Stable Enzyme Research Articles

A Thermostable Heme Protein Fold Adapted for Stereoselective C-H Bond Hydroxylation Using Peroxygenase Activity.

Thermostable protein folds of natural and synthetic origin are highly sought-after templates for biocatalyst generation due to their enhanced stability to elevated temperatures which overcomes one of the major limitations of applying enzymes for synthesis. Cytochrome P450 enzymes (CYPs) are a family of heme-thiolate monooxygenases that catalyse the oxidation of their substrates in a highly stereo- and regio-selective manner. The CYP enzyme (CYP107PQ1) from the thermophilic bacterium Meiothermus ruber binds the norisoprenoid β-ionone and was employed as a scaffold for catalyst design. The I-helix was modified to convert this enzyme from a monooxygenase into a peroxygenase (CYP107PQ1QE), enabling the enantioselective oxidation of β-ionone to (S)-4-hydroxy-β-ionone (94 % e.e.). The enzyme was resistant to 20 mM H2O2, 20 % (v/v) of organic solvent, supported over 1700 turnovers and was fully functional after incubation at 60 °C for 1 h and 30 °C for 365 days. The reaction was scaled-up to generate multi milligram quantities of the product for characterisation. Overall, we demonstrate that sourcing a CYP protein fold from an extremophile enabled the design of a highly stable enzyme for stereoselective C-H bond activation only using H2O2 as the oxidant, providing a viable strategy for future biocatalyst design.

Preclinical efficacy and safety of adeno-associated virus 5 alpha-galactosidase A gene therapy for Fabry disease

We developed a novel adeno-associated virus 5 gene therapy (AAV5-GLA) expressing human alpha-galactosidase A (GLA) under the control of a novel, small and strong, liver-restricted promoter. We assessed the preclinical potential of AAV5-GLA for treating Fabry disease, an X-linked hereditary metabolic disorder resulting from mutations in the gene encoding alpha-galactosidase A that lead to accumulation of the substrates globotriaosylceramide and globotriaosylsphingosine, causing heart, kidney, and central nervous system dysfunction. Effects of intravenously administered AAV5-GLA were evaluated in 1) alpha-galactosidase A-knockout mice aged 7–8 weeks (early in disease) and 20 weeks (nociception phenotype manifestation) and 2) cynomolgus macaques during an 8-week period. In both species, AAV5-GLA was observed as safe, generated detectable vector DNA and mRNA levels in liver, and produced stable enzyme activity in liver and plasma. In mice, dose-dependent transgene enzyme activity, cross-correction (substrate reduction) in kidney and heart, and improved nociception lasted over 6 months. Moreover, after delayed administration when animals displayed the nociception phenotype, target organ enzyme activity was present, and accumulated substrates were reduced. Given the strong, durable expression of active GLA with this promoter and favorable profile of adeno-associated virus-based gene therapy in humans, AAV5-GLA warrants further investigation in clinical trials for Fabry disease.

Manufacturing of the highly active thermophile PETases PHL7 and PHL7mut3 using Escherichia coli

BackgroundThe global plastic waste crisis requires combined recycling strategies. One approach, enzymatic degradation of PET waste into monomers, followed by re-polymerization, offers a circular economy solution. However, challenges remain in producing sufficient amounts of highly active PET-degrading enzymes without costly downstream processes.ResultsUsing the growth-decoupled enGenes eX-press V2 E. coli strain, pH, induction strength and feed rate were varied in a factorial-based optimization approach, to find the best-suited production conditions for the PHL7 enzyme. This led to a 40% increase in activity of the fermentation supernatant. Optimization of the expression construct resulted in a further 4-fold activity gain. Finally, the identified improvements were applied to the production of the more active and temperature stable enzyme variant, PHL7mut3. The unpurified fermentation supernatant of the PHL7mut3 fermentation was able to completely degrade our PET film sample after 16 h of incubation at 70 °C at an enzyme loading of only 0.32 mg enzyme per g of PET.ConclusionsIn this research, we present an optimized process for the extracellular production of thermophile and highly active PETases PHL7 and PHL7mut3, eliminating the need for costly purification steps. These advancements support large-scale enzymatic recycling, contributing to solving the global plastic waste crisis.

B-295 Sensitive and Rapid Homogeneous Immunoassay for the Detection of Zolpidem and its Major Metabolite in Urine

Abstract Background Zolpidem, a schedule IV controlled substance under the Controlled Substances Act, is commonly known as Ambien and Ambien CR, a prescription only treatment for insomnia. Zolpidem is one of the most common prescribed sleep aids in the U.S. due to its rapid sedative effects. The recommended duration of treatment is between two days and four weeks, as long-term use of zolpidem increases the risk of habit formation. Addiction to zolpidem can cause severe side effects, including memory loss, delusion, and in extreme cases, fatal overdose. From 2005-2010, Zolpidem-related emergency room visits doubled, underscoring the need for accurate testing as zolpidem testing plays an important role in cases of misuse, diversion, and forensics. The sole commercially available homogeneous immunoassay for the detection of zolpidem in urine has a relatively high cutoff at 20 ng/mL and poor cross-reactivity (0.02%) to major metabolite zolpidem 4-carboxylic acid. ARK Diagnostics has developed the ARK™ Zolpidem Assay to detect zolpidem at a cutoff concentration of 10 ng/mL with high cross-reactivity to its metabolite, Zolpidem phenyl 4-carboxylic acid and no cross-reactivity to zaleplon and zopiclone at concentrations below 100,000 ng/mL. Methods The ARK™ Zolpidem Assay is a liquid stable homogeneous enzyme immunoassay, consisting of two reagents, with a cutoff concentration of 10 ng/mL and semi-quantitative range up to 40 ng/mL. Preliminary performance characterization for this assay was evaluated on the Beckman Coulter AU680 Automated Clinical Chemistry Analyzer. Precision, analytical recovery, specificity, Histogram Overlap Analysis of ±50% controls and the cutoff, and method comparison with LC-MS/MS were evaluated. Results In semi-quantitative mode, total precision ranged from 2.6 to 3.1% CV. Spiked recovery of zolpidem ranged from 88.6% (2.5 ng/mL) to 96.8% (10 ng/mL). The major metabolite, zolpidem phenyl-4-carboxylic acid, showed an approximate equivalence to the 10 ng/mL zolpidem cutoff at 30 ng/mL (33.3% cross-reactivity). Histogram overlap analysis showed no overlap between cutoff and control levels. Method correlation with LC-MS/MS using authentic urine samples showed an excellent agreement with a specificity of 100% and sensitivity of 100% (15 positives and 100 negatives). Conclusions The ARK™ Zolpidem Assay measures zolpidem and its major metabolite zolpidem phenyl-4-carboxylic acid in human urine with acceptable performance. The assay is sensitive, rapid, and applicable to a wide range of clinical chemistry analyzers.

Novel Insights into Enzymatic Thermostability: The “Short Board” Theory and Zero‐Shot Hamiltonian Model

AbstractUnderstanding the mechanism underlying thermostabilization in naturally stable enzymes and enhancing the thermostability of unstable enzymes are crucial aspects in enzyme engineering. Despite the development of various engineering methods, there remains substantial scope for improvement. In this study, a novel concept termed as the “short board” theory is proposed, which conceptualizes proteins as barrels with each component representing a jagged board. Notably, optimizing modifications to the shortest board yields optimal enhancements in terms of thermostability performance. To validate this theory, α‐amylase, an industrial bulk enzyme with multiple domains, is employed as a model enzyme. The existence of “short boards” and their impact on thermostability modification are demonstrated at the domain, residue, and atomic levels through experimental confirmation using domain substitution. Furthermore, a novel thermostable design and prediction model called Zero‐Shot Hamiltonian (ZSH) is established and evaluated on α‐amylase. This coevolutionary approach based on thermostability and deep learning exhibits remarkable success exclusively when applied to enzymes with fixed short boards. The integration of the “short board” theory with the ZSH model presents an innovative tool for enhancing enzymatic thermostability.

Thermophilic Hemicellulases Secreted by Microbial Consortia Selected from an Anaerobic Digester.

The rise of agro-industrial activities over recent decades has exponentially increased lignocellulose biomasses (LCB) production. LCB serves as a cost-effective source for fermentable sugars and other renewable chemicals. This study explores the use of microbial consortia, particularly thermophilic consortia, for LCB deconstruction. Thermophiles produce stable enzymes that retain activity under industrial conditions, presenting a promising approach for LCB conversion. This research focused on two microbial consortia (i.e., microbiomes) that were analyzed for enzyme production using a cheap medium, i.e., a mixture of spent mushroom substrate (SMS) and digestate. The secreted xylanolytic enzymes were characterized in terms of temperature and pH optima, thermal stability, and hydrolysis products from LCB-derived polysaccharides. These enzymes showed optimal activity aligning with common biorefinery conditions and outperformed a formulated enzyme mixture in thermostability tests in the digestate. Phylogenetic and genomic analyses highlighted the genetic diversity and metabolic potential of these microbiomes. Bacillus licheniformis was identified as a key species, with two distinct strains contributing to enzyme production. The presence of specific glycoside hydrolases involved in the cellulose and hemicellulose degradation underscores these consortia's capacity for efficient LCB conversion. These findings highlight the potential of thermophilic microbiomes, isolated from an industrial environment, as a robust source of robust enzymes, paving the way for more sustainable and cost-effective bioconversion processes in biofuel and biochemical production and other biotechnological applications.

Multilevel Systematic Optimization To Achieve Efficient Integrated Expression of Escherichia coli.

Genomic integration of heterologous genes is the preferred approach in industrial fermentation-related strains due to the drawbacks associated with plasmid-mediated microbial fermentation, including additional growth burden, genetic instability, and antibiotic contamination. Synthetic biology and genome editing advancements have made gene integration convenient. Integrated expression is extensively used in the field of biomanufacturing and is anticipated to become the prevailing method for expressing recombinant proteins. Therefore, it is pivotal to strengthen the expression of exogenous genes at the genome level. Here, we systematically optimized the integrated expression system of Escherichia coli from 3 aspects. First, the integration site slmA with the highest expression activity was screened out of 18 sites in the ORI region of the E. coli BL21 (DE3) genome. Second, we characterized 16 endogenous promoters in E. coli and combined them with the T7 promoter. A constitutive promoter, Plpp-T7, exhibited significantly higher expression strength than the T7 promoter, achieving a 3.3-fold increase in expression levels. Finally, to further enhance the T7 expression system, we proceeded with overexpression of T7 RNA polymerase at the chassis cell level. The resulting constitutive efficient integrated expression system (CEIES_Ecoli) showed a 2-fold increase in GFP expression compared to the pET3b recombinant plasmid. Therefore, CEIES_Ecoli was applied to the integrated expression of nitrilase and hyaluronidase, achieving stable and efficient enzyme expression, with enzyme activities of 22.87 and 12,195 U·mL-1, respectively, comparable to plasmid levels. Overall, CEIES_Ecoli provides a stable and efficient method of gene expression without the need for antibiotics or inducers, making it a robust tool for synthetic biology, enzyme engineering, and related applications.

Ultrathin Film Antimony-Doped Tin Oxide Prevents [NiFe] Hydrogenase Inactivation at High Electrode Potentials.

For hydrogenases to serve as effective electrocatalysts in hydrogen biotechnological devices, such as enzymatic fuel cells, it is imperative to design electrodes that facilitate stable and functional enzyme immobilization, efficient substrate accessibility, and effective interfacial electron transfer. Recent years have seen considerable advancements in this area, particularly concerning hydrogenases. However, a significant limitation remains: the inactivation of hydrogenases at high oxidative potentials across most developed electrodes. Addressing this issue necessitates a thorough understanding of the interactions between the enzyme and the electrode surface. In this study, we employ ATR-IR spectroscopy combined with electrochemistry in situ to investigate the interaction mechanisms, electrocatalytic behavior, and stability of the oxygen-tolerant membrane-bound [NiFe] hydrogenase from Cupriavidus necator (MBH), which features a His-tag on its small subunit C-terminus. Antimony-doped tin oxide (ATO) thin films were selected as electrodes due to their protein compatibility, suitable potential window, conductivity, and transparency, making them an ideal platform for spectroelectrochemical measurements. Our comprehensive examination of the physiological and electrochemical processes of [NiFe] MBH on ATO thin film electrodes demonstrates that by tuning the electron transport properties of the ATO thin film, we can prevent MBH inactivation at extended oxidative potentials while maintaining direct electron transfer between the enzyme and the electrode.

Identification, screening and molecular mechanisms of natural stable angiotensin-converting enzyme (ACE) inhibitory peptides from foxtail millet protein hydrolysates: a combined in silico and in vitro study.

The stability of bioactive peptides under various food processing conditions is the basis for their use in industrial manufacturing. This study aimed to identify natural ACE inhibitors with excellent stability and investigate their physicochemical properties and putative molecular mechanisms. Five novel ACE inhibitory peptides (QDPLFPL, FPGVSPF, SPAQLLPF, LVPYRP, and WYWPQ) were isolated and identified using RP-HPLC and Nano LC-MS/MS with foxtail millet protein hydrolysates as the raw material. These peptides are non-toxic and exhibit strong ACE inhibitory activity in vitro (IC50 values between 0.13 mg mL-1 and 0.56 mg mL-1). In addition to QDPLFPL, FPGVSPF, SPAQLLPF, LVPYRP, and WYWPQ have excellent human intestinal absorption. Compared to FPGVSPF and SPAQLLPF, the stable helical structure of LVPYRP and WYWPQ allows them to maintain high stability under conditions that mimic gastrointestinal digestion and various food processing (temperatures, pH, sucrose, NaCl, citric acid, sodium benzoate, Cu2+, Zn2+, K+, Mg2+, Ca2+). The results of molecular docking and molecular dynamics simulation suggest that LVPYRP has greater stability and binding capacity to ACE than WYWPQ. LVPYRP might attach to the active pockets (S1, S2, and S1') of ACE via hydrogen bonds and hydrophobic interactions, then compete with Zn2+ in ACE to demonstrate its ACE inhibitory activity. The binding of LVPYRP to ACE enhances the rearrangement of ACE's active structural domains, with electrostatic and polar solvation energy contributing the most energy to the binding. Our findings suggested that LVPYRP derived from foxtail millet protein hydrolysates has the potential to be incorporated into functional foods to provide antihypertensive benefits.

Computational stabilization of a non-heme iron enzyme enables efficient evolution of new function.

Directed evolution has emerged as a powerful tool for engineering new biocatalysts. However, introducing new catalytic residues can be destabilizing, and it is generally beneficial to start with a stable enzyme parent. Here we show that the deep learning-based tool ProteinMPNN can be used to redesign Fe(II)/αKG superfamily enzymes for greater stability, solubility, and expression while retaining both native activity and industrially-relevant non-native functions. For the Fe(II)/αKG enzyme tP4H, we performed site-saturation mutagenesis with both the wild-type and stabilized design variant and screened for activity increases in a non-native C-H hydroxylation reaction. We observed substantially larger increases in non-native activity for variants obtained from the stabilized scaffold compared to those from the wild-type enzyme. ProteinMPNN is user-friendly and widely-accessible, and straightforward structural criteria were sufficient to obtain stabilized, catalytically-functional variants of the Fe(II)/αKG enzymes tP4H and GriE. Our work suggests that stabilization by computational sequence redesign could be routinely implemented as a first step in directed evolution campaigns for novel biocatalysts.

A diverse proteome is present and enzymatically active in metabolite extracts

Metabolite extraction is the critical first-step in metabolomics experiments, where it is generally regarded to inactivate and remove proteins. Here, arising from efforts to improve extraction conditions for polar metabolomics, we discover a proteomic landscape of over 1000 proteins within metabolite extracts. This is a ubiquitous feature across several common extraction and sample types. By combining post-resuspension stable isotope addition and enzyme inhibitors, we demonstrate in-extract metabolite interconversions due to residual transaminase activity. We extend these findings with untargeted metabolomics where we observe extensive protein-mediated metabolite changes, including in-extract formation of glutamate dipeptide and depletion of total glutathione. Finally, we present a simple extraction workflow that integrates 3 kDa filtration for protein removal as a superior method for polar metabolomics. In this work, we uncover a previously unrecognized, protein-mediated source of observer effects in metabolomics experiments with broad-reaching implications across all research fields using metabolomics and molecular metabolism.

Efficient and rapid digestion of proteins with a dual-enzyme microreactor featuring 3-D pores formed by dopamine/polyethyleneimine/acrylamide-coated KIT-6 molecular sieve

The microreactor with two types of immobilized enzymes, exhibiting excellent orthogonal performance, represents an effective approach to counteract the reduced digestion efficiency resulting from the absence of a single enzyme cleavage site, thereby impacting protein identification. In this study, we developed a hydrophilic dual-enzyme microreactor characterized by rapid mass transfer and superior enzymatic activity. Initially, we selected KIT-6 molecular sieve as the carrier for the dual-IMER due to its three-dimensional network pore structure. Modification involved co-deposition of polyethyleneimine (PEI) and acrylamide (AM) as amine donors, along with dopamine to enhance material hydrophilicity. Remaining amino and double bond functional groups facilitated stepwise immobilization of trypsin and Glu-C. Digestion times for bovine serum albumin (BSA) and bovine hemoglobin (BHb) on the dual-IMER were significantly reduced compared to solution-based digestion (1 min vs. 36 h), resulting in improved sequence coverage (91.30% vs. 82.7% for BSA; 90.24% vs. 89.20% for BHb). Additionally, the dual-IMER demonstrated excellent durability, retaining 96.08% relative activity after 29 reuse cycles. Enhanced protein digestion efficiency can be attributed to several factors: (1) KIT-6’s large specific surface area, enabling higher enzyme loading capacity; (2) Its three-dimensional network pore structure, facilitating faster mass transfer and substance diffusion; (3) Orthogonality of trypsin and Glu-C enzyme cleavage sites; (4) The spatial effect introduced by the chain structure of PEI and glutaraldehyde’s spacing arm, reducing spatial hindrance and enhancing enzyme–substrate interactions; (5) Mild and stable enzyme immobilization. The KIT-6-based dual-IMER offers a promising technical tool for protein digestion, while the PDA/PEI/AM-KIT-6 platform holds potential for immobilizing other proteins or active substances.

Codon optimization, expression, and purification of the reverse transcriptase enzyme variant MMLV RT2 in <i>Escherichia coli</i>

Moloney leukemia virus reverse transcriptase (MMLV RT) is an important enzyme widely utilized in molecular biology research and various diagnostic testing kits. A previous study demonstrated that variants of MMLV RT containing specific mutations contribute to enhanced stability and increased capability for RNA detection. However, the optimization and increased expression level of MMLV RT variants for large-scale production have not been extensively studied. In this research, the gene segment encoding the MMLV RT variant (E286R/E302K/L435R/D524A) was codon optimized and subcloned into pET-M expression vector. The obtained pET-M MMLV RT2 was transformed into E. coli BL21(DE3) host cell system for over-expression of the MMLV RT2 enzyme. The recombinant enzyme was then purified using nickel affinity chromatography and quantified using the Bradford method. The results revealed significant increase in recombinant enzyme yield, achieving 0.25 g/L in the culture medium. This represents a 1.4-fold increase compared to the 0.175 g/L yield reported in the previous study. Enzyme MMLV RT2 was also evaluated for its activity and was found to have comparable activity to the commercial enzyme Promega and another variant, enzyme MMLV RT1, which carries the D524A mutation and has previously been evaluated for activity. This result suggests that a high expression level of a stable MMLV-RT enzyme variant can be achieved which is beneficial for further improving the production of this important enzyme. The high yield and sustained activity of the MMLV RT2 variant indicate its potential as a reliable and cost-effective alternative for various applications in research and diagnostics. The successful optimization and expression in E. coli highlight the feasibility of large-scale production, potentially lowering costs and improving accessibility for molecular biology applications.

Activation mechanism of fatty acids on membrane-bound polyphenol oxidase in peach fruit: Conformational change analysis and molecular docking simulation

Polyphenol oxidase (PPO) is the main enzyme initiating internal browning in peach fruit stored at low temperature. Previous studies showed fatty acids activated PPO, but its specific effect and mechanism of activation on PPO are still not clear. Thus, the effects of fatty acids on the biochemical properties and structural conformation of PPO were studied, with sodium dodecyl sulfate used as comparison. The highest degree of activation (5 folds) of membrane-bound PPO (mPPO) was found with 2.2 mM linoleic acid (LA) and its optimal activity was observed also around pH 7.0. Further structure analysis showed LA led to an increase of α-helix content of mPPO from 23.53% to 52.33%; the maximum emission wavelength of treated mPPO shifted from 338 to 340 nm with the fluorescence intensity increased from 446 to 510. LA also can form hydrogen bonds and hydrophobic interactions with mPPO. These results indicated that LA could contribute to a more stable and open enzyme structure, thus activated mPPO. The results will provide more information for PPO activation that highly probably occurs in postharvest peach fruit and its relationship with internal browning.

Metformin-induced and Mitochondrial Stress-mediated Apoptosis in Schizosaccharomyces pombe

Metformin, a widely used first-line medication in the treatment of type II diabetes, has been proposed to have a second indication in the treatment of cancers and aging. However, its accounting mechanisms in cellular physiology were not clearly understood. Therefore, its cytotoxicity and underlying physiological mechanisms should be explained. Schizosaccharomyces pombe was evaluated as a single-cell cytotoxicity model and was treated with metformin and grown on YEL media at 30 °C and 180 rpm. 0,1-20 mM metformin caused dose-dependent apoptosis and necrosis demonstrated by using Annexin V-FITC/PI and DAPI staining. Surprisingly, metformin reduced ROS levels with stable antioxidant enzyme levels, but the mitochondrial transmembrane potential was significantly increased indicating a differential regulation by the dual character of metformin. In addition, a possible role can be attributed to Cnx1 in apoptotic cell death; which showed a dramatic increase in transcription, however, three other potential apoptotic genes, Rad9, Pca1, and Aif1 were stable. To conclude, the dual effect of metformin was clarified, and related cellular physiological effects with accompanying mechanisms (particularly Cnx1-mediated) were shown using S. pombe.

Physiologically-based pharmacokinetic models versus allometric scaling for prediction of tyrosine-kinase inhibitor exposure from adults to children

PurposeModel-based methods can predict pediatric exposure and support initial dose selection. The aim of this study was to evaluate the performance of allometric scaling of population pharmacokinetic (popPK) versus physiologically based pharmacokinetic (PBPK) models in predicting the exposure of tyrosine kinase inhibitors (TKIs) for pediatric patients (≥ 2 years), based on adult data. The drugs imatinib, sunitinib and pazopanib were selected as case studies due to their complex PK profiles including high inter-patient variability, active metabolites, time-varying clearances and non-linear absorption.MethodsPediatric concentration measurements and adult popPK models were derived from the literature. Adult PBPK models were generated in PK-Sim® using available physicochemical properties, calibrated to adult data when needed. PBPK and popPK models for the pediatric populations were translated from the models for adults and were used to simulate concentration-time profiles that were compared to the observed values.ResultsTen pediatric datasets were collected from the literature. While both types of models captured the concentration-time profiles of imatinib, its active metabolite, sunitinib and pazopanib, the PBPK models underestimated sunitinib metabolite concentrations. In contrast, allometrically scaled popPK simulations accurately predicted all concentration-time profiles. Trough concentration (Ctrough) predictions from the popPK model fell within a 2-fold range for all compounds, while 3 out of 5 PBPK predictions exceeded this range for the imatinib and sunitinib metabolite concentrations.ConclusionBased on the identified case studies it appears that allometric scaling of popPK models is better suited to predict exposure of TKIs in pediatric patients ≥ 2 years. This advantage may be attributed to the stable enzyme expression patterns from 2 years old onwards, which can be easily related to adult levels through allometric scaling. In some instances, both methods performed comparably. Understanding where discrepancies between the model methods arise, can further inform model development and ultimately support pediatric dose selection.

Controllable radical polymerization of TEMPO redox for stable and sensitive enzyme electrode interface

Assembling functional molecules on the surface of an enzyme electrode is the most basic technique for constructing a biosensor. However, precise control of electron transfer interface or electron mediator on the electrode surface remains a challenge, which is a key step that affects the stability and sensitivity of enzyme-based biosensors. In this study, we propose the use of controllable free radical polymerization to grow stable 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) polymer as electron mediator on enzyme surface for the first time. Through scanning electron microscopy (SEM), Raman spectroscopy, electrode surface coverage measurement, static contact angle (SCA), and a series of electrochemical methods, it has been demonstrated that the TEMPO-based enzyme electrode exhibits a uniform hydrophilic morphology and stable electrochemical performance. Furthermore, the results show that the sensor demonstrates high sensitivity for detecting glucose biomolecules in artificial sweat and serum. Attributing to the quantitative and controllable radical polymerization of TEMPO redox assembled enzyme electrode surface, the as-proposed biosensor providing a use, storage, and inter-batch sensing stability, providing a vital platform for wearable/implantable biochemical sensors.

Construction and characterization of a novel fusion alginate lyase with endolytic and exolytic cleavage activity for industrial preparation of alginate oligosaccharides

Alginate lyases with high activity and good thermostability are lacking for the preparation of alginate oligosaccharides (AOS) with various biological activities. We constructed a fusion alginate lyase with both endo-and exo-activities. AlyRm6A-Zu7 was successfully constructed by connecting the highly thermostable AlyRm6A to a new exotype lyase, AlyZu7. The fusion enzyme exhibited high catalytic activity and thermostability. It transformed sodium alginate into oligosaccharides with degrees of polymerization (DP) of 2–4 while producing 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). The maximum reducing sugar, AOS, and DP1 + DEH yields were 75 %, 45 %, and 40 %, respectively. Molecular docking confirmed the formation of a stable complex between the substrate and AlyRm6A-Zu7. Protein interactions increased the thermostability of AlyZu7. This work provides new insights into the industrial formation of AOS and monosaccharide DEH using thermally stable fusion enzymes, which has a positive effect in the fields of functional oligosaccharide production and biofuel formation.

Simultaneous ocean acidification and warming do not alter the lipid-associated biochemistry but induce enzyme activities in an asterinid starfish

Ocean acidification and warming affect marine ecosystems from the molecular scale in organismal physiology to broad alterations of ecosystem functions. However, knowledge of their combined effects on tropical-subtropical intertidal species remains limited. Pushing the environmental range of marine species away from the optimum initiates stress impacting biochemical metabolic characteristics, with consequences on lipid-associated and enzyme biochemistry. This study investigates lipid-associated fatty acids (FAs) and enzyme activities involved in biomineralization of the tropical-subtropical starfish Aquilonastra yairi in response to projected near-future global change. The starfish were acclimatized to two temperature levels (27 °C, 32 °C) crossed with three pCO2 concentrations (455 μatm, 1052 μatm, 2066 μatm). Total lipid (ΣLC) and FAs composition were unaffected by combined elevated temperature and pCO2, but at elevated temperature, there was an increase in ΣLC, SFAs (saturated FAs) and PUFAs (polyunsaturated FAs), and a decrease in MUFAs (monounsaturated FAs). However, temperature was the sole factor to significantly alter SFAs composition. Positive parabolic responses of Ca-ATPase and Mg-ATPase enzyme activities were detected at 27 °C with elevated pCO2, while stable enzyme activities were observed at 32 °C with elevated pCO2. Our results indicate that the lipid-associated biochemistry of A. yairi is resilient and capable of coping with near-future ocean acidification and warming. However, the calcification-related enzymes Ca-ATPase and Mg-ATPase activity appear to be more sensitive to pCO2/pH changes, leading to vulnerability concerning the skeletal structure.

Two Fluorescent Probes for Recognition of Acetylcholinesterase: Design, Synthesis, and Comparative Evaluation.

In this study, two "on-off" probes (BF2-cur-Ben and BF2-cur-But) recognizing acetylcholinesterase (AChE) were designed and synthesized. The obtained probes can achieve recognition of AChE with good selectivity and pH-independence with a linear range of 0.5~7 U/mL and 0.5~25 U/mL respectively. BF2-cur-Ben has a lower limit of detection (LOD) (0.031 U/mL), higher enzyme affinity (Km = 16 ± 1.6 μM), and higher inhibitor sensitivity. A responsive mechanism of the probes for AChE was proposed based on HPLC and mass spectra (MS) experiments, as well as calculations. In molecular simulation, BF2-cur-Ben forms more hydrogen bonds (seven, while BF2-cur-But has only four) and thus has a more stable enzyme affinity, which is mirrored by the results of the comparison of Km values. These two probes could enable recognition of intracellular AChE and probe BF2-cur-Ben has superior cell membrane penetration due to its higher log p value. These probes can monitor the overexpression of AChE during apoptosis of lung cancer cells. The ability of BF2-cur-Ben to monitor AChE in vivo was confirmed by a zebrafish experiment.