Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) as a compensatory protease inhibitor in hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) is a genetic disorder that manifests as recurrent angioedema attacks, most frequently due to absent or reduced C1 inhibitor (C1INH) activity. C1INH is a crucial regulator of enzymatic cascades in the complement, fibrinolytic, and contact systems. Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) is an abundant plasma protease inhibitor that can inhibit enzymes in the proteolytic pathways associated with HAE. Nothing is known about its role in HAE. OBJECTIVE: We investigated ITIH4 activation in HAE, establishing it as a potential biomarker, and explored its involvement in HAE-associated proteolytic pathways. METHODS: Specific immunoassays for noncleaved ITIH4 (intact ITIH4) and an assay detecting both intact and cleaved ITIH4 (total ITIH4) were developed. We initially tested serum samples from HAE patients (n = 20), angiotensin-converting enzyme inhibitor-induced edema patients (ACEI) (n = 20), and patients with HAE of unknown cause (HAE-UNK) (n = 20). Validation involved an extended cohort of 80 HAE patients (60 with HAE-C1INH type 1, 20 with HAE-C1INH type 2), including samples taken during attack and quiescent disease periods, as well as samples from 100 healthy controls. RESULTS: In 63% of HAE patients, intact ITIH4 assay showed lower signals than total ITIH4 assay. This difference was not observed in ACEI and HAE-UNK patients. Western blot analysis confirmed cleaved ITIH4 with low intact ITIH4 samples. In serum samples lacking intact endogenous ITIH4, we observed immediate cleavage of added recombinant ITIH4, suggesting continuous enzymatic activity in the serum. Confirmatory HAE cohort analysis revealed significantly lower intact ITIH4 levels in both type 1 and type 2 HAE patients compared to controls, with consistently low intact/total ITIH4 ratios during clinical HAE attacks. CONCLUSION: The disease-specific low intact ITIH4 levels highlight its unique nature in HAE. ITIH4 may exhibit compensatory mechanisms in HAE, suggesting its utility as a diagnostic and prognostic biomarker. The variations during quiescent and active disease periods raise intriguing questions about the dynamics of proteolytic pathways in HAE.

Photoelectrochemical Solution Gated Graphene Field-Effect Transistor Functionalized by Enzymatic Cascade Reaction for Organophosphate Detection.

Solution Gated Graphene Field-Effect Transistors (SGGT) are eagerly anticipated as an amplification platform for fabricating advanced ultra-sensitive sensors, allowing significant modulation of the drain current with minimal gate voltage. However, few studies have focused on light-matter interplay gating control for SGGT. Herein, this challenge is addressed by creating an innovative photoelectrochemical solution-gated graphene field-effect transistor (PEC-SGGT) functionalized with enzyme cascade reactions (ECR) for Organophosphorus (OPs) detection. The ECR system, consisting of acetylcholinesterase (AChE) and CuBTC nanomimetic enzymes, selectively recognizes OPs and forms o-phenylenediamine (oPD) oligomers sediment on the PEC electrode, with layer thickness related to the OPs concentration, demonstrating time-integrated amplification. Under light stimulation, the additional photovoltage generated on the PEC gate electrode is influenced by the oPD oligomers sediment layer, creating a differentiated voltage distribution along the gate path. PEC-SGGT, inherently equipped with built-in amplification circuits, sensitively captures gate voltage changes and delivers output with an impressive thousandfold current gain. The seamless integration of these three amplification modes in this advanced sensor allows a good linear range and highly sensitive detection of OPs, with a detection limit as low as 0.05 pm. This work provides a proof-of-concept for the feasibility of light-assisted functionalized gate-controlled PEC-SGGT for small molecule detection.

One-Pot Enzymatic Cascade for Toxicant Degradation and Sugar Acid Production.

This study introduces a novel one-pot enzymatic cascade approach for converting toxicants and continuously generating an electron acceptor for production of sugar acids. This method offers a promising solution to concerns about pesticide toxicity and environmental contamination by transforming hazardous substances into a useful electron acceptor. This acceptor is then utilized to produce valuable chemicals with broad industrial applications, particularly in the food and pharmaceutical sectors. The cascade reaction employs organophosphate hydrolase (OPD) to convert pesticides into 4-nitrophenol (4-NP), which is subsequently transformed into 1,4-benzoquinone by HadA monooxygenase (HadA). 1,4-benzoquinone serves as an electron acceptor in the catalysis of sugar acid formation via pyranose dehydrogenase (PDH). The results indicate that this cascade reaction effectively converts lactose to lactobionic acid and xylose to 2-keto-xylonic acid. The latter can be further processed into xylonic acid through NaBH4 reduction. Notably, the one-pot reaction yields up to 10% higher compared to the direct addition of 1,4-benzoquinone. The synthesized xylonic acid exhibits exceptional water uptake properties in hydrogels, and the synthesized lactobionic acid shows antioxidant activity comparable to well-established antioxidants. These findings demonstrate the technological viability of these reaction cascades for various applications.

Investigation on production and reaction conditions of sucrose synthase based glucosylation cascade towards flavonoid modification.

Enzyme-based glycosylation is of great interest in the context of natural products decoration. Yet, its industrial application is hindered by optimisation difficulties and hard-to-standardise productivities. In this study, five sugar nucleotide-dependent glucosyltransferases from different origins (bacterial, plant and fungal) were coupled with soy sucrose synthase (GmSuSy) to create a set of diverse cascade biocatalysts for flavonoid glucosylation, which evaluation brought new insights into the field. Investigations into co-expression conditions and reaction settings enabled to define optimal induction temperature (25 °C) and uridine diphosphate (UDP) concentration (0.5 mM) for all tested pairs of enzymes. Moreover, the influence of pH and substrate concentration on the monoglucosylated product distribution was detected and analysed. The utilisation of crude protein extracts as a cost-effective source of catalysts unveiled their glycosidase activity against flavonoid glucosides, resulting in decreased productivity, which, to our knowledge, has not previously been discussed in such a context. Additionally, examination of the commercially available EziG immobilisation resins showed that selection of suitable carrier for solid catalyst production can be problematic and not only enzyme's but also reagent's properties have to be considered. Flavonoids, due to their complexation and hydrophobic properties, can adsorb on different types of surfaces, including divalent metal ions required for IMAC based immobilisation, necessitating detailed examination of the resins while the catalysis design.

Self-Assembled Copper-Based Nanoparticles for Glutathione Activated and Enzymatic Cascade-Enhanced Ferroptosis and Immunotherapy in Cancer Treatment.

As an emerging cancer treatment strategy, ferroptosis is greatly restricted by excessive glutathione (GSH) in tumor microenvironment (TME) and low reactive oxygen species (ROS) generation efficiency. Here, this work designs self-assembled copper-alanine nanoparticles (CACG) loaded with glucose oxidase (GOx) and cinnamaldehyde (Cin) for in situ glutathione activated and enzymatic cascade-enhanced ferroptosis and immunotherapy. In response to GSH-rich and acidic TME, CACG allows to effectively co-deliver Cu2+ , Cin, and GOx into tumors. Released Cin consumes GSH through Michael addition, accompanying with the reduction of Cu2+ into Cu+ for further GSH depletion. With the cascade of Cu+ -catalyzed Fenton reactions and enzyme-catalyzed reactions by GOx, CACG could get rid of the restriction of insufficient hydrogen peroxide in TME, leading to a robust and constant generation of ROS. With the high efficiency of GSH depletion and ROS production, ferroptosis is significantly enhanced by CACG in vivo. Moreover, elevated oxidative stress triggers robust immune responses by promoting dendritic cells maturation and T cell infiltration. The in vivo results prove that CACG could efficiently inhibit tumor growth in 4T1 tumor-bearing mouse model without causing obvious systemic toxicity, suggesting the great potential of CACG in enhancing ferroptosis and immunotherapy for effective cancer treatment.

Engineering an Enzymatic Cascade Catalytic Smartphone-Based Sensor for Onsite Visual Ratiometric Fluorescence-Colorimetric Dual-Mode Detection of Methyl Mercaptan.

Precise and reliable onsite detection of methyl mercaptan (CH3SH) is of great significance for environmental surveillance. Here, we synthesized a novel blue fluorescence nanozyme CeO2@TPE with high peroxidase-like activity by employing aggregation-induced emission (AIE) tetraphenylethene (TPE) to embed into hollow CeO2 nanospheres. In the presence of ethanol oxidase (AOX) and o-phenylenediamine (OPD), we engineered an enzymatic cascade activation ratiometric fluorescence-colorimetric dual-mode system AOX/CeO2@TPE + OPD toward CH3SH. In this design, CH3SH initiated AOX catalytic activity to convert it into H2O2 for activating the peroxidase-like activity of CeO2@TPE, producing •OH for oxidizing the naked-eye colorless OPD into deep yellow 2,3-diaminophenazine (DAP) with an absorption enhancement at ∼425 nm, companied by a new emission peak at ∼550 nm to match with the intrinsic emission at ∼441 nm for observing ratiometric fluorescence response, enabling a ratiometric fluorescence-colorimetric dual-mode analysis. Interestingly, both the ratiometric fluorescence and colorimetric signals could be gathered for being converted into the hue parameter on a smartphone-based sensor, achieving the onsite visual fluorescence-colorimetric dual-mode detection of CH3SH in real environmental media with acceptable results. This study gave a novel insight into designing target-responsive enzymatic cascade activation system-based efficient and reliable dual-mode point-of-care sensors for safeguarding environmental health.

Cu-MOF derived CuO@g-C3N4 nanozyme for cascade catalytic colorimetric sensing.

The use of peroxidase mimics has great potential for various real applications due to their strong catalytic activity. Herein, a facile strategy was proposed to directly prepare CuO@g-C3N4 by Cu-MOF derivatization and demonstrated its efficacy in constructing a multiple enzymatic cascade system by loading protein enzymes onto it. The resulting CuO@g-C3N4 possessed high peroxidase-like activity, with a Michaelis constant (Km) of 0.25 and 0.16 mM for H2O2 and 3,3',5,5'-tetramethylbenzidine (TMB), respectively. Additionally, the high surface area of CuO@g-C3N4 facilitated the loading of protein enzymes and maintained their activity over an extended period, expanding the potential applications of CuO@g-C3N4. To test its feasibility, CuO@g-C3N4/protein oxidase complex was prepared and used to sense the ripeness and freshness of fruits and meat, respectively. The mechanism relied on the fact that the ripeness of fruits increased and freshness of food decreased with the release of marked targets, such as glucose and xanthine, which could produce H2O2 when digested by the corresponding oxidase. The peroxidase mimics of CuO@g-C3N4 could then sensitively colorimetric detect H2O2 in present of TMB. The obtained CuO@g-C3N4/oxidase complex exhibited an excellent linear response to glucose or xanthine in the range of 1.0-120 µmol/L or 8.0-350 µmol/L, respectively. Furthermore, accurate quantification of glucose and xanthine in real samples is achieved with spiked recoveries ranging from 80.2% to 120.0% and from 94.2% to 112.0%, respectively. Overall, this work demonstrates the potential of CuO@g-C3N4 in various practical applications, such as food freshness detection.

Multi-Enzymatic Cascade for Efficient Deracemization of dl-Pantolactone into d-Pantolactone.

d-pantolactone is an intermediate in the synthesis of d-pantothenic acid, which is known as vitamin B5. The commercial synthesis of d-pantolactone is carried out through the selective resolution of dl-pantolactone catalyzed by lactone hydrolase. In contrast to a kinetic resolution approach, the deracemization of dl-pantolactone is a simpler, greener, and more sustainable way to obtain d-pantolactone with high optical purity. Herein, an efficient three-enzyme cascade was developed for the deracemization of dl-pantolactone, using l-pantolactone dehydrogenase from Amycolatopsis methanolica (AmeLPLDH), conjugated polyketone reductase from Zygosaccharomyces parabailii (ZpaCPR), and glucose dehydrogenase from Bacillus subtilis (BsGDH). The AmeLPLDH was used to catalyze the dehydrogenated l-pantolactone into ketopantolactone; the ZpaCPR was used to further catalyze the ketopantolactone into d-pantolactone; and glucose dehydrogenase together with glucose fulfilled the function of coenzyme regeneration. All three enzymes were co-expressed in E. coli strain BL21(DE3), which served as the whole-cell biocatalyst. Under optimized conditions, 36 h deracemization of 1.25 M dl-pantolactone d-pantolactone led to an e.e.p value of 98.6%, corresponding to productivity of 107.7 g/(l·d).

Development of an enzymatic cascade to systematically utilize lignocellulosic monosaccharide.

BACKGROUND: The fermentation valorization of two main lignocellulosic monosaccharides, glucose and xylose, is extensively developed; however, it is restricted by limited yield and process complexity. An in vitro enzymatic cascade reaction can be an alternative approach. RESULTS: In this study, a three-stage, five-enzyme cascade was developed to convert pretreated biomass to valuable chemicals. First, a ribose-5-phosphate isomerase B mutant isomerized xylose to d-xylulose with high substrate specificity, and a d-arabinose dehydrogenase continued to reduce d-xylulose to d-arabitol. Simultaneously, glucose was utilized for the coenzyme regeneration catalyzed by a glucose dehydrogenase, generating useful gluconic acid and achieving 73% of total conversion rate after 36 h. Then, six kinds of pretreated biomass lignocellulose were hydrolyzed by cellulase and hemicellulase, and corn cob was identified as the initial substrate for providing the highest monosaccharide content. A 65% conversion rate of the lignocellulosic xylose was obtained after 24 h. CONCLUSIONS: This study presents a proof of concept to convert main lignocellulosic monosaccharides systematically by an enzymatic cascade at stoichiometric ratio. © 2022 Society of Chemical Industry.

Efficient synthesis of 2'-deoxyguanosine in one-pot cascade by employing an engineered purine nucleoside phosphorylase from Brevibacterium acetylicum.

2'-deoxyguanosine is a key medicinal intermediate that could be used to synthesize anti-cancer drug and biomarker in type 2 diabetes. In this study, an enzymatic cascade using thymidine phosphorylase from Escherichia coli (EcTP) and purine nucleoside phosphorylase from Brevibacterium acetylicum (BaPNP) in a one-pot whole cell catalysis was proposed for the efficient synthesis of 2'-deoxyguanosine. BaPNP was semi-rationally designed to improve its activity, yielding the best triple variant BaPNP-Mu3 (E57A/T189S/L243I), with a 5.6-fold higher production of 2'-deoxyguanosine than that of wild-type BaPNP (BaPNP-Mu0). Molecular dynamics simulation revealed that the engineering of BaPNP-Mu3 resulted in a larger and more flexible substrate entrance channel, which might contribute to its catalytic efficiency. Furthermore, by coordinating the expression of BaPNP-Mu3 and EcTP, a robust whole cell catalyst W05 was created, capable of producing 14.8 mM 2'-deoxyguanosine (74.0% conversion rate) with a high time-space yield (1.32 g/L/h) and therefore being very competitive for industrial applications.

Cascaded Biocatalysis and Bioelectrocatalysis: Overview and Recent Advances.

Enzyme cascades are plentiful in nature, but they also have potential in artificial applications due to the possibility of using the target substrate in biofuel cells, electrosynthesis, and biosensors. Cascade reactions from enzymes or hybrid bioorganic catalyst systems exhibit extended substrate range, reaction depth, and increased overall performance. This review addresses the strategies of cascade biocatalysis and bioelectrocatalysis for (a) CO2 fixation, (b) high value-added product formation, (c) sustainable energy sources via deep oxidation, and (d) cascaded electrochemical enzymatic biosensors. These recent updates in the field provide fundamental concepts, designs of artificial electrocatalytic oxidation-reduction pathways (using a flexible setup involving organic catalysts and engineered enzymes), and advances in hybrid cascaded sensors for sensitive analyte detection.

Engineering cascade biocatalysis in whole cells for bottom-up synthesis of cello-oligosaccharides: flux control over three enzymatic steps enables soluble production.

BACKGROUND: Soluble cello-oligosaccharides (COS, ß-1,4-D-gluco-oligosaccharides with degree of polymerization DP 2-6) have been receiving increased attention in different industrial sectors, from food and feed to cosmetics. Development of large-scale COS applications requires cost-effective technologies for their production. Cascade biocatalysis by the three enzymes sucrose-, cellobiose- and cellodextrin phosphorylase is promising because it enables bottom-up synthesis of COS from expedient substrates such as sucrose and glucose. A whole-cell-derived catalyst that incorporates the required enzyme activities from suitable co-expression would represent an important step towards making the cascade reaction fit for production. Multi-enzyme co-expression to reach distinct activity ratios is challenging in general, but it requires special emphasis for the synthesis of COS. Only a finely tuned balance between formation and elongation of the oligosaccharide precursor cellobiose results in the desired COS. RESULTS: Here, we show the integration of cellodextrin phosphorylase into a cellobiose-producing whole-cell catalyst. We arranged the co-expression cassettes such that their expression levels were upregulated. The most effective strategy involved a custom vector design that placed the coding sequences for cellobiose phosphorylase (CbP), cellodextrin phosphorylase (CdP) and sucrose phosphorylase (ScP) in a tricistron in the given order. The expression of the tricistron was controlled by the strong T7lacO promoter and strong ribosome binding sites (RBS) for each open reading frame. The resulting whole-cell catalyst achieved a recombinant protein yield of 46% of total intracellular protein in an optimal ScP:CbP:CdP activity ratio of 10:2.9:0.6, yielding an overall activity of 315 U/g dry cell mass. We demonstrated that bioconversion catalyzed by a semi-permeabilized whole-cell catalyst achieved an industrial relevant COS product titer of 125 g/L and a space-time yield of 20 g/L/h. With CbP as the cellobiose providing enzyme, flux into higher oligosaccharides (DP ≥ 6) was prevented and no insoluble products were formed after 6 h of conversion. CONCLUSIONS: A whole-cell catalyst for COS biosynthesis was developed. The coordinated co-expression of the three biosynthesis enzymes balanced the activities of the individual enzymes such that COS production was maximized. With the flux control set to minimize the share of insolubles in the product, the whole-cell synthesis shows a performance with respect to yield, productivity, product concentration and quality that is promising for industrial production.

Continuous process technology for bottom-up synthesis of soluble cello-oligosaccharides by immobilized cells co-expressing three saccharide phosphorylases.

BACKGROUND: Continuous processing with enzyme reuse is a well-known engineering strategy to enhance the efficiency of biocatalytic transformations for chemical synthesis. In one-pot multistep reactions, continuous processing offers the additional benefit of ensuring constant product quality via control of the product composition. Bottom-up production of cello-oligosaccharides (COS) involves multistep iterative ß-1,4-glycosylation of glucose from sucrose catalyzed by sucrose phosphorylase from Bifidobacterium adeloscentis (BaScP), cellobiose phosphorylase from Cellulomonas uda (CuCbP) and cellodextrin phosphorylase from Clostridium cellulosi (CcCdP). Degree of polymerization (DP) control in the COS product is essential for soluble production and is implemented through balance of the oligosaccharide priming and elongation rates. A whole-cell E. coli catalyst co-expressing the phosphorylases in high yield and in the desired activity ratio, with CdP as the rate-limiting enzyme, was reported previously. RESULTS: Freeze-thaw permeabilized E. coli cells were immobilized in polyacrylamide (PAM) at 37-111 mg dry cells/g material. PAM particles (0.25-2.00 mm size) were characterized for COS production (~ 70 g/L) in mixed vessel with catalyst recycle and packed-bed reactor set-ups. The catalyst exhibited a dry mass-based overall activity (270 U/g; 37 mg cells/g material) lowered by ~ 40% compared to the corresponding free cells due to individual enzyme activity loss, CbP in particular, caused by the immobilization. Temperature studies revealed an operational optimum at 30 °C for stable continuous reaction (~ 1 month) in the packed bed (volume: 40 mL; height: 7.5 cm). The optimum reflects the limits of PAM catalyst structural and biological stability in combination with the requirement to control COS product solubility in order to prevent clogging of the packed bed. Using an axial flow rate of 0.75 cm- 1, the COS were produced at ~ 5.7 g/day and ≥ 95% substrate conversion (sucrose 300 mM). The product stream showed a stable composition of individual oligosaccharides up to cellohexaose, with cellobiose (48 mol%) and cellotriose (31 mol%) as the major components. CONCLUSIONS: Continuous process technology for bottom-up biocatalytic production of soluble COS is demonstrated based on PAM immobilized E. coli cells that co-express BaScP, CuCbP and CcCdP in suitable absolute and relative activities.

CunO/Au heterostructure dendrimer anchored on Cu foam as dual functional catalytic nanozyme for glucose sensing by enzyme mimic cascade reaction.

Multifunctional catalytic performance plays a crucial role in bio-applications through the diversity and durability of artificial nanozymes. An effective synergy with sufficient accessible active sites and high specific surface area is a challenge for composite catalysts, especially to avoid uncontrollable aggregation and structural instability. Here, we fabricated a CunO/Au heterostructure dendrimer on copper foam (CunO/Au HD/CF) as dual functional catalytic nanozyme to achieve enzyme mimic cascade reactions for efficient colorimetric analysis. A highly porous CF skeleton-based CuO nanowire array (CuO NWA) with a large specific surface area supported an efficient load capacity to assemble sufficient CunO/Au HD by electrodeposition. The bimetallic Au-Cu nanozyme successfully achieved an oxidase-like and peroxidase-like cascade catalysis by a target-responsive sensing mechanism. Due to the confirmed catalytic performance of selectivity, anti-interference ability, and reproducibility, a CunO/Au HD/CF-based quantitative analytical method was developed for glucose detection with a wide linear range and considerable detection limit of 8.4 µM. The robust nonenzymatic catalytic strategy for colorimetric detection not only confirmed the dual functional catalytic activity of CunO/Au HD/CF, but also showed great potential for applications in clinical diagnostics and biochemical analysis.

Real-time and simultaneous assay of monophenolase and diphenolase activity in tyrosinase catalyzed cascade reactions by combination of three-way calibration and excitation-emission matrix fluorescence.

A real-time assay for multiple enzyme activities in cascade reactions is required for research on metabolism and bioengineering. Tyrosinase has the bifunctional activity of monophenolase and diphenolase. A combined strategy of three-way calibration with excitation-emission matrix (EEM) fluorescence was developed for real-time and simultaneous determination of monophenolase and diphenolase activity with tyrosine as a substrate. Mathematical separation and second-order advantage were utilized to solve spectral overlapping and uncalibrated interferents during complex dynamic enzymatic processes. Kinetic evolution profiles of EEM were monitored to stack a fusion three-way data array together with static samples. Using a parallel factor analysis (PARAFAC) algorithm, pseudo-univariate calibration curves with limits of detection (LODs) of 3.00 µM and 0.85 µM were established to simultaneously and real-time measure tyrosine and DOPA. Progress curves for tyrosine consumption by monophenolase and DOPA consumption by diphenolase were obtained using the law of mass conservation to calculate the initial velocity. The LODs for monophenolase and diphenolase were 0.0232 U⋅mL-1 and 0.0316 U⋅mL-1. The method achieved real-time and simultaneous assays of multiple enzyme activities in cascade reactions. It showed potential application in the metabolic pathway and biochemical industry.

Functional Characterization and Screening of Promiscuous Kinases and Isopentenyl Phosphate Kinases for the Synthesis of DMAPP via a One-Pot Enzymatic Cascade.

Dimethylallyl diphosphate (DMAPP) is a key intermediate metabolite in the synthesis of isoprenoids and is also the prenyl donor for biosynthesizing prenylated flavonoids. However, it is difficult to prepare DMAPP via chemical and enzymatic methods. In this study, three promiscuous kinases from Shigella flexneri (SfPK), Escherichia coli (EcPK), and Saccharomyces cerevisiae (ScPK) and three isopentenyl phosphate kinases from Methanolobus tindarius (MtIPK), Methanothermobacter thermautotrophicus str. Delta H (MthIPK), and Arabidopsis thaliana (AtIPK) were cloned and expressed in Escherichia coli. The enzymatic properties of recombinant enzymes were determined. The Kcat/Km value of SfPK for DMA was 6875 s-1 M-1, which was significantly higher than those of EcPK and ScPK. The Kcat/Km value of MtIPK for DMAP was 402.9 s-1 M-1, which was ~400% of that of MthIPK. SfPK was stable at pH 7.0-9.5 and had a 1 h half-life at 65 °C. MtIPK was stable at pH 6.0-8.5 and had a 1 h half-life at 50 °C. The stability of SfPK and MtIPK was better than that of the other enzymes. Thus, SfPK and MtIPK were chosen to develop a one-pot enzymatic cascade for producing DMAPP from DMA because of their catalytic efficiency and stability. The optimal ratio between SfPK and MtIPK was 1:8. The optimal pH and temperature for the one-pot enzymatic cascade were 7.0 and 35 °C, respectively. The optimal concentrations of ATP and DMA were 10 and 80 mM, respectively. Finally, maximum DMAPP production reached 1.23 mM at 1 h under optimal conditions. Therefore, the enzymatic method described herein for the biosynthesis of DMAPP from DMA can be widely used for the synthesis of isoprenoids and prenylated flavonoids.

Torque-Driven Orientation Motion of Chemotactic Colloidal Motors.

We report a direct experimental observation of the torque-driven active reorientation of glucose-fueled flasklike colloidal motors to a glucose gradient exhibiting a positive chemotaxis. These streamlined flasklike colloidal motors are prepared by combining a hydrothermal synthesis and a vacuum infusion and can be propelled by an enzymatic cascade reaction in the glucose fuel. Their flasklike architecture can be used to recognize their moving posture, and thus the dynamic glucose-gradient-induced alignment and orientation-dependent motility during positive chemotaxis can be examined experimentally. The chemotactic mechanism is that the enzymatic reactions inside lead to the glucose acid gradient and the glucose gradient which generate two phoretic torques at the bottom and the opening respectively, and thus continuously steer it to the glucose gradient. Such glucose-fueled flasklike colloidal motors resembling the chemotactic capability of living organisms hold considerable potential for engineering active delivery vehicles in response to specific chemical signals.

Chemoenzymatic Production of Enantiocomplementary 2-Substituted 3-Hydroxycarboxylic Acids from L-α-Amino Acids.

A two-enzyme cascade reaction plus in situ oxidative decarboxylation for the transformation of readily available canonical and non-canonical L-α-amino acids into 2-substituted 3-hydroxy-carboxylic acid derivatives is described. The biocatalytic cascade consisted of an oxidative deamination of L-α-amino acids by an L-α-amino acid deaminase from Cosenzaea myxofaciens, rendering 2-oxoacid intermediates, with an ensuing aldol addition reaction to formaldehyde, catalyzed by metal-dependent (R)- or (S)-selective carboligases namely 2-oxo-3-deoxy-l-rhamnonate aldolase (YfaU) and ketopantoate hydroxymethyltransferase (KPHMT), respectively, furnishing 3-substituted 4-hydroxy-2-oxoacids. The overall substrate conversion was optimized by balancing biocatalyst loading and amino acid and formaldehyde concentrations, yielding 36-98% aldol adduct formation and 91- 98% ee for each enantiomer. Subsequent in situ follow-up chemistry via hydrogen peroxide-driven oxidative decarboxylation afforded the corresponding 2-substituted 3-hydroxycarboxylic acid derivatives.

An ADH toolbox for raspberry ketone production from natural resources via a biocatalytic cascade.

Raspberry ketone is a widely used flavor compound in food and cosmetic industry. Several processes for its biocatalytic production have already been described, but either with the use of genetically modified organisms (GMOs) or incomplete conversion of the variety of precursors that are available in nature. Such natural precursors are rhododendrol glycosides with different proportions of (R)- and (S)-rhododendrol depending on the origin. After hydrolysis of these rhododendrol glycosides, the formed rhododendrol enantiomers have to be oxidized to obtain the final product raspberry ketone. To be able to achieve a high conversion with different starting material, we assembled an alcohol dehydrogenase toolbox that can be accessed depending on the optical purity of the intermediate rhododendrol. This is demonstrated by converting racemic rhododendrol using a combination of (R)- and (S)-selective alcohol dehydrogenases together with a universal cofactor recycling system. Furthermore, we conducted a biocatalytic cascade reaction starting from naturally derived rhododendrol glycosides by the use of a glucosidase and an alcohol dehydrogenase to produce raspberry ketone in high yield. KEY POINTS: • LB-ADH, LK-ADH and LS-ADH oxidize (R)-rhododendrol • RR-ADH and ADH1E oxidize (S)-rhododendrol • Raspberry ketone production via glucosidase and alcohol dehydrogenases from a toolbox.

Handheld pH meter-assisted immunoassay for C-reactive protein using glucose oxidase-conjugated dendrimer loaded with platinum nanozymes.

A simple and feasible pH meter-based immunoassay is reported for detection of C-reactive protein (CRP) using glucose oxidase (GOD)-conjugated dendrimer loaded with platinum nanozyme. Initially, platinum nanozymes were loaded into the dendrimers through an in situ synthetic method. Then, GOD and monoclonal anti-CRP antibody with a high molar ratio were covalently conjugated onto carboxylated dendrimers via typical carbodiimide coupling. The immunoreaction was carried out with a competitive mode in a CRP-coated microplate. Along with formation of immunocomplex, the added glucose was oxidized into gluconic acid and hydrogen peroxide by GOD, and the latter was further decomposed by platinum nanozyme, thus accelerating chemical reaction in the positive direction. The produced gluconic acid changed the pH of detection solution, which was determined using a handheld pH meter. Under optimum conditions, the pH meter-based immunoassay gave a good signal toward target CRP from 0.01 to 100 ng mL-1. The limit of detection was 5.9 pg mL-1. An intermediate precision ≤ 11.2% was acquired with batch-to-batch identification. No nonspecific adsorption was observed during a series of procedures to detect target CRP, and the cross-reaction against other biomarkers was very low. Importantly, our system gave well-matched results for analysis of human serum samples relative to a referenced ELISA kit.Graphical abstract.