Importance of C-Terminal Region for Thermostability of GH11 Xylanase from Streptomyces lividans

The amino acid sequences of xylanase B (XlnB) and xylanase C (XlnC) from Streptomyces lividans show significant homology. However, the temperature optima and stabilities of the two enzymes are quite different. XlnB exhibits an optimum temperature of 40 °C and retains 50% of its maximum activity at 43 °C, whereas the corresponding values for XlnC are 60 and 70 °C. To analyze these properties further, as well as to study the effect of the exchange of homologous segments in the C-terminal region, four chimeras designated as BSC, BFC, CSB, and CFB were constructed by substituting segments from the C-terminal homologous region of XlnB gene with that of XlnC and in turn substituting XlnC gene with that of XlnB. The purified chimeric enzymes were characterized with respect to pH/temperature activity, stability, and kinetic parameters. Most of enzymatic properties of chimeras were admixtures of those of the two parents. The chimeric enzymes were optimally active at 45–55 °C and pH 7.0. Both K _m and k _cat values of chimeric enzymes for p-nitrophenyl-β-d-cellobioside were admixtures of both parental enzymes, except that the k _cat value of chimeric BFC (2.79 s⁻¹) was higher than that of parental XlnC (1.99 s⁻¹). Notably, thermal stability of chimeric BSC and BFC was increased by 25 and 13 °C separately, as compared to one of parental XlnB, whereas the thermal stability of chimeric CSB and CFB was decreased by 23 and 21 °C, respectively, as compared to another parental XlnC. These results suggest that homologous C-terminal region in S. lividans GH11 xylanase appears to play an important role in determining enzyme characteristics, and exchanging of different segments of gene in this region might significantly alter or improve the enzymatic properties such as thermal stability.     

The Application of Engineered Glucose Dehydrogenase to a Direct Electron–Transfer‐Type Continuous Glucose Monitoring System and a Compartmentless Biofuel Cell

Abstract

Continuous glucose monitoring (CGM) is expected to become an ideal way to monitor glycemic levels in diabetic patients. On the other hand, biofuel cells can be used as an alternative energy source in future implantable devices, such as implantable glucose sensors in the artificial pancreas. Glucose dehydrogenase from Acinetobacter calcoaceticus, which harbors pyrroloquinoline quinone as the prosthetic group (PQQGDH), is one of the enzymes most attractive as a glucose sensor constituent and as the anode enzyme in biofuel cells, due to its high catalytic activity and insensitivity to oxygen. However, the application of PQQGDH for these purposes is inherently limited because an electron mediator is required for the electron transfer to the electrode.

We have recently reported on the development of an engineered enzyme, quinohemoprotein glucose dehydrogenase (QH‐GDH), in which the cytochrome c domain of the quinohemoprotein ethanol dehydrogenase (QH‐EDH) was fused with PQQGDH, to enable electron transfer to the electrode in the absence of an artificial mediator. In this study, we constructed a direct electron‐transfer‐type CGM system employing QH‐GDH. This CGM system showed sufficient current response and high operational stability. Furthermore, we successfully constructed a compartmentless biofuel cell employing QH‐GDH.     

Recognition of Artificial Nucleobases by E. coli Purine Nucleoside Phosphorylase versus its Ser90Ala Mutant in the Synthesis of Base‐Modified Nucleosides

A wide range of natural purine analogues was used as probe to assess the mechanism of recognition by the wild‐type (WT) E. coli purine nucleoside phosphorylase (PNP) versus its Ser90Ala mutant. The results were analyzed from viewpoint of the role of the Ser90 residue and the structural features of the bases. It was found that the Ser90 residue of the PNP 1) plays an important role in the binding and activation of 8‐aza‐7‐deazapurines in the synthesis of their nucleosides, 2) participates in the binding of α‐D ‐pentofuranose‐1‐phosphates at the catalytic site of the PNP, and 3) catalyzes the dephosphorylation of intermediary formed 2‐deoxy‐α‐D ‐ribofuranose‐1‐phosphate in the trans‐2‐deoxyribosylation reaction. 5‐Aza‐7‐deazaguanine manifested excellent substrate activity for both enzymes, 8‐amino‐7‐thiaguanine and 2‐aminobenzothiazole showed no substrate activity for both enzymes. On the contrary, the 2‐amino derivatives of benzimidazole and benzoxazole are substrates and are converted into the N1‐ and unusual N2‐glycosides, respectively. 9‐Deaza‐5‐iodoxanthine showed moderate inhibitory activity of the WT E. coli PNP, whereas 9‐deazaxanthine and its 2′‐deoxyriboside are weak inhibitors.     

Enhanced stability of glucoamylase through chemical crosslinking

A series of bifunctional chemical modification reagents, presenting variations in both the chemistry of the functional groups and in the length of the spacer between the two reactive groups, have been evaluated as agents for enhancing the thermal stability of purifiedAspergillus niger amyloglucosidase by means of intramolecular cross-linking. Several chemical modifiers (e.g., diimidoesters) were identified that more than double the half-life of this industrially important enzyme during incubation at 65°C in the absence of substrate. The increased stability of the modified enzymes has been correlated with changes in the fluorescence-monitored thermal denaturation curves of the modified enzymes, relative to that of the native enzyme.     

Nonnucleosidic Base Surrogates: The Effect of 1,2‐Disubstituted Phenanthrenes on DNA Duplex Stability

Phenanthrene‐1,2‐dimethanol was incorporated into oligodeoxynucleotides via formation of phosphodiester bonds (cf. Scheme 1). If placed at internal positions in a DNA duplex, a strong reduction of duplex stability is observed (Table 1). Terminal attachment of stretches of phenanthrene residues, however, leads to a substantial increase in stability. The stabilization is attributed to a cooperative interaction of the phenanthrene residues of the two strands rather than to dangling end effects. Chimeric oligomers containing a stretch of six phenanthrene residues show two separate transitions (Table 2): one arising from the denaturation of the DNA stem (observable by a hyperchromic effect at 260 nm) and a second one from the denaturation of the phenanthrene part (observable by temperature‐dependant gel mobility assays). Based on these findings, a model of the chimeric hybrids is proposed, in which the phenanthrene residues stack in a zipper‐like manner on top of the DNA base pairs without disrupting the B‐form of the DNA stem (see Fig. 7).     

The role of residues Arg169 and Arg220 in intersubunit interactions of yeast D-amino acid oxidase

D-Amino acid oxidase from the yeast Trigonopsis variabilis (EC 1.4.3.3, TvDAAO) exists as a dimer consisting of two identical subunits. The dimeric structure of the enzyme is stabilized by 12 (six pairs) hydrogen bonds, the residues Arg169 and Arg220 of each subunit being involved in eight hydrogen bonds. The Arg169Glu and Arg(169,220)Ala mutants of TvDAAO were prepared. Both mutant enzymes were expressed in E. coli cells as insoluble but catalytically active inclusion bodies. The introduction of amino acid substitutions at the intersubunit interface resulted in a change in the substrate specificity profile and a strong decrease in thermal stability.     

Thermal inactivation of alkali phosphatases under various conditions

The thermal inactivation of alkali phosphatases from bacteria Escherichia coli (ECAP), bovine intestines (bovine IAP), and chicken intestines (chicken IAP) was studied in different buffer solutions and in the solid state. The conclusion was made that these enzymes had maximum stability in the solid state, and, in a carbonate buffer solution, their activity decreased most rapidly. It was found that the bacterial enzyme was more stable than animal phosphatases. It was noted that, for ECAP, four intermediate stages preceded the loss of enzyme activity, and, for bovine and chicken IAPs, three intermediate stages were observed. The activation energy of thermal inactivation of ECAP over the range 25–70°C was determined to be 80 kJ/mol; it corresponded to the dissociation of active dimers into inactive monomers. Higher activation energies (∼200 kJ/mol) observed at the initial stage of thermal inactivation of animal phosphatases resulted from the simultaneous loss of enzyme activity caused by dimer dissociation and denaturation. It was shown that the activation energy of denaturation of monomeric animal alkali phosphatases ranged from 330 to 380 kJ/mol depending on buffer media. It was concluded that the inactivation of solid samples of alkali phosphatases at 95°C was accompanied by an about twofold decrease in the content of β structures in protein molecules. Original Russian Text ? L.F. Atyaksheva, B.N. Tarasevich, E.S. Chukhrai, O.M. Poltorak, 2009, published in Zhurnal Fizicheskoi Khimii, 2009, Vol. 83, No. 2, pp. 391–396.     

Expression and characterization of mutant forms of penicillin acylase from Alcaligenes faecalis

The sequencing of six plasmids carrying a gene of penicillin acylase from Alcaligenes faecalis VKM B1518 (AfPA) revealed the presence of random mutations in the gene; they occurred during a polymerase chain reaction. Six mutant AfPAs and a wild-type enzyme were expressed in E. coli cells. The activity assay of mutant AfPAs in E. coli cells indicated that several amino acid substitutions affect the expression level of the AfPA gene and the rate of cell growth. Four mutant AfPAs were purified; their catalytic properties and thermal stability were studied. It is shown that the amino acid substitutions under study do not affect the catalytic efficiency value. Within the experimental error, the βQ133R and βK184E (the AfPA M2 mutant) substitutions had no effect on the thermal stability of the enzyme; in the case of mutants AfPA M4 (βY90H), M5 (αD132G, βR97C), and M6 (αV5E, αN183S, and βE439G), the inactivation rate constant increased 2.4, 2.75, and 8.3 times, respectively, as compared to that of the wild-type enzyme.     

A conductometric immunosensor based on functionalized magnetite nanoparticles for E. coli detection

We report a new approach for immunoassays based on magnetite nanoparticles for Escherichia coli (E. coli) detection using conductometric measurements. Biotinylated antibodies, anti-E. coli, were immobilized on streptavidin modified magnetite nanoparticles by biotin–streptavidin interaction. A layer of functionalized nanoparticles were directly immobilized on the conductometric electrode using glutaraldehyde cross-linking.The specific test with E. coli cells and the non specific test using Staphylococcus epidermidis (S. epidermidis) were investigated by conductometric measurements. Results show a good response as a function of antigen additions. The detection of 1 CFU/ml of E. coli induces a conductivity variation of 35 μS. The negative test shows good selectivity using the conductometric immunosensor. Conductometric measurements allow to detect 500 CFU/l.     

Heterologous Expression of Two Ferulic Acid Esterases from Penicillium funiculosum

Two recombinant ferulic acid esterases from Penicillium funiculosum produced in Aspergillus awamori were evaluated for their ability to improve the digestibility of pretreated corn stover. The genes, faeA and faeB, were cloned from P. funiculosum and expressed in A. awamori using their native signal sequences. Both enzymes contain a catalytic domain connected to a family 1 carbohydrate-binding module by a threonine-rich linker peptide. Interestingly, the carbohydrate binding-module is N-terminal in FaeA and C-terminal in FaeB. The enzymes were purified to homogeneity using column chromatography, and their thermal stability was characterized by differential scanning microcalorimetry. We evaluated both enzymes for their potential to enhance the cellulolytic activity of purified Trichoderma reesei Cel7A on pretreated corn stover.     

Norvancomycin-capped silver nanoparticles: Synthesis and antibacterial activities against E. coli

The synthesis of norvancomycin (NVan)-capped silver nanoparticles (Ag@NVan) and their notable in vitro antibacterial activities against E. coli, a Gram-negative bacterial strain (GNB), are reported here. Mercaptoacetic acid-stabilized spherical silver nanoparticles with a diameter of 16±4 nm are prepared by a simple chemical reaction. The formation process of the silver nanoparticles is investigated by UV-visible (UV-vis) spectroscopy and transmission electron microscopy (TEM). NVan is then grafted to the terminal carboxyl of the mercaptoacetic acid in the presence of N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC). The TEM images of single bacteria treated with Ag@NVan show that plenty of Ag@NVan aggregate in the cell wall of E. coli. A possible antibacterial mechanism is proposed that silver nanoparticles may help destroy the stability of the outer membrane of E. coli, which makes NVan easier to bind to the nether part of the peptidoglycan structure. The antibacterial activities of silver nanoparticles on their own, together with the rigid polyvalent interaction between Ag@NVan and cell wall, enables Ag@NVan to be an effective inhibitor of GNB. This kind of bionanocomposites might be used as novel bactericidal materials and we also provide an effective synthesis method for preparing functional bioconjugated nanoparticles here. Supported by the National Natural Science Foundation of China (Grant No. 50373036) and Fok Ying Tung Education Foundation (Grant No. J20040212)     

A Facile Method for Producing Selenocysteine‐Containing Proteins

Selenocysteine (Sec, U) confers new chemical properties on proteins. Improved tools are thus required that enable Sec insertion into any desired position of a protein. We report a facile method for synthesizing selenoproteins with multiple Sec residues by expanding the genetic code of Escherichia coli. We recently discovered allo‐tRNAs, tRNA species with unusual structure, that are as efficient serine acceptors as E. coli tRNA^Ser. Ser‐allo‐tRNA was converted into Sec‐allo‐tRNA by Aeromonas salmonicida selenocysteine synthase (SelA). Sec‐allo‐tRNA variants were able to read through five UAG codons in the fdhF mRNA coding for E. coli formate dehydrogenase H, and produced active FDH_H with five Sec residues in E. coli. Engineering of the E. coli selenium metabolism along with mutational changes in allo‐tRNA and SelA improved the yield and purity of recombinant human glutathione peroxidase 1 (to over 80 %). Thus, our allo‐tRNA^UTu system offers a new selenoprotein engineering platform.     

Synthesis,Characterization, and Repair of a Flexible O6‐2′‐Deoxyguanosine‐alkylene‐O6‐2′‐deoxyguanosine Intrastrand Cross‐Link

Oligonucleotides tethered by an alkylene linkage between the O⁶‐atoms of two consecutive 2′‐deoxyguanosines, which lack a phosphodiester linkage between these residues, have been synthesized as a model system of intrastrand cross‐linked (IaCL) DNA. UV thermal denaturation studies of duplexes formed between these butylene‐ and heptylene‐linked oligonucleotides with their complementary DNA sequences revealed about 20 °C reduction in stability relative to the unmodified duplex. Circular dichroism spectra of the model IaCL duplexes displayed a signature characteristic of B‐form DNA, suggesting minimal global perturbations are induced by the lesion. The model IaCL containing duplexes were investigated as substrates of O⁶‐alkylguanine DNA alkyltransferase (AGT) proteins from human and E. coli (Ada‐C and OGT). Human AGT was found to repair both model IaCL duplexes with greater efficiency towards the heptylene versus butylene analog adding to our knowledge of substrates this protein can repair.     

Boron increases the transition temperature and enhances thermal stability of heme proteins

Transition temperature and thermal stability of proteins were studied in the presence and absence of boron. The observed midpoint of thermal denaturation (T _m) of cytochrome c (Cyt c) at pH 9.2 was 68.8 °C, which in the presence of boron increased to 71.0 °C. For metmyoglobin, T _m increased from 79.7 °C in the absence of boron to 83.5 °C in the presence of boron. Boron caused an increase of 10% in the reversibility of thermal denaturation of cytochrome c when compared with control. Activity measurements of the heat treated proteins and T _m suggest an increased thermal stability toward inactivation and denaturation of heme proteins in the presence of boron.     

O‐Mannosylation Affords a Glycopeptide Hydrogel with Inherent Antibacterial Activities against E. coli via Multivalent Interactions between Lectins and Supramolecular Assemblies

Multivalent carbohydrate–lectin interactions play a crucial role in bacterial infection. Biomimicry of multivalent glycosystems represents a major strategy in the repression of bacterial growth. In this study, a new kind of glycopeptide (Naphthyl‐Phe‐Phe‐Ser‐Tyr, NMY) scaffold with mannose modification is designed and synthesized, which is able to perform supramolecular self‐assembly with the assistance of catalytic enzyme, and present multiple mannose ligands on its self‐assembled structure to target mannose‐binding proteins. Relying on multivalent carbohydrate–lectin interactions, the glycopeptide hydrogel is able to bind Escherichia coli (E. coli) in high specificity, and result in bacterial adhesion, membrane disruption and subsequent cell death. In vivo wound healing assays reveal that this glycopeptide hydrogel exhibits considerable potentials for promoting wound healing and preventing E. coli infection in a full‐thickness skin defect mouse model. Therefore, through a specific mannose–lectin interaction, a biocompatible hydrogel with inherent antibacterial activity against E. coli is achieved without the need to resort to antibiotic or antimicrobial agent treatment, highlighting the potential role of sugar‐coated nanomaterials in wound healing and control of bacterial pathogenesis.     

Temperature-induced denaturation of Aes acetyl-esterase from Escherichiacoli

The thermal stability of the Aes acetyl esterase from Escherichia coli has been investigated by means of differential scanning calorimetry and circular dichroism measurements. The calorimetric curves show a denaturation temperature of 68 °C for Aes and 61 °C for the single point mutant V20D-Aes. The same values are obtained from CD denaturation curves of the two proteins recorded in both the far-UV and near-UV regions. Even if the denaturation process is irreversible and characterized by a single calorimetric peak and a single inflection point in both far- and near-UV CD curves, the overall data indicate that the process is more complex than a two-state transition. This is in line with the presence of two structural domains in the 3D model of Aes, according to homology modelling. A comparison of the thermal stability of Aes with those of the homologous thermophilic EST2 and hyperthermophilic AFEST suggests that the optimization of charge-charge interactions should not be so effective in the case of the mesophilic enzyme.     

Preferential solvation of lysozyme by dimethyl sulfoxide in binary solutions of water and dimethyl sulfoxide

To reveal the denaturation mechanism of lysozyme by dimethyl sulfoxide (DMSO), thermal stability of lysozyme and its preferential solvation by DMSO in binary solutions of water and DMSO was studied by differential scanning calorimetry (DSC) and using densities of ternary solutions of water (1), DMSO (2) and lysozyme (3) at 298.15 K. A significant endothermic peak was observed in binary solutions of water and DMSO except for a solution with a mole fraction of DMSO (x ₂) of 0.4. As x ₂ was increased, the thermal denaturation temperature T _m decreased, but significant increases in changes in enthalpy and heat capacity for denaturation, ΔH _cal and ΔC _p, were observed at low x ₂ before decreasing. The obtained amount of preferential solvation of lysozyme by DMSO (∂g ₂/∂g ₃) was about 0.09 g g⁻¹ at low x ₂, indicating that DMSO molecules preferentially solvate lysozyme at low x ₂. In solutions with high x ₂, the amount of preferential solvation (∂g ₂/∂g ₃) decreased to negative values when lysozyme was denatured. These results indicated that DMSO molecules do not interact directly with lysozyme as denaturants such as guanidine hydrochloride and urea do. The DMSO molecules interact indirectly with lysozyme leading to denaturation, probably due to a strong interaction between water and DMSO molecules.     

Display of Escherichia coli Phytase on the Surface of Bacillus subtilis Spore Using CotG as an Anchor Protein

Escherichia coli phytase (AppA) has been widely used as an exogenous feed enzyme for monogastric animals; however, the production of this enzyme has been examined primarily in E. coli and yeast expression systems. As an alternative to production of soluble phytase, an enzyme immobilization method using the Bacillus subtilis spore outer-coat protein CotG as an anchoring motif for the display of the AppA was attempted. Using this motif, AppA was successfully produced on the spore surface of B. subtilis as verified by Western blot analysis and phytase activity measurements. Analysis of the pH stability indicated that more than 50% activity was retained after incubation at four different pH values (2.0, 4.0, 7.0, and 8.0) for up to 12 h, with maximum activity observed at pH 4.5. The highest enzyme activity seen at 55 °C and thermal stability measurements demonstrated that more than 30% activity remained after 30 min incubation at 60 °C. The spore surface-displayed AppA was resistant to pepsin, and more stable than phytase produced previously using a yeast expression system. Furthermore, we present data indicating that the use of peptide linkers may help improve the bioactivity of displayed enzymes on the spore surface of B. subtilis.

     

Disposable Immunosensor for Escherichia Coli O157:H7 Based on a Multi-Walled Carbon Nanotube-Sodium Alginate Nanocomposite Film Modified Screen-Printed Carbon Electrode

A disposable immunosensor for the detection of Escherichia coli O157:H7 based on a multiwalled carbon nanotube–sodium alginate nanocomposite film was constructed. The nanocomposite was placed on a screen-printed carbon electrode, and horseradish peroxidase-labeled antibodies were immobilized to E. coli O157:H7 on the modified electrode to construct the immunosensor. The modification procedure was characterized by atomic force microscopy and cyclic voltammetry. Under optimal conditions, the proposed immunosensor exhibited good electrochemical sensitivity to E. coli O157:H7 in a concentration range of 10³–10¹⁰ cfu/mL, with a relatively low detection limit of 2.94 × 10² cfu/mL (S/N = 3). This immunosensor exhibited satisfactory specificity, reproducibility, stability, and accuracy, making it a potential alternative tool for early assessment of E. coli O157:H7.     

Decontamination of vegetables sprayed with organophosphate pesticides by organophosphorus hydrolase and carboxylesterase (B1)

A genetically engineered Escherichia coli cell expressing both organophosphorus hydrolase (OPH) and carboxyl esterase (CaE) B1 intracellularly was constructed and cultivated. The harvested wet cells were vacuum dried, and the storage stability of the dried cell powder was determined in terms of OPH activity. Over a period of 5 mo, the dried cells showed no significant decrease in the activities of the detoxifying enzymes. The crude enzymes in 50 mM citrate-phosphate buffer (pH 8.0) were able to degrade approx 97% of the organophosphate pesticides sprayed on cabbage. The detoxification efficiency was superior to that of the treatments of water, detergent, and a commercially available enzyme product. Additionally, the products of pesticide hydrolysis generated by treatment with the enzyme extract were determined to be virtually nontoxic.