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In this work, lipase from Candida rugosa was bound with the multi-covalent bonds attachment to the amino-functionalized magnetic Fe3O4 nanoparticles by glutaraldehyde as a coupling agent. The protein assay showed that the enzyme binding efficiency was 77.65% in the immobilization process. The catalytic activity of the immobilized lipase was compared with the free lipase in enzymatic epoxidation of free fatty acids. The results showed that the immobilized enzyme had better stability than the free system during the reaction in the presence of hydrogen peroxide, as an inactivating substrate, especially at extreme conditions of temperature and pH. The statistical study on effects of temperature and pH by response surface methodology specified that the highest epoxidation activity for the immobilized system was observed at temperature 52.2 °C and pH of 6.7. At the optimum condition, the immobilized lipase showed a good reusability, where 100% and 80.97% of origin catalytic epoxidation activity were, respectively, maintained after sixth and tenth cycles. Also, the storage stability of the immobilized lipase was investigated in room temperate and 4 °C. The experiments revealed that the immobilized lipase had an excellent long-term storage stability after 152 and 203 days at 4 °C after being bound to magnetic nanoparticles.

Holliday junction (HJ) resolution by its resolving enzymes is essential for chromosome segregation and recombination-mediated DNA repair. HJs undergo two types of structural dynamics that determine the outcome of recombination: conformer exchange between two isoforms and branch migration. However, it is unknown how the preferred branch-point and conformer are achieved between enzyme binding and HJ resolution given the extensive binding interactions seen in static crystal structures. Single molecule fluorescence resonance energy transfer analysis of resolving-enzymes from bacteriophages (T7 endonuclease I), bacteria (RuvC), fungi (GEN1) and humans (hMus81-Eme1) showed that both types of HJ dynamics still occur after enzyme binding. These dimeric enzymes use their multivalent interactions to achieve this, going through a partially-dissociated intermediate in which the HJ undergoes nearly unencumbered dynamics. This evolutionarily conserved property of HJ resolving-enzymes provides previously unappreciated insight on how junction resolution, conformer exchange and branch migration may be coordinated.

Ultrasensitive determination of some ultra-low abundance biological molecules closely related to diseases is currently a wide concern and urgent issue to be addressed. Here, a spherical poly(acrylic acid)–alkaline phosphatase (SP–AKP) signal amplification block using spherical poly(acrylic acid) brush nanoparticles (SP) as the immobilized carriers was designed and synthesized optimally first. The results show that a single SP–AKP with high enzyme binding capacity and high catalytic ability (up to about 4800 effective free AKP per SP–AKP) has much greater fluorescence signal amplification ability than a single free AKP or SiO2–COOH–AKP. Then, a droplet generation microfluidic chip was prepared successfully, and the SP–AKP was loaded and confined in a 14 pL droplet by adjusting its concentration to ensure at most one SP–AKP was encapsulated in each droplet according to Poisson's theory. Finally, the fluorescence signals produced by 4-methylumbelliferyl phosphate (4-MUP) catalyzed via SP–AKP within 6 min were sufficient to be detected by a fluorescence microscope. Thus, the digital signal distribution of “1/0” (signal/background) was obtained, making this SP–AKP signal amplification block a promising enzyme label for potential high sensitivity digital biological detection applications.

Objective. To predict and explore the potential mechanism of Yinchensini decoction (YCSND) based on systemic pharmacology. Method. TCMSP database was searched for the active constituents and related target proteins of YCSND. Cytoscape 3.5.1 was used to construct the active ingredient-target interaction of YCSND and network topology analysis, with STRING online database for protein-protein interaction (PPI) network construction and analysis; and collection from the UniProt database of target protein gene name, with the DAVID database for the gene ontology (GO) functional analysis, KEGG pathway enrichment analysis mechanism and targets of YCSND. Results. The results indicate the core compounds of YCSND, namely, kaempferol, 7-Methoxy-2-methyl isoflavone, and formononetin. And its core targets are prostaglandin G/H synthase 2, estrogen receptor, Calmodulin, heat shock protein HSP 90, etc. PPI network analysis shows that the key components of the active ingredients of YCSND are JUN, TP53, MARK1, RELA, MYC, and so on. The results of the GO analysis demonstrate that extracellular space, cytosol, and plasma membrane are the main cellular components of YCSND. Its molecular functions are mainly acting on enzyme binding, protein heterodimerization activity, and drug binding. The biological process of YCSND is focused on response to drug, positive regulation of transcription from RNA polymerase II promoter, the response to ethanol, etc. KEGG results suggest that the pathways, including pathways in cancer, hepatitis B, and pancreatic cancer, play a key role in YCSND. Conclusion. YCSND exerts its drug effect through various signaling pathways and acts on kinds of targets. By system pharmacology, the potential role of drugs and the mechanism of action can be well predicted.

Although many studies have investigated the enzyme susceptibility of bean starches, the factors that reduce the lower hydrolysis of bean starch are not well understood. In the present study, we isolated starches from eight commercial bean varieties and established a causal relationship between micro and macro-structure with α-amylase susceptibility. The in vitro starch digestion kinetic profiles were monitored at a fixed enzyme concentration, and the data were fitted into the Logarithm of Slope model to obtain first-order rate coefficients and extent of digestion. Among eight varieties, pinto bean starch showed the lowest digestion rate (0.0013 min−1) and extent (40.7%), whereas red kidney bean starch has the highest rate (0.0042min−1) and extent (69.2%). In order to probe the interdependability of digestion kinetic parameters with starch microstructure, the enzyme binding capacity, molecular size, crystalline structure as well as thermal parameters of eight bean starches were correlated as Pearson's matrix. Whilst, significant correlation was not observed between digestion rate/extent and the structural parameters, the residual enzyme activity after binding with starch granules at non-hydrolyzing condition had significant negative correlation with the digestion rate and extent. Similarly, positive correlation was observed for degree of starch damage and enzyme susceptibility. Thus, it is concluded that for bean starches with similar biological origins, the available sites for initial binding of enzymes predominates the succeeding catalysis rate.

RNA processing by ribonucleases and RNA modifying enzymes often involves sequential reactions of the same enzyme on a single precursor transcript. In Escherichia coli, processing of polycistronic tRNA precursors involves separation into individual pre-tRNAs by one of several ribonucleases followed by 5′ end maturation by ribonuclease P. A notable exception are valine and lysine tRNAs encoded by three polycistronic precursors that follow a recently discovered pathway involving initial 3′ to 5′ directional processing by RNase P. Here, we show that the dicistronic precursor containing tRNAvalV and tRNAvalW undergoes accurate and efficient 3′ to 5′ directional processing by RNase P in vitro. Kinetic analyses reveal a distributive mechanism involving dissociation of the enzyme between the two cleavage steps. Directional processing is maintained despite swapping or duplicating the two tRNAs consistent with inhibition of processing by 3′ trailer sequences. Structure-function studies identify a stem–loop in 5′ leader of tRNAvalV that inhibits RNase P cleavage and further enforces directional processing. The results demonstrate that directional processing is an intrinsic property of RNase P and show how RNA sequence and structure context can modulate reaction rates in order to direct precursors along specific pathways.

Most native polygalacturonases are glycosylated. Owing to the influence of attached glycans on the function of proteins, an immobilized non-glycosylated polygalacturonase is fabricated for the degradation of pectin-rich biomass pectin into fermentable sugars. Recombinant non-glycosylated and glycosylated PGAs expressed by E. coli and P. pastoris, respectively, were prepared. As application model, recombinant polygalacturonases in immobilized enzyme membrane reactors were tested. The electrospun chitosan-based nanofibrous membrane was selected as the support matrix. The maximum enzyme loading efficiency and enzyme binding efficiency of non-glycosylated polygalacturonase and glycosylated polygalacturonase were 89.12 mg/g and 84.13% and 34.16 mg/g and 87.18%, respectively, suggesting that the glycosylation of polygalacturonases had a serious effect on its immobilization. The membranes were characterized by scanning electron microscope, Fourier transform infrared spectroscopy and X-ray diffraction. Furthermore, the glycosylation can significantly enhance the stability of recombinant polygalacturonases. Meanwhile, the glycosylation influenced their specific activity and degradation pattern. Most interesting of all, some d -galacturonic acid monomers occurred in the hydrolysate after the degradation of pectin by the non-glycosylated polygalacturonase, and the yield of reducing sugar could be 31.98 mg after 60min, with a conversion rate of 63.96%. Thus, the constructed reactor has potential in using pectin-rich biomass as the feedstock.

Background Cervical cancer (CC) is one of the most common gynaecological malignancies all around the world. The mechanisms of cervical carcinoma formation remain under close scrutiny. The long non-coding RNAs (lncRNA) and microRNAs (miRNAs) play important roles in controlling gene expression and promoting the development and progression of cervical cancer by acting as competitive endogenous RNA (ceRNA). However, the roles of lncRNA associated with ceRNAs in cervical carcinogenesis remains unknown. In this study, the expression of long non-coding RNA HOTAIR was investigated in HPV16 positive cervical cancer cells, the candidate miRNAs and target genes were identified to clarify putative ceRNAs of HOTAIR/miRNA in cervical cancer cells. Methods The proliferation ability of cells was measured by CCK8 and EdU incorporation assays and cell apoptosis was analyzed by flow cytometry. The expression of HOTAIR, miR-214-3p, HPV16 E7 mRNA were detected by qRT-PCR. As for searching for the interaction between miR-214-3p and HOTAIR, the binding sites for miR-214-3p on HOTAIR was predicted by starbase v2.0 database, then dual-luciferase assay was used to verify the binding sites. In addition, Gene Ontology (GO) and protein–protein interaction (PPI) network analysis of target genes of miR-214-3p were performed with bioinformatics analysis. The potential signal pathway regulated by HOTAIR/miR-214-3p was predicted by KEGG enrichment analysis and confirmed by qPCR and WB analysis in cervical cancer cells. Results Our results showed that expression of HOTAIR was up-regulated, while that of miR-214-3p was down-regulated in HPV16-positive cervical cancer cells. The expression status of HPV16 E7 played an important role in regulating expression of HOTAIR or miR-214-3p in cervical cancer cells. HOTAIR knockdown could significantly inhibited cell proliferate ability and promote cellular apoptosis, whereas the inhibition of miR-214-3p expression partially reversed such results. Bioinformatics analysis identified 1451 genes as target genes of miR-214-3p. The Gene ontology (GO) and KEGG Pathway enrichment analysis showed that these target genes were mainly related to regulation of cell communication, protein binding, enzyme binding and transferase activity, and Wnt ligand biogenesis. Pathway enrichment analysis results showed that the predicted target genes were significantly enriched in Wnt/β-catenin signaling pathway. Finally, our results confirmed that miR-214-3p could significantly inhibit β-catenin expression in HPV16 positive cancer cells by qPCR and WB analysis. Conclusion HOTAIR could act as a ceRNA through binding to miR-214-3p, promote cell proliferation and inhibit the apoptosis of HPV16 positive cervical cancer. HOTAIR/miR-214-3p/Wnt/β-catenin signal pathway might played important regulated roles in HPV16 positive cervical cancer. Our results provided new insight into defining novel biomarkers for cervical cancer.

DNA damage has been implicated in numerous human diseases, particularly cancer, and the aging process. Single-base lesions and mismatches in DNA can be cytotoxic or mutagenic and are recognized by a DNA glycosylase during the process of base excision repair. Altered local dynamics and conformational properties in damaged DNAs have previously been suggested to assist in recognition and specificity. Herein, we use solution nuclear magnetic resonance to quantify changes in BI-BII backbone conformational dynamics due to the presence of single-base lesions in DNA, including uracil, dihydrouracil, 1,N6-ethenoadenine, and T:G mismatches. Stepwise changes to the %BII and ΔG of the BI-BII dynamic equilibrium compared to those of unmodified sequences were observed. Additionally, the equilibrium skews toward endothermicity for the phosphates nearest the lesion/mismatched base pair. Finally, the phosphates with the greatest alterations correlate with those most relevant to the repair of enzyme binding. All of these results suggest local conformational rearrangement of the DNA backbone may play a role in lesion recognition by repair enzymes.

Hepatic uptake of triglyceride-rich remnant lipoproteins is mediated by the low-density lipoprotein receptor, a low-density lipoprotein receptor related protein and the heparan sulfate proteoglycan, syndecan-1. Heparan sulfate proteoglycan also mediates low-density lipoprotein receptor degradation by a regulator of cholesterol homeostasis, proprotein convertase subtilisin kexin type 9 (PCSK9), thereby hampering triglyceride-rich remnant lipoproteins uptake. In this study, we investigated the effects of proteinuria on PCSK9, hepatic heparan sulfate proteoglycan and plasma triglyceride-rich remnant lipoproteins. Adriamycin-injected rats developed proteinuria, elevated triglycerides and total cholesterol (all significantly increased). Proteinuria associated with triglycerides and total cholesterol and serum PCSK9 (all significant associations) without loss of the low-density lipoprotein receptor as evidenced by immunofluorescence staining and western blotting. In proteinuric rats, PCSK9 accumulated in sinusoids, whereas in control rats PCSK9 was localized in the cytoplasm of hepatocytes. Molecular profiling revealed that the heparan sulfate side chains of heparan sulfate proteoglycan to be hypersulfated in proteinuric rats. Competition assays revealed sulfation to be a major determinant for PCSK9 binding. PCSK9 partly colocalized with hypersulfated heparan sulfate in proteinuric rats, but not in control rats. Hence, proteinuria induces hypersulfated hepatic heparan sulfate proteoglycans, increasing their affinity to PCSK9. This might impair hepatic triglyceride-rich remnant lipoproteins uptake, causing proteinuria-associated dyslipidemia. Thus, our study reveals PCSK9/heparan sulfate may be a novel target to control dyslipidemia.

The extensive use of antibiotics over the last decades is responsible for the emergence of multidrug-resistant (MDR) microorganisms that are challenging health care systems worldwide. The use of alternative antimicrobial materials could mitigate the selection of new MDR strains by reducing antibiotic overuse. This paper describes the design of enzyme-based antimicrobial cellulose beads containing a covalently coupled glucose oxidase from Aspergillus niger (GOx) able to release antimicrobial concentrations of hydrogen peroxide (H2O2) (≈ 1.8 mM). The material preparation was optimized to obtain the best performance in terms of mechanical resistance, shelf life, and H2O2 production. As a proof of concept, agar inhibition halo assays (Kirby-Bauer test) against model pathogens were performed. The two most relevant factors affecting the bead functionalization process were the degree of oxidation and the pH used for the enzyme binding process. Slightly acidic conditions during the functionalization process (pH 6) showed the best results for the GOx/cellulose system. The functionalized beads inhibited the growth of all the microorganisms assayed, confirming the release of sufficient antimicrobial levels of H2O2. The maximum inhibition efficiency was exhibited toward Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli), although significant inhibitory effects toward methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus were also observed. These enzyme-functionalized cellulose beads represent an inexpensive, sustainable, and biocompatible antimicrobial material with potential use in many applications, including the manufacturing of biomedical products and additives for food preservation.

Protein labeling strategies have been explored for decades to study protein structure, function, and regulation. Fluorescent labeling of a protein enables the study of protein-protein interactions through biophysical methods such as microscale thermophoresis (MST). MST measures the directed motion of a fluorescently labeled protein in response to microscopic temperature gradients, and the protein's thermal mobility can be used to determine binding affinity. However, the stoichiometry and site specificity of fluorescent labeling are hard to control, and heterogeneous labeling can generate inaccuracies in binding measurements. Here, we describe an easy-to-apply protocol for high-stoichiometric, site-specific labeling of a protein at its N-terminus with N-hydroxysuccinimide (NHS) esters as a means to measure protein-protein interaction affinity by MST. This protocol includes guidelines for NHS ester labeling, fluorescent-labeled protein purification, and MST measurement using a labeled protein. As an example of the entire workflow, we additionally provide a protocol for labeling a ubiquitin E3 enzyme and testing ubiquitin E2-E3 enzyme binding affinity. These methods are highly adaptable and can be extended for protein interaction studies in various biological and biochemical circumstances. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Labeling a protein of interest at its N-terminus with NHS esters through stepwise reaction Alternate Protocol: Labeling a protein of interest at its N-terminus with NHS esters through a one-pot reaction Basic Protocol 2: Purifying the N-terminal fluorescent-labeled protein and determining its concentration and labeling efficiency Basic Protocol 3: Using MST to determine the binding affinity of an N-terminal fluorescent-labeled protein to a binding partner. Basic Protocol 4: NHS ester labeling of ubiquitin E3 ligase WWP2 and measurement of the binding affinity between WWP2 and an E2 conjugating enzyme by the MST binding assay.

The current focus for many researchers has turned to the development of therapeutics that have the potential for serving as broad-spectrum inhibitors that can target numerous viruses, both within a particular family, as well as to span across multiple viral families. This will allow us to build an arsenal of therapeutics that could be used for the next outbreak. In that regard, nucleosides have served as the cornerstone for antiviral therapy for many decades. As detailed herein, many nucleosides have been shown to inhibit multiple viruses due to the conserved nature of many viral enzyme binding sites. Thus, it is somewhat surprising that up until very recently, many researchers focused more on "one bug one drug," rather than trying to target multiple viruses given those similarities. This attitude is now changing due to the realization that we need to be proactive rather than reactive when it comes to combating emerging and reemerging infectious diseases. A brief summary of prominent nucleoside analogues that previously exhibited broad-spectrum activity and are now under renewed interest, as well as new analogues, that are currently under investigation against SARS-CoV-2 and other viruses is discussed herein.

Using structure-based design on the herbicidal target enzyme protoporphyrinogen IX oxidase (PPO), the new herbicide Tirexor could be identified. Tirexor shows broad-spectrum herbicidal activity in pre- and postemergence applications, as well as in preplant burndown, with selectivities in wheat, corn, and soybean in preemergence application. Due to its unique enzyme binding properties, it shows also high activity on many weeds resistant to other PPO inhibitors. The identification of tolerance traits for the potential use in Tirexor-tolerant crops could also be demonstrated.

The use of hydrogen peroxide-releasing enzymes as a component to produce alternative and sustainable antimicrobial materials has aroused interest in the scientific community. However, the preparation of such materials requires an effective enzyme binding method that often involves the use of expensive and toxic chemicals. Here, we describe the development of an enzyme-based hydrogen peroxide-producing regenerated cellulose film (RCF) in which a cellobiohydrolase (TrCBHI) and a cellobiose dehydrogenase (MtCDHA) were efficiently adsorbed, 90.38 ± 2.2 and 82.40 ± 5.7%, respectively, without making use of cross-linkers. The enzyme adsorption kinetics and binding isotherm experiments showed high affinity of the proteins possessing cellulose-binding modules for RCF, suggesting that binding on regenerated cellulose via specific interactions can be an alternative method for enzyme immobilization. Resistance to compression and porosity at a micrometer scale were found to be tunable by changing cellulose concentration prior to film regeneration. The self-degradation process, triggered by stacking TrCBHI and MtCDHA (previously immobilized onto separate RCF), produced 0.15 nmol/min·cm2 of H2O2. Moreover, the production of H2O2 was sustained for at least 24 h reaching a concentration of ∼2 mM. The activity of MtCDHA immobilized on RCF was not affected by reuse for at least 3 days (1 cycle/day), suggesting that no significant enzyme leakage occurred in that timeframe. In the material herein designed, cellulose (regenerated from a 1-ethyl-3-methylimidazolium acetate/dimethyl sulfoxide (DMSO) solution) serves both as support and substrate for the immobilized enzymes. The sequential reaction led to the production of H2O2 at a micromolar-millimolar level revealing the potential use of the material as a self-degradable antimicrobial agent.

Lignin is known to limit the enzyme-mediated hydrolysis of biomass by both restricting substrate swelling and binding to the enzymes. Pretreated mechanical pulp (MP) made from Aspen wood chips was incubated with either 16% sodium sulfite or 32% sodium percarbonate to try to incorporate similar amounts of sulfonic and carboxylic acid groups onto the lignin (60mmol/kg substrate) present in the pulp without resulting in significant delignification. When Simon’s stain was used to assess potential enzyme accessibility to the cellulose, it was apparent that both post-treatments enhanced accessibility and cellulose hydrolysis. To try to further elucidate how acid group addition might influence potential enzyme binding to lignin, Protease Treated Lignin (PTL) was isolated from the original and modified mechanical pulps and added to a cellulose rich, delignified Kraft pulp. As anticipated, the PTLs from both the oxidized and sulfonated substrates proved less inhibitory and adsorbed less enzymes than did the PTL derived from the original pulp. Subsequent analyses indicated that both the sulfonated and oxidized lignin samples contained less phenolic hydroxyl groups, resulting in enhanced hydrophilicity and a more negative charge which decreased the non-productive binding of the cellulase enzymes to the lignin.

Background Obesity and its related diseases are increasing worldwide. One of the best therapeutic strategies for obesity management is through the inhibition of pancreatic lipase (PL) enzyme. So far orlistat is the only FDA approved PL inhibitor, but with unpleasant side effects. New efficacious anti-obesity drugs are needed to achieve a successful reduction in the incidence and prevalence of obesity. Many microbial metabolites have PL inhibitory activity. Screening soil inhabitants for PL inhibitors could help in increasing the available anti-obesity drugs. We aimed to isolate and identify alternative PL inhibitors from soil flora. Results We screened the crude mycelial methanolic extracts of 39 soil samples for PL inhibitory activity by the quantitative lipase colorimetric assay, using the substrate p-nitrophenyl palmitate and orlistat as positive control. AspsarO, a PL inhibitor producer, was isolated from an agricultural field soil in Giza, Egypt. It was identified as Aspergillus oryzae using colony morphology, microscopical characteristics, 18S rDNA sequencing, and molecular phylogeny. Increasing the PL inhibitor activity, in AspsarO cultures, from 25.9 ± 2% to 61.4 ± 1.8% was achieved by optimizing the fermentation process using a Placket–Burman design. The dried 100% methanolic fraction of the AspsarO culture had an IC50 of 7.48 μg/ml compared to 3.72 μg/ml for orlistat. It decreased the percent weight gain, significantly reduced the food intake and serum triglycerides levels in high-fat diet-fed Sprague–Dawley rats. Kojic acid, the active metabolite, was identified using several biological guided chromatographic and 1H and 13C NMR techniques and had an IC50 of 6.62 μg/ml. Docking pattern attributed this effect to the interaction of kojic acid with the key amino acids (Lys80, Trp252, and Asn84) in PL enzyme binding site. Conclusion Combining the results of the induced obesity animal model, in silico molecular docking and the lipase inhibitory assay, suggests that kojic acid can be a new therapeutic option for obesity management. Besides, it can lower serum triglycerides in obese patients.

Neutrophil elastases are deposited in azurophilic granules interspace of neutrophils and tightly associated with inflammatory ailments. The root barks of Artocarpus elasticus had a strong inhibitory potential against human neutrophil elastase (HNE). The responsible components for HNE inhibition were confirmed as alkylated flavones (2–4, IC50 = 14.8 ~ 18.1 μM) and dihydrobenzoxanthones (5–8, IC50 = 9.8 ~ 28.7 μM). Alkyl groups on flavone were found to be crucial functionalities for HNE inhibition. For instance, alkylated flavone 2 (IC50 = 14.8 μM) was 20-fold potent than mother compound norartocarpetin (1, IC50 > 300 μM). The kinetic analysis showed that alkylated flavones (2–4) were noncompetitive inhibition, while dihydrobenzoxanthones (5–8) were a mixed type I (KI KIS) inhibitors, which usually binds with free enzyme better than to complex of enzyme–substrate. Inhibitors and HNE enzyme binding affinities were examined by fluorescence quenching effect. In the result, the binding affinity constants (KSV) had a significant correlation with inhibitory potencies (IC50).

Enzymatic digestibility of an acid-pretreated poplar (AP, 42.9%) was superior to that of a similarly acid-pretreated larch (AL, 12.5%). Effects of lignin-related recalcitrance on enzymatic hydrolysis were comprehensively investigated by disrupting the two predominant lignin fractions present in acid-pretreated material (extractable lignin and bulk lignin). Lignin removal and bovine serum albumin (BSA) addition were performed to estimate the relative contributions of lignin towards physical blocking and enzyme binding on enzymatic hydrolysis. The lignin physical blocking played a more significant role in limiting the enzymatic hydrolysis of AL. BSA addition improved enzymatic hydrolysis of AP more significantly than AL. Moreover, the effects of lignin embedded in the lignocellulosic matrix on enzyme non-productive binding were compared with the isolated lignin. It indicated that the lignin distribution would influence the lignin effects on enzyme non-productive binding during enzymatic hydrolysis. Results will give insights towards improvement of enzymatic hydrolysis on acid-pretreated woody biomass.

Fungal infections are a global issue affecting over 150 million people worldwide annually, with 750 000 of these caused by invasive Candida infections. Azole drugs are the frontline treatment against fungal infections; however, resistance to current azole antifungals in C. albicans poses a threat to public health. Two series of novel azole derivatives, short and extended derivatives, have been designed, synthesised and investigated for CYP51 inhibitory activity, binding affinity and minimum inhibitory concentration (MIC) against C. albicans strains. The short derivatives were more potent against the C. albicans strains (e. g., MIC 2‐(4‐chlorophenyl)‐N‐(2,4‐dichlorobenzyl)‐3‐(1H‐imidazol‐1‐yl)propanamide (5 f), Two series of novel azole derivatives, short and long, have been designed, synthesised and investigated for CYP51 inhibitory activity, binding affinity and minimum inhibitory concentration against C. albicans strains. The short derivatives were more potent than the extended derivatives, but both displayed comparable enzyme binding and inhibition. Molecular modelling and biological and selectivity profiles provide useful SAR for further development.

We assessed the enzymatic activation of four different biochars produced from pyrolyzing swine manure and poultry litter, and by co-pyrolyzing these livestock residues with agricultural spent mulch plastic film wastes (plastichars). Enzymatic activation consisted of incubating biochars in soil inoculated with earthworms (Lumbricus terrestris), which acted as biological vectors to facilitate retention of extracellular enzymes onto biochar surface. The activity of carboxylesterase ‒a pesticide-detoxifying enzyme‒ was measured in non-bioturbed soils (reference), linings of the burrows created by earthworms, casts (feces) and biochar particles recovered from the soil. Our results revealed that: 1) biochar increased soil carboxylesterase activity respect to biochar-free (control) soils, which was more prominent in the presence of earthworms. 2) The maximum enzyme activity was found in soils amended with plastichars. 3) The plastichars showed higher enzyme binding capacities than that of the biochars produced from animal manure alone, corroborating the pattern of enzyme distribution found in soil. 4) The presence of earthworms in soil significantly increased the potential of the plastichars for enzymatic activation. These findings suggest that the plastichars are suitable for increasing and stabilizing soil enzyme activities with no toxicity on earthworms.

Dissociation of non-productively bound cellulolytic enzymes from cellulose is hypothesized to be a key rate-limiting factor impeding cost-effective biomass conversion to fermentable sugars. However, the role of carbohydrate-binding modules (CBMs) in enabling non-productive enzyme binding is not well understood. Here, we examine the subtle interplay of CBM binding and cellulose hydrolysis activity for three model Type-A CBMs (families 1, 3a, and 64) tethered to a multifunctional endoglucanase (CelE) on two distinct cellulose allomorphs (i.e., cellulose I and III). We generated a small-library of mutant CBMs with varying cellulose affinity, as determined by equilibrium binding assays, followed by monitoring cellulose hydrolysis activity of CelE-CBM fusion constructs. Finally, kinetic binding assays using quartz crystal microbalance with dissipation (QCM-D) were employed to measure CBM adsorption and desorption rate constants kon and koff , respectively, towards nanocrystalline cellulose derived from both allomorphs. Overall, our results indicate that reduced CBM equilibrium binding affinity towards cellulose I alone, resulting from increased desorption rates (koff ) and reduced effective adsorption rates (nkon ), is correlated to overall improved endocellulase activity. Future studies could employ similar approaches to unravel the role of CBMs in non-productive enzyme binding and develop improved cellulolytic enzymes for industrial applications. This article is protected by copyright. All rights reserved.

Dissociation of non-productively bound cellulolytic enzymes from cellulose is hypothesized to be a key rate-limiting factor impeding cost-effective biomass conversion to fermentable sugars. However, the role of carbohydrate-binding modules (CBMs) in enabling non-productive enzyme binding is not well understood. Here, we examine the subtle interplay of CBM binding and cellulose hydrolysis activity for three model Type-A CBMs (families 1, 3a, and 64) tethered to a multifunctional endoglucanase (CelE) on two distinct cellulose allomorphs (i.e., cellulose I and III). We generated a small-library of mutant CBMs with varying cellulose affinity, as determined by equilibrium binding assays, followed by monitoring cellulose hydrolysis activity of CelE-CBM fusion constructs. Finally, kinetic binding assays using quartz crystal microbalance with dissipation (QCM-D) were employed to measure CBM adsorption and desorption rate constants Kon and Koff, respectively, towards nanocrystalline cellulose derived from both allomorphs. Overall, our results indicate that reduced CBM equilibrium binding affinity towards cellulose I alone, resulting from increased desorption rates (Koff) and reduced effective adsorption rates (nKon), is correlated to overall improved endocellulase activity. Future studies could employ similar approaches to unravel the role of CBMs in non-productive enzyme binding and develop improved cellulolytic enzymes for industrial applications.

Design of enzyme binding pocket to accommodate substrates with different chemical structure is a great challenge. Traditionally, thousands even millions of mutants have to be screened in wet-lab experiment to find a ligand-specific mutant and large amount of time and resources is consumed. To accelerate the screening process, here we propose a novel workflow through integration of molecular modeling and data-driven machine learning method to generate mutant libraries with high enrichment ratio for recognition of specific substrate. M. jannaschii tyrosyl-tRNA synthetase (Mj. TyrRS) is used as an example system to give a proof of concept since the sequence and structure of many unnatural amino acid specific Mj. TyrRS mutants have been reported. Based on the crystal structures of different Mj. TyrRS mutants and Rosetta modeling result, we find D158G/P is the critical residue which influences the backbone disruption of helix with residue 158-163. Our results show that compared with random mutation, Rosetta modeling and score function calculation can elevate the enrichment ratio of desired mutants by 2-fold in a test library having 687 mutants, while after calibration by machine learning model trained using known data of Mj. TyrRS mutants and ligand, the enrichment ratio can be elevated by 11-fold. This molecular modeling and machine learning-integrated workflow is anticipated to significantly benefit to the Mj. tyrRS mutant screening and substantially reduce the time and cost of web-lab experiment. Besides, this novel process will have broad application in the field of computational protein design.CCS Concepts• Applied computing • Life and medical sciences • Computational biology • Molecular structural biology

Graphical abstract, Highlights • The rising epidemics of SARS-CoV2 has globally made a serious concern. • Thus, understanding the virus-host relationship remains a serious concern. • Here, we adopted the in-silico approach to picturized the similarities between the miRNAs of SARS-CoV2 genomes and human. • Further, the assessments and prediction of miRNAs helped us to analyze and understand the mechanism of pathogenesis., Background The progressive SARS-CoV2 outbreaks worldwide have evoked global investigation. Despite the numerousin-silico approaches, the virus-host relationship remains a serious concern. MicroRNAs are the small non-coding RNAs that help in regulating gene profiling. The current study utilized miRNA prediction tools along with the PANTHER classification system to demonstrate association and sequence similarities shared between miRNAs of SARS-CoV2 and human host. Method An in-silico approach was carried out using Vmir analyzer to predict miRNAs from SARS-CoV2 viral genomes. Predicted miRNAs from SARS-CoV2 viral genomes were used for effective hybridization sequence identification along the nucleotide similarities with human miRNAs from miRbase database. Further, it was proceeded to analyze the gene ontology using miRDB with PANTHER classification. Result Based on the prediction and analysis, we have identified 22 potential miRNAs from five genomes of SARS-CoV2 linked with 12 human miRNAs. Analysis of human miRNAs hsa-mir-1267, hsa-mir-1-3p, hsa-mir-5683 were found shared between all the five viral SARS-CoV2 miRNAs. Further, PANTHER classification analyzed the gene-ontology being carried by these associations showed that 44 genes were involved in biological functions that includes genes specific for signaling pathway, immune complex generation, enzyme binding with effective role in the virus-host relationship. Conclusion Our analysis concludes that the genes identified in this study can be effective in analyzing the virus-host interaction. It also provides a new direction to understand viral pathogenesis with a probable new way to link, that can be used to understand and relate the miRNAs of the virus to the host conditions.

Background α-Amylase inhibitors (α-AIs) belong to the discrete classes, and exhibited differential specificities against α-amylases from various sources. Several α-amylases and their complexes with inhibitors at the molecular level have been studied in detail. Interestingly, some α-AIs depict specific and selective interactions amid different insect α-amylases. Scope of review There are studies to understand evolutionary variability and functional differentiation of insect α-amylases and their cognate inhibitors. We have examined sequence, structural, and interaction diversity between various α-amylases and α-AIs. Based on these analyses, we are providing a potential basis for the functional differentiation among certain insect α-amylases concerning mammalian counterparts and their interactions with different proteinaceous α-AIs. Major conclusions Insect α-amylases have conserved domain architecture with differences in length, number of disulfide bonds, and secondary structure. Furthermore, few of them exhibit variable characteristics like chloride dependent activity, the presence of N-terminal glutamine residue to protect against proteolytic degradation, and loop variations near the enzyme active site. Conformation of α-AI protein could be an essential factor for their specificity and binding affinities towards target α-amylase(s). Furthermore, variation into the enzyme binding pocket residues might contribute to differential interactions with inhibitors. General significance Molecular insights in the interactions between insect α-amylases and plant α-AI will provide the details of mechanisms assisting the inhibitor specificity. Furthermore, this information will help to design potent and effective α-AIs against specific α-amylase.

Depression is a common affective disorder characterized by significant and persistent low mood. Ketamine, an N-methyl-D-aspartate receptor (NMDAR) antagonist, is reported to have a rapid and durable antidepressant effect, but the mechanisms are unclear. Protein phosphorylation is a post-translational modification that plays a crucial role in cell signaling. Thus, we present a phosphoproteomics approach to investigate the mechanisms underlying stress-induced depression and the rapid antidepressant effect of ketamine in mice. We analyzed the phosphoprotein changes induced by chronic unpredictable mild stress (CUMS) and ketamine treatment in two known mood control centers, the medial prefrontal cortex (mPFC) and the nucleus accumbens (NAc). We initially obtained >8,000 phosphorylation sites. Quantitation revealed 3,988 sites from the mPFC and 3,196 sites from the NAc. Further analysis revealed that changes in synaptic transmission-related signaling are a common feature. Notably, CUMS-induced changes were reversed by ketamine treatment, as shown by the analysis of commonly altered sites. Ketamine also induced specific changes, such as alterations in synapse organization, synaptic transmission, and enzyme binding. Collectively, our findings establish a signaling framework for stress-induced depression and the rapid antidepressant effect of ketamine.

Designing side chain substituents complementary to enzyme binding pockets is of great importance in the construction of potent and selective phosphinic dipeptide inhibitors of metallo-aminopeptidases. Proper structure selection makes inhibitor construction more economic, as the development process typically consists of multiple iterative preparation/bioassay steps. On the basis of these principles, using noncomplex computation and modeling methodologies, we comprehensively screened 900 commercial precursors of the P1&prime, residues of phosphinic dipeptide and dehydrodipeptide analogs to identify the most promising ligands of 52 metallo-dependent aminopeptidases with known crystal structures. The results revealed several nonproteinogenic residues with an improved energy of binding compared with the best known inhibitors. The data are discussed taking into account the selectivity and stereochemical implications of the enzymes. Using this approach, we were able to identify nontrivial structural elements substituting the recognized phosphinic peptidomimetic scaffold of metallo-aminopeptidase inhibitors.

COVID-19 outbreak is still threatening the public health. Therefore, in the middle of the pandemic, all kind of knowledge on SARS-CoV-2 may help us to find the solution. Determining the 3D structures of the proteins involved in host-pathogen interactions are of great importance in the fight against infection. Besides, post-translational modifications of the protein on 3D structure should be revealed in order to understand the protein function since these modifications are responsible for the host-pathogen interaction. Based on these, we predicted O-glycosylation and phosphorylation positions using full amino acid sequence of S1 protein. Candidate positions were further analyzed with enzyme binding activity, solvent accessibility, surface area parameters and the positions determined with high accuracy rate were used to design full 3D glycoprotein structure of the S1 protein using carbohydrate force field. In addition, the interaction between the C-type lectin CD209L and α-mannose residues was examined and carbohydrate recognition positions were predicted. We suggest these positions as a potential target for the inhibition of the initial binding of SARS-CoV-2 S1 protein to the host cell.

Phospholipase A(2) (PLA(2)) is a membrane lytic enzyme that is present in many organisms. Human PLA(2) has emerged as a potential biomarker as well as a therapeutic target for several diseases including cancer, cardiovascular diseases, and some inflammatory diseases. The current study focuses on the development of lipo-beads that are very reactive and highly sensitive to PLA(2). To develop the best supported lipid bilayer formulation, several lipid combinations were investigated using 10 μm porous silica beads. The reactivity of PLA(2) was monitored via the decrease in particle fluorescence because of the release of entrapped fluorescent dye from the particle pores or the disintegration of a fluorescent lipid constituted on the bilayer upon lipid hydrolysis using flow cytometry. The enzyme binding studies indicate that lipo-beads with bulky fluorescent tags in the lipid head group and anionic lipids produce a more pronounced response. The kinetic studies suggest that these lipo-beads are very reactive with PLA(2) and can generate a detectable signal in less than 5 min. The enzyme inhibition studies were also conducted with two known PLA(2) inhibitors, varespladib and quercetin. We find that quercetin can hydrolyze the supported membrane, and thus inhibition of PLA(2) is not observed; however, varespladib has shown significant PLA(2) inhibition on lipo-beads. We have demonstrated that our lipo-bead-based approach can detect annexin-3, a known disease biomarker, as low as 10 nM within 5 min after incubation.

Pro-inflammatory activation of caspase-1 in the neurodegenerative pathway has been associated with age-dependent cognitive impairment and Alzheimer's disease (AD) in humans. A recent report highlighted 2,4-diaminopyrimidine ring as an essential fragment in the inhibition of human caspase-1. However, the role of the ring and its enzyme inhibitory mechanism is not thoroughly investigated at the molecular level. The purpose of this study is therefore in twofold: (1) to understand the enzyme binding mechanism of the 2,4-diaminopyrimidine ring and (2) to search for more potent caspase-1 inhibitors that contain the ring, using integrative per-residue energy decomposition (PRED) pharmacophore modeling. Ligand interaction profile of a reference compound revealed a peculiar hydrogen formation of the amino group of 2,4-diaminopyrimidine with active site residue Arg341, possibly forming the bases for its inhibitory prowess against caspase-1. A generated pharmacophore model for structure-based virtual screening identified compounds, ZINC724667, ZINC09908119, and ZINC09933770, as potential caspase-1 inhibitors that possessed desirable pharmacokinetic and physiochemical properties. Further analyses revealed active site residues, Arg179, Ser236, Cys285, Gln283, Ser339, and Arg341, as crucial to inhibitor binding by stabilizing and forming hydrogen bonds, hydrophobic, and pi-pi interactions with the 2,4-diaminopyrimidine rings. Common interaction patterns of the hits could have accounted for their selective and high-affinity ligand binding, which was characterized by notable disruptions in caspase-1 structural architecture. These compounds could further be explored as potential leads in the development of novel caspase-1 inhibitors.

One-pot reactions that combine non-enzymatic and biocatalytic transformations represent an emerging strategy in chemical synthesis. Some of the most powerful chemoenzymatic methodologies, although uncommon, are those that form a carbon–carbon (C–C) bond and a stereocenter at one of the reacting carbons, thereby streamlining traditional retrosynthetic disconnections. Here we report the one-pot, chemoenzymatic conversion of amides to enantioenriched alcohols. This transformation combines a nickel-catalyzed Suzuki–Miyaura coupling of amides in aqueous medium with an asymmetric, biocatalytic reduction to provide diarylmethanol derivatives in high yields and enantiomeric excesses. The synthetic utility of this platform is underscored by the formal syntheses of both antipodes of the pharmaceutical orphenadrine, which rely on ketoreductase enzymes that instill complementary stereoselectivities. We provide an explanation for the origins of stereoselectivity based on an analysis of the enzyme binding pockets.

Enzyme immobilization is a powerful tool not only as a protective agent against harsh reaction conditions but also for the enhancement of enzyme activity, stability, reusability, and for the improvement of enzyme properties as well. Herein, immobilization of β-glucosidase from Thermotoga maritima (Tm-β-Glu) on magnetic nanoparticles (MNPs) functionalized with chitin (Ch) was investigated. This technology showed a novel thermostable chitin-binding domain (Tt-ChBD), which is more desirable in a wide range of large-scale applications. This exclusive approach was fabricated to improve the Galacto-oligosaccharide (GOS) production from a cheap and abundant by-product such as lactose through a novel green synthesis route. Additionally, SDS-PAGE, enzyme activity kinetics, transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR) revealed that among the immobilization strategies for Thermotoga maritime-β-Glucosidase thermostable chitin-binding domain (Tm-β-Glu-Tt-ChBD) on the attractive substrate; Ch-MNPs had the highest enzyme binding capacity and GOS production ratio when compared to the native enzyme. More interestingly, a magnetic separation technique was successfully employed in recycling the immobilized Tm-β-Glu for repetitive batch-wise GOS without significant loss or reduction of enzyme activity. This immobilization system displayed an operative stability status under various parameters, for instance, temperature, pH, thermal conditions, storage stabilities, and enzyme kinetics when compared with the native enzyme. Conclusively, the GOS yield and residual activity of the immobilized enzyme after the 10th cycles were 31.23% and 66%, respectively. Whereas the GOS yield from native enzyme synthesis was just 25% after 12 h in the first batch. This study recommends applying Tt-ChBD in the immobilization process of Tm-β-Glu on Ch-MNPs to produce a low-cost GOS as a new eco-friendly process besides increasing the biostability and efficiency of the immobilized enzyme.

Curcumin is a dietary compound with accrued evidence of antiviral activity. Poor solubility and permeation renders curcumin a good applicant for incorporation into proniosomes. The intent of this study was to formulate curcumin proniosomal gel for topical application and the evaluation of its in-vitro, ex-vivo activities against Herpes Simplex virus type 1 (HSV-1), as well as molecular docking studies on HSV-1 thymidine kinase proteins. Coacervation phase separation tactic, using 23 full factorial design, was used in the preparation of different proniosomes. Cytotoxicity of the selected formulae (F4 and F8) was evaluated on the Vero cell line. Optimal formulae (F4 and F8) showed entrapment efficiency of 97.15 &plusmn, 2.47% and 95.85 &plusmn, 2.9%, vesicle size of 173.7 &plusmn, 2.26 nm and 206.15 &plusmn, 4.17 nm and percentages curcumin released after 3 h of 51.9 &plusmn, 1.4% and 50.5 &plusmn, 1.1%, respectively. Ex-vivo permeation studies demonstrated that the optimal formulae markedly improved the dermal curcumin delivery. Curcumin proniosomal gel formulae exhibited 85.4% reduction of HSV-1 replication. The ability of curcumin to interact with the key amino acids in the enzyme binding sites of 1KI7, 1KI4, and 1E2P, as indicated by its docking pattern, rationalized its observed activity. Therefore, curcumin proniosomes could be considered as a successful topical delivery system for the treatment of HSV-1.

The ability to switch on the activity of an enzyme through its spontaneous reconstitution has proven to be a valuable tool in fundamental studies of enzyme structure/reactivity relationships or in the design of artificial signal transduction systems in bioelectronics, synthetic biology, or bioanalytical applications. In particular, those based on the spontaneous reconstitution/activation of the apo-PQQ-dependent soluble glucose dehydrogenase (sGDH) from Acinetobacter calcoaceticus were widely developed. However, the reconstitution mechanism of sGDH with its two cofactors, i.e. the pyrroloquinoline quinone (PQQ) and Ca2+, remains unknown. The objective here is to elucidate this mechanism by stopped-flow kinetics under single-turnover conditions. The reconstitution of sGDH exhibited biphasic kinetics, characteristic of a square reaction scheme associated to two activation pathways. From a complete kinetic analysis, we were able to fully predict the reconstitution dynamic, but also to demonstrate that when PQQ first binds to the apo-sGDH, it strongly impedes the access of Ca2+ to its enclosed position at the bottom of the enzyme binding site, thereby greatly slowing down the reconstitution rate of sGDH. This slow calcium insertion may purposely be accelerated by providing more flexibility to the Ca2+ binding loop through the specific mutation of the calcium coordinating P248 proline residue, reducing thus the kinetic barrier to calcium ion insertion. The dynamic nature of the reconstitution process is also supported by the observation of a clear loop shift and a reorganization of the hydrogen bonding network and van der Waals interactions observed in both active sites of the apo and holo forms, a structural change modulation that was revealed from the refined X-ray structure of apo-sGDH (PDB:5MIN)., 45 pages, 11 figures, 6 tables

In mammalian females, the transition between quiescent primordial follicles and follicular development is critical for maintaining ovarian function and reproductive longevity. In primary oocytes of mouse quiescent primordial follicles, Golgi complexes are organized into a spherical structure, the Balbiani body. Here, we show that the structure of the B-body is maintained by microtubules and actin. The B-body stores mRNA-capping enzyme and 597 mRNAs associated with mRNA-decapping enzyme 1A. Proteins encoded by these mRNAs function in enzyme binding, cellular component organization and packing of telomere ends. Pharmacological disassembly of the B-body triggers translation of stored mRNAs and activates primordial follicles in culture and in vivo mouse model. Thus, primordial follicle quiescence is maintained by the B-body, and translationally inactive B-body-stored mRNAs may be regulated by 5’-capping.

Multiphoton microscopy of reduced Nicotinamide Adenine Dinucleotide(phosphate) (NAD(P)H) is an excellent tool for exploring metabolism at the single-cell level. We present the application of fluorescence anisotropy in multiphoton autofluorescence imaging. We explore time-resolved methods that can be used for metabolic interpretations: 1) the time-resolved anisotropy, 2) time-gated anisotropy and 3) steady-state anisotropy methods. These imaging schemes are achieved using a time-correlated single-photon counting microscope, allowing simultaneous pixel-wise registration of fluorescence intensity, lifetime, and anisotropy. This tool is well-suited to identify the enzyme binding states and offer new biophysical definitions for NAD(P)H imaging. We demonstrate this hyperdimensional imaging modality under an open-sourced imaging platform.

Graphene oxide (GO) coated electrodes provide an excellent platform for enzymatic glucose sensing, induced by the presence of glucose oxidase and an electrochemical transduction. Here, we show that the sensitivity of GO layers for glucose detection redoubles upon blending GO with chitosan (GO+Ch) and increases up to eight times if covalent binding of chitosan to GO (GO−Ch) is exploited. In addition, the conductivity of the composite material GO−Ch is suitable for electrochemical applications without the need of GO reduction, which is generally required for GO based coatings. Covalent modification of GO is achieved by a standard carboxylic activation/amidation approach by exploiting the abundant amino pendants of chitosan. Successful functionalization is proved by comparison with an ad-hoc synthesized control sample realized by using non-activated GO as precursor. The composite GO−Ch was deposited on standard screen-printed electrodes by a drop-casting approach. Comparison with a chitosan-GO blend and with pristine GO demonstrated the superior reliability and efficiency of the electrochemical response for glucose as a consequence of the high number of enzyme binding sites and of the partial reduction of GO during the carboxylic activation synthetic step.

Boronic acid transition state inhibitors (BATSIs) are known reversible covalent inhibitors of serine β-lactamases. The selectivity and high potency of specific BATSIs bearing an amide side chain mimicking the β-lactam's amide side chain are an established and recognized synthetic strategy. Herein, we describe a new class of BATSIs where the amide group is replaced by a bioisostere triazole; these compounds were designed as molecular probes. To this end, a library of 26 α-triazolylmethaneboronic acids was synthesized and tested against the clinically concerning Acinetobacter-derived cephalosporinase, ADC-7. In steady state analyses, these compounds demonstrated Ki values ranging from 90 nM to 38 μM (±10%). Five compounds were crystallized in complex with ADC-7 β-lactamase, and all the crystal structures reveal the triazole is in the putative amide binding site, thus confirming the triazole-amide bioisosterism. The easy synthetic access of these new inhibitors as prototype scaffolds allows the insertion of a wide range of chemical groups able to explore the enzyme binding site and provides insights on the importance of specific residues in recognition and catalysis. The best inhibitor identified, compound 6q (Ki 90 nM), places a tolyl group near Arg340, making favorable cation-π interactions. Notably, the structure of 6q does not resemble the natural substrate of the β-lactamase yet displays a pronounced inhibition activity, in addition to lowering the minimum inhibitory concentration (MIC) of ceftazidime against three bacterial strains expressing class C β-lactamases. In summary, these observations validate the α-triazolylboronic acids as a promising template for further inhibitor design.

This work is focused on immobilization of laccase from Myceliophthora thermophila expressed in Aspergillus oryzae (Novozym 51003® laccase) on amino modified fumed nano-silica (AFNS) and the possible use in bioremediation. Hereby, for the first time, factors affecting the immobilization of Novozym 51003® laccase on AFNS were investigated for defining the immobilization mechanism and optimizing the utilization of AFNS as support for laccase immobilization. The highest specific activity (13.1 IU·mg−1 proteins) was achieved at offered 160 mg per g of AFNS and for the same offered protein concentration the highest activity immobilization yield, reaching 68.3% after the equilibrium time, at optimum pH 5.0, was obtained. Laccase immobilization occurs by adsorption as monolayer enzyme binding in 40 min, following pseudo-first-order kinetics. The possible use of obtained immobilized preparation was investigated in degradation of pesticide lindane. Within 24 h, lindane concentration was reduced to 56.8% of initial concentration and after seven repeated reuses it retained 70% of the original activity.

In this chapter, we report a simple and facile method to armor enzymes with carbon nanotubes (CNTs) which are exfoliated, and debundled using bovine serum albumin (BSA). The fabricated CNT/BSA dispersions are biofriendly, biocompatible, defect-free, and highly stable solutions. BSA gives maximum exfoliation efficiency, exceeding the 4 mg/mL of CNT concentration compared to any previous reports. Further, the produced bioCNT dispersions were characterized by UV-visible, Raman, circular dichroism spectroscopy, and scanning electron microscopy (SEM). Exfoliation and debundling of the bioCNT dispersions is possible due to the π–π interaction, hydrogen bonding, hydrophobic interaction, and electrostatic attractive forces driving the adsorption of BSA on CNTs surface. Protein adsorption then makes a highly stable suspension in water that can be stored for a prolonged period. CNT dispersions are stable over a wide range of pH from 3 to 10 and at 4 °C or 25 °C for more than 2 months. Here, we also report the facile, inexpensive and green-chemistry method to fabricate a buckypaper (CNT paper), composed of the high packing density, self-assembled and randomly oriented bioCNTs, and these assemblies could be used in many emerging applications like air and water purification, nanocomposites, energy storage, and biosensing. Moreover, the CNT dispersions stabilized by BSA were successfully used in enzyme binding and kinetic studies and bound enzyme retained substantial catalytic activity. The current approach may facilitate bulk production of water dispersed CNTs in both academic and industrial laboratories. This is done by a simple method of stirring, which provides new opportunities for a wider range of CNT applications.

LinB is a haloalkane dehalogenase found in Sphingobium indicum B90A, an aerobic bacterium isolated from contaminated soils of hexachlorocyclohexane (HCH) dumpsites. We showed that this enzyme also converts hexabromocyclododecanes (HBCDs). Here we give new insights in the kinetics and stereochemistry of the enzymatic transformation of δ-HBCD, which resulted in the formation of two pentabromocyclododecanols (PBCDols) as first- (P1δ, P2δ) and two tetrabromocyclododecadiols (TBCDdiols) as second-generation products (T1δ, T2δ). Enzymatic transformations of δ-HBCD, α1-PBCDol, one of the transformation products, and α2-PBCDol, its enantiomer, were studied and modeled with Michaelis-Menten (MM) kinetics. Respective MM-parameters KM, vmax, kcat/KM indicated that δ-HBCD is the best LinB substrate followed by α2- and α1-PBCDol. The stereochemistry of these transformations was modeled in silico, investigating respective enzyme-substrate (ES) and enzyme-product (EP) complexes. One of the four predicted ES-complexes led to the PBCDol product P1δ, identical to α2-PBCDol with the 1R,2R,5S,6R,9R,10S-configuration. An SN2-like substitution of bromine at C6 of δ-HBCD by Asp-108 of LinB and subsequent hydrolysis of the alkyl-enzyme led to α2-PBCDol. Modeling results further indicate that backside attacks at C1, C9 and C10 are reasonable too, selectively binding leaving bromide ions in a halide pocket found in LinB. Docking with α2-PBCDol, also allowed productive enzyme binding. A TBCD-1,5-diol with the 1S,2S,5R,6R,9S,10R-configuration is the predicted second-generation product T1δ. In conclusion, in vitro- and in silico findings now allow a detailed description of step-wise enzymatic dehalohydroxylation reactions of δ-HBCD to specific PBCDols and TBCDdiols at A-resolution and predictions of their stereochemistry.

A series of triazolo-thiadiazepines 4a&ndash, k were synthesized with excellent yields using dehydrated PTSA as a catalyst in toluene. Two triazolo-thiadiazines were obtained, 8a was formed directly by reflux in ethanol, whereas, PTSA promoted the formation of 8b. The molecular structure of the formed triazolo-thiadiazepines is identical to the imine-form 4a&ndash, k and not the enamine-tautomer 6a&ndash, k. The structures of the newly synthesized triazolo-thiadiazepines 4a&ndash, k and triazolo-thiadiazines 8a&ndash, b were elucidated using NMR (1H, and 13C), 2D NMR, HRMS, and X-ray single crystal. Furthermore, 4a was deduced using X-ray single crystal diffraction analysis. These new thiadiazepine hits represent an optimized series of previously synthesized indole-triazole derivatives for the inhibition of EGFR. The cytotoxicity activity against two cancer cell lines including human liver cancer (HEPG-2) and breast cancer (MCF-7) was promising, with IC50 between 12.9 to 44.6 &micro, g/mL and 14.7 to 48.7 &micro, g/mL for the tested cancer cell lines respectively, compared to doxorubicin (IC50 4.0 &micro, g/mL). Docking studies revealed that the thiadiazepine scaffold presented a suitable anchor, allowing good interaction of the various binding groups with the enzyme binding regions and sub-pockets.