Immobilization of glycolate oxidase from Medicago falcata on magnetic nanoparticles for application in biosynthesis of glyoxylic acid

Glycolate oxidase was isolated from Medicago falcata Linn. after a screening from 13 kinds of C₃ plant leaves, with higher specific activity than the enzyme from spinach. The M. falcata glycolate oxidase (MFGO) was partially purified and then immobilized onto hydrothermally synthesized magnetic nanoparticles via physical adsorption. The magnetic nanoparticles were characterized with scanning electron microscope (SEM), transmission electron microscopy (TEM) and Fourier transform infrared (FT-IR) spectroscopy. The maximum load of MFGO was 56 mg/g support and the activity recovery was 45%. Immobilization of MFGO onto magnetic nanoparticles enhanced the enzyme stability, and the optimum temperature was significantly increased from 15 °C to 30 °C. The immobilized biocatalyst was successfully used in a batch reactor for repeated oxidization of glycolic acid to synthesize glyoxylic acid, retaining ca. 70% of its initial activity after 4 cycles of reaction at 30 °C for nearly 70 h, and its half-life was calculated to be 117 h.     

Kinetic analysis for suicide-substrate inactivation of microperoxidase-11: A modified model for bisubstrate enzymes in the presence of reversible inhibitors

Kinetics of microperoxidase-11 (MP-11) as a heme–peptide enzyme model in oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied in the presence of amino acids, taking into account the inactivation of MP-11 during reaction by its suicide substrate, H₂O₂. Reliability of the kinetic equation was evaluated by non-linear mathematical fitting. Fitting of experimental data into a new integrated kinetic relation showed a close match between the kinetic model and the experimental data. Indeed, it was found that the mechanism of suicide-peroxide inactivation of MP-11 in the presence of amino acids is different from MP-11 and/or horseradish peroxidase. In this mechanism, amino acids compete with hydrogen peroxide for the sixth co-ordination position of iron atom in the heme group through a competitive inhibition mechanism.The proposed model can successfully determine the kinetic parameters including inactivation by hydrogen peroxide as well as the inhibitory rate constants by the amino acid inhibitor.Kinetic parameters of inactivation including the initial activity of MP-11, α₀, the apparent inactivation rate constant, k_i and the apparent inhibition rate constant for cysteine, k_I were obtained 0.282 ± 0.006 min^?1, 0.497 ± 0.013 min^?1 and 1.374 ± 0.007 min^?1 at [H₂O₂] = 1.0 mM, 27 °C, phosphate buffer 5.0 mM, pH 7.0. Results showed that inactivation and inhibition of microperoxidase as a peroxidase model enzyme occurred simultaneously even at low concentrations of hydrogen peroxide (0.4 mM). This kinetic analysis based on the suicide-substrate inactivation of microperoxidase-11, provides a tool and model for studying peroxidase models in the presence of reversible inhibitors. The introduced inhibition procedure can be used in designing activity tunable and specific protected enzyme models in the hidden and reversibly inhibited forms, which do not undergo inactivation.     

Stability of phycocyanin extracted from Spirulina sp.: Influence of temperature,pH and preservatives

Temperature and pH play an important role in the stability of phycocyanin, a natural blue colorant. Systematic investigations showed the maximum stability of phycocyanin was in the pH range 5.5–6.0. Incubation at temperatures between 47 and 64 °C caused the concentration (C_R) and half-life of phycocyanin in solution to decrease rapidly. The C_R value remained at approximately 50% after incubating for 30 min at 59 °C. After heating at 60 °C for 15 min, the C_R value of phycocyanin at pH 7.0 was maintained at around 62–70% when 20–40% glucose or sucrose was added, and the half-life increased from 19 min to 30–44 min. 2.5% sodium chloride was found to be an effective preservative for phycocyanin at pH 7.0 as a C_R value of 76% was maintained and the half-life of 67 min was increased.     

Relative larvicidal potentiality of nano-encapsulated Temephos and Imidacloprid against Culex quinquefasciatus

Encapsulation of temephos ranging from 1% to 16% and imidacloprid from 1% to 8% within biodegradable and biocompatible, polyethylene glycol in different ratios was done by using melt-dispersion method. The efficacy of encapsulated forms was evaluated and compared with their non-capsulated forms against larvae of Culex quinquefasciatus. The encapsulated temephos was more toxic than the encapsulated imidacloprid with LC₅₀ values of 0.013, 0.010 and 0.003 mg/L after 24, 48 and 72 h, respectively. No doubt, the non-capsulated temephos and imidacloprid were more effective as compared to their encapsulated forms. However, the same mortality rate was achieved by the slow release of lesser amount of pesticides after encapsulation, e.g., 0.003 mg/L for 8% temephos formulation and 0.019 mg/L for 4% imidacloprid as compared to their non-capsulated form of temephos and imidacloprid (0.004 and 0.021 mL/L) after 72 h of exposure. Thus, encapsulated forms are more economical and eco-friendly due to controlled slow release of their nanoparticles.     

Immobilization and characterization of human carbonic anhydrase I on amine functionalized magnetic nanoparticles

In this study, we synthesized magnetic nanoparticles (MNPs) by co-precipitation method. After that, silica coating with tetraethyl orthosilicate (TEOS) (SMNPs), amine functionalization of silica coated MNPs (ASMNPs) by using 3-aminopropyltriethoxysilane (APTES) were performed, respectively. After activation with glutaraldehyde (GA) of ASMNPs, human carbonic anhydrase (hCA I) was immobilized on ASMNPs. The characterization of nanoparticles was performed by transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD) and vibrating sample magnetometer (VSM). The immobilization conditions such as GA concentration, activation time of support with GA, enzyme amount, enzyme immobilization time were optimized. In addition of that, optimum conditions for activity, kinetic parameters (K_m, V_max, k_cat, kcat/K_m), thermal stability, storage stability and reusability of immobilized enzyme were determined.The immobilized enzyme activity was optimum at pH 8.0 and 25 °C. The K_m value of the immobilized enzyme (1.02 mM) was higher than the free hCA I (0.48 mM). After 40 days incubation at 4 °C and 25 °C, the immobilized hCA I sustained 89% and 85% of its activity, respectively. Also, it sustained 61% of its initial activity after 13 cycles. Such results revealed good potential of immobilized enzyme for various applications.     

Ethyl isovalerate synthesis using Candida rugosa lipase immobilized on silica nanoparticles prepared in nonionic reverse micelles

Uniform and monodispersed silica nanoparticles were synthesized with a mean diameter of 100 ± 20 nm as analyzed by Transmission Electron Microscopy (TEM). Glutaraldehyde was used as a coupling agent for efficient binding of the lipase onto the silica nanoparticles. For the hydrolysis of pNPP at pH 7.2, the activation energy within 25–40 °C for free and immobilized lipase was 7.8 and 1.25 KJ/mol, respectively. The V_max and K_m of immobilized lipase at 25 °C for pNPP hydrolysis were found to be 212 μmol/min/mg and 0.3 mM, whereas those for free lipase were 26.17 μmol/min and 1.427 mM, respectively. The lower activation energy of immobilized lipase in comparison to free lipase suggests a change in conformation of the enzyme leading to a requirement for lower energy on the surface of the nanoparticles. A better yield (7 fold higher) of ethyl isovalerate was observed using lipase immobilized onto silica nanoparticles in comparison to free lipase.     

Reactivation of the alternative oxidase of Yarrowia lipolytica by nucleoside monophosphates

The study of the effect of nucleoside phosphates on the activity of cyanide-resistant oxidase in the mitochondria and submitochondrial particles of Yarrowia lipolytica showed that adenosine monophosphate (5′-AMP, AMP) did not stimulate the respiration of intact mitochondria. The incubation of mitochondria at room temperature (25 °C) for 3–5 h or their treatment with ultrasound, phospholipase A, and the detergent Triton X-100 at a low temperature inactivated the cyanide-resistant alternative oxidase. The inactivated alternative oxidase could be reactivated with AMP. The reactivating effect of AMP was enhanced by azolectin. Some other nucleoside phosphates also showed reactivating ability in the following descending order: AMP = GMP > GDP > GTP > XMP > IMP. The apparent K_m values for AMP in reactivation of the alternative oxidase of submitochondrial particles or mitochondria treated with Triton X-100 and incubated at 25 °C were calculated. Physiological aspects of activation of the alternative oxidase are discussed in connection with the impairment of electron transfer through the cytochrome pathway.     

Expression,characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus

An FAD-dependent glucose dehydrogenase (FAD-GDH) from Aspergillus terreus NIH2624 was expressed in Escherichia coli with a yield of 228 ± 16 U/L of culture. Co-expression with chaperones DnaK/DnaJ/GrpE and osmotic stress induced by simple carbon sources enhanced productivity significantly, improving the yield to 23883 ± 563 U/L after optimization. FAD-GDH was purified in two steps with the specific activity of 604 U/mg. Using d-glucose as substrate, the optimal pH and temperature for FAD-GDH were determined to be 7.5 and 50 °C, respectively. Activity was stable across the pH range 3.5–9.0, and the half-life was 52 min at 42 °C. K_m and V_max were calculated as 86.7 ± 5.3 mM and 928 ± 35 U/mg, and the molecular weight was approximately 65.6 kDa based on size exclusion chromatography, indicating a monomeric structure. The 3D structure of FAD-GDH was simulated by homology modelling using the structure of A. niger glucose oxidase (GOD) as template. From the model, His551, His508, Asn506 and Arg504 were identified as key residues, and their importance was verified by site-directed mutagenesis. Furthermore, three additional mutants (Arg84Ala, Tyr340Phe and Tyr406Phe) were generated and all exhibited a higher degree of substrate specificity than the native enzyme. These results extend our understanding of the structure and function of FAD-GDH, and could assist potential commercial applications.     

Immobilization of glucose oxidase onto cobalt based on silica core/shell nanoparticles as carrier

Co–B/SiO₂/NH₂ magnetic nanoparticles (NPs) were prepared from a silica shell-coated Co–B core using the Stöber method and amine-modification on the surface. Glucose oxidase (GOD) was covalently immobilized on the surface of Co–B/SiO₂/NH₂ NPs using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) as an activating agent. The magnetic NPs characteristics, such as the synthesis of Co–B/SiO₂/NH₂ NPs, effect of pH, temperature, and concentration of buffer for enzyme immobilization, were investigated. The optimal reaction conditions for immobilization were determined to be 0.1 M of phosphate buffer solution, pH 7.0, and 5 °C. In the case of immobilized GOD without d-glucose and with 0.1 M of d-glucose for blocking, 22.98 U/g and 24.83 U/g of their original activity were retained after 7 reuses, respectively.     

Sequence-based screening and characterization of cytosolic mandelate oxidase using oxygen as electron acceptor

Sequence-based screening was carried out to find a type of cytosolic mandelate oxidase that converted l-mandelate to phenylglyoxylate using oxygen as the final electron acceptor. The sequence features of the cytosolic mandelate oxidase were summarized, and were used in the screening process. Mandelate oxidases from Streptomyces coelicolor (Hmo_SC) and Amycolatopsis orientalis (Hmo_AO) were screened and then they were heterologously expressed and characterized. At pH 7.3 40 °C, the Hmo_AO showed k_cat and K_m values of 140 min⁻¹ and 10.2 mM, the Hmo_SC showed k_cat and K_m values of 105.1 min⁻¹ and 2.06 mM. The Hmo_SC was thermal stable and retained its 90% activity at 60 °C for up to 5 h, while Hmo_AO lost most of its activity at this temperature. The Hmo_SC could effectively catalyze the conversion of l-mandelate to phenylglyoxylate at higher temperature using oxygen as the final electron acceptor.     

Cell-associated acid β-xylosidase production by Penicillium sclerotiorum

In recent decades, β-xylosidases have been used in many processing industries. In this work, the study of xylosidase production by Penicillium sclerotiorum and its characterization are reported. Optimal production was obtained in medium supplemented with oat spelts xylan, pH 5.0, at 30 °C, under stationary condition for six days. The optimum activity temperature was 60 °C and unusual optimum pH 2.5. The enzyme was stable at 50 and 55 °C, with half-life of 240 and 232 min, respectively. High pH stability was verified from pH 2.0 to 4.0 and 7.5. The β-xylosidase was strongly inhibited by divalent cations, sensitive to denaturing agents SDS, EDTA and activated by thiol-containing reducing agents. The apparent V_max and K_m values was 0.48 μmol PNXP min^?1 mg^?1 protein and 0.75 mM, respectively. The enzyme was xylose tolerant with a K_i of 28.7. This enzyme presented interesting characteristics for biotechnological process such as animal feed, juice and wine industries.     

Structural characterization of thermostable,solvent tolerant,cytosafe tannase from Bacillus subtilis PAB2

Tannase production by Bacillus subtilis PAB2, was investigated under solid state fermentation using tamarind seed as sole carbon source and it was found as the highest titer (73.44 U/gds). The enzyme was purified to homogeneity, which showed the molecular mass around 52 kDa (K_m = 0.445 mM, V_max = 125.8 mM/mg/min and K_cat = 2.88 min^–1). The enzyme was found stable in a range of pH (3.0–8.0) and temperature (30–70 °C) with an optimal activity at pH 5.0, pI of 4.4 and at 40 °C temperature. It exhibited half-life (t_1/2) of 4.5 h at 60 °C. The enzyme comprised a typical secondary structure containing α-helix (9.3%), β-pleated sheet (33.6%) and β-turn (17.2%). The native conformation of the enzyme was alike a 44 nm spherical nanoparticle upon aggregation. Thermodynamic parameters of tannase revealed that it was stable at 40 °C and showed Q₁₀, ΔG_d and ΔS_d values of 2.08, 99.37 KJ/mol and 252.38 J mol⁻¹ K⁻¹, respectively. Organic solvents were stimulatory with regard to enzyme activity. Moreover, the altered enzyme activity was determined to be correlated with the changes in structural conformation in presence of inducer and inhibitor. Tannase was explored to have no cytotoxicity on Vero cell line as well as rat model study.     

Application of chitosan beads immobilized Rhodococcus sp. NCIM 2891 cholesterol oxidase for cholestenone production

The cell-bound cholesterol oxidase from the Rhodococcus sp. NCIM 2891 was purified three fold by diethylaminoethyl–sepharose chromatography. The estimated molecular mass (SDS-PAGE) and K_m of the enzyme were ∼55.0 kDa and 151 μM, respectively. The purified cholesterol oxidase was immobilized on chitosan beads by glutaraldehyde cross-linking reaction and immobilization was confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy and energy dispersive X-ray analysis. The optimum temperature (45 °C, 5 min) for activity of the enzyme was increased by 5 °C after immobilization. Both the free and immobilized cholesterol oxidases were found to be stable in many organic solvents except for acetone. Fe²⁺ and Pb²⁺ at 0.1 mM of each acted as inhibitors, while Ag⁺, Ca²⁺, Ni²⁺ and Zn²⁺ activated the enzyme at similar concentration. The biotransformation of cholesterol (3.75 mM) with the cholesterol oxidase immobilized beads (3.50 U) leads to ∼88% millimolar yield of cholestenone in a reaction time of 9 h at 25 °C. The immobilized enzyme retains ∼67% activity even after 12 successive batches of operation. The biotransformation method thus, shows a great promise for the production of pharmaceutically important cholestenone.     

β-Galactosidase of Aspergillus oryzae immobilized in an ion exchange resin combining the ionic-binding and crosslinking methods: Kinetics and stability during the hydrolysis of lactose

In this work, the hydrolysis kinetics of lactose by Aspergillus oryzae β-galactosidase was studied using the ionic exchange resin Duolite A568 as a carrier. The enzyme was immobilized using a β-galactosidase concentration of 16 g/L in pH 4.5 acetate buffer and an immobilization time of 12 h at 25 ± 0.5 °C. Next, the immobilized β-galactosidase was crosslinked using glutaraldehyde concentration of 3.5 g/L for 1.5 h. The influence of lactose concentration was studied for a range of 5–140 g/L, and the Michaelis–Menten model was fitted well to the experimental results with V_m and K_m values of 0.71 U and 35.30 mM, respectively. The influence of the product galactose as an inhibitor on the hydrolysis reaction was studied. The model that was best fitted to the experimental results was the competitive inhibition by galactose with V_m, K_m and K_i values of 0.77 U, 35.30 mM and 27.44 mM, respectively. The influence of temperature on the enzymatic activity of the immobilized enzyme was studied in the range of 10–80 °C, in which the temperature of the maximum activity was 60 °C, with an activation energy of 5.32 kcal/mol of lactose, using an initial concentration of lactose of 50 g/L in a pH 4.5 sodium acetate buffer solution. The thermal stability of the immobilized biocatalyst was determined to be in the range 55–65 °C. The first-order model described well the kinetics of thermal deactivation for all the temperatures studied. The activation energy of thermal deactivation from immobilized biocatalyst was 66.48 kcal/mol with a half-life of 8.9 h at 55 °C.     

Co-immobilization of oxalate oxidase and catalase in films for scavenging of oxygen or oxalic acid

Oxalate oxidase has potential to act as an oxygen scavenger in active packaging to increase the shelf-life of food and beverages, while simultaneously producing the protective packaging gas carbon dioxide. This study shows that oxalate oxidase from barley can be immobilized with retained catalytic activity through entrapment in a latex polymer matrix. Conditions for formation of film containing oxalate oxidase have been evaluated as well as effects of storage and latex on enzyme activity, migration of enzyme in films, and the ability of the latex films to resist higher temperatures. Drying of enzyme-containing latex films at 75 °C prior to conditioning at 30 °C resulted in higher activity than drying solely at 30 °C, or drying at 95 °C or 105 °C followed by conditioning at 30 °C. Storage of films in air at 4 °C for 14 days did not negatively affect the enzymatic activity. Inclusion of catalase in films with oxalate oxidase effectively prevented release of hydrogen peroxide. The results suggest that the immobilized enzyme can successfully be used both as an oxygen scavenger and as an oxalic-acid scavenger.     

Hen egg white as a feeder protein for lipase immobilization

Enzyme stabilization via immobilization is one of the preferred processes as it provides the advantages of recovery and reusability. In this study, Thermomyces lanuginosus lipase has been immobilized through crosslinking using 2% glutaraldehyde and hen egg white, as an approach towards CLEA preparation. The immobilization efficiency and the properties of the immobilized enzyme in terms of stability to pH, temperature, and denaturants was studied and compared with the free enzyme. Immobilization efficiency of 56% was achieved with hen egg white. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0 whereas the pH optimum for free enzyme was at pH 6.0. The immobilized enzyme was stable at higher temperature retaining about 83% of its maximum activity as compared to the free enzyme retaining only 41% activity at 70 °C. The denaturation of lipase in free form was rapid with a half-life of 2 h at 60 °C and 58 min at 70 °C as compared to 12 h at 60 °C and 2 h at 70 °C for the immobilized enzyme. The effect of denaturants, urea and guanidine hydrochloride on the free and immobilized enzyme was studied and the immobilized enzyme was found to be more stable towards denaturants retaining 74% activity in 8 M urea and 98% in 6 M GndHCl as compared to 42% and 33% respectively in the case of free enzyme. The apparent K_m (2.08 mM) and apparent V_max (0.95 μmol/min) of immobilized enzyme was lower as compared to free enzyme; K_m (8.0 mM) and V_max (2.857 μmol/min). The immobilized enzyme was reused several times for the hydrolysis of olive oil.     

Stability and activity of Dictyoglomus thermophilum GH11 xylanase and its disulphide mutant at high pressure and temperature

The functional properties of extremophilic Dictyoglomus thermophilum xylanase (XYNB) and the N-terminal disulphide-bridge mutant (XYNB-DS) were studied at high pressure and temperature. The enzymes were quite stable even at the pressure of 500 MPa at 80 °C. The half-life of inactivation in these conditions was over 30 h. The inactivation at 80 °C in atmospheric pressure was only 3-times slower. The increase of pressure up to 500 MPa at 80 °C decreased only slightly the enzyme's stability, whereas in 500 MPa the increase of temperature from 22 to 80 °C decreased significantly more the enzyme's stability. While the high temperature (80–100 °C) decreased the enzyme reaction with short xylooligosaccharides (xylotetraose and xylotriose), the high pressure (100–300 MPa) had an opposite effect. The temperature of 100 °C strongly increased the K_m but did not affect the k_cat to the same extent, thus indicating that the interaction of the substrate with the active site suffers before the catalytic reaction begins to decrease as the temperature rises. Circular dichroism spectroscopy showed the high structural stability of XYNB and XYNB-DS at 93 °C.     

Cyanide hydratase from Aspergillus niger K10: Overproduction in Escherichia coli,purification, characterization and use in continuous cyanide degradation

A cyanide hydratase from Aspergillus niger K10 was expressed in Escherichia coli and purified. Apart from HCN, it transformed some nitriles, preferentially 2-cyanopyridine and fumaronitrile. V_max and K_m for HCN were ca. 6.8 mmol min⁻¹ mg⁻¹ protein and 109 mM, respectively. V_max for fumaronitrile and 2-cyanopyridine was two to three orders of magnitude lower than for HCN (ca. 18.8 and 10.3 μmol min⁻¹ mg⁻¹, respectively) but K_m was also lower (ca. 14.7 and 3.7 mM, respectively). Both cyanide hydratase and nitrilase activities were abolished in truncated enzyme variants missing 18–34 C-terminal aa residues. The enzyme exhibited the highest activity at 45 °C and pH 8–9; it was unstable at over 35 °C and at below pH 5.5. The operational stability of the whole-cell catalyst was examined in continuous stirred membrane reactors with 70-mL working volume. The catalyst exhibited a half-life of 5.6 h at 28 °C. A reactor loaded with an excess of the catalyst was used to degrade 25 mM KCN. A conversion rate of over 80% was maintained for 3 days.     

Alkyl-substituted methoxysilanes enhance the activity and stability of d-amino acid oxidase encapsulated in biomimetic silica

Several alkyl-substituted methoxysilanes were evaluated as potential activity and stability enhancing agents for biomimetic silicification of Rhodosporidium toruloides D-amino acid oxidase (RtDAO). When methyl-substituted silanes along with tetramethoxysilane were used as silicic acid precursors for polyallylamine (PAA)--or R5 peptide-catalyzed silicic encapsulation, the RtDAO activity increased with the degree of substitution and the molar ratio up to 15 % of methyl-substituted silanes added. In the presence of 15 mol% trimethylmethoxysilane, the specific activities of encapsulated RtDAO catalyzed by PAA and R5 increased by 1.4- and 4.8-fold, respectively. For PAA-catalyzed encapsulation, a 2.4-fold increase occurred with 30 mol% n-propyltrimethoxysilane; this modification increased the T (m) value by 10 °C and gave a threefold longer half-life in the presence of 10 mM H(2)O(2) as compared to the encapsulation using tetramethoxysilane only.     

In vitro and in sacco digestibility of wheat straw treated with calcium oxide and sodium hydroxide alone or with hydrogen peroxide

A series of five factorial experiments examined the effects of sodium hydroxide (NaOH) and calcium oxide (CaO) alone or together with hydrogen peroxide (H₂O₂, 27.5% w/w) at pH of about 11.5 (AHP) on in vitro (IVDMD) and in sacco (ISDMD) dry matter digestibility of wheat straw. The effects of different temperatures (20°C, 40°C and 60°C), various times (2, 3, 4, 6 and 27 h), pre-soaking, filtration and washing on the efficacy of the above levels of chemicals in improving IVDMD and ISDMD were tested in separate experiments. AHP improved IVDMD (P<0.001) of straws when pH was regulated to around 11.5 using NaOH. In contrast, AHP was ineffective or depressive (P<0.001) when CaO was used to regulate pH to around 11.5. However, CaO alone increased IVDMD to a similar extent as did NaOH. Washing, filtration and temperature were ineffective in improving the IVDMD of CaO-treated straw. AHP was most effective when 130 g H₂O₂ was applied to each kg DM of straw after soaking it with 3 l solution containing 80 g NaOH for a period of 27 h. The nutritional value of low quality forages can be enhanced for ruminants by using alkalis provided conditions as described above are maintained during alkali treatments.