Directed evolution of stereo‐, regio‐, and chemoselective enzymes constitutes a unique way to generate biocatalysts for synthetically interesting transformations in organic chemistry and ...biotechnology. In order for this protein engineering technique to be efficient, fast, and reliable, and also of relevance to synthetic organic chemistry, methodology development was and still is necessary. Following a description of early key contributions, this review focuses on recent developments. It includes optimization of molecular biological methods for gene mutagenesis and the design of efficient strategies for their application, resulting in notable reduction of the screening effort (bottleneck of directed evolution). When aiming for laboratory evolution of selectivity and activity, second‐generation versions of Combinatorial Active‐Site Saturation Test (CAST) and Iterative Saturation Mutagenesis (ISM), both involving saturation mutagenesis (SM) at sites lining the binding pocket, have emerged as preferred approaches, aided by in silico methods such as machine learning. The recently proposed Focused Rational Iterative Site‐specific Mutagenesis (FRISM) constitutes a fusion of rational design and directed evolution. On‐chip solid‐phase chemical gene synthesis for rapid library construction enhances library quality notably by eliminating undesired amino acid bias, the future of directed evolution?
Take the structure‐guided route: Application of advanced gene mutagenesis methods and strategies ensures speed and reliability in laboratory evolution of selective enzymes as catalysts in organic chemistry and biotechnology. Recent developments in novel methodology from sequence‐based to structure‐guided methods are presented in this Review.
•Occurrence of antibiotics in wastewater and seawater from Antarctic was evaluated.•Quinolones and macrolides were the most found in wastewater from several stations.•Detailed study made to ensure ...quality control of analysis during sample collection and treatment, and shipping of samples.•Bacteria resistance (E. Coli) was observed for several groups of antibiotics.•Periodical monitoring of pharmaceuticals in waters and additional treatments in WWTPs are required.
The potential presence of introduced antibiotics in the aquatic environment is a hot topic of concern, particularly in the Antarctic, a highly vulnerable area protected under the Madrid protocol. The increasing presence of human population, especially during summer, might led to the appearance of pharmaceuticals in wastewater. The previous discovery of Escherichia coli strains resistant to antibiotics in sea water and wastewater collected in King George Island motivated our investigation on antibiotics occurrence in these samples. The application of a multi-residue LCMS/MS method for 20 antibiotics, revealed the presence of 8 compounds in treated wastewater, mainly the quinolones ciprofloxacin and norfloxacin (92% and 54% of the samples analyzed, average concentrations 0.89 μg/L and 0.75 μg/L, respectively) and the macrolides azithromycin and clarithromycin (15% positive samples, and average concentrations near 0.4 μg/L), and erythromycin (38% positive samples, average concentration 0.003 μg/L). Metronidazole and clindamycin were found in one sample, at 0.17 and 0.1 μg/L, respectively; and trimethoprim in two samples, at 0.001 μg/L. Analysis of sea water collected near the outfall of the wastewater discharges also showed the sporadic presence of 3 antibiotics (ciprofloxacin, clindamycin, trimethoprim) at low ng/L level, illustrating the impact of pharmaceuticals consumption and the poor removal of these compounds in conventional WWTPs. The most widespread antibiotic in sea water was ciprofloxacin, which was found in 15 out of 34 sea water samples analyzed, at concentrations ranging from 4 to 218 ng/L. Bacteria resistance was observed for some antibiotics identified in the samples (e.g. trimetropim and nalidixic acid –a first generation quinolone). However, resistance to some groups of antibiotics could not be correlated to their presence in the water samples due to analytical limitations (penicillins, tetraciclines). On the contrary, for some groups of antibiotics detected in samples (macrolides), the antibacterial activity against E. Coli was not investigated because these antibiotics do not include this bacterial species in their spectrum of activity.
Our preliminary data demonstrate that antibiotics occurrence in the Antarctic aquatic environment is an issue that needs to be properly addressed. Periodical monitoring of water samples and the implementation of additional treatments in the WWTPs are recommended as a first step to prevent potential problems related to the presence of antibiotics and other emerging contaminants in the near future in Antarctica.
The goal of xenobiology is to design biological systems endowed with unusual biochemical functions, whereas enzymology concerns the study of enzymes, the workhorses of biocatalysis. Biocatalysis ...employs enzymes and organisms to perform useful biotransformations in synthetic chemistry and biotechnology. During the past few years, the effects of incorporating noncanonical amino acids (ncAAs) into enzymes with potential applications in biocatalysis have been increasingly investigated. In this Review, we provide an overview of the effects of new chemical functionalities that have been introduced into proteins to improve various facets of enzymatic catalysis. We also discuss future research avenues that will complement unnatural mutagenesis with standard protein engineering to produce novel and versatile biocatalysts with applications in synthetic organic chemistry and biotechnology.
Unnatural design: The goal of xenobiology is the design of biological systems endowed with unusual biochemical units. For example, engineering the genetic code of enzymes to contain unnatural amino acids and cofactors can lead to unique properties that make the enzymes useful biocatalysts.
Steroidal C7β alcohols and their respective esters have shown significant promise as neuroprotective and anti‐inflammatory agents to treat chronic neuronal damage like stroke, brain trauma, and ...cerebral ischemia. Since C7 is spatially far away from any functional groups that could direct C−H activation, these transformations are not readily accessible using modern synthetic organic techniques. Reported here are P450‐BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7 regioselectivities and β stereoselectivities, as well as high activities. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil‐Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high‐resolution X‐ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio‐ and stereoselectivity.
One mutant, six targets: The regio‐ and stereoselective C−H activation for hydroxylation of six different steroids with formation of C7β alcohols was accomplished using a single P450 mutant evolved by protein library construction based on a special DNA assembly and cloning procedure. The C7β steroidal alcohols, not readily accessible by synthetic reagents or catalysts, are of intense interest as therapeutic drugs.
Aim
To determine the bacterial microbiota in root canals associated with persistent apical periodontitis and their relationship with the clinical characteristics of patients using next‐generation ...sequencing (NGS).
Methodology
Bacterial samples from root canals associated with teeth having persistent apical periodontitis were taken from 24 patients undergoing root canal retreatment. Bacterial DNA was extracted, and V3–V4 variable regions of the 16S rRNA gene were amplified. The amplification was deep sequenced by Illumina technology to establish the metagenetic relationships among the bacterial species identified. The composition and diversity of microbial communities in the root canal and their relationships with clinical features were analysed. Parametric and nonparametric tests were used to analyse differences between patient characteristics and microbial data.
Results
A total of 86 different operational taxonomic units (OTUs) were identified and Good's nonparametric coverage estimator method indicated that 99.9 ± 0.00001% diversity was recovered per sample. The largest number of bacteria belonged to the phylum Proteobacteria. According to the medical history from the American Society of Anesthesiologists (ASA) Classification System, ASA II–III had higher richness estimates and distinct phylogenetic relationships compared to ASA I individuals (P < 0.05). Periapical index (PAI) score 5 was associated with increased microbiota diversity in comparison to PAI score 4, and this index was reduced in symptomatic patients.
Conclusions
Based on the findings of this study, it is possible to suggest a close relationship between several clinical features and greater microbiota diversity with persistent endodontic infections. This work provides a better understanding on how microbial communities interact with their host and vice versa.
Allosteric information transfer in proteins has been linked to distinct vibrational energy transfer (VET) pathways in a number of theoretical studies. Experimental evidence for such pathways, ...however, is sparse because site‐selective injection of vibrational energy into a protein, that is, localized heating, is required for their investigation. Here, we solved this problem by the site‐specific incorporation of the non‐canonical amino acid β‐(1‐azulenyl)‐l‐alanine (AzAla) through genetic code expansion. As an exception to Kasha's rule, AzAla undergoes ultrafast internal conversion and heating after S1 excitation while upon S2 excitation, it serves as a fluorescent label. We equipped PDZ3, a protein interaction domain of postsynaptic density protein 95, with this ultrafast heater at two distinct positions. We indeed observed VET from the incorporation sites in the protein to a bound peptide ligand on the picosecond timescale by ultrafast IR spectroscopy. This approach based on genetically encoded AzAla paves the way for detailed studies of VET and its role in a wide range of proteins.
The ultrafast vibrational energy injector β‐(1‐azulenyl)‐l‐alanine was genetically encoded and applied in a protein interaction domain (PDZ3 of PSD‐95) to study site‐resolved vibrational energy transfer (VET). VET pathways in proteins had previously been proposed by various theoretical studies to be linked to allosteric communication and information transfer, and can now be mapped experimentally in proteins.
Ascorbic acid (AA) may contribute to restoring hemostatic balance after mental stress (MS) in overweight/obese adults. We aimed to determine the effects of AA administration on hemostatic responses ...to MS in overweight/obese men. Fourteen overweight/obesity men (27 ± 7 years; BMI: 29.7 ± 2.6 kg m
) performed the Stroop color-word stress task for 5 min after non-simultaneous infusion of placebo (PL, 0.9% NaCl) and AA (3 g). Blood was collected at baseline, during MS, and 60 min after MS to measure: activated partial thromboplastin time, prothrombin time, and fibrinogen concentration, by coagulometer; platelet-derived microvesicles (PMV, mv/μL), by flow cytometry; nitrite (μM), by chemiluminescence. In PL session, MS led to decreases in PTs (stress, p = 0.03; 60 min, p < 0.001), PT-INR (stress, p < 0.001; 60 min, p < 0.01), aPTTs (60 min, p = 0.03), aPTT ratio (60 min, p = 0.04) and fibrinogen (60 min, p = 0.04), while increased PT activity (60 min, p = 0.01) when compared to baseline. Furthermore, AA increased PTs (60 min, p < 0.001), PT-INR (60 min, p = 0.03) and decreased PT activity (60 min, p < 0.001) and fibrinogen (stress, p = 0.04) when compared to PL. Nitrite was increased in response to stress during AA session (p < 0.001 vs PL). There was no difference in PMV. Ascorbic acid prevented the impaired hemostatic profile and improved nitrite response to stress in the overweight and obese adults.
Chylous ascites (CA), also called chyloperitoneum, is a rare form of ascites in the neonate. It results from the leakage of lymph into the peritoneal cavity. There are congenital and acquired forms ...of CA. CA may occur during fetal life, and the prognosis will depend on its volume, gestational age at the onset, and the association with other anomalies. Lymphangiectasia is the most common congenital cause, and acquired forms are mainly traumatic and/or post-operative. This review aims to gather the most current information on CA and addresses important aspects regarding etiology, pathophysiology, clinic, diagnostic tools, and treatment.
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