Low-Pass Filters for a Temperature Drift Correction Method for Electromagnetic Induction Systems.

Electromagnetic induction (EMI) systems are used for mapping the soil's electrical conductivity in near-surface applications. EMI measurements are commonly affected by time-varying external environmental factors, with temperature fluctuations being a big contributing factor. This makes it challenging to obtain stable and reliable data from EMI measurements. To mitigate these temperature drift effects, it is customary to perform a temperature drift calibration of the instrument in a temperature-controlled environment. This involves recording the apparent electrical conductivity (ECa) values at specific temperatures to obtain a look-up table that can subsequently be used for static ECa drift correction. However, static drift correction does not account for the delayed thermal variations of the system components, which affects the accuracy of drift correction. Here, a drift correction approach is presented that accounts for delayed thermal variations of EMI system components using two low-pass filters (LPF). Scenarios with uniform and non-uniform temperature distributions in the measurement device are both considered. The approach is developed using a total of 15 measurements with a custom-made EMI device in a wide range of temperature conditions ranging from 10 °C to 50 °C. The EMI device is equipped with eight temperature sensors spread across the device that simultaneously measure the internal ambient temperature during measurements. To parameterize the proposed correction approach, a global optimization algorithm called Shuffled Complex Evolution (SCE-UA) was used for efficient estimation of the calibration parameters. Using the presented drift model to perform corrections for each individual measurement resulted in a root mean square error (RMSE) of <1 mSm-1 for all 15 measurements. This shows that the drift model can properly describe the drift of the measurement device. Performing a drift correction simultaneously for all datasets resulted in a RMSE <1.2 mSm-1, which is considerably lower than the RMSE values of up to 4.5 mSm-1 obtained when using only a single LPF to perform drift corrections. This shows that the presented drift correction method based on two LPFs is more appropriate and effective for mitigating temperature drift effects.

Model-Based Correction of Temperature-Dependent Measurement Errors in Frequency Domain Electromagnetic Induction (FDEMI) Systems.

Data measured using electromagnetic induction (EMI) systems are known to be susceptible to measurement influences associated with time-varying external ambient factors. Temperature variation is one of the most prominent factors causing drift in EMI data, leading to non-reproducible measurement results. Typical approaches to mitigate drift effects in EMI instruments rely on a temperature drift calibration, where the instrument is heated up to specific temperatures in a controlled environment and the observed drift is determined to derive a static thermal apparent electrical conductivity (ECa) drift correction. In this study, a novel correction method is presented that models the dynamic characteristics of drift using a low-pass filter (LPF) and uses it for correction. The method is developed and tested using a customized EMI device with an intercoil spacing of 1.2 m, optimized for low drift and equipped with ten temperature sensors that simultaneously measure the internal ambient temperature across the device. The device is used to perform outdoor calibration measurements over a period of 16 days for a wide range of temperatures. The measured temperature-dependent ECa drift of the system without corrections is approximately 2.27 mSm-1K-1, with a standard deviation (std) of only 30 µSm-1K-1 for a temperature variation of around 30 K. The use of the novel correction method reduces the overall root mean square error (RMSE) for all datasets from 15.7 mSm-1 to a value of only 0.48 mSm-1. In comparison, a method using a purely static characterization of drift could only reduce the error to an RMSE of 1.97 mSm-1. The results show that modeling the dynamic thermal characteristics of the drift helps to improve the accuracy by a factor of four compared to a purely static characterization. It is concluded that the modeling of the dynamic thermal characteristics of EMI systems is relevant for improved drift correction.

Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation.

We previously engineered the ß-subunit of tryptophan synthase (TrpB), which catalyzes the condensation of l-serine and indole to l-tryptophan, to synthesize a range of noncanonical amino acids from l-serine and indole derivatives or other nucleophiles. Here we employ directed evolution to engineer TrpB to accept 3-substituted oxindoles and form C-C bonds leading to new quaternary stereocenters. Initially, the variants that could use 3-substituted oxindoles preferentially formed N-C bonds on N1 of the substrate. Protecting N1 encouraged evolution toward C-alkylation, which persisted when protection was removed. Six generations of directed evolution resulted in TrpB Pfquat with a 400-fold improvement in activity for alkylation of 3-substituted oxindoles and the ability to selectively form a new, all-carbon quaternary stereocenter at the γ-position of the amino acid products. The enzyme can also alkylate and form all-carbon quaternary stereocenters on structurally similar lactones and ketones, where it exhibits excellent regioselectivity for the tertiary carbon. The configurations of the γ-stereocenters of two of the products were determined via microcrystal electron diffraction (MicroED), and we report the MicroED structure of a small molecule obtained using the Falcon III direct electron detector. Highly thermostable and expressed at >500 mg/L E. coli culture, TrpB Pfquat offers an efficient, sustainable, and selective platform for the construction of diverse noncanonical amino acids bearing all-carbon quaternary stereocenters.

Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1.

Synthetic peptides derived from ethylene-insensitive protein 2 (EIN2), a central regulator of ethylene signalling, were recently shown to delay fruit ripening by interrupting protein-protein interactions in the ethylene signalling pathway. Here, we show that the inhibitory peptide NOP-1 binds to the GAF domain of ETR1 - the prototype of the plant ethylene receptor family. Site-directed mutagenesis and computational studies reveal the peptide interaction site and a plausible molecular mechanism for the ripening inhibition.

Pyrazolidine-3,5-dione-based inhibitors of phosphoenolpyruvate carboxylase as a new class of potential C4 plant herbicides.

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the C4 photosynthetic pathway of many of the world's worst weeds and a valuable target to develop C4 plant-selective herbicides. By virtual screening, analog synthesis, and in vitro validation, we identified pyrazolidine-3,5-diones as a new class of small molecules with inhibitory potential down to the submicromolar range against C4 PEPC and a selectivity factor of up to 16 over C3 PEPC. No other biological activity has yet been reported for the best compound, (3-bromophenyl)-4-(3-hydroxybenzylidene)-pyrazolidine-3,5-dione. A systematic variation in the substituents allowed the derivation of a qualitative structure-activity relationship. These findings make this compound class highly interesting for further investigations toward generating potent, C4 plant-selective herbicides with a low potential for unwanted effects.

Probing the acetaldehyde-sensitivity of 2-deoxy-ribose-5-phosphate aldolase (DERA) leads to resistant variants.

The 2-deoxy-d-ribose-5-phosphate aldolase (DERA) is a synthetically attractive enzyme because of its ability to perform CC-couplings stereoselectively, the enzyme uses acetaldehyde as nucleophile and thus produces true aldols rather than ketols, and may add two acetaldehyde molecules onto one electrophile. However, DERA produces crotonaldehyde as side reaction from acetaldehyde which is then an irreversible inhibitor forming a covalent Michael-adduct within the active site in particular with cysteine 47 (Dick et al., 2016). This inhibition can be resolved by mutating C47 to non-nucleophile amino acids. Still, the inhibition is not an on-off-feature and the present mutagenesis study illustrates that there must be a C47-independent inactivation mechanism. As a practical result: The virtually fully resistant mutant C47L was found, which shows no loss in stereoselectivity, - this renders this variant as promising catalyst.

Immobilization of 2-Deoxy-d-ribose-5-phosphate Aldolase in Polymeric Thin Films via the Langmuir-Schaefer Technique.

A synthetic protocol for the fabrication of ultrathin polymeric films containing the enzyme 2-deoxy-d-ribose-5-phosphate aldolase from Escherichia coli (DERAEC) is presented. Ultrathin enzymatically active films are useful for applications in which only small quantities of active material are needed and at the same time quick response and contact times without diffusion limitation are wanted. We show how DERA as an exemplary enzyme can be immobilized in a thin polymer layer at the air-water interface and transferred to a suitable support by the Langmuir-Schaefer technique under full conservation of enzymatic activity. The polymer in use is a poly(N-isopropylacrylamide-co-N-2-thiolactone acrylamide) (P(NIPAAm-co-TlaAm)) statistical copolymer in which the thiolactone units serve a multitude of purposes including hydrophobization of the polymer, covalent binding of the enzyme and the support and finally cross-linking of the polymer matrix. The application of this type of polymer keeps the whole approach simple as additional cocomponents such as cross-linkers are avoided.

Trading off stability against activity in extremophilic aldolases.

Understanding enzyme stability and activity in extremophilic organisms is of great biotechnological interest, but many questions are still unsolved. Using 2-deoxy-D-ribose-5-phosphate aldolase (DERA) as model enzyme, we have evaluated structural and functional characteristics of different orthologs from psychrophilic, mesophilic and hyperthermophilic organisms. We present the first crystal structures of psychrophilic DERAs, revealing a dimeric organization resembling their mesophilic but not their thermophilic counterparts. Conversion into monomeric proteins showed that the native dimer interface contributes to stability only in the hyperthermophilic enzymes. Nevertheless, introduction of a disulfide bridge in the interface of a psychrophilic DERA did confer increased thermostability, suggesting a strategy for rational design of more durable enzyme variants. Constraint network analysis revealed particularly sparse interactions between the substrate pocket and its surrounding α-helices in psychrophilic DERAs, which indicates that a more flexible active center underlies their high turnover numbers.

Mechanism-based inhibition of an aldolase at high concentrations of its natural substrate acetaldehyde: structural insights and protective strategies.

2-Deoxy-d-ribose-5-phosphate aldolase (DERA) is used in organic synthesis for the enantioselective reaction between acetaldehyde and a broad range of other aldehydes as acceptor molecules. Nevertheless, its application is hampered by a poor tolerance towards high concentrations of acetaldehyde, its natural substrate. While numerous studies have been performed searching for new, more acetaldehyde-resistant DERAs, the mechanism underlying this deactivation process has remained elusive. By using NMR spectroscopy on both the protein and the small-molecule scale, we could show that a reaction product binds to the inner part of the enzyme, and that this effect can be partly reversed via heating. The crystal structure of DERA before and after acetaldehyde incubation was determined at high resolution, revealing a covalently bound reaction product bridging the catalytically active lysine (K167) to a nearby cysteine (C47) in the deactivated enzyme. A reaction mechanism is proposed where crotonaldehyde as the aldol product of two acetaldehyde molecules after water elimination forms a Schiff base with the lysine side chain, followed by Michael addition of the cysteine thiol group to the Cß atom of the inhibitor. In support of this mechanism, direct incubation of DERA with crotonaldehyde results in a more than 100-fold stronger inhibition, compared to acetaldehyde, whereas mutation of C47 gives rise to a fully acetaldehyde-resistant DERA. Thus this variant appears perfectly suited for synthetic applications. A similar diagnostic and preventive strategy should be applicable to other biocatalysts suffering from mechanism-based inhibition by a reactive substrate, a condition that may be more common than currently appreciated in biotechnology.