Immobilization of fungal (Trametes versicolor) laccase onto Amberlite IR-120 H beads: Optimization and characterization

Laccase from Trametes versicolor was immobilized on Amberlite IR-120 H beads. Maximum immobilization obtained was 78.7% at pH = 4.5 and temperature T = 45 °C. Kinetic parameters, K_m and V_max values, were determined respectively as 0.051 mM and 2.77 × 10^?2 mM/s for free and 4.70 mM and 5.27 × 10^?3 mM/s for immobilized laccase. The Amberlite–laccase system showed a 30% residual activity after 7 cycles. On the other hand, the loss of activity for free laccase after 7 days of storage at 4 °C was 18.5% in comparison to Amberlite–laccase system with a loss of 1.4%, during the same period. Improved operational, thermal and storage stabilities of the immobilized laccase were obtained compared to the free counterpart. Therefore, the use of low-cost matrices, like Amberlite for enzyme immobilization represents a promising product for enzymatic industrial applications.     

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.     

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.     

Surface functionalized mesoporous activated carbon for the immobilization of acidic lipase and their application to hydrolysis of waste cooked oil: Isotherm and kinetic studies

This study deals with the surface functionalization of mesoporous activated carbon, using ethylenediamine and glutaraldehyde to facilitate the strong immobilization of acidic lipase (AL) onto MAC. The AL was produced from Pseudomonas gessardii by using slaughterhouse lipid waste as the substrate. The AL immobilized on functionalized mesoporous activated carbon (ALFMAC) was applied for the hydrolysis of waste cooked oil (WCO). The optimum conditions for the immobilization of AL onto functionalized mesoporous activated carbon (FMAC) were 90 min; pH 3.5; and 35 °C; which resulted at the maximum immobilization of 5440 U/g of FMAC (3.693 mg of AL/g of FMAC or the yield 2.7% or the expressed activity 103.7% or the activity per unit area of FMAC 1.08 mg of AL/m²). The ALFMAC showed better thermal and storage stabilities than the free AL. The ALFMAC retained a 98% and a 92% initial activity at 40 °C and 50 °C, respectively, while the AL showed the thermal stability (residual activities) 65% and 38%, respectively. The storage stability of ALFMAC at 4 °C showed 100% initial activity up to 15 days from the initial day of the storage, whereas AL showed only 88% initial activity up to 15 days. The FMAC and ALFMAC were characterized by using scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, and X-ray diffraction (XRD) analysis. The K_m values of the ALFMAC and AL were 0.112 mM and 0.411 mM, respectively. The v_max values of the ALFMAC and AL were 1.26 mM/min and 0.53 mM/min, respectively. Immobilization of AL onto FMAC obeyed the Freundlich and Redlich–Peterson isotherm models. The non-linear models of pseudo first, and second order, intra-particle diffusion, Bangham, and Boyd plot were also performed to understand the dynamic mechanism of immobilization. ALFMAC showed a 100% hydrolysis of WCO up to 21 cycles of reuse, and 60% up to 45 cycles. The hydrolysis of WCO was confirmed by using FT-IR spectra.     

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.     

Immobilization of acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon for the hydrolysis of olive oil

Mesoporous activated carbon (MAC) derived from rice husk is used for the immobilization of acidic lipase (ALIP) produced from Pseudomonas gessardii. The purified acidic lipase had the specific activity and molecular weight of 1473 U/mg and 94 kDa respectively. To determine the optimum conditions for the immobilization of lipase onto MAC, the experiments were carried out by varying the time (10–180 min), pH (2–8), temperature (10–50 °C) and the initial lipase activity (49 × 10³, 98 × 10³, 147 × 10³ and 196 × 10³ U/l in acetate buffer). The optimum conditions for immobilization of acidic lipase were found to be: time—120 min; pH 3.5; temperature—30 °C, which resulted in achieving a maximum immobilization of 1834 U/g. The thermal stability of the immobilized lipase was comparatively higher than that in its free form. The free and immobilized enzyme kinetic parameters (K_m and V_max) were found using Michaelis–Menten enzyme kinetics. The K_m values for free enzyme and immobilized one were 0.655 and 0.243 mM respectively. The immobilization of acidic lipase onto MAC was confirmed using Fourier Transform-Infrared Spectroscopy, X-ray diffraction analysis and scanning electron microscopy.     

A novel method for the immobilization of glucoamylase onto polyglutaraldehyde-activated gelatin

A novel method was developed for the immobilization of glucoamylase from Aspergillus niger. The enzyme was immobilized onto polyglutaraldehyde-activated gelatin particles in the presence of polyethylene glycol and soluble gelatin, resulting in 85% immobilization yield. The immobilized enzyme has been fully active for 30 days. In addition, the immobilized enzyme retained 90 and 75% of its activity in 60 and 90 days, respectively. The enzyme optimum conditions were not affected by immobilization and the optimum pH and temperature for free and immobilized enzyme were 4 and 65 °C, respectively. The kinetic parameters for the hydrolysis of maltodextrin by free and immobilized glucoamylase were also determined. The K_m values for free and immobilized enzyme were 7.5 and 10.1 g maltodextrin/l, respectively. The V_max values for free and immobilized enzyme were estimated as 20 and 16 μmol glucose/(min μl enzyme), respectively. The newly developed method is simple yet effective and could be used for the immobilization of some other enzymes.     

Bioconversion of phenylpyruvic acid to l-phenylalanine by mixed-gel immobilization of Escherichia coli EP8-10

A mixed-gel of κ-carrageenan and gelatin was used in l-phenylalanine production. The mixed-gel, containing 87.5% κ-carrageenan and 12.5% gelatin [the total gel concentration was 4 wt%], showed the best performance and was selected for further study with Escherichia coli EP8-10. The optimum pH and temperature were 8.5 and 37 °C, respectively. The effects of trehalose and Mg²⁺ were studied in the mixed-gel immobilization. Their optimum concentrations were 5 × 10^?2 and 2 × 10^?3 mol/L, respectively. Under the optimal conditions, 98.3% of the phenylpyruvic acid (PPA) was converted to l-phenylalanine. The activity recovery of the transaminase enzyme in the mixed-gel immobilization was higher than that in single κ-carrageenan immobilization, which was 93.6%. The total PPA conversion rate was over 80% in all 15 batches, suggesting great sustainability in the mixed-gel immobilization. The maximum reaction rate (r_max) was calculated to be 4.75 × 10^?2 mol/(L g h).     

Hyperuricemia influences tryptophan metabolism via inhibition of multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP)

Hyperuricemia is related to a variety of pathologies, including chronic kidney disease (CKD). However, the pathophysiological mechanisms underlying disease development are not yet fully elucidated. Here, we studied the effect of hyperuricemia on tryptophan metabolism and the potential role herein of two important uric acid efflux transporters, multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP). Hyperuricemia was induced in mice by treatment with the uricase inhibitor oxonic acid, confirmed by the presence of urate crystals in the urine of treated animals. A transport assay, using membrane vesicles of cells overexpressing the transporters, revealed that uric acid inhibited substrate-specific transport by BCRP at clinically relevant concentrations (calculated IC₅₀ value: 365 ± 13 μM), as was previously reported for MRP4. Moreover, we identified kynurenic acid as a novel substrate for MRP4 and BCRP. This finding was corroborated by increased plasma levels of kynurenic acid observed in Mrp4^?/? (107 ± 19 nM; P = 0.145) and Bcrp^?/? mice (133 ± 10 nM; P = 0.0007) compared to wild type animals (71 ± 11 nM). Hyperuricemia was associated with > 1.5 fold increase in plasma kynurenine levels in all strains. Moreover, hyperuricemia led to elevated plasma kynurenic acid levels (128 ± 13 nM, P = 0.005) in wild type mice but did not further increase kynurenic acid levels in knockout mice. Based on our results, we postulate that elevated uric acid levels hamper MRP4 and BCRP functioning, thereby promoting the retention of other potentially toxic substrates, including kynurenic acid, which could contribute to the development of CKD.     

Immobilization of urease on vapour phase stain etched porous silicon

Porous silicon layers fabricated by the reaction-induced vapor phase stain etch method were coated with 5% polyethylenimine. Urease from Canavalia brasiliensis beans was immobilized on this support through covalent linking with 2.5% glutaraldehyde. The pH and temperature profile of the immobilized and free urease exhibited higher activity at pH 6.5 and 37 °C. After being stored for 30 days at 4 °C, the immobilized enzyme had 75% of the initial activity. The maximum apparent Michaelis constant for free urease (K_m) was 94.33 mM whereas for immobilized urease was 53.04 mM. The maximum reaction velocity (V_max) for free urease was 3.51 mmol/min and for immobilized urease was 1.57 mmol/min.     

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.     

Immobilization of phytase on epoxy-activated Sepabead EC-EP for the hydrolysis of soymilk phytate

In this work, an active phytase concentrated extract from soybean sprout was immobilized on a polymethacrylate-based polymer Sepabead EC-EP which is activated with epoxy groups. The immobilized enzyme exhibited an activity of 0.1 U/g of carrier and activity yield of 64.7%. The optimum temperature and pH for the activity of both free and immobilized enzymes were found as 60 °C and pH 5.0, respectively. The immobilized enzyme was more stable than free enzyme in the range of pH 3.0–8.0 and more than 70% of the original activity was recovered. Both the enzymes completely retained nearly about 84% of their original activity at 65 °C. The K_m and V_max values were measured as 5 mM and 0.63 U/mg for free enzyme and 12.5 mM and 0.71 U/mg for immobilized enzyme, respectively. Free and immobilized soybean sprout phytase enzymes were also used in the biodegradation of soymilk phytate. The immobilized enzyme hydrolysed 92.5% of soymilk phytate in 7 h at 60 °C, as compared with 98% hydrolysis observed for the native enzyme over the same period of time. The immobilization procedure on Sepabead EC-EP is very cheap and also easy to carry out, and the features of the immobilized enzyme are very attractive that the potential for practical application is considerable.     

Reversible immobilization of catalase on fibrous polymer grafted and metal chelated chitosan membrane

Poly(itaconic acid) grafted and/or Fe(III) ions incorporated chitosan membranes were used for reversible immobilization of catalase (from bovine liver) via adsorption. The influences of pH and initial catalase concentration on the immobilization capacities of the CH-g-poly(IA) and CH-g-poly(IA)-Fe(III) membranes have been investigated in a batch system. Maximum catalase adsorption onto CH-g-poly(IA) and CH-g-poly(IA)-Fe(III) membrane were found to be 6.3 and 37.8 mg/g polymer at pH 5.0 and 6.5, respectively. The CH-g-poly(IA)-Fe(III) membrane with high catalase adsorption capacity was used in the rest of the study. The K_m value for immobilized catalase on CH-g-poly(IA)-Fe(III) (25.8 mM) was higher about 1.6-fold than that of free enzyme (13.5 mM). Optimum operational temperature was observed at 40 °C, a 5 °C higher than that of the free enzyme and was significantly broader. The optimum operational pH was same for both free and immobilized catalase (pH 7.0). Thermal stability was found to increase with immobilization. Free catalase lost all its activity within 20 days whereas immobilized catalase lost 23% of its activity during the same incubation period. It was observed that the same support enzyme can be repeatedly used for immobilization of catalase after regeneration without significant loss in adsorption capacity or enzyme activity. In addition, the CH-g-poly(IA)-Fe(III) membrane prepared in this work showed promising potential for various biotechnological applications.     

Characterization and PCR application of a new high-fidelity DNA polymerase from Thermococcus waiotapuensis

The family B DNA polymerase gene from the euryarchaeon Thermococcus waiotapuensis (Twa) contains an open reading frame of 4404 bases that encodes 1467 amino acid residues. The gene is split by two intein-coding sequences that forms a continuous open reading frame with the three polymerase exteins. Twa DNA polymerase genes with (whole gene) and without (genetically intein-spliced) inteins were expressed in Escherichia coli Rosetta(DE3)pLysS. The inteins of the expressed whole gene were easily spliced during purification. The molecular mass of the purified Twa DNA polymerase was about 90 kDa, as estimated by SDS-PAGE. The optimal pH for Twa DNA polymerase activity was 6.0 and the optimal temperature was 75 °C. The enzyme was activated by magnesium ions. The half-life of the enzyme at 99 °C was about 4 h. The optimal buffer for PCR with Twa DNA polymerase was 50 mM Tris–HCl (pH 8.2), 2.0 mM MgCl₂, 30 mM KCl, 2.0 mM (NH₄)₂SO₄, 0.01% Triton X-100, and 0.005% BSA. The PCR fidelity of Twa DNA polymerase was higher than Pfu, KOD and Vent DNA polymerases. A ratio of 15:1 Taq:Twa DNA polymerase efficiently facilitated long-range PCR.     

Immobilization of α-galactosidase on galactose-containing polymeric beads

Industrial application of α-galactosidase requires efficient methods to immobilize the enzyme, yielding a biocatalyst with high activity and stability compared to free enzyme. An α-galactosidase from tomato fruit was immobilized on galactose-containing polymeric beads. The immobilized enzyme exhibited an activity of 0.62 U/g of support and activity yield of 46%. The optimum pH and temperature for the activity of both free and immobilized enzymes were found as pH 4.0 and 37 °C, respectively. Immobilized α-galactosidase was more stable than free enzyme in the range of pH 4.0–6.0 and more than 85% of the initial activity was recovered. The decrease in reaction rate of the immobilized enzyme at temperatures above 37 °C was much slower than that of the free counterpart. The immobilized enzyme shows 53% activity at 60 °C while free enzyme decreases 33% at the same temperature. The immobilized enzyme retained 50% of its initial activity after 17 cycles of reuse at 37 °C. Under same storage conditions, the free enzyme lost about 71% of its initial activity over a period of 7 months, whereas the immobilized enzyme lost about only 47% of its initial activity over the same period. Operational stability of the immobilized enzyme was also studied and the operational half-life (t_1/2 was determined as 6.72 h for p-nitrophenyl α-d-galactopyranoside (PNPG) as substrate. The kinetic parameters were determined by using PNPG as substrate. The K_m and V_max values were measured as 1.07 mM and 0.01 U/mg for free enzyme and 0.89 mM and 0.1 U/mg for immobilized enzyme, respectively. The synthesis of the galactose-containing polymeric beads and the enzyme immobilization procedure are very simple and also easy to carry out.     

Glutathione-supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa

In this study we evaluated the effects of semen extender supplementation with different concentrations of glutathione (GSH) on buffalo (Bubalus bubalis) bull sperm motility, plasma membrane integrity, viability and DNA integrity as well as in vivo fertility. Semen from three Nili-Ravi buffalo bulls was collected, and qualified semen ejaculates (n = 18) were split into five aliquots for dilution (37 °C; 50 × 10⁶ spermatozoa ml^?1) with experimental tris-citric acid extender containing 0, 0.5, 1.0, 1.5 or 2.0 mM GSH. Extended semen was cooled to 4 °C, equilibrated and filled in French straws. The straws were kept on liquid nitrogen vapors (5 cm above the LN₂ level) for 10 min and plunged in liquid nitrogen for storage. Sperm motility (%), plasma membrane integrity (%), viability (%) and DNA integrity (%) were assessed at 0, 2 and 4 h post-thawing (37 °C). Extender supplementation with GSH (0.5, 1.0, 1.5 and 2.0 mM) increased sperm motility, plasma membrane integrity and viability in a dose dependent manner. Sperm DNA integrity was higher (p < 0.05) in all experimental extenders containing GSH when compared to the control extender (0 mM GSH). The in vivo fertility rate of cryopreserved buffalo bull (n = 2) spermatozoa was higher (p < 0.05) in extender containing 2.0 mM GSH compared to that of control. In summary, tris-citric acid extender supplemented with glutathione improved the freezability of buffalo bull spermatozoa in a dose dependant manner. Moreover, the addition of 2.0 mM GSH to the extender enhanced the in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa.     

Overexpression and characterization of a glucose-tolerant β-glucosidase from Thermotoga thermarum DSM 5069T with high catalytic efficiency of ginsenoside Rb1 to Rd

The β-glucosidase gene Tt-bgl from Thermotoga thermarum DSM 5069T was cloned and overexpressed in Escherichia coli. A simple strategy, induction at 37 °C with no IPTG, was explored to reduce the inclusion bodies, by which the activity of Tt-BGL was 13 U/mL in LB medium. Recombinant Tt-BGL was purified by heat treatment followed by Ni–NTA affinity. The optimal activity was at pH 4.8 and 90 °C. The activity of Tt-BGL was significantly enhanced by methanol and Al³⁺. The enzyme was stable over pH range of 4.4–8.0, and had a 2-h half life at 90 °C. The V_max for p-nitrophenyl-β-d-glucopyranoside and ginsenoside Rb1 was 142 U/mg and 107 U/mg, while the K_m was 0.59 mM and 0.15 mM, respectively. The activity of the enzyme was not inhibited by ginsenoside Rb1 (36 g/L). It was activated by glucose at concentrations lower that 400 mM. With glucose further increasing, the activity of Tt-BGL was gradually inhibited, but remained 50% of the original value in even as high as 1500 mM glucose. Under the optimal conditions, Tt-BGL transformed ginsenoside Rb1 (36 g/L) to Rd by 95% in 1 h.     

Elicitation with abiotic stresses improves pro-health constituents,antioxidant potential and nutritional quality of lentil sprouts

Phenolic content and antioxidant potential of lentil sprouts may be enhanced by treatment of seedlings in abiotic stress conditions without any negative influence on nutritional quality.The health-relevant and nutritional quality of sprouts was improved by elicitation of 2-day-old sprouts with oxidative, osmotic, ion-osmotic and temperature stresses. Among the sprouts studied, those obtained by elicitation with osmotic (600 mM mannitol) and ion-osmotic (300 mM NaCl) shocks had the highest total phenolic content levels: 6.52 and 6.56 mg/g flour, respectively. Oxidative stress significantly enhanced the levels of (+)-catechin and p-coumaric acid. A marked elevation of the chlorogenic and gallic acid contents was also determined for sprouts induced at 4 °C and 40 °C. The elevated phenolic content was translated into the antioxidant potential of sprouts, especially the ability to reduce lipid oxidation. A marked elevation of this ability was determined for seedlings treated with 20 mM, 200 mM H₂O₂ (oxidative stress) and 600 mM mannitol (osmotic stress); about a 12-fold, 8-fold and 9.5-fold increase in respect to control sprouts. The highest ability to quench free radicals was observed in sprouts induced by osmotic stress (IC₅₀- 4.91 and 5.12 mg/ml for 200 mM and 600 mM mannitol, respectively). The highest total antioxidant activity indexes were determined for sprouts elicited with 20 mM H₂O₂ and 600 mM mannitol: 4.0 and 3.4, respectively. All studied growth conditions, except induction at 40 °C, caused a significant elevation of resistant starch levels which was also affected in a subsequent reduction of starch digestibility.Improvement of sprout quality by elicitation with abiotic stresses is a cheap and easy biotechnology and it seems to be an alternative to conventional techniques applied to improve the health promoting phytochemical levels and bioactivity of low-processed food.     

Immobilization of horseradish peroxidase on activated wool

This work is aimed to immobilize partially purified horseradish peroxidase (HRP) on wool activated by multifunctional reactive center, namely cyanuric chloride. The effect of cyanuric chloride concentration, pH and enzyme concentration on immobilization of HRP was studied. FT-IR and SEM analyses were detected for wool, activated wool and immobilized wool-HRP. The wool-HRP, prepared at 2% (w/v) cyanuric chloride and pH 5.0, retained 50% of initial activity after seven reuses. The wool-HRP showed broad optimum pH at 7.0 and 8.0, which was higher than that of the soluble HRP (pH 6.0). The soluble HRP had an optimum temperature of 30 °C, which was shifted to 40 °C for immobilized enzyme. The soluble and wool-HRP were stable up to 30 and 40 °C after incubation for 1 h, respectively. The apparent kinetic constant values (K_ms) of wool-HRP were 10 mM for guiacol and 2.5 mM for H₂O₂, which were higher than that of soluble HRP. The wool-HRP was remarkably more stable against proteolysis mediated by trypsin. The wool-HRP exhibited more resistance to heavy metal induced inhibition. The wool-HRP was more stable to the denaturation induced by urea, Triton X-100, isopropanol, butanol and dioxan. The wool-HRP was found to be the most stable under storage. In conclusion, the wool-HRP could be more suitable for several industrial and environmental purposes.     

Purification of a laccase from fungus combs in the nest of Odontotermes formosanus

A new enzyme was isolated from the fungus combs in the nest of Odontotermes formosanus and identified as a laccase. The single laccase was purified with a purification factor of 16.83 by ammonium sulphate precipitation and anion exchange chromatography, to a specific activity of 211.11 U mg⁻¹. Its molecular mass was 65 kDa. The optimum pH value and temperature were 4.0 °C and 10 °C with ABTS as the substrate, respectively. The enzyme activity stabilized at temperatures between 10 °C and 30 °C and decreased rapidly when the temperature was above 30 °C. The V_max and K_m values were 3.62 μmol min⁻¹ mg⁻¹ and 119.52 μM, respectively. Ethanol concentration affected laccase activity, inhibiting 60% of enzyme activity at a concentration of 70%. Metal ions of Mg²⁺, Ba²⁺ and Fe²⁺ showed inhibition on enzyme activity of 17.2%, 5.3% and 9.4%, respectively, with the increase of metal ions concentration from 1 mM to 5 mM. Especially Fe²⁺ strongly inhibited enzyme activity up to 89% inhibition at a concentration of 1 mM.