Tailoring surface properties of functionalized graphene papers aiming to enzyme immobilization

The use of enzymes as catalysts requires recovery and reuse to make the process viable. Enzymatic immobilization changes enzyme stability, activity, and specificity. It is very important to explore new substrates for immobilization with appropriate composition and structure to improve the efficiency of the immobilized enzymes. This work explores the use of two different graphene oxide papers, one produced by oxidation route (GO) and the other by electrochemical synthesis (EG), aiming for β-galactosidase immobilization. The chemical and structural properties of these two papers were characterized by Raman spectroscopy, X-ray photoelectron spectroscopy and X-ray diffraction. Atomic force microscopy images showed that EG paper ensured more efficient immobilization of the enzymes on the surface of the paper. Cyclic voltammetry was used to monitor the reaction of conversion of lactose to glucose in the free enzyme solution and graphene paper immobilized enzyme solutions. The cyclic voltammetry analysis showed that immobilized enzymes on GO paper showed an improvement in the activity of β-galactose when compared to free enzyme solution, as well as enzyme immobilized on a glassy carbon electrode.

Engineering a Pseudo-26-kDa Schistosoma Glutathione Transferase from bovis/haematobium for Structure, Kinetics, and Ligandin Studies

Glutathione transferases (GSTs) are the main detoxification enzymes in schistosomes. These parasitic enzymes tend to be upregulated during drug treatment, with Schistosoma haematobium being one of the species that mainly affect humans. There is a lack of complete sequence information on the closely related bovis and haematobium 26-kDa GST isoforms in any database. Consequently, we engineered a pseudo-26-kDa S. bovis/haematobium GST (Sbh26GST) to understand structure–function relations and ligandin activity towards selected potential ligands. Sbh26GST was overexpressed in Escherichia coli as an MBP-fusion protein, purified to homogeneity and catalyzed 1-chloro-2,4-dinitrobenzene-glutathione (CDNB-GSH) conjugation activity, with a specific activity of 13 μmol/min/mg. This activity decreased by ~95% in the presence of bromosulfophthalein (BSP), which showed an IC50 of 27 µM. Additionally, enzyme kinetics revealed that BSP acts as a non-competitive inhibitor relative to GSH. Spectroscopic studies affirmed that Sbh26GST adopts the canonical GST structure, which is predominantly α-helical. Further extrinsic 8-anilino-1-naphthalenesulfonate (ANS) spectroscopy illustrated that BSP, praziquantel (PZQ), and artemisinin (ART) might preferentially bind at the dimer interface or in proximity to the hydrophobic substrate-binding site of the enzyme. The Sbh26GST-BSP interaction is both enthalpically and entropically driven, with a stoichiometry of one BSP molecule per Sbh26GST dimer. Enzyme stability appeared enhanced in the presence of BSP and GSH. Induced fit ligand docking affirmed the spectroscopic, thermodynamic, and molecular modelling results. In conclusion, BSP is a potent inhibitor of Sbh26GST and could potentially be rationalized as a treatment for schistosomiasis.

Poly(vinyl alcohol)-based Electrospun Nanofibers: Characterization and Phytase Immobilization

The aim of this study is to immobilization phytase obtained from cowpea seeds into nanofiber-based on poly(vinyl alcohol) (PVA) and to investigate kinetic properties, optimal pH, and temperature of free and immobilized phytase. The structural analysis and morphological properties of the nanofibers are carried out via SEM and XRD. The results indicated that enzyme stability, pH, and thermal stability are increased after immobilizing phytase into the nanofiber. The optimum pH and temperature of the free and immobilized phytase are found as pH 5.0 and 45-65 oC, respectively. These results indicated that the immobilized phytase could be a good candidate for agriculture, animal feed, food, and medical applications.

Towards a Synthetic Biology Toolset for Metallocluster Enzymes in Biosynthetic Pathways: What We Know and What We Need

Microbes are routinely engineered to synthesize high-value chemicals from renewable materials through synthetic biology and metabolic engineering. Microbial biosynthesis often relies on expression of heterologous biosynthetic pathways, i.e., enzymes transplanted from foreign organisms. Metallocluster enzymes are one of the most ubiquitous family of enzymes involved in natural product biosynthesis and are of great biotechnological importance. However, the functional expression of recombinant metallocluster enzymes in live cells is often challenging and represents a major bottleneck. The activity of metallocluster enzymes requires essential supporting pathways, involved in protein maturation, electron supply, and/or enzyme stability. Proper function of these supporting pathways involves specific protein–protein interactions that remain poorly characterized and are often overlooked by traditional synthetic biology approaches. Consequently, engineering approaches that focus on enzymatic expression and carbon flux alone often overlook the particular needs of metallocluster enzymes. This review highlights the biotechnological relevance of metallocluster enzymes and discusses novel synthetic biology strategies to advance their industrial application, with a particular focus on iron-sulfur cluster enzymes. Strategies to enable functional heterologous expression and enhance recombinant metallocluster enzyme activity in industrial hosts include: (1) optimizing specific maturation pathways; (2) improving catalytic stability; and (3) enhancing electron transfer. In addition, we suggest future directions for developing microbial cell factories that rely on metallocluster enzyme catalysis.

Rational Protein Engineering to Increase the Activity and Stability of IsPETase Using the PROSS Algorithm

Polyethylene terephthalate (PET) is the most widely used polyester plastic, with applications in the textile and packaging industry. Currently, re-moulding is the main path for PET recycling, but this eventually leads to an unsustainable loss of quality; thus, other means of recycling are required. Enzymatic hydrolysis offers the possibility of monomer formation under mild conditions and opens up alternative and infinite recycling paths. Here, IsPETase, derived from the bacterium Ideonella sakaiensis, is considered to be the most active enzyme for PET degradation under mild conditions, and although several studies have demonstrated improvements to both the stability and activity of this enzyme, stability at even moderate temperatures is still an issue. In the present study, we have used sequence and structure-based bioinformatic tools to identify mutations to increase the thermal stability of the enzyme so as to increase PET degradation activity during extended hydrolysis reactions. We found that amino acid substitution S136E showed significant increases to activity and stability. S136E is a previously unreported variant that led to a 3.3-fold increase in activity relative to wild type.

Extraction, Purification and Characterization of Transglutaminase From Some Plants

The present study aimed to Isolate trans-glutaminase EC:2.3.2.13 from some plants sources and Purified it and Studied it’s Charctarestics as well as it’s practical applications in the production of sausage.The enzyme was extracted from four types of plants (rosemary, chard, radish, arugula) using nine extraction solutions that included distilled water,Sodium chloride 3% solution, sodium chloride 5% solution, sodium phosphate solution 0.1 M and an pH 6.5, sodium phosphate 0.1 M and an pH of 7.5, Tris - Hcl solution 0.2 M and pH 7, Tris - Hcl solution 0.2 M and an pH 8, Tris - Hcl solution 0.1 M and an pH 7 and Tris - Hcl solution 0.1 M and an pH 8 in order to find out the best source of enzyme and the best extraction solution. chard was the best source of enzyme compared with other sources, Tirs -HCl 0.1M, pH 8 solution was the best extraction solution which gave the highest specific activity 8.104 unit/mg.Protein content for the crude enzyme extracts were concentrated using saturated ammonium sulfate in arrange 20-60%, Dialysis was done using distilled water. Then, the purification steps of the enzyme were completed using the gel filtration in the Sephadex G-100 Purification Folds 13.91 time and the yield was 20.04% %. Electrophoresise process using poly acryl amid gel in the absence of SDS observe the presence of one protein band which indicates the complete purification of transglutaminase. transglutaminase molecular weight was 42,660 Dalton when it was evaluated using poly acryl amide electrophoresis in the presence of SDS. the optimum pH for enzyme activity and enzyme stability was 7, while the optimum temperature for enzyme activity was 55°C, and the optimum temperature for enzyme stability was between 25-45°C.

Enzymatic Activity and Physicochemical Properties of Soil Profiles of Luvisols

Most studies on soil enzymes are focused on the upper horizons of the soil profile, even though they transform the soil organic matter at every depth of the soil profile. The aim of this work was to investigate the distribution of β-glucosidase (GLU), nitrate reductase (NR), urease (UR), phosphatase (PHA), dehydrogenase (DHA) and catalase (CAT) activity through 14 trunked soil profiles of the Luvisols formed from a glacial till. The content of microbial biomass carbon (MBC) as well as physicochemical properties such as organic carbon (CORG), total nitrogen (NTOT), available P, K and Mg, soil density and porosity, pH in KCl and fractional composition were also studied. In general, enzymatic activity was highest in the top 30 cm layer of the profiles and decreased progressively towards the deeper horizons. The exceptions were the NR activity, which was active only in the Ap horizon and whose activity decreased sharply to nearly zero in the Bt horizon and parent rock, and the PHA activity, which was highly active even in the parent rock depth. The decreased availability of carbon and nutrients was the main driver of decreases in microbial abundance and enzymatic activity with depth. The enzymatic activity, when expressed on a CORG and MBC basis, behaves differently compared to the activity expressed on a soil mass basis. The activity decreased (NR), increased (PHA, UR), showed no clear pattern (GLU) or the changes were not significant (DHA, CAT). The content of CORG, NTOT, K and PAVAIL generally decreased with depth, while for Mg, there was no clear direction in the profile distribution. Future studies to characterize the substrate distribution within the soil profile and enzyme stability will provide further insight into the controls on nutrient cycling and related enzymes throughout the soil profiles.

The Circadian Rhythms of STAT3 in the Rat Pineal Gland and Its Involvement in Arylalkylamine-N-Acetyltransferase Regulation

In rodents, the melatonin production by the pineal gland is controlled through adrenergic signaling from the suprachiasmatic nuclei and regulation of the principal enzyme in its synthesis, arylalkylamine-N-acetyltransferase (AANAT). In the present study, we identified increased isoprenaline-induced aa-nat expression and nocturnal AANAT activity in the pineal glands in response to the silencing of the signal transducer and activator of transcription 3 (STAT3) with siRNA or STAT3 inhibitors WP1066 and AZD1480. This AANAT activity enhancement in vivo did not interfere with light-induced AANAT suppression. Systemic or in vitro lipopolysaccharide (LPS) administration markedly increased Stat3 expression and STAT3 phosphorylation, but it did not significantly affect AANAT expression or activity. Simultaneous LPS administration and Stat3 silencing enhanced the aa-nat transcription and AANAT activity to a similar extent as Stat3 inhibition without LPS co-administration. Furthermore, we describe the circadian rhythmicity in Stat3 expression and the phosphorylated form of STAT3 protein in the rat pineal gland. Our data suggest that the higher nocturnal endogenous level of STAT3 in the pineal gland decelerates or hampers the process of NA-induced AANAT activation or affects the AANAT enzyme stability.

Active site characterization and activity of the human aspartyl (asparaginyl) β-hydroxylase

Human aspartyl/asparaginyl beta-hydroxylase (HAAH) is a member of the superfamily of nonheme Fe2+/α-ketoglutarate (αKG) dependent oxygenase enzymes with a noncanonical active site. HAAH hydroxylates epidermal growth factor (EGF) like domains to form the β-hydroxylated product from substrate asparagine or aspartic acid and has been suggested to have a negative impact in a variety of cancers. In addition to iron, HAAH also binds divalent calcium, although the role of the latter is not understood. Herein, the metal binding chemistry and influence on enzyme stability and activity have been evaluated by a combined biochemical and biophysical approach. Metal binding parameters for the HAAH active site were determined by use of isothermal titration calorimetry, demonstrating a high-affinity regulatory binding site for Ca2+ in the catalytic domain in addition to the catalytic Fe2+ cofactor. We have analyzed various active site derivatives, utilizing LC-MS and a new HPLC technique to determine the role of metal binding and the second coordination sphere in enzyme activity, discovering a previously unreported residue as vital for HAAH turnover. This analysis of the in vitro biochemical function of HAAH furthers the understanding of its importance to cellular biochemistry and metabolic pathways.

L-asparaginase: therapeutic use and applications in the food industry – a review

L-asparaginase (L-asnase) is an amino hydrolase that has been used in the last decades for leukemia treatment, which boosted scientific studies on production, purification and immobilization of this enzyme. More recently, L-asnase has called food industry attention because of its effect on acrylamide formation in fried and baked foods. Several studies have been carried out in order to evaluate the effect of L-asnase in reducing acrylamide formation in different food models. This review brings up an overview in L-asnase kinetic parameters from different sources, immobilization methods, its therapeutic use in leukemia treatment and food processing applications. This review also discusses acrylamide formation in fried and baked foods. Commercial L-asnase is produced by two microorganisms, Escherichia coli and Erwinia sp. However, studies using different microorganisms have shown the possibility of producing this enzyme from different sources, obtaining enzymes with interesting kinetic properties. Immobilization strategies have provided enzymes with greater activity and stability, which could contribute to maintain L-asnase activity in the body for longer periods. Researches applying L-asnase in food products have shown significant reduction in acrylamide production, above 90% in some cases. For this purpose, during enzyme application some variables must be taken into account, as enzyme dose, food matrix, pretreatment, processing time and temperature. Medical and food applications make L-asnase a multipurpose enzyme. Reducing prices, improving enzyme stability and reducing co-lateral effects in leukemia treatment are still challenges to overcome.