scholarly journals Novel Bifunctional Acylase from Actinoplanes utahensis: A Versatile Enzyme to Synthesize Antimicrobial Compounds and Use in Quorum Quenching Processes

Antibiotics ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 922
Author(s):  
Lara Serrano-Aguirre ◽  
Rodrigo Velasco-Bucheli ◽  
Begoña García-Álvarez ◽  
Ana Saborido ◽  
Miguel Arroyo ◽  
...  
Keyword(s):  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Homoserine Lactones ◽  
Natural Analog ◽  
Acylhomoserine Lactone ◽  
Penicillin V ◽  
Communication Processes ◽  
Hydrolysis Of ◽  
Potential Use

Many intercellular communication processes, known as quorum sensing (QS), are regulated by the autoinducers N-acyl-l-homoserine lactones (AHLs) in Gram-negative bacteria. The inactivation of these QS processes using different quorum quenching (QQ) strategies, such as enzymatic degradation of the autoinducers or the receptor blocking with non-active analogs, could be the basis for the development of new antimicrobials. This study details the heterologous expression, purification, and characterization of a novel N-acylhomoserine lactone acylase from Actinoplanes utahensis NRRL 12052 (AuAHLA), which can hydrolyze different natural penicillins and N-acyl-homoserine lactones (with or without 3-oxo substitution), as well as synthesize them. Kinetic parameters for the hydrolysis of a broad range of substrates have shown that AuAHLA prefers penicillin V, followed by C12-HSL. In addition, AuAHLA inhibits the production of violacein by Chromobacterium violaceum CV026, confirming its potential use as a QQ agent. Noteworthy, AuAHLA is also able to efficiently synthesize penicillin V, besides natural AHLs and phenoxyacetyl-homoserine lactone (POHL), a non-natural analog of AHLs that could be used to block QS receptors and inhibit signal of autoinducers, being the first reported AHL acylase capable of synthesizing AHLs.

scholarly journals Characterization of a Phosphotriesterase-Like Lactonase fromSulfolobus solfataricusand Its Immobilization for Disruption of Quorum Sensing

2010 ◽  
Vol 77 (4) ◽  
pp. 1181-1186 ◽  
Author(s):  
Filomena S. W. Ng ◽  
Daniel M. Wright ◽  
Stephen Y. K. Seah
Keyword(s):  
Quorum Sensing ◽  
Virulence Factors ◽  
Acyl Chain ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Homoserine Lactones ◽  
Specificity Constant ◽  
Chain Lengths ◽  
Quorum Sensing Signals

ABSTRACTSsoPox, a bifunctional enzyme with organophosphate hydrolase andN-acyl homoserine lactonase activities from the hyperthermophilic archaeonSulfolobus solfataricus, was overexpressed and purified from recombinantPseudomonas putidaKT2440 with a yield of 9.4 mg of protein per liter of culture. The enzyme has a preference forN-acyl homoserine lactones (AHLs) with acyl chain lengths of at least 8 carbon atoms, mainly due to lowerKmvalues for these substrates. The highest specificity constant obtained was forN-3-oxo-decanoyl homoserine lactone (kcat/Km= 5.5 × 103M−1·s−1), but SsoPox can also degradeN-butyryl homoserine lactone (C4-HSL) andN-oxo-dodecanoyl homoserine lactone (oxo-C12-HSL), which are important for quorum sensing in ourPseudomonas aeruginosamodel system. WhenP. aeruginosaPAO1 cultures were grown in the presence of SsoPox-immobilized membranes, the production of C4-HSL- and oxo-C12-HSL-regulated virulence factors, elastase, protease, and pyocyanin were significantly reduced. This is the first demonstration that immobilized quorum-quenching enzymes can be used to attenuate the production of virulence factors controlled by quorum-sensing signals.

scholarly journals EVALUATION OF ALKALINE PRETREATMENT ON THE ENZYMATIC HYDROLYSIS OF CARNAUBA STRAW RESIDUE

2019 ◽  
Vol 8 (3) ◽  
Author(s):  
Alan De Oliveira Campos ◽  
Francinaldo Leite Da Silva ◽  
Emilianny Rafaely Batista Magalhães ◽  
Gorete Ribeiro De Macedo ◽  
Everaldo Silvino Dos Santos
Keyword(s):  
Enzymatic Hydrolysis ◽  
Cellulosic Ethanol ◽  
Chemical Characterization ◽  
Crystallinity Index ◽  
Biomass Pretreatment ◽  
Cellulose Conversion ◽  
Hydrolysis Of ◽  
Potential Use ◽  
Second Generation Ethanol

Carnauba (Copernicia prunifera) straw residue generated from production of its wax is rich in cellulose, thus showing a potential use in the production of second generation ethanol. However, the high lignin and hemicellulose load associated with cellulose makes it difficult the enzymatic attack, thus having the need of an adequate pretreatment of this material. The objective of this study was to optimize the enzymatic hydrolysis of carnauba straw residue, focusing on the alkaline biomass pretreatment. Therefore, NaOH solutions at concentrations of 1.0% (w/v) (PA1), 2.0% (w/v) (PA2), 3.0% (w/v) (PA3) and 4.0% (w/v) (PA4) were used. The chemical and physical characterization of natural and pre-treated carnauba straw were according to the NREL, and DRX and FTIR performed analyzes. The materials chemical characterization showed that all the used pretreatments were able to remove a significant amount of lignin and hemicellulose, which can improve the enzymes access, favoring the increase of cellulose conversion. In relation to DRX analysis an increase in crystallinity index happens reaching up to 55.15% after the pretreatment PA4, which may be associated to the removal of hemicellulose and amorphous lignin, related to cellulose. After a period of 96 hours of enzymatic hydrolysis, the PA4 pretreated residue showed the best performance with a cellulosic conversion of 78%. Spite of a slightly lower performance of the residue that presented higher cellulose conversion, the pretreated material PA2 is an alternative to reduce costs in the cellulosic ethanol production.

scholarly journals Fast, Continuous, and High-Throughput (Bio)Chemical Activity Assay forN-Acyl-l-Homoserine Lactone Quorum-Quenching Enzymes

2016 ◽  
Vol 82 (14) ◽  
pp. 4145-4154 ◽  
Author(s):  
Daniel Last ◽  
Georg H. E. Krüger ◽  
Mark Dörr ◽  
Uwe T. Bornscheuer
Keyword(s):  
High Throughput ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Chemical Activity ◽  
Reaction Products ◽  
Activity Assay ◽  
Gram Negative ◽  
Content Type ◽  
Homoserine Lactones ◽  
Activity Measurements

ABSTRACTQuorum sensing, the bacterial cell-cell communication by small molecules, controls important processes such as infection and biofilm formation. Therefore, it is a promising target with several therapeutic and technical applications besides its significant ecological relevance. Enzymes inactivatingN-acyl-l-homoserine lactones, the most common class of communication molecules among Gram-negative proteobacteria, mainly belong to the groups of quorum-quenching lactonases or quorum-quenching acylases. However, identification, characterization, and optimization of these valuable biocatalysts are based on a very limited number of fundamentally different methods with their respective strengths and weaknesses. Here, a (bio)chemical activity assay is described, which perfectly complements the other methods in this field. It enables continuous and high-throughput activity measurements of purified and unpurified quorum-quenching enzymes within several minutes. For this, the reaction products released by quorum-quenching lactonases and quorum-quenching acylases are converted either by a secondary enzyme or by autohydrolysis tol-homoserine. In turn,l-homoserine is detected by the previously described calcein assay, which is sensitive to α-amino acids with free N and C termini. Besides its establishment, the method was applied to the characterization of three previously undescribed quorum-quenching lactonases and variants thereof and to the identification of quorum-quenching acylase-expressingEscherichia coliclones in an artificial library. Furthermore, this study indicates that porcine aminoacylase 1 is not active towardN-acyl-l-homoserine lactones as published previously but instead converts the autohydrolysis productN-acyl-l-homoserine.IMPORTANCEIn this study, a novel method is presented for the identification, characterization, and optimization of quorum-quenching enzymes that are active towardN-acyl-l-homoserine lactones. These are the most common communication molecules among Gram-negative proteobacteria. The activity assay is a highly valuable complement to the available analytical tools in this field. It will facilitate studies on the environmental impact of quorum-quenching enzymes and contribute to the development of therapeutic and technical applications of this promising enzyme class.

N-Acylhomoserine lactone quorum-sensing signalling in antagonistic phenazine-producing Pseudomonas isolates from the red cocoyam rhizosphere

Microbiology ◽  
2011 ◽  
Vol 157 (2) ◽  
pp. 459-472 ◽  
Author(s):  
K. De Maeyer ◽  
J. D'aes ◽  
G. K. H. Hua ◽  
M. Perneel ◽  
L. Vanhaecke ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Regulatory System ◽  
Homoserine Lactone ◽  
Antagonistic Activity ◽  
Signal Molecules ◽  
Pseudomonas Chlororaphis ◽  
Homoserine Lactones ◽  
Acylhomoserine Lactone ◽  
Phenazine Production ◽  
Phenazine Antibiotics

Forty fluorescent Pseudomonas strains isolated from white and red cocoyam roots were tested for their ability to synthesize N-acyl-l-homoserine lactones (acyl-HSLs). Remarkably, only isolates from the red cocoyam rhizosphere that were antagonistic against the cocoyam root rot pathogen Pythium myriotylum and synthesized phenazine antibiotics produced acyl-HSLs. This supports the assumption that acyl-HSL production is related to the antagonistic activity of the strains. After detection, the signal molecules were identified through TLC-overlay and liquid chromatography-multiple MS (LC-MS/MS) analysis. In our representative strain, Pseudomonas CMR12a, production of the signal molecules could be assigned to two quorum-sensing (QS) systems. The first one is the QS system for phenazine production, PhzI/PhzR, which seemed to be well conserved, since it was genetically organized in the same way as in the well-described phenazine-producing Pseudomonas strains Pseudomonas fluorescens 2-79, Pseudomonas chlororaphis PCL1391 and Pseudomonas aureofaciens 30-84. The newly characterized genes cmrI and cmrR make up the second QS system of CMR12a, under the control of the uncommon N-3-hydroxy-dodecanoyl-homoserine lactone (3-OH-C12-HSL) and with low similarity to other Pseudomonas QS systems. No clear function could yet be assigned to the CmrI/CmrR system, although it contributes to the biocontrol capability of CMR12a. Both the PhzI/PhzR and CmrI/CmrR systems are controlled by the GacS/GacA two-component regulatory system.

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