Production of a novel opine dehydrogenase

Opine dehydrogenases are a family of NAD(P)H dependent oxidoreductases, whichcatalyze the reductive condensation of an α amino group from an amino acid with an αketo acid during anaerobic glycolysis by regenerating NAD. They are widespread incephalopods and mollusks. Opines are associated with crown gall tumor pathogenesiscaused by A. tumefaciens providing nutrients to the pathogen, and novel opine compoundsacting as metallophores have been identified. Besides, opine-type secondary aminedicarboxylic acids are chiral intermediates of angiotensin-converting enzyme inhibitors. Anovel enzyme originating from an extremophile bacterium, with assumed opinedehydrogenase function was successfully expressed in Escherichia coli STAR cells andpurified by affinity chromatography. Molecular mass determined by SDS-PAGE wasapproximately 40 kDa. The activity was measured by using pyruvate and alanine assubstrates, by which proved that it has opine dehydrogenase activity

In Silico and In Vitro Inhibition of SARS-CoV-2 PLpro with Gramicidin D

Finding an effective drug to prevent or treat COVID-19 is of utmost importance in tcurrent pandemic. Since developing a new treatment takes a significant amount of time, drug repurposing can be an effective option for achieving a rapid response. This study used a combined in silico virtual screening protocol for candidate SARS-CoV-2 PLpro inhibitors. The Drugbank database was searched first, using the Informational Spectrum Method for Small Molecules, followed by molecular docking. Gramicidin D was selected as a peptide drug, showing the best in silico interaction profile with PLpro. After the expression and purification of PLpro, gramicidin D was screened for protease inhibition in vitro and was found to be active against PLpro. The current study’s findings are significant because it is critical to identify COVID-19 therapies that are efficient, affordable, and have a favorable safety profil

In Silico and In Vitro Inhibition of SARS-CoV-2 PLpro with Gramicidin D

Finding an effective drug to prevent or treat COVID-19 is of utmost importance in tcurrent pandemic. Since developing a new treatment takes a significant amount of time, drug repurposing can be an effective option for achieving a rapid response. This study used a combined in silico virtual screening protocol for candidate SARS-CoV-2 PLpro inhibitors. The Drugbank database was searched first, using the Informational Spectrum Method for Small Molecules, followed by molecular docking. Gramicidin D was selected as a peptide drug, showing the best in silico interaction profile with PLpro. After the expression and purification of PLpro, gramicidin D was screened for protease inhibition in vitro and was found to be active against PLpro. The current study’s findings are significant because it is critical to identify COVID-19 therapies that are efficient, affordable, and have a favorable safety profile

Inhibition of SARS-CoV-2 Mpro with Vitamin C, L-Arginine and a Vitamin C/L-Arginine Combination

Drug resistance is a critical problem in health care that affects therapy outcomes and requires new approaches to drug design. SARS-CoV-2 Mpro mutations are of concern as they can potentially reduce therapeutic efficacy. Viral infections are amongst the many disorders for which nutraceuticals have been employed as an adjunct therapy. The aim of this study was to examine the potential in vitro activity of L-arginine and vitamin C against SARS-CoV-2 Mpro. Methods: The Mpro inhibition assay was developed by cloning, expression, purification, and characterization of Mpro. Selected compounds were then screened for protease inhibition. Results: Larginine was found to be active against SARS-CoV-2 Mpro, while a vitamin C/L-arginine combination had a synergistic antiviral action against Mpro. These findings confirm the results of our previous in silico repurposing study that showed L-arginine and vitamin C were potential Mpro inhibitors. Moreover, they suggest a possible molecular mechanism to explain the beneficial effect of arginine in COVID patients. Conclusions: The findings of the current study are important because they help to identify COVID-19 treatments that are efficient, inexpensive, and have a favorable safety profile. The results of this study also suggest a possible adjuvant nutritional strategy for COVID-19 that could be used in conjunction with pharmacological agents

In Silico and In Vitro Inhibition of SARS-CoV-2 PLpro with Gramicidin D

Finding an effective drug to prevent or treat COVID-19 is of utmost importance in tcurrentpandemic. Since developing a new treatment takes a significant amount of time, drug repurposingcan be an effective option for achieving a rapid response. This study used a combined in silico virtualscreening protocol for candidate SARS-CoV-2 PLpro inhibitors. The Drugbank database was searchedfirst, using the Informational Spectrum Method for Small Molecules, followed by molecular docking.Gramicidin D was selected as a peptide drug, showing the best in silico interaction profile with PLpro.After the expression and purification of PLpro, gramicidin D was screened for protease inhibitionin vitro and was found to be active against PLpro. The current study’s findings are significantbecause it is critical to identify COVID-19 therapies that are efficient, affordable, and have a favorablesafety profile.This article belongs to the Special Issue Molecular Interactions and Mechanisms of COVID-19 Inhibition 2.

Inhibition of SARS-CoV-2 Mpro with Vitamin C, L-Arginine and a Vitamin C/L-Arginine Combination

Drug resistance is a critical problem in health care that affects therapy outcomes and requires new approaches to drugdesign. SARS-CoV-2 Mpro mutations are of concern as they can potentially reduce therapeutic efficacy. Viral infections are amongst themany disorders for which nutraceuticals have been employed as an adjunct therapy. The aim of this study was to examine the potentialin vitro activity of L-arginine and vitamin C against SARS-CoV-2 Mpro. Methods: The Mpro inhibition assay was developed by cloning,expression, purification, and characterization of Mpro. Selected compounds were then screened for protease inhibition. Results: Largininewas found to be active against SARS-CoV-2 Mpro, while a vitamin C/L-arginine combination had a synergistic antiviral actionagainst Mpro. These findings confirm the results of our previous in silico repurposing study that showed L-arginine and vitamin C werepotential Mpro inhibitors. Moreover, they suggest a possible molecular mechanism to explain the beneficial effect of arginine in COVIDpatients. Conclusions: The findings of the current study are important because they help to identify COVID-19 treatments that areefficient, inexpensive, and have a favorable safety profile. The results of this study also suggest a possible adjuvant nutritional strategyfor COVID-19 that could be used in conjunction with pharmacological agents

Heterologous Expression and Partial Characterization of a Putative Opine Dehydrogenase from a Metagenomic Sequence of Desulfohalobium retbaense

The aim of this research was to prove the function of the putative opine dehydrogenase from Desulfohalobium retbaense and to characterize the enzyme in terms of functional and kinetic parameters. A putative opine dehydrogenase was identified from a metagenomic library by a sequence-based technique search of the metagenomic library, and afterward was successfully heterologously produced in Escherichia coli. In order to examine its potential for applications in the synthesis of secondary amines, first the substrate specificity of the enzyme towards different amino donors and amino acceptors was determined. The highest affinity was observed towards small amino acids, preferentially L-alanine, and when it comes to α-keto acids, pyruvate proved to be a preferential amino acceptor. The highest activity was observed at pH 6.5 in the absence of salts. The enzyme showed remarkable stability in a wide range of experimental conditions, such as broad pH stability (from 6.0–11.0 after 30 min incubation in buffers at a certain pH), stability in the presence of NaCl up to 3.0 M for 24 h, it retained 80% of the initial activity after 1 h incubation at 45°C, and 65% of the initial activity after 24 h incubation in 30% dimethyl sulfoxide

Immobilization of ArRMut11 omega-transaminase for increased operational stability and reusability in the synthesis of 3α-amino-5α-androstan-17β-ol

The aim of this research was to improve the operational stability and enable the reusability of ω-transaminase for synthesis of new enantiopure chiral amines of steroids. Dihydrotestosterone was used to optimize the synthetic procedure of corresponding amino-steroid on a larger scale. The obtained product 3α-amino-5α-androstan-17β-ol was isolated and characterized. The enzyme was immobilized on a methacrylate-based carrier, giving the specific activity of 1.84 U/g of dry polymer. Higher residual activity of the immobilized enzyme in comparison to the soluble form (100 % versus 35%) after 24 h incubation in 35 % dimethylformamide (DMF) was obtained. The soluble enzyme retained 19 % of the initial activity after 2 h incubation in 35 % DMF at 70 °C, while the activity of the immobilized enzyme decreased only to 75 %. Immobilized retained 85 % of initial activity after ten consecutive cycles of 3α-amino-5α-androstan-17β-ol synthesis. We have tested the specificity of the ArRMut11 variant, further increased its stability by immobilization, and used it in several cycles for the synthesis of 3α-amino-5α-androstan-17β-ol. We showed that the enzyme previously evolved for higher stability as the immobilized variant showed more increased stability and high reusability that can more effectively be applied for the biosynthesis of amino steroids

Immobilization of ArRMut11 omega-transaminase for increased operational stability and reusability in the synthesis of 3α-amino-5α-androstan-17β-ol

The aim of this research was to improve the operational stability and enable the reusability of ω-transaminase for synthesis of new enantiopure chiral amines of steroids. Dihydrotestosterone was used to optimize the synthetic procedure of corresponding amino-steroid on a larger scale. The obtained product 3α-amino-5α-androstan-17β-ol was isolated and characterized. The enzyme was immobilized on a methacrylate-based carrier, giving the specific activity of 1.84 U/g of dry polymer. Higher residual activity of the immobilized enzyme in comparison to the soluble form (100 % versus 35%) after 24 h incubation in 35 % dimethylformamide (DMF) was obtained. The soluble enzyme retained 19 % of the initial activity after 2 h incubation in 35 % DMF at 70 °C, while the activity of the immobilized enzyme decreased only to 75 %. Immobilized retained 85 % of initial activity after ten consecutive cycles of 3α-amino-5α-androstan-17β-ol synthesis. We have tested the specificity of the ArRMut11 variant, further increased its stability by immobilization, and used it in several cycles for the synthesis of 3α-amino-5α-androstan-17β-ol. We showed that the enzyme previously evolved for higher stability as the immobilized variant showed more increased stability and high reusability that can more effectively be applied for the biosynthesis of amino steroids