In this study, a rapid, inexpensive, and accurate colorimetric sensor for detecting psychrophilic bacteria was designed, comprising gold (Au) nanoparticles (NPs) modified by d-amino acid (D-AA) as color-metric probes. Based on the aggregation of Au NPs induced by psychrophilic bacteria, a noticeable color shift occurred within 6 h. Depending on the various metabolic behaviors of bacteria to different D-AA, four primary psychrophilic bacteria in raw milk were successfully distinguished by learning the response patterns. Furthermore, the quantification of single bacteria and the practical application in milk samples could be realized. Notably, a rapid colorimetric method was constructed by combining Au/D-AA with antibiotics for the minimum inhibitory concentration of psychrophilic bacteria, which relied on differences in bacteria metabolic activity in response to diverse antibiotic treatments. Therefore, the method enables the rapid detection and susceptibility evaluation of psychrophilic bacteria, promoting clinical practicability and antibiotic management.

Pseudomonas aeruginosa is an opportunistic gram-negative pathogenic microorganism that poses a significant challenge in clinical treatment. Antibiotics exhibit limited efficacy against mature biofilm, culminating in an increase in the number of antibiotic-resistant strains. Therefore, novel strategies are essential to enhance the effectiveness of antibiotics against Pseudomonas aeruginosa biofilms. D-histidine has been previously identified as a prospective anti-biofilm agent. However, limited attention has been directed towards its impact on Pseudomonas aeruginosa. Therefore, this study was undertaken to explore the effect of D-histidine on Pseudomonas aeruginosa in vitro. Our results demonstrated that D-histidine downregulated the mRNA expression of virulence and quorum sensing (QS)-associated genes in Pseudomonas aeruginosa PAO1 without affecting bacterial growth. Swarming and swimming motility tests revealed that D-histidine significantly reduced the motility and pathogenicity of PAO1. Moreover, crystal violet staining and confocal laser scanning microscopy demonstrated that D-histidine inhibited biofilm formation and triggered the disassembly of mature biofilms. Notably, D-histidine increased the susceptibility of PAO1 to amikacin compared to that in the amikacin-alone group. These findings underscore the efficacy of D-histidine in combating Pseudomonas aeruginosa by reducing biofilm formation and increasing biofilm disassembly. Moreover, the combination of amikacin and D-histidine induced a synergistic effect against Pseudomonas aeruginosa biofilms, suggesting the potential utility of D-histidine as a preventive strategy against biofilm-associated infections caused by Pseudomonas aeruginosa.

Microbiologically influenced corrosion (MIC) caused by sulfate reducing bacteria (SRB) is a serious challenge in many industries, but biofilm greatly decreases the toxicity of bactericides to cell inside. d-amino acids are potential enhancers for bactericides due to their excellent performance on biofilm inhibition. However, the mechanism of d-amino acid cooperating with bactericides for MIC inhibition is still unknown. In this study, d-tyrosine（D-Tyr）and disoctyl dimethyl ammonium chloride (DDAC) were selected as the typical d-amino acid and bactericide, respectively, to evaluate their synergetic inhibition on the corrosion caused by Desulfovibrio vulgaris. D-Tyr obviously enhanced the role of DDAC in inhibiting corrosion with high corrosion inhibition efficiency at 77.23 %. The attachment of EPS and live cells on the coupon surface decreased in the presence of D-Try, leading to more cells directly exposed to DDAC. Besides, D-Try decreased the amount of live cells on the surface and thus reduced the utilization of Fe by SRB and corrosion current. Moreover, dead cells settling to the coupon surface may form a protective lay to retard the contact between live SRB and Fe, leading to slow cathode reaction and less corrosion. Therefore, D-Tyr can reduce the coverage of biofilm, thereby reducing its protective effect on SRB and achieving better corrosion inhibition effect. This work provides a new strategy for improving bactericides and inhibiting MIC.

Enzymes with expanded substrate specificity are good starting points for the design of biocatalysts for target reactions. However, the structural basis of the expanded substrate specificity is still elusive, especially in the superfamily of pyridoxal-5\'-phosphate-dependent transaminases, which are characterized by a conserved organization of both the active site and functional dimer. Here, we analyze the structure-function relationships in a non-canonical D-amino acid transaminase from Blastococcus saxobsidens, which is active towards D-amino acids and primary (R)-amines. A detailed study of the enzyme includes a kinetic analysis of its substrate scope and a structural analysis of the holoenzyme and its complex with phenylhydrazine-a reversible inhibitor and analogue of (R)-1-phenylethylamine-a benchmark substrate of (R)-selective amine transaminases. We suggest that the features of the active site of transaminase from B. saxobsidens, such as the flexibility of the R34 and R96 residues, the lack of bulky residues in the β-turn at the entrance to the active site, and the short O-pocket loop, facilitate the binding of substrates with and without α-carboxylate groups. The proposed structural determinants of the expanded substrate specificity can be used for the design of transaminases for the stereoselective amination of keto compounds.

Since the identified standard genetic code contains 61 triplet codons of three bases for the 20 L-proteinogenic amino acids (AAs), no D-AA should be found in natural products. This is not what is observed in the living world. D-AAs are found in numerous natural compounds produced by bacteria, algae, fungi, or marine animals, and even vertebrates. A review of the literature indicated the existence of at least 132 peptide natural compounds in which D-AAs are an essential part of their structure. All compounds are listed, numbered and described herein. The two biosynthetic routes leading to the presence of D-AA in natural products are: non-ribosomal peptide synthesis (NRPS), and ribosomally synthesized and post-translationally modified peptide (RiPP) synthesis which are described. The methods used to identify the AA chirality within naturally occurring peptides are briefly discussed. The biological activity of an all-L synthetic peptide is most often completely different from that of the D-containing natural compounds. Analyzing the selected natural compounds showed that D-Ala, D-Val, D-Leu and D-Ser are the most commonly encountered D-AAs closely followed by the non-proteinogenic D-allo-Thr. D-Lys and D-Met were the least prevalent D-AAs in naturally occurring compounds.

The D-amino acid content of Ishizuchi-kurocha, a post-fermented tea produced in Ehime, Japan, was measured. Ishizuchi-kurocha mainly contains D-glutamic acid and D-alanine, but it also contains a small amount of D-aspartic acid. Two types of lactic acid bacteria, Lactiplantibacillus plantarum and Levilactobacillus brevis, are the main species involved in lactic acid fermentation during the tea fermentation process. Therefore, the D-amino acid-producing abilities of strains of these two species isolated from Ishizuchi-kurocha were examined. Specifically, the production of D-aspartic acid, D-alanine, and D-glutamic acid by L. brevis and L. plantarum strains was observed. The amount of D-aspartic acid produced by L. plantarum was low. D-glutamine was detected in culture supernatant but not in bacterial cells. D-arginine was detected in bacterial cells of the L. plantarum strains but not in the culture supernatant. Both the L. brevis and L. plantarum strains possessed at least three kinds of putative racemase genes: alanine racemase, glutamate racemase, and aspartate racemase. However, their expression and enzyme activity remain unknown. L. plantarum and L. brevis could play an important role in the production of D-amino acids in Ishizuchi-kurocha. In fact, Ishizuchi-kurocha is expected to possess the effective physiological activities of D-amino acids.

D-amino acids, the chiral counterparts of protein L-amino acids, were primarily produced and utilized by microbes, including those in the human gut. However, little was known about how orally administered or microbe-derived D-amino acids affected the gut microbial community or gut disease progression.
The ratio of D- to L-amino acids was analyzed in feces and blood from patients with ulcerative colitis (UC) and healthy controls. Also, composition of microbe was analyzed from patients with UC. Mice were treated with D-amino acid in dextran sulfate sodium colitis model and liver cholangitis model.
The ratio of D- to L-amino acids was lower in the feces of patients with UC than that of healthy controls. Supplementation of D-amino acids ameliorated UC-related experimental colitis and liver cholangitis by inhibiting growth of Proteobacteria. Addition of D-alanine, a major building block for bacterial cell wall formation, to culture medium inhibited expression of the ftsZ gene required for cell fission in the Proteobacteria Escherichia coli and Klebsiella pneumoniae, thereby inhibiting growth. Overexpression of ftsZ restored growth of E. coli even when D-alanine was present. We found that D-alanine not only inhibited invasion of pathological K. pneumoniae into the host via pore formation in intestinal epithelial cells but also inhibited growth of E. coli and generation of antibiotic-resistant strains.
D-amino acids might have potential for use in novel therapeutic approaches targeting Proteobacteria-associated dysbiosis and antibiotic-resistant bacterial diseases by means of their effects on the intestinal microbiota community.

Peptides have unique characteristics that make them highly desirable as therapeutic agents. The physicochemical and proteolytic stability profiles determine the therapeutic potential of peptides. Multiple strategies to enhance the therapeutic profile of peptides have emerged. They include chemical modifications, such as cyclization, substitution with d-amino acids, peptoid formation, N-methylation, and side-chain halogenation, and incorporation in delivery systems. There have been recent advances in approaches to discover peptides having these modifications to attain desirable therapeutic properties. We critically review these recent advancements in therapeutic peptide development.

The in silico prediction of T cell epitopes within any peptide or biologic drug candidate serves as an important first step for assessing immunogenicity. T cell epitopes bind human leukocyte antigen (HLA) by a well-characterized interaction of amino acid side chains and pockets in the HLA molecule binding groove. Immunoinformatics tools, such as the EpiMatrix algorithm, have been developed to screen natural amino acid sequences for peptides that will bind HLA. In addition to commonly occurring in synthetic peptide impurities, unnatural amino acids (UAA) are also often incorporated into novel peptide therapeutics to improve properties of the drug product. To date, the HLA binding properties of peptides containing UAA are not accurately estimated by most algorithms. Both scenarios warrant the need for enhanced predictive tools. The authors developed an in silico method for modeling the impact of a given UAA on a peptide\'s likelihood of binding to HLA and, by extension, its immunogenic potential. In silico assessment of immunogenic potential allows for risk-based selection of best candidate peptides in further confirmatory in vitro, ex vivo and in vivo assays, thereby reducing the overall cost of immunogenicity evaluation. Examples demonstrating in silico immunogenicity prediction for product impurities that are commonly found in formulations of the generic peptides teriparatide and semaglutide are provided. Next, this article discusses how HLA binding studies can be used to estimate the binding potentials of commonly encountered UAA and \"correct\" in silico estimates of binding based on their naturally occurring counterparts. As demonstrated here, these in vitro binding studies are usually performed with known ligands which have been modified to contain UAA in HLA anchor positions. An example using D-amino acids in relative binding position 1 (P1) of the PADRE peptide is presented. As more HLA binding data become available, new predictive models allowing for the direct estimation of HLA binding for peptides containing UAA can be established.

The yeast Vanrija (previously Cryptococcus) humicola strain UJ1 produces d-aspartate oxidase (DDO) only in the presence of d-aspartate in culture media. This article provides RNA-sequencing data to identify the differentially expressed genes (DEGs) in the yeast cells grown between l- and d-aspartate. RNA samples were prepared from the yeast cells grown in a culture medium containing 30 mM d-aspartate or l-aspartate as the sole carbon source and subjected to RNA sequencing on Illumina NovaSeq6000 platform. The clean reads obtained by removing adaptor sequences and low-quality reads from raw reads were submitted to the Sequence Read Archive (SRA) database of the National Center for Biotechnology Information (NCBI) under the BioProject accession number PRJDB13570. The clean reads were subjected to differential gene expression analysis using DEGSeq to provide data on the upregulated and downregulated DEGs in the cells grown on d-aspartate. The DEGs were subjected to gene ontology (GO) and KEGG pathway enrichment analyses using GOSeq and KOBAS, respectively, to provide data on the possible biological functions of the DEGs. The data set obtained in this project might be helpful for further investigation of the effects of d-aspartate on cellular processes in yeast cells and other eukaryotic organisms.