Microfluidic devices for drug assays

In this review, we give an overview of the current state of microfluidic-based high-throughput drug assays. In this highly interdisciplinary research field, various approaches have been applied to high-throughput drug screening, including microtiter plate, droplets microfluidics as well as continuous flow, diffusion and concentration gradients-based microfluidic drug assays. Therefore, we reviewed over 100 recent publications in the field and sorted them according to their microfluidic approach. As a result, we are showcasing, comparing and discussing broadly applied approaches as well as singular promising ones that might contribute to shaping the future of this field

Pathway of human AS3MT arsenic methylation

A synthetic gene encoding human As(III) S-adenosylmethionine (SAM) methyltransferase (hAS3MT) was expressed, and the purified enzyme was characterized. The synthetic enzyme is considerably more active than a cDNA-expressed enzyme using endogenous reductants thioredoxin (Trx), thioredoxin reductase (TR), NADPH, and reduced glutathione (GSH). Each of the seven cysteines (the four conserved residues, Cys32, Cys61, Cys156, and Cys206, and nonconserved, Cys72, Cys85, and Cys250) was individually changed to serine. The nonconserved cysteine derivates were still active. None of the individual C32S, C61S, C156S, and C206S derivates were able to methylate As(III). However, the C32S and C61S enzymes retained the ability to methylate MAs(III). These observations suggest that Cys156 and Cys206 play a different role in catalysis than that of Cys32 and Cys61. A homology model built on the structure of a thermophilic orthologue indicates that Cys156 and Cys206 form the As(III) binding site, whereas Cys32 and Cys61 form a disulfide bond. Two observations shed light on the pathway of methylation. First, binding assays using the fluorescence of a single-tryptophan derivative indicate that As(GS)3 binds to the enzyme much faster than inorganic As(III). Second, the major product of the first round of methylation is MAs(III), not MAs(V), and remains enzyme-bound until it is methylated a second time. We propose a new pathway for hAS3MT catalysis that reconciles the hypothesis of Challenger ((1947) Sci. Prog., 35, 396-416) with the pathway proposed by Hayakawa et al. ((2005) Arch. Toxicol., 79, 183-191). The products are the more toxic and more carcinogenic trivalent methylarsenicals, but arsenic undergoes oxidation and reduction as enzyme-bound intermediates

Pathway of Human AS3MT ArsenicMethylation

A synthetic gene encoding human As(III) S-adenosylmethionine (SAM) methyltransferase (hAS3MT) was expressed, and the purified enzyme was characterized. The synthetic enzyme is considerably more active than a cDNA-expressed enzyme using endogenous reductants thioredoxin (Trx), thioredoxin reductase (TR), NADPH, and reduced glutathione (GSH). Each of the seven cysteines (the four conserved residues, Cys32, Cys61, Cys156, and Cys206, and nonconserved, Cys72, Cys85, and Cys250) was individually changed to serine. The nonconserved cysteine derivates were still active. None of the individual C32S, C61S, C156S, and C206S derivates were able to methylate As(III). However, the C32S and C61S enzymes retained the ability to methylate MAs(III). These observations suggest that Cys156 and Cys206 play a different role in catalysis than that of Cys32 and Cys61. A homology model built on the structure of a thermophilic orthologue indicates that Cys156 and Cys206 form the As(III) binding site, whereas Cys32 and Cys61 form a disulfide bond. Two observations shed light on the pathway of methylation. First, binding assays using the fluorescence of a single-tryptophan derivative indicate that As(GS)3 binds to the enzyme much faster than inorganic As(III). Second, the major product of the first round of methylation is MAs(III), not MAs(V), and remains enzyme-bound until it is methylated a second time. We propose a new pathway for hAS3MT catalysis that reconciles the hypothesis of Challenger ((1947) Sci. Prog., 35, 396?416) with the pathway proposed by Hayakawa et al. ((2005) Arch. Toxicol., 79, 183?191). The products are the more toxic and more carcinogenic trivalent methylarsenicals, but arsenic undergoes oxidation and reduction as enzyme-bound intermediates

Rational design of a (S)-selective-transaminase for asymmetric synthesis of (1S)-1-(1,1′-biphenyl-2-yl)ethanamine

Amine transaminases offer an environmentally sustainable synthesis route for the production of pure chiral amines. However, their catalytic efficiency toward bulky ketone substrates is greatly limited by steric hindrance and therefore presents a great challenge for industrial synthetic applications. We hereby report an example of rational transaminase enzyme design to help alleviate these challenges. Starting from the Vibrio fluvialis amine transaminase that has no detectable catalytic activity toward the bulky aromatic ketone 2-acetylbiphenyl, we employed a rational design strategy combining in silico and in vitro studies to engineer the transaminase enzyme with a minimal number of mutations, achieving an high catalytic activity and high enantioselectivity. We found that, by introducing two mutations W57G/R415A, detectable enzyme activity was achieved. The rationally designed variant, W57F/R88H/V153S/K163F/I259M/R415A/V422A, showed an improvement in reaction rate by more than 1716-fold toward the bulky ketone under study, producing the corresponding enantiomeric pure (S)-amine (enantiomeric excess (ee) value of >99%)

Structural basis of terephthalate recognition by solute binding protein TphC

From Springer Nature via Jisc Publications RouterHistory: received 2021-03-24, accepted 2021-10-06, registration 2021-10-12, pub-electronic 2021-10-29, online 2021-10-29, collection 2021-12Publication status: PublishedFunder: Commonwealth Scholarship Commission (CSC); doi: https://doi.org/10.13039/501100000867; Grant(s): INCN-2018-57Funder: RCUK | Engineering and Physical Sciences Research Council (EPSRC); doi: https://doi.org/10.13039/501100000266; Grant(s): EP/M013219/1, EP/023755/1Funder: RCUK | Biotechnology and Biological Sciences Research Council (BBSRC); doi: https://doi.org/10.13039/501100000268; Grant(s): BB/M011208/1, BB/M011208/1, BB/P01738X/1Abstract: Biological degradation of Polyethylene terephthalate (PET) plastic and assimilation of the corresponding monomers ethylene glycol and terephthalate (TPA) into central metabolism offers an attractive route for bio-based molecular recycling and bioremediation applications. A key step is the cellular uptake of the non-permeable TPA into bacterial cells which has been shown to be dependent upon the presence of the key tphC gene. However, little is known from a biochemical and structural perspective about the encoded solute binding protein, TphC. Here, we report the biochemical and structural characterisation of TphC in both open and TPA-bound closed conformations. This analysis demonstrates the narrow ligand specificity of TphC towards aromatic para-substituted dicarboxylates, such as TPA and closely related analogues. Further phylogenetic and genomic context analysis of the tph genes reveals homologous operons as a genetic resource for future biotechnological and metabolic engineering efforts towards circular plastic bio-economy solutions

An integrated portable system for single chip simultaneous measurement of multiple disease associated metabolites

Metabolites, the small molecules that underpin life, can act as indicators of the physiological state of the body when their abundance varies, offering routes to diagnosis of many diseases. The ability to assay for multiple metabolites simultaneously will underpin a new generation of precision diagnostic tools. Here, we report the development of a handheld device based on complementary metal oxide semiconductor (CMOS) technology with multiple isolated micro-well reaction zones and integrated optical sensing allowing simultaneous enzyme-based assays of multiple metabolites (choline, xanthine, sarcosine and cholesterol) associated with multiple diseases. These metabolites were measured in clinically relevant concentration range with minimum concentrations measured: 25 μM for choline, 100 μM for xanthine, 1.25 μM for sarcosine and 50 μM for cholesterol. Linking the device to an Android-based user interface allows for quantification of metabolites in serum and urine within 2 min of applying samples to the device. The quantitative performance of the device was validated by comparison to accredited tests for cholesterol and glucose

Purification and Properties of Amycolatopsis Mediterranei DSM 43304 Lipase and Its Potential in Flavour Ester Synthesis

An extracellular thermostable lipase from Amycolatopsis mediterranei DSM 43304 has been purified to homogeneity using ammonium sulphate precipitation followed by anion exchange chromatography and hydrophobic interaction chromatography. This protocol resulted in 398 fold purification with 36% final recovery. The purified A. mediterranei DSM 43304 lipase (AML) has an apparent molecular mass of 33 kDa. The N-terminal sequence, AANPYERGPDPTTASIEATR, showed highest similarity to a lipase from Streptomyces exfoliatus. The values of and for p-nitrophenyl palmitate (p-NPP) under optimal temperature (60°C) and pH (8.0) conditions were 0.10 ± 0.01 mM and 2.53 ± 0.06 mmol/minmg, respectively. The purified AML displayed significant activity towards a range of short and long chain triglycerides. It was most active on triolein and a wide range of p-nitrophenyl esters, with a preference for an acyl chain length of C8:0. Hydrolysis of glycerol esterbonds occurred non-specifically. The purified AML displayed significant stability in the presence of organic solvents (40% v/v) and catalyzed the synthesis of flavour ester isoamyl acetate in free and immobilized states

Purification and properties of Amycolatopsis mediterranei DSM 43304 lipase and its potential in flavour ester synthesis

An extracellular thermostable lipase from Amycolatopsis mediterranei DSM 43304 has been purified to homogeneity using ammonium sulphate precipitation followed by anion exchange chromatography and hydrophobic interaction chromatography. This protocol resulted in a 398-fold purification with 36% final recovery. The purified A. mediterranei DSM 43304 lipase (AML) has an apparent molecular mass of 33 kDa. The N-terminal sequence, AANPYERGPDPTTASIEATR, showed highest similarity to a lipase from Streptomyces exfoliatus. The values of K(m)(app) and V(max)(app) for p-nitrophenyl palmitate (p-NPP) at the optimal temperature (60°C) and pH (8.0) were 0.099±0.010 mM and 2.53±0.06 mmol/min mg, respectively. The purified AML displayed significant activity towards a range of short and long chain triglyceride substrates and p-nitrophenyl esters. Hydrolysis of glycerol ester bonds occurred non-specifically. The purified AML displayed significant stability in the presence of organic solvents (40%, v/v) and catalyzed the synthesis of the flavour ester isoamyl acetate in free and immobilized states

Purification and characterization of an extracellular lipase from a novel strain Penicillium sp. DS-39 (DSM 23773)

A newly isolated fungal strain, Penicillium sp. DS-39 (DSM 23773), was found to produce an inducible extracellular lipase when grown in a medium containing 1.0% (v/v) olive oil. Maximum lipase activity was obtained after 120 h of incubation at 28 °C. The lipase was purified 129-fold with a final specific activity of 308.73 IU/mg. The molecular weight of the homogeneous lipase was 43 kDa as determined by SDS-PAGE. It was optimally active at pH 5.5 and 45 °C. The lipase was most active on triolein and exhibited a broad substrate range with a preference for triacylglycerols containing long chain unsaturated fatty acids. It showed no regio-specificity for the ester bond in triolein. It was activated by Ca2+ and Mn2+, while significant inhibition was observed with Hg2+ and Zn2+. The lipase showed significant stability and activation in the presence of organic solvents with log P ≥ 2.0. These features render Penicillium sp. DS-39 lipase (PEL) a potential biocatalyst for applications such as biodiesel production, enzymatic restructuring, by interesterification of different oils and fats, and biodegradation of oil spills in the environment

A disulfide-bond cascade mechanism for arsenic(III) S-adenosylmethionine methyltransferase

Methylation of the toxic metalloid arsenic is widespread in nature. Members of every kingdom have arsenic(III) S-adenosylmethionine (SAM) methyltransferase enzymes, which are termed ArsM in microbes and AS3MT in animals, including humans. Trivalent arsenic(III) is methylated up to three times to form methylarsenite [MAs(III)], dimethylarsenite [DMAs(III)] and the volatile trimethylarsine [TMAs(III)]. In microbes, arsenic methylation is a detoxification process. In humans, MAs(III) and DMAs(III) are more toxic and carcinogenic than either inorganic arsenate or arsenite. Here, new crystal structures are reported of ArsM from the thermophilic eukaryotic alga Cyanidioschyzon sp. 5508 (CmArsM) with the bound aromatic arsenicals phenylarsenite [PhAs(III)] at 1.80 Å resolution and reduced roxarsone [Rox(III)] at 2.25 Å resolution. These organoarsenicals are bound to two of four conserved cysteine residues: Cys174 and Cys224. The electron density extends the structure to include a newly identified conserved cysteine residue, Cys44, which is disulfide-bonded to the fourth conserved cysteine residue, Cys72. A second disulfide bond between Cys72 and Cys174 had been observed previously in a structure with bound SAM. The loop containing Cys44 and Cys72 shifts by nearly 6.5 Å in the arsenic(III)-bound structures compared with the SAM-bound structure, which suggests that this movement leads to formation of the Cys72-Cys174 disulfide bond. A model is proposed for the catalytic mechanism of arsenic(III) SAM methyltransferases in which a disulfide-bond cascade maintains the products in the trivalent state