New extracellular thermostable oxalate oxidase produced from endophytic Ochrobactrum intermedium CL6: Purification and biochemical characterization

Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide with the reduction of molecular oxygen to hydrogen peroxide. Oxalate oxidase found its application in clinical assay for oxalate in blood and urine. This study describes the purification and biochemical characterization of an oxalate oxidase produced from an endophytic bacterium, Ochrobactrum intermedium CL6. The cell-free fermentation broth was subjected to two-step enzyme purification, which resulted in a 58.74-fold purification with 83% recovery. Specific activity of the final purified enzyme was 26.78 U?mg^?1 protein. The enzyme displayed an optimum pH and temperature of 3.8 and 80°C, respectively, and high stability at 4–80°C for 6?h. The enzymatic activity was not influenced by metal ions and chemical agents (K⁺, Na⁺, Zn²⁺, Fe³⁺, Mn²⁺, Mg²⁺, glucose, urea, lactate) commonly found in serum and urine, with Cu²⁺ being the exception. The enzyme appears to be a metalloprotein stimulated by Ca²⁺ and Fe²⁺. Its K_m and K_cat for oxalate were found to be 0.45?mM and 85?s^?1, respectively. This enzyme is the only known oxalate oxidase which did not show substrate inhibition up to a substrate concentration of 50?mM. Thermostability, kinetic properties, and the absence of substrate inhibition make this enzyme an ideal candidate for clinical applications.     

A novel nanobiosensor for the detection of paraoxon using chitosan-embedded organophosphorus hydrolase immobilized on Au nanoparticles

Organophosphorus (OP) compounds are one of the most hazardous chemicals used as insecticides/pesticide in agricultural practices. A large variety of OP compounds are hydrolyzed by organophosphorus hydrolases (OPH; EC 3.1.8.1). Therefore, OPHs are among the most suitable candidates that could be used in designing enzyme-based sensors for detecting OP compounds. In this work, a novel nanobiosensor for the detection of paraoxon was designed and fabricated. More specifically, OPH was covalently embedded onto chitosan and the enzyme–chitosan bioconjugate was then immobilized on negatively charged gold nanoparticles (AuNPs) electrostatically. The enzyme was immobilized on AuNPs without chitosan as well, to compare the two systems in terms of detection limit and enzyme stability under different pH and temperature conditions. Coumarin 1, a competitive inhibitor of the enzyme, was used as a fluorogenic probe. The emission of coumarin 1 was effectively quenched by the immobilized Au-NPs when bound to the developed nanobioconjugates. However, in the presence of paraoxon, coumarin 1 left the nanobioconjugate, leading to enhanced fluorescence intensity. Moreover, compared to the immobilized enzyme without chitosan, the chitosan-immobilized enzyme was found to possess decreased K_m value by more than 50%, and increased V_max and K_cat values by around 15% and 74%, respectively. Higher stability within a wider range of pH (2–12) and temperature (25–90°C) was also achieved. The method worked in the 0 to 1050?nM concentration ranges, and had a detection limit as low as 5?×?10^?11 M.     

Application and characterization of magnetic chitosan microspheres for enhanced immobilization of cellulase

In this study, a unique carrier magnetic chitosan microspheres (MCTS) was simply synthesized by anchoring Fe₃O₄ onto chitosan for direct immobilization of cellulases cross-linked by gluteraldehye. The structure and morphology were characterized using FT-IR, TGA, VSM and SEM. The optimum immobilization conditions were investigated: immobilized pH 7.0, amount of enzyme 15?mL (0.1?mg/mL), immobilization temperature 30?°C, immobilization time 5?h. At optimum conditions, MCTS achieved maximum enzyme solid loading rate of 73.5?mg/g, while recovery of enzyme activity approached to 71.6%. In the recycle test, immobilized cellulases operated without significant loss in its initial performances after 3 cycles, which indicated that immobilized cellulases can be regenerated and reused. The immobilized enzyme has better values of thermal and storage stability than that of free enzyme. Therefore, MCTS may be considered as a candidate with potential value of application in large-scale operations for cellulases immobilization.     

β-xylosidase from Selenomonas ruminantium: Immobilization,stabilization, and application for xylooligosaccharide hydrolysis

The tetrameric β-xylosidase from Selenomonas ruminantium is very stable in alkaline pH allowing it to easily immobilize by multipoint covalent attachments on highly activated glyoxyl agarose gels. Initial immobilization resulted only in slight stabilization in relation to the free enzyme, since involvement of all subunits was not achieved. Coating the catalyst with aldehyde-dextran or polyethylenimine, fully stabilized the quaternary structure of the enzyme rendering much more stabilization to the biocatalyst. The catalyst coated with polyethylenimine of molecular weight 1300 is the most stable one exhibiting an interesting half-life of more than 10 days at pH 5.0 and 50?°C, being, therefore, 240-fold more stable than free enzyme. Optimum activity was observed in the pH range 4.0–6.0 and at 55?°C. The catalyst retained its side activity against p-nitrophenyl α-l-arabinofuranoside and it was inhibited by xylose and glucose. Kinetic parameters with p-nitrophenyl β-d-xylopyranoside as substrate were V_max 0.20?μmol.min^?1?mg?prot.^?1, K_m 0.45?mM, K_cat 0.82?s^?1, and K_cat/K_m 1.82?s^?1?mM^?1. Xylose release was observed from the hydrolysis of xylooligosaccharides with a decrease in the rate of xylose release by increasing substrate chain-length. Due to the high thermostability and the complete stability after five reuse cycles, the applicability of this biocatalyst in biotechnological processes, such as for the degradation of lignocellulosic biomass, is highly increased.     

Amine Oxidases of Microorganisms

The amine oxidase was found to be formed in mycelia of fungi when they were grown on monoamines or diamines as sole nitrogen sources. The maximal formation of enzyme was observed in the initial stage of growth, then the enzyme disappeared semilogarithmically. Other sources of nitrogen, such as ammonia, nitrate, urea and amino acids, were fully inactive for the enzyme formation. Furthermore, ammonia repressed the enzyme formation by fungi. The amine oxidase of fungi resembled in substrate specificity the monoamine oxidase of animal tissues. The enzyme oxidized preferentially aliphatic monoamines of C₃–C₆. Agmatine and histamine were also oxidized but in lower rates. Benzylamine was well oxidized by the enzymes of Aspergillus niger and Penicillium chrysogenum, but not by the enzymes of Monascus anka and Fusarium bulbigenum. Polyamines were not oxidized by the fungal enzymes.     

Locating the binding sites of retinol and retinoic acid with milk β-lactoglobulin

β-lactoglobulin (β-LG) is a member of lipocalin superfamily of transporters for small hydrophobic molecules such as retinoids. We located the binding sites of retinol and retinoic acid on β-LG in aqueous solution at physiological conditions, using FTIR, CD, fluorescence spectroscopic methods, and molecular modeling. The retinoid-binding sites and the binding constants as well as the effect of retinol and retinoic acid complexation on protein stability and secondary structure were determined. Structural analysis showed that retinoids bind strongly to β-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K _{retinol- β -LG?}=?6.4 (±?.6)?×?10⁶?M^?1 and K _{retinoic acid- β -LG?}=?3.3 (±?.5)?×?10⁶?M^?1. The number of retinoid molecules bound per protein (n) is 1.1 (±?.2) for retinol and 1.5 (±?.3) for retinoic acid. Molecular modeling showed the participation of several amino acids in the retinoid–protein complexes with the free binding energy of ?8.11?kcal/mol for retinol and ?7.62?kcal/mol for retinoic acid. Protein conformation was altered with reduction of β-sheet from 59 (free protein) to 52–51% and a major increase in turn structure from 13 (free protein) to 24–22%, in the retinoid–β-LG complexes, indicating a partial protein destabilization.     

The inducible microsomal fatty alcohol oxidase of Candida (Torulopsis) apicola

In the transition phase of Candida apicola IMET 43747 from logarithmic to stationary growth a pyridine-nucleotide-independent alcohol oxidase was induced coinciding with the beginning of sophorose lipid production. This enzyme was not repressed by glucose and was measurable in stationary cells grown on glucose or on a mixture of n-hexadecane and glucose. An NAD⁺-dependent aldehyde dehydrogenase behaved in the same way. Both enzymes were localized in the microsomal fraction. The alcohol oxidase accepted long-chain (fatty) aliphatic alcohols (C₈ to at least C₁₆) and diols starting from decanediol. Trace activities were found with -hydroxy fatty acids. Aromatic, secondary and tertiary alcohols were not oxidized. In the stationary growth phase the substrate specificity of the alcohol oxidase tends to be changed to more hydrophobic substrates. The physiological role of both enzymes, the alcohol oxidase and aldehyde dehydrogenase, is discussed including their possible involvement in the synthesis of sophorose lipid. Correspondence to: R. K. Hommel     

Effect of abiotic resistance inducers, γ-amino-n-butyric acid (GABA), ascorbic acid and chitosan on certain enzyme activities of eggplant inoculated with root-knot nematode,Meloidogyne incognita

Treatments with γ-amino-n-butyric acid (GABA), ascorbic acid (vitamin C) and chitosan by foliar spray or root dipping technique to eggplant growing under greenhouse conditions before and after inoculation of Meloidogyne incognita showed a generalised increase in the activity of the enzymes, peroxidase (POX), polyphenol oxidase (PPO) and chitinase as compared with the infected non treated control. The maximum increase in POX activity occurred after 10?days of nematode inoculation. The relative PPO activity with chitosan at 2500?ppm, GABA at 5000?ppm and ascorbic acid at 10?ppm using root dipping was found to be 375, 338 and 175% of control, respectively. As for PPO oxidase, the maximum activity was observed after five?days of nematode inoculation by using ascorbic acid at 10?ppm followed by GABA at 5000?ppm and chitosan at 2500?ppm by root dipping (800, 767 and 600% of control), respectively, while the highest chitinase enzyme activity (281% of control) was observed using chitosan at 2500?ppm after 10?days of inoculation.     

Improved octyl glucoside synthesis using immobilized β-glucosidase on PA-M with reduced glucose surplus inhibition

A β-glucosidase extracted from bitter almond (Prunus dulcis var. amara) was immobilized on polyamine microspheres (PA-M) for catalytic octyl glucoside (OG) synthesis from glucose and octanol through reversed hydrolysis. The immobilization increased the activity of enzyme at pH 6.0–7.0, and the optimal reaction temperature for immobilized enzyme was identical to the free enzyme. The thermal stability and solvent tolerance of enzyme were increased by its immobilization. In the co-solvent system using 10% t-butyl alcohol and 10% (v/v) water, the yield of OG was increased by 1.7-fold compared to the yield from the system without co-solvent. Based on dynamic and Dixon plot analyses, the initial reaction velocity (V₀) increased approximately three-fold on immobilization and the OG synthesis was inhibited by surplus glucose. The inhibition dissociation constants for free and immobilized enzyme were 219?mM and 116?mM, respectively. A fed-batch mode was applied in the OG synthesis to minimize substrate inhibition. After 336?h of reaction, the OG yield and the conversion rate of glucose reached 134?mM and 59.6%, respectively. Compared to the batch operation, the fed-bath operation increased the OG yield and the conversion rate of glucose by 340% and 381%, respectively.     

Immobilizing cholesterol oxidase in chitosan–alginic acid network

Proton conducting biopolymer networks have potential use for bio-sensors. The cost-effective, non-hazardous and environmentally safe biopolymer, such as chitosan, is an attractive feature for bio-sensors. Cholesterol oxidase was immobilized in conducting network via complexation of chitosan with alginic acid. A method for the preparation of the complex along with characterization by elemental analysis, FTIR spectroscopy, TGA and DSC were reported. The proton conductivity chitosan–alginic acid network was studied via impedance spectroscopy under humidified condition. The complex polymer electrolyte with x = 1 exhibited maximum proton conductivity of 1.4 × 10^?3 S/cm at RT, RH ～ 50%. The potential use of this network in enzyme immobilization was studied by manufacturing cholesterol oxidase entrapped polymer networks. Additionally, the maximum reaction rate (V_max) and Michaelis–Menten constant (K_m) were investigated for the immobilized cholesterol oxidase. Also, temperature and pH optimization studies were performed, and operational stability and shelf life of the polymer network were examined.     

Stimulation of microbial extracellular enzyme activities by elevated CO2 depends on soil aggregate size

Increased belowground carbon (C) transfer by plant roots at elevated CO₂ may change properties of the microbial community in the rhizosphere. Previous investigations that focused on total soil organic C or total microbial C showed contrasting results: small increase, small decrease or no changes. We evaluated the effect of 5 years of elevated CO₂ (550 ppm) on four extracellular enzymes: β‐glucosidase, chitinase, phosphatase, and sulfatase. We expected microorganisms to be differently localized in aggregates of various sizes and, therefore analyzed microbial biomass (C_mic by SIR) and enzyme activities in three aggregate‐size classes: large macro‐ (> 2 mm), small macro‐ (0.25–2 mm), and microaggregates (< 0.25 mm). To estimate the potential enzyme production, we activated microorganisms by substrate (glucose and nutrients) amendment. Although C_total and C_mic as well as the activities of β‐glucosidase, phosphatase, and sulfatase were unaffected in bulk soil and in aggregate‐size classes by elevated CO₂, significant changes were observed in potential enzyme production after substrate amendment. After adding glucose, enzyme activities under elevated CO₂ were 1.2–1.9‐fold higher than under ambient CO₂. This indicates the increased activity of microorganisms, which leads to accelerated C turnover in soil under elevated CO₂. Significantly higher chitinase activity in bulk soil and in large macroaggregates under elevated CO₂ revealed an increased contribution of fungi to turnover processes. At the same time, less chitinase activity in microaggregates underlined microaggregate stability and the difficulties for fungal hyphae penetrating them. We conclude that quantitative and qualitative changes of C input by plants into the soil at elevated CO₂ affect microbial community functioning, but not its total content. Future studies should therefore focus more on the changes of functions and activities, but less on the pools.     

Development of urea biosensor using non-covalent complexes of urease with aldehyde derivative of PEG and analysis on serum samples

Abstract

Non-covalent complexes of urease/polyethylene glycol (PEG)-aldehyde were synthesized using regular molar ratios of urease and PEG-aldehyde at room temperature. The physical properties of the non-covalent complexes were analyzed in order to investigate the impact of coupling ratio, temperature, pH, storage stability, and thermal stability. Urease activity was analyzed by UV–Vis spectrophotometer at 630?nm. The results showed that the strongest thermal resistance was obtained using n_U/n_PEG:1/1 (mg/mL) complex within all molar ratios tested. The enzymatic activity of n_U/n_PEG:1/1 complex doubled the activity of the free enzyme. Therefore, this complex was chosen to be used in the analyses. When coupled with PEG-aldehyde, urease exhibited improved activity between pH 4.0–9.0 and the optimum pH was found to be 7.0. The thermal inactivation results of the complex demonstrated that higher activity remained (40%) when compared with the free enzyme (10%) at 60?°C. The storage stability of the non-covalent complex was 4 weeks which was greater than the storage stability of the free enzyme. A kinetic model was suggested in order to reveal the mechanism of enzymatic conversion. Potentiometric urea biosensor was prepared using two different membranes: carboxylated poly vinyl chloride (PVC) and palmitic acid containing PVC. The potentiometric responses of both sensors were tested against pH and temperature and the best results were obtained at pH 7.0 and 20–30?°C. Also, selectivity of the suggested biosensors toward Na⁺, Li⁺ Ca²⁺, and K⁺ ions was evaluated and the reproducibility responses of the urea biosensors were measured with acceptable results.     

Cumulative Title Index

Abstract

Quantitative data on the speciation of chitosan (310 kDa) with low and high molecular weight carboxylates in aqueous solution are reported. The following carboxylic ligands were considered: monocarboxylate (butyrate); dicarboxylates (malonate, succinate, azelate); tricarboxylate (1,2,3-propa-netricarboxylate); tetracarboxylate (1,2,3,4-butanetetracarboxylate); polyacrylates (2.0 and 20 kDa); polymethacrylate (5.4 kDa). The investigation was performed by potentiometry at t =25°C, at low ionic strength (without addition of supporting electrolyte) and at I =0.15mol L^?1 (NaCl). For all the systems the formation of (chitosan)LH_i species was found (L = carboxylic ligand; i = 1 to 4 depending on the carboxylic ligand considered). The stability of proton–chitosan–carboxylate species depends on the number of carboxylic groups involved in the complexation, and it was possible to calculate a rough free energy value per bond ΔG_n = ?15±2kJ mol^?1. By using the stability data, the quantitative sequestering capacity of chitosan towards the carboxylates here considered [expressed as the-log(total chitosan concentration) necessary to bind 50% of carboxylate, i.e., pL₅₀] was calculated for different pH values, at low ionic strength and at I =0.15 mol L^?1. The pL₅₀ values, ranging from 3 to 7, show that chitosan is quite a strong sequestering agent towards carboxylates. Evidences were also obtained for the different behaviour between low and high molecular weight carboxylates.     

Preparation and Some Properties of Chitosan Bound Enzymes

An immobilization method using chitosan prepared from chitin as an insoluble carrier was investigated. Glucose isomerase, urease, glucamylase, trypsin and glucose oxidase were attached to chitosan by the aid of water soluble carbodiimide. Their activity yields were as follows; glucose isomerase 32%, urease 44%, glucamylase 8%, trypsin 10%, glucose oxidase 37%.

Immobilized glucose isomerase showed no significant changes in optimal temperature and heat stability. But pH optimum of reaction and pH stability range were somewhat lowered. The inhibitory effects of bivalent metal ions were considerably reduced by immobilization and similar tendency was observed for buffer reagents such as Tris or veronal. Immobilized glucose isomerase was inhibited by 8 m urea or 6 m guanidine hydrochloride in nearly the same way as free enzyme. With SDS, cysteine or mercaptoethanol free glucose isomerase was scarcely affected by these reagents, while immobilized enzyme considerably suffered to a loss of its activity.     

Effects of bleaching wastewater irrigation on soil quality of constructed reed wetlands

Constructed reed wetland microcosms (CRWs) in a lab of east China have been irrigated with bleaching wastewater per month for a reed growth season. The soil physicochemical properties, enzyme activities (i.e. urease, invertase, polyphenol oxidase, alkaline phosphatase and cellulase) and soil microbial diversity were assayed before and after the exposure experiment. Compared to the river water irrigated controls (CKs), bleaching wastewater application has no marked influence on soil pH, but significantly increased soil Na⁺, total halogen and absorbable organic halogen (AOX) contents, which induced the increasing of soil electrical conductivity. Furthermore, soil enzyme activities displayed significant variation (except for polyphenol oxidase). Bleaching wastewater irrigation decreased Sorenson’s pairwise similarity coefficient (C_s), which indicated the changes of the structure of bacterial and fungal communities. However, only the diversity of bacterial community was inhibited and has no effect on the diversity of fungal community, as evidenced by the calculated Shannon–Wiener index (H).     

Purification and properties of glycolate oxidase from plants with different photosynthetic pathways: Distinctness of C4 enzyme from that of a C3 species and a C3–C4 intermediate

Glycolate oxidase (GO; EC 1.1.3.1) was purified from the leaves of three plant species:Amaranthus hypochondriacus L.(NAD-ME type C₄ dicot),Pisum sativum L. (C₃ species) andParthenium hysterophorus L. (C₃–C₄. intermediate). A flavin moiety was present in the enzyme from all the three species. The enzyme from the C₄ plant had a low specific activity, exhibited lower K_M for glycolate, and required a lower pH for maximal activity, compared to the C₃ enzyme. The enzyme from the C₄ species oxidized glyoxylate at <10% of the rate with glycolate, while the GO from the C₃ plant oxidized glyoxylate at a rate of about 35 to 40% of that with glycolate. The sensitivity of GO from C₄ plant to -hydroxypyridinemethane sulfonate, 2-hydroxy-3-butynoate and other inhibitors was less than that of the enzyme from C₃ source. The properties of GO fromParthenium hysterophorus, were similar to those of the enzyme fromPisum sativum. The characteristics of glycolate oxidase from leaves of a C₄ plant,Amaranthus hypochondriacus are different from those of the C₃ species or the C₃–C₄ intermediate.     

Stereochemistry involved in the mechanism of action of dextransucrase in the synthesis of dextran and the formation of acceptor products

A proposed sequence of events in the synthesis of dextran and in the formation of acceptor products by dextransucrase from Leuconostoc mesenteroides B-512F has been developed with molecular models. The following mechanism is postulated: (1) two nucleophiles at the active site displace fructose from two sucrose molecules, giving two β-glucosyl intermediates; (2) these two β-glucosyl units rotate together so that the C₆-hydroxyl of each is apposed to the α-side of C₁ of the other; (3) one glucosyl unit assumes a boat conformation in which the bond to the enzyme is axial; (4) the C₆-hydroxyl oxygen of the other glucosyl unit makes a nucleophilic attack on C₁ of the first, displacing the enzyme nucleophile and making an α-1,6 bond; (5) rotations about the new α-1,6 linkage remove the transferred glucose from the active site. The free enzyme nucleophile attacks another sucrose as in step (1), and then steps (2)–(5) are repeated as the reducing-end glucosyl unit of the growing chain assumes the boat conformation and is attacked by the C₆-hydroxyl of the new glucosyl unit, which displaces the enzyme nucleophile and forms another α-1,6 linkage, about which rotations occur to remove the growing dextran chain from the active site. An additional feature of the mechanism presented here is a pair of enzymic proton-exchange groups, which protonate the glycosidic oxygen of sucrose to facilitate cleavage, and then remove a proton from the attacking C₆ hydroxyl during the polymerization reaction.Acceptors are polyhydroxy compounds which are capable of nucleophilic attack on enzyme-bound β-glucosyl or dextranosyl units to give α-glucosides or dextranosides. Noting the broad acceptor specificity of the enzyme and the unusual structure of some of the acceptor products, we have proposed that acceptor specificity is determined not by an enzymic binding site per se, but by the formation of hydrogen-bonded complexes between the acceptors and the glucosyl or dextranosyl enzyme intermediates. The acceptor attack on C₁ of the β-glucosyl enzyme is mediated by the same proton-exchange group as that proposed for catalysis of polymerization. It is shown that specific multiple hydrogen bonding to the glucosyl-enzyme intermediate can account for the formation of the observed acceptor products from α-methyl-d-glucoside, d-fructopyranose, isomaltose, maltose, β-d-mannopyranose, β-d-galactofuranose, cellobiose, lactose, β,β-trehalose, α,β-trehalose, and raffinose.     

Mixed substrate growth of methylotrophic yeasts in chemostat culture: Influence of the dilution rate on the utilisation of a mixture of glucose and methanol

The growth of Hansenula polymorpha and Kloeckera sp. 2201 with a mixture of glucose and methanol (38.8%/61.2%, w/w) and the regulation of the methanol dissimilating enzymes alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase were studied in chemostat culture, as a function of the dilution rate. Both organisms utilized and assimilated glucose and methanol simultaneously up to dilution rates of 0.30 h^-1 (H. polymorpha) and 0.26h^-1, respectively (Kloeckera sp. 2201) which significantly exceeded _max found for the two yeasts with methanol as the only source of carbon. At higher dilution rates methanol utilisation ceased and only glucose was assimilated. Over the whole range of mixed-substrate growth both carbon sources were assimilated with the same efficiency as during growth with glucose or methanol alone.In cultures of H. polymorpha, however, the growth yield for glucose was lowered by the unmetabolized methanol at high dilution rates. During growth on both carbon sources the repression of the synthesis of all catabolic methanol enzymes which is normally caused by glucose was overcome by the inductive effect of the simultaneously fed methanol. In both organisms the synthesis of alcohol oxidase was found to be regulated differently as compared to catalase, formaldehyde and formate dehydrogenase. Whereas increasing repression of the synthesis of alcohol oxidase was found with increasing dilution rates as indicated by gradually decreasing specific activities of this enzyme in cell-free extracts, the specific activities of this enzyme in cell-free extracts, the specific activities of catalase and the dehydrogenases increased with increasing growth rates until repression started. The results indicate similar patterns of the regulation of the synthesis of methanol dissimilating enzymes in different methylotrophic yeasts.Abbreviations and Terms C₁ Methanol - C₆ glucose; D dilution rate (h^-1) - D _c critical dilution rate (h^-1) - q _s specific, rate of substrate consumption (g substrate [g cell dry weight]^-1 h^-1) - q _CO2 and q _O2 are the specific rates of carbon dioxide release and oxygen consumption (mmol [g cell dry weight]^-1 h^-1) - RQ respiration quotient (q _CO2 q _O2 ¹ ) - s ₀(C₁) and s ₀(C₆) are the concentrations of methanol and glucose in the inflowing medium (g l^-1) - s residual substrate concentration in the culture liquid (g l^-1) - Sp. A. enzyme specific activity - x cell dry weight concentration (gl^-1) - Y _X/C6 growth yield on glucose (g cell dry weight [g substrate]^-1     

An explorative study on Staphylococcus aureus MurE inhibitor: induced fit docking,binding free energy calculation,and molecular dynamics

Staphylococcus aureus MurE enzyme catalyzes the addition of l-lysine as third residue of the peptidoglycan peptide moiety. Due to the high substrate specificity and its ubiquitous nature among bacteria, MurE enzyme is considered as one of the potential target for the development of new therapeutic agents. In the present work, induced fit docking (IFD), binding free energy calculation, and molecular dynamics (MD) simulation were carried out to elucidate the inhibition potential of 2-thioxothiazolidin-4-one based inhibitor 1 against S. aureus MurE enzyme. The inhibitor 1 formed majority of hydrogen bonds with the central domain residues Asn151, Thr152, Ser180, Arg187, and Lys219. Binding free-energy calculation by MM-GBSA approach showed that van der Waals (ΔG_vdW, ?57.30?kcal/mol) and electrostatic solvation (ΔG_solv, ?36.86?kcal/mol) energy terms are major contributors for the inhibitor binding. Further, 30-ns MD simulation was performed to validate the stability of ligand–protein complex and also to get structural insight into mode of binding. Based on the IFD and MD simulation results, we designed four new compounds D1–D4 with promising binding affinity for the S. aureus MurE enzyme. The designed compounds were subjected to the extra-precision docking and binding free energy was calculated for complexes. Further, a 30-ns MD simulation was performed for D1/4C13 complex.     

Partial purification and characterization of lipase from Geobacillus stearothermophilus AH22

The lipase was partially purified by ion exchange chromatography and gel filtration column chromatography, and was characterized from Geobacillus stearothermophilus AH22 strain. The lipase was purified 18.3-folds with 19.7% recovery. The lipase activity was determined by using p-nitrophenyl esters (C₂–C₁₂) as substrates. The K_m values of the enzyme for these substrates were found as 0.16, 0.02, 0.19 and 0.55?mM, respectively, while V_max values were 0.52, 1.03, 0.72 and 0.15?U?mg^?1. The enzyme showed maximum activity at 50?°C and between pH 8.0 and 9.0. The enzyme was found to be quite stable at pH range of 4.0–10.0, and thermal stability between 50 and 60?°C. It was found that the best inhibitory effect of the enzyme activity was of Hg²⁺. The inhibitory effect as orlistat, catechin, propyl paraben, p-coumaric acid, 3,4-dihydroxy hydro-cinnamic acid was examined. These results suggest that G. stearothermophilus AH22 lipase presents very suitable properties for industrial applications.