of the benchmarking and independent datasets. Full article

(This article belongs to the Special Issue Computing and Artificial Intelligence for Visual Data Analysis)

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11 pages, 4643 KiB

Open AccessArticle

Design, Synthesis, and Evaluation of Novel Phenolic Acid/Dipeptide/Borneol Hybrids as Potent Angiotensin Converting Enzyme (ACE) Inhibitors with Anti-hypertension Activity

by Ying Sun, Yujun Bai, Xirui He, Yajun Bai, Pei Liu, Zefeng Zhao, Xufei Chen and Xiaohui Zheng

Molecules 2017, 22(11), 1739; https://doi.org/10.3390/molecules22111739 - 3 Nov 2017

Cited by 10 | Viewed by 6151 | Correction

Abstract

Under the guidance of combination of traditional Chinese medicine chemistry (CTCMC), this study describes the preparation of a phenolic acid/dipeptide/borneol hybrid consisting of phenolic acid and a bornyl moiety connected to the dipeptide N-terminal and C-terminal respectively. It also evaluates their angiotensin converting enzyme (ACE) inhibitory and synergistic antihypertensive activities. Briefly, a series of novel phenolic acid/dipeptide/borneol hybrids were prepared and investigated for their ability to inhibit ACE. The influence of the phenolic acid and bornyl moiety on subsite selectivity is also demonstrated. Among all the new compounds, two compounds—7a and 7g—reveal good inhibition potency in in vitro ACE-inhibitory tests. Interestingly, favorable binding results in molecular docking studies also supported the in vitro results. Additionally, the bioassay showed that oral administration of the two compounds displayed high and long-lasting antihypertensive activity both in acute antihypertensive tests and in therapeutic antihypertensive tests by non-invasive blood pressure measurements in spontaneously hypertensive rats. Full article

(This article belongs to the Section Medicinal Chemistry)

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