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Promiscuous enzymes can be modified by protein engineering, which enables the catalysis of non-native substrates. γ-lactamase Sspg from Sulfolobus solfataricus is an enzyme with high activity, high stability, and pronounced tolerance of high concentrations of the γ-lactam substrate. These characteristics suggest Sspg as a robust enzymatic catalyst for the preparation of optically pure γ-lactam. This study investigated the modification of this enzyme to expand its application toward resolving chiral esters. γ-Lactamase-esterase conversion was performed by employing a three-step method: initial sequence alignment, followed by substrate screening, and protein engineering based on the obtained substrate-enzyme docking results. This process of fine-tuning of chemical groups on substrates has been termed \"substrate screening.\" Steric hindrance and chemical reactivity of the substrate are major concerns during this step, since both are determining factors for the enzyme-substrate interaction. By employing this three-step method, γ-lactamase Sspg was successfully converted into an esterase with high enantioselectivity towards phenylglycidate substrates (E value > 300). However, since both wild-type Sspg and Sspg mutants did not hydrolyze para-nitrophenyl substrates (pNPs), this esterase activity was termed \"atypical esterase activity.\" The γ-lactamase activity and stability of the Sspg mutants were not severely compromised. The proposed method can be applied to find novel multi-functional enzyme catalysts within existing enzyme pools.

In this paper, the combined effect of electronic and substrate screening on impurity states in inversion layers is investigated theoretically. An explicit expression of the screened impurity interaction potential with an effective screening parameter, depending on the material and structural parameters, is established analytically for the first time. The main physical results are (a) an enhancement of the carrier saturation effect and (b) the dependence of the nature of the screening mechanism on the dielectric type (low-κ and high-κ) of the oxide layer. An experimentally measurable impurity binding energy is studied and numerically presented for realistic InSb/SiO2/SiO2/metal (ll-) and InSb/S(sulfur)/HfO2/metal (lh-κ type) multi-layer structures. A substantial enhancement of the binding energy is obtained with the non-degenerate Q2D EG for the ll-κ-type structure, reaching an almost fourfold value of the InSb bulk sample (~0.66 meV).

Rhamnolipids (RLs) are one of the most promising eco-friendly green alternatives to commercially viable fossil fuel-based surfactants. However, the current bioprocess practices cannot meet the required affordability, quantity, and biocompatibility within an industrially relevant framework. To circumvent these issues, our study aims to develop a sustainable biorefinery approach using post-consumption food waste as a second-generation feedstock. In-depth substrate screening revealed that food waste hydrolysate (FWH) was rich in readily assimilable carbohydrates, volatile fatty acids, and amino acids. The fermentative valorization of FWH as a sole carbon and energy source with Burkholderis thailandensis E264 in a bioreactor showed active RLs biosynthesis of up to 0.6–0.8 g/L (34–40 mg/g FWH) in a short duration (72 h). In terms of the kinetic parameters, the FWH-RLs outperformed other supplemented pure/waste streams. Interestingly, the recovered RLs had a long chain length, with Rha-Rha-C12-C14 being the predominant isoform and exhibiting a strong emulsification ability (E24, 54.6%). To the best of our knowledge, this study is the first to prove bioreactor-level RLs production and their abundance in food waste. Moreover, the feasibility of this developed process could propel next-generation biosurfactants, lower waste burdens, and increase the industrial applicability of RLs, thereby significantly contributing to the development of a circular bioeconomy.

The objective of this study was to establish a fast approach (< 1 h) for the evaluation of technical enzyme preparations (TEPs). An automated photometric analyzer (GalleryTM Plus) was equipped with 32 synthetic and natural substrates to measure aminopeptidase, carboxypeptidase, dipeptidyl peptidase and endopeptidase activities distinguishably and the proteolytic activity towards lupine protein of TEPs. The established so-called “activity fingerprints” (AFPs) delivered detailed information about the substrate spectra and peptidase side activities, noticing furthermore batch variations of Flavourzyme1000L. Based on their AFPs, particular TEPs were selected for lupine protein hydrolysis and the hydrolysates were analyzed regarding the degree of hydrolysis and the free amino acids. It was demonstrated that the information of the AFPs were applicable to predict important properties of the resulting hydrolysates. Consequently, the hydrolysis efficiency was improved (increase of 47%). The system introduced enables the targeted selection of TEPs for enzymatic protein hydrolysis, resulting in specific food protein hydrolysates.

Hydrogels are intensively investigated biomaterials due to their useful physicochemical and biological properties in bioengineering. In particular, naturally occurring hydrogels are being deployed as carriers for bio-compounds. We used two approaches to develop a plate colourimetric test by immobilising (1) ABTS or (2) laccase from Trametes versicolor in the gelatine-based hydrogel. The first system (1) was applied to detect laccase in aqueous samples. We investigated the detection level of the enzyme between 0.05 and 100 µg/mL and pH ranging between 3 and 9; the stability of ABTS in the solution and the immobilised form, as well as the retention functional property of the hydrogel in 4 °C for 30 days. The test can detect laccase within 20 min in the concentration range of 2.5–100 µg/mL; is effective at pH 3–6; preserves high stability and functionality under storage and can be also successfully applied for testing samples from a microbial culture. The second system with the immobilised laccase (2) was tested in terms of substrate specificity (ABTS, syringaldazine, guaiacol) and inhibitor (NaN3) screening. ABTS appeared the most proper substrate for laccase with detection sensitivity CABTS > 0.5 mg/mL. The NaN3 tested in the range of 0.5–100 µg/mL showed a distinct inhibition effect in 20 min for 0.5 µg/mL and total inhibition for ≥75 µg/mL.

Carbon monoxide (CO) is a key component of syngas and an important intermediate in the chemical, metallurgical, heavy, and food industries. Although mainly associated with thermochemical processes, CO can also be generated during composting, offering an environmentally friendly biological alternative. This study assessed the potential for CO production during laboratory-scale composting of seven selected organic waste fractions: coffee grounds, green tea leaves/grounds, wheat straw, grass cuttings, branches, food waste, and a biowaste mixture with an optimal C/N ratio. Composting was carried out under laboratory conditions at 45 °C for 14 days, with daily passive aeration and monitoring of CO, CO2, and O2 concentrations in the reactor headspace. CO production kinetics were calculated for each substrate, and the CO mass yield was determined in each bioreactor. The study confirmed the CO generation potential of the analyzed organic waste fractions. The highest CO production was observed for grass cuttings (max. 2000 ppm, 1.21 mg), biowaste mix (2000 ppm, 0.82 mg), and wheat straw (1180 ppm, 0.24 mg). Grass cuttings exhibited the highest average reaction rate (3991.1 ppm·d−1) and the most rapid process (2.920 d−1). Fungal colonization was visibly present in the most CO-productive reactors, suggesting a role of fungal metabolism in CO formation.

To develop a comprehensive substrate-screening method for the ATP-binding cassette (ABC) transporter, and identify new substrates for multidrug resistance-associated protein 4 (MRP4/ABCC4). Human MRP4-expressing membrane vesicles were incubated with a mixture of 50 compounds, including methotrexate, a known MRP4 substrate. The amounts transported were simultaneously determined by liquid chromatography-tandem mass spectrometry. From 49 compounds, 12 were identified as substrate candidates for MRP4 in the first screening. The second screening was performed involving the uptake of mixture using single quadrupole multichannel mode, and the third screening was performed involving the uptake of individual compounds using multiple reaction monitoring multichannel mode. As a result, eight substrate candidates were additionally identified. Subsequently, in the fourth step, osmotic pressure-dependent transport was demonstrated for 18 compounds (cefmetazole, piperacillin, rebamipide, tetracycline, ampicillin, benzylpenicillin, bumetanide, cephalosporin C, enalapril, pipemidic acid, furosemide, ceftazidime, pravastatin, hydrochlorothiazide, sulbactam, baclofen, bezafibrate and alacepril) among the 20 substrate candidates, thereby confirming them as MRP4 substrates. By contrast, the uptakes of meloxicam and nateglinide did not depend on osmolarity, indicating that these compounds were not substrates, but bound to MRP4. The new comprehensive substrate-screening method for ABC transporters allowed the identification of 18 new substrates for MRP4.

CYP102A1 is an efficient medium- to long-chain fatty acid hydroxylase that is able to accept a wide range of non-natural substrates which bear no resemblance to the natural ones. 4-Hexylbenzoic acid (HBA) and 4-nonyloxybenzoic acid (NOBA) were identified as CYP102A1 substrates via screening studies using the BD Oxygen Biosensor System. Spectroscopic binding studies showed that these two substrates bind in the active site of CYP102A1 with K ^sub d^ values of 2.6±0.1 μM for HBA and 1.9±0.2 μM for NOBA. NADPH consumption rates in the presence of HBA and NOBA were 45±1 min^sup -1^ and 61±1 min^sup -1^, respectively. The coupling efficiency for NADPH was 57% for NOBA, while it was 77% for HBA. During whole-cell biotransformations, HBA was converted into ω-1- and ω-2-hydroxyhexylbenzoic acid, whereas NOBA was oxidized to ω-2-hydroxynonyloxybenzoic acid and ω-2,ω-4-dihydroxynonyloxybenzoic acid. HBA was used as a fatty acid mimic to compare whole-cell biotransformations with cell-free extracts. Whole-cell biotransformations carried out in a biphasic system resulted in 86% conversion of 5 mM HBA, producing 3.8 mM ω-2- and 0.5 mM ω-1-hydroxyhexylbenzoic acid in 4 h with a turnover number of 4.1 min^sup -1^, whereas 100% conversion of 5 mM HBA was obtained in 1 h with crude cell extracts and a cofactor regeneration system, giving a turnover number of 10.5 min^sup -1^. [PUBLICATION ABSTRACT]

We have previously developed the WST-8 method as a simple and rapid screening test for detection of glucose-6-phosphate dehydrogenase (G6PD) deficiency accomplished by the naked eye. However, it was little difficult to distinguish between faint orange colors developed by heterozygous females and pink colors of normal hemolyzed blood, since both have similar tones. To solve this problem, we established a new and simple screening method that utilizes another formazan substrate, MTT (3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2H tetrazolium bromide) in combination with a hydrogen carrier, 1-methoxy phenazine methosulfate. MTT formazan exhibits a purple color, thus allowing for the ability to easily distinguish the pink colors of hemolyzed blood. However, MTT has been reported to react with hemoglobin non-specifically and to interfere with the interpretation of the color reaction. In our examinations by mixing MTT with hemolyzed blood, we found that the non-specific reaction was very slow, and that the addition of a small amount of blood (5~10 μl) into a reaction mixture (800 μl) did not interfere with the reaction of G6PD activity. In this new MTT method, a strong purple color was generated in normal blood samples at 20~30 min after incubation, which could be distinguished by the naked eye from G6PD-deficient blood samples with less than 50% residual activity. In addition, quantitative measurement using a spectrophotometer was also possible despite the fact that MTT formazan is water-insoluble.

Research in the field of asymmetric catalysis over the past half century has resulted in landmark advances, enabling the efficient synthesis of chiral building blocks, pharmaceuticals and natural products 1 – 3 . A small number of asymmetric catalytic reactions have been identified that display high selectivity across a broad scope of substrates; not coincidentally, these are the reactions that have the greatest impact on how enantioenriched compounds are synthesized 4 – 8 . We postulate that substrate generality in asymmetric catalysis is rare not simply because it is intrinsically difficult to achieve, but also because of the way chiral catalysts are identified and optimized 9 . Typical discovery campaigns rely on a single model substrate, and thus select for high performance in a narrow region of chemical space. Here we put forth a practical approach for using multiple model substrates to select simultaneously for both enantioselectivity and generality in asymmetric catalytic reactions from the outset 10 , 11 . Multisubstrate screening is achieved by conducting high-throughput chiral analyses by supercritical fluid chromatography–mass spectrometry with pooled samples. When applied to Pictet–Spengler reactions, the multisubstrate screening approach revealed a promising and unexpected lead for the general enantioselective catalysis of this important transformation, which even displayed high enantioselectivity for substrate combinations outside of the screening set.  The analytical workflow outlined in this study allows multiple crude reaction mixtures to be analysed simultaneously, with substantial reductions in method development and analysis time, and maximizes the chances of finding catalytic systems with broad substrate scope.