Genetic code reprogramming allows for ribosomal incorporation of exotic amino acids, such as d-α- and d-β-amino acids, into peptides. However, their incorporation efficiency remains much lower than that of canonical l-amino acids, making their multiple/consecutive incorporations difficult. The side chain of d-α-amino acids clashes with U2506 of 23S rRNA, hindering the incorporation of d-α-amino acids with bulky side chains; therefore, consecutive incorporation has been limited to small d-α-amino acids. To overcome this limitation, we screened ribosomes from phylogenetically diverse bacterial species to identify variants that enable consecutive incorporation of bulky d-amino acids. The Alteromonas macleodii (AM) ribosome is capable of incorporating d-α-, d-β-, and α,α-disubstituted amino acids with efficiencies superior to the Escherichia coli (EC) ribosome. It elongates six consecutive d-Ser and two consecutive 1-aminocyclobutane-1-carboxylic acid with yields 6.1- and 7.3-fold higher than the EC ribosome. Moreover, consecutive incorporation of nine types of bulky d-α-amino acids (d-Asn, d-Asp, d-Gln, d-Met, d-Phe, d-Thr, d-Trp, d-Tyr, d-Val) was achieved for the first time. A model macrocyclic peptide containing four d-amino acids and one α,α-disubstituted amino acid was also synthesized by the AM ribosome. These results expand the potential of ribosomal synthesis for peptide libraries containing structurally diverse, bulky d-amino acids.

The intestine is considered a habitat for both bacteria and parasites. In this study, many fecal bacterial isolates and the protozoan Blastocystis sp. were recovered from stool samples of individuals with gastrointestinal conditions. Isolated bacteria were tested for extracellular protease production, and the most potent producer was identified by 16SrDNA gene sequencing as P. aeruginosa FZM498. The enzyme was extracted and purified to electrophoretic homogeneity by the DEAE-Sepharose ion-exchanger and SDS-PAGE revealed a major band at 42.15 KDa. It exhibited maximal activity at 35 °C with thermostability at 60 °C (T1/2 = 200.04 min). It was most active at pH 8.0 and stable at 5.0-9.5. Enzymatic activity was greatly stimulated in the presence of Fe[2+] ions, but was repressed by Zn[2+] and Hg[2+] ions. Inhibition by PMSF, TLCK, aprotinin, benzamidine, and SBTI protease reagents suggests a serine protease family. The Vmax and Km dynamic constants against azocasein were 36.232 U/mL and 0.0072 mM, respectively. It exhibited the lowest Km value against the synthetic substrate D-Val-Leu-Lys-pNA among all substrates, indicating a plasmin-like activity. Interestingly, when tested against Blastocystis sp., cysts appeared progressively shrunken, ruptured, and mycelial-like, indicating complete structural collapse with leakage of intracellular contents. The importance of this research is that it is the first study to test the anti-Blastocystis activity of an extracted bacterial serine protease from the gut. This could be a promising, eco-friendly, natural alternative as an anti-Blastocystis agent. The objective of this study was to isolate, purify, and biochemically characterize an extracellular serine protease produced by gut-associated bacteria, as well as to assess its in vitro anti-Blastocystis efficacy as a potential natural and ecologically friendly antiparasitic therapy.

In this work, a dual-parameter quantification protocol was successfully developed for determining the enzymatic activity of human urinary kallidinogenase (KN) using reversed-phase high-performance liquid chromatography (RP-HPLC). KN serves as a hydrolytic enzyme to cleave the substrate S-2266 (H-D-Val-Leu-Arg-pNA). Effective separation of the product, substrate, and KN was achieved under optimized chromatographic conditions. By minimizing matrix interference through HPLC separation and using multi-wavelength detection, both selectivity and sensitivity were significantly improved. Moreover, the dual-parameter quantification method, which simultaneously measured the formation of reaction products and the consumption of substrates to reflect KN enzyme activity, markedly improved the reliability of the determination. This method was successfully applied to the activity assay of actual KN samples. The established method has been demonstrated to be effective and reliable with comparable greenness. It provides a powerful tool for the quantitative monitoring of KN activity in biological products and paves the way for its application in complex biological matrices.

An ultra-high-performance liquid chromatography with fluorescence detection (UPLC-FL) method was developed and applied for the quantitative analysis of D-alanine (D-Ala) and L-alanine (L-Ala), D-leucine (D-Leu) and L-leucine (L-Leu), and D-valine (D-Val) and L-valine (L-Val) in human saliva. Enantiomers were derivatized using the fluorescent reagent R-(-)-4-(3-isothiocyanatepyrrolidine-l-methyl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R-(-)-DBD-PyNCS] at 55°C in the presence of 3% triethylamine for 20 min, yielding aminothiocarbonyl diastereomeric derivatives. The derivatized products were detected using UPLC-FL at an excitation wavelength (Ex) of 460 nm and a fluorescence wavelength (Em) of 550 nm. The limit of detection was 0.010-0.200 μM, and the resolution (Rs) > 1.5 within 20 min. The R[2] of D-type amino acids in the range of 0.5-50 μM and L-type amino acids in the range of 1.0-500 μM was greater than or equal to 0.9988. Intraday and interday assay precisions were below 6.94%, with average recoveries ranging from 95.20% to 109.6%. This method was succeeded in quantifying amino acid enantiomers in the saliva of healthy volunteers and patients with diabetes. Significant differences in D-Ala, L-Ala, L-Leu, D-Val, and L-Val levels were observed between the two groups (p < 0.01). The proposed method holds potential for diagnosing diabetes and may assist as an early warning indicator of diabetes.

d-Amino acids (d-AAs) have wide applications in industries such as pharmaceutical, food, and cosmetics due to their unique properties. Currently, the production of d-AAs has relied on chemical synthesis or enzyme catalysts, and it is challenging to produce d-AAs via direct fermentation from glucose. We observed that Corynebacterium glutamicum exhibits a remarkable tolerance to high concentrations of d-Ala, a crucial characteristic for establishing a successful fermentation process. By optimizing meso-diaminopilmelate dehydrogenases in different C. glutamicum strains and successively deleting l-Ala biosynthetic pathways, we developed an efficient d-Ala fermentation system. The d-Ala titer was enhanced through systems metabolic engineering, which involved strengthening glucose assimilation and pyruvate supply, reducing the formation of organic acid byproducts, and attenuating the TCA cycle. During fermentation in a 5-L bioreactor, a significant accumulation of l-Ala was observed in the broth, which was subsequently diminished by introducing an l-amino acid deaminase. Ultimately, the engineered strain DA-11 produced 85 g/L d-Ala with a yield of 0.30 g/g glucose, accompanied by an optical purity exceeding 99%. The fermentation platform has the potential to be extended for the synthesis of other d-AAs, as demonstrated by the production of d-Val and d-Glu.

The Phe-incorporated cyclic peptide [cyclo(-Phe1-oxazoline2-d-Val3-thiazole4-Ile5-oxazoline6-d-Val7-thiazole8-)] is in a conformational equilibrium between square and folded forms in solution. In the folded form, a CH⋯π interaction between the Phe1 aromatic ring and the Oxz2 methyl group is observed. We endeavored to control the local conformation and thus modulate the CH⋯π interaction and flexibility of the Phe1 side chain by controlling the electronic substituent effects at the 4-position of the aromatic ring of the Phe1 residue. The effect of the 4-substituent on the global conformation was indicated by the linear relationship between the conformational free energies (ΔGo) determined through NMR-based quantification and the Hammett constants (σ). Electron-donating substituents, which had relatively strong CH⋯π interactions, promoted peptide folding by restraining the loss in entropy. Local control by the 4-substituent effects suggested that the Phe side chain exerts an entropic influence on the folding of these cyclic peptides.

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.

Two kahalalide analogues were isolated from a Bryopsis sp. marine green alga. Even though our initial structure determination of the peptides by NMR and MS identified them as kahalalide Z1 (KZ1; 3) and Z2 (KZ2; 4), the absolute configuration of the Thr residues by Marfey's analysis was different from those found in kahalalide F (KF), 3, and 4. To ascertain the absolute configuration of the amino acid residues genetically, we conducted a metagenomic analysis for symbiotic bacteria in the alga, leading to the biosynthetic gene cluster (BGC) responsible for producing the kahalalides named kahalalides Z3 (KZ3; 1) and Z4 (KZ4; 2). The identification of amino acid residues based on the A-domain suggested these peptides possess the amino acid sequence d-allo-Thr-l-Val-l-Val-d-Val residues at the N-terminus, instead of the d-Val-l-Thr-l-Val-d-Val residues found in KF, 3, and 4. The N-terminal amino acid sequence including absolute configuration was unambiguously determined by a comparison of LCMS data of synthetic tetrapeptides and the hydrolysates derived from 1 and 2. This structural difference is caused by swapping the substrate specificities of the first two A-domains.

In this work, 1,8-naphthalimide (NMI)-conjugated three hybrid dipeptides constituted of a β-amino acid and an α-amino acid have been designed, synthesized, and purified. Here, in the design, the chirality of the α-amino acid was varied to study the effect of molecular chirality on the supramolecular assembly. Self-assembly and gelation of three NMI conjugates were studied in mixed solvent systems [water and dimethyl sulphoxide (DMSO)]. Interestingly, chiral NMI derivatives [NMI-βAla-[l]Val-OMe (NLV) and NMI-βAla-[d]Val-OMe (NDV)] formed self-supported gels, while the achiral NMI derivative [NMI-βAla-Aib-OMe, (NAA)] failed to form any kind of gel at 1 mM concentration and in a mixed solvent (70% water in DMSO medium). Self-assembly processes were thoroughly investigated using UV-vis spectroscopy, nuclear magnetic resonance (NMR), fluorescence, and circular dichroism (CD) spectroscopy. A J-type molecular assembly was observed in the mixed solvent system. The CD study indicated the formation of chiral assembled structures for NLV and NDV, which were mirror images of one another, and the self-assembled state by NAA was CD-silent. The nanoscale morphology of the three derivatives was studied using scanning electron microscopy (SEM). In the case of NLV and NDV, left- and right-handed fibrilar morphologies were observed, respectively. In contrast, a flake-like morphology was noticed for NAA. The DFT study indicated that the chirality of the α-amino acid influenced the orientation of π-π stacking interactions of naphthalimide units in the self-assembled structure that in turn regulated the helicity. This is a unique work where molecular chirality controls the nanoscale assembly as well as the macroscopic self-assembled state.

Ascidiacyclamide [cyclo(-Ile-oxazoline-D-Val-thiazole-)2] is a cytotoxic cyclic peptide from ascidian. We examined the potential of the CH⋯π interaction at the diagonal position of ascidiacyclamide by comparing the interactions of Ile, Val, Abu (2-aminobutyric acid) or Ala with Ile, Chg (cyclohexylglycine) or Phg (phenylglycine). In solution, ascidiacyclamides are in a conformational equilibrium between square and folded forms. The CH⋯π interaction is expected to contribute to stabilization of the square form, which enhances the peptides' cytotoxicity. The distances between the alkyl side chain of Xaa and the π-plane of Phg were estimated from the crystal structures. The conformational free energies (ΔG°) determined through NMR-based quantitation indicated remarkable stabilization of the square form upon incorporation of Phg. These observations were consistent with the circular dichroism (CD) spectral measurements. Chemical shift perturbation studies suggested that stabilization of the square form of Phg-incorporated peptides was due to the CH⋯π interaction with the alkyl side chain of Xaa. Greater enthalpic losses were caused during the folding process of Phg-incorporated peptides than Ile- or Chg-incorporated peptides. It is suggested that these enthalpic losses are relevant to the CH⋯π interaction energies, which must be disrupted during folding. In addition, the CH⋯π interactions in the Phg-incorporated peptides increased cytotoxicity.

The functionalization of materials for ultrasensitive detection of heavy metal ions (HMIs) in the environment is crucial. Herewith, we have functionalized inexpensive and environmentally friendly Fe3O4 nanoparticles with D-valine (Fe3O4-D-Val) by a simple co-precipitation synthetic approach characterized by XRD, FE-SEM, and FTIR spectroscopy. The Fe3O4-D-Val sensor was used for the ultrasensitive detection of Cd+2, Pb+2, and Cu+2 in water samples. This sensor shows a very low detection limit of 11.29, 4.59, and 20.07 nM for Cd+2, Pb+2, and Cu+2, respectively. The detection limits are much lower than the values suggested by the world health Organization. The real water samples were also analyzed using the developed sensor.