Design and characterisation of an enzyme system for inulin hydrolysis

The optimal conditions for inulin hydrolysis using a commercial inulinase preparation, either free or immobilised in activated Amberlite were established by factorial design and surface response methodology. The immobilised biocatalyst displayed highest activity at pH 5.5 and 50 °C, whereas the optimum pH for the free form was slightly more acidic (4.5), and the optimum temperature was a little higher (55 °C). The model system estimated optimal pH and temperature values of 5.4 and 52 °C for the immobilised system and 4.9 and 56 °C for the free system. Michaelis–Menten type kinetics adequately described both free and immobilised bioconversion systems, which were evaluated under the respective optimal pH and temperature conditions. The use of a non-linear regression method for the determination of the kinetic parameters provided a best fit to the experimental data, as compared to a conventional Lineweaver–Burk linearisation. The K_m for inulin of the free biocatalyst was 153 g l⁻¹ at 55 °C and pH 4.5, whereas the apparent K_m for inulin of the immobilised biocatalyst was 108 g l⁻¹ at pH 5.5 and 50 °C. The reutilisation of the immobilised biocatalyst throughout consecutive batches was evaluated. A significant decrease of enzyme activity was observed in the first two batches, after which the system exhibited significant stability. The low cost of the support, the stability of the immobilised biocatalyst towards pH and temperature and its high affinity for the substrate suggests its potential for inulin hydrolysis.     

Chitosan-tethered the silica particle from a layer-by-layer approach for pectinase immobilization

“Spherical polyelectrolyte brush” of core–shell structure were prepared by grafting poly (sodium 4-styrenesulphonate) (PSStNa) from SiO₂ nanoparticle via the surface-initiated atom transfer radical polymerization strategy. The colloidal stability was not impeded by the adsorbed proteins despite the fact that up to 316.8 mg of enzyme was adsorbed per gram of the carrier particles. The immobilized pectinase revealed acceptable pH stability over a broad experimental range of 3.0–4.5. The activity half lives for native and bound states of enzyme were found as 13.5 d and 30 d, respectively. The activity of immobilized pectinase adsorbed onto these particles was analyzed in terms of the Michaelis–Menten parameters. Kinetic parameters were calculated as 8.28 and 9.98 g pectin ml⁻¹ for K_m and 1.165 × 10⁻³ g pectin s⁻¹ g enzyme⁻¹ 1.124 × 10⁻³ g pectin s⁻¹ g particle⁻¹ for V_max in the case of free and immobilized enzymes, respectively. Enzyme activity was found to be approximately 49.7% for immobilized enzyme after storage for 1 month.     

Hydrolysis of sucrose by invertase immobilized on nylon-6 microbeads

A commercial extracellular invertase (EC 3.2.1.26) from Saccharomyces cerevisiae has been inmobilized by covalent bonding on novel microbeads of nylon-6 using glutaraldehyde. The enzyme was strongly bound on the support, immobilized with an efficiency factor of 0.93. The biocatalyst showed a maximum enzyme activity when immobilized at pH 5.0, but optimum pH activity for both immobilized and free invertases was 5.5. The optimum temperatures for immobilized and free enzymes were 60 and 65 °C, respectively. Kinetic parameters were determined for immobilized and free invertases: V_max values were 1.37 and 1.06 mmol min⁻¹ mg⁻¹, respectively. The K_m and K_i values were 0.029 and 0.71 M for immobilized invertase and 0.024 and 0.69 M for free invertase. It was found that the thermal stability of the immobilized invertase with regard to the free one increased by 25% at 50 °C, 38% at 60 °C and 750% at 70 °C. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous sucrose hydrolysis. The effects of two different sugar concentrations and three flow rates on the productivity of the reactor and on the specific productivity of the biocatalyst were studied. The system demonstrated a very good productivity up to 2.0 M sugar concentration, with conversion factors of 0.95 and 0.97, depending on sucrose concentration in the feeding. This approach may serve as a simple technique and can be a feasible alternative to continuous sucrose hydrolysis in a fixed bed reactor for the preparation of fructose-rich syrup.     

Alcoholysis of salicornia oil using free and covalently bound lipase onto chitosan beads

Porcine pancreatic lipase was immobilized on chitosan by covalent binding and retention of its activity was examined. The activities of free and immobilized lipase were determined using olive oil as substrate. The free and immobilized enzymes showed pH 9 as optimum and retained 50% of activity after five cycles. When the substrate concentration was kept constant and enzyme concentration was varied, the K_m and V_max were observed to be 4.0 × 10⁻⁷ and 0.32, and 3.32 × 10⁻⁷ and 0.32, respectively, for free and covalently bound enzyme. This indicates that there is no possibility of conformational change during immobilization. Immobilized enzyme showed improved thermal and storage stability. Alcoholysis of salicornia oil, mediated by free and immobilized lipase, was carried out at 25 °C using methanol in hexane and acetone. Free and immobilized enzyme in hexane produced, respectively, 45% and 55% of fatty acid methyl ester after 12 h.     

Formation of biopolymer particles by thermal treatment of β-lactoglobulin–pectin complexes

The purpose of this study was to prepare and characterize biopolymer particles based on thermal treatment of protein–polysaccharide electrostatic complexes formed from a globular protein (β-lactoglobulin) and an anionic polysaccharide (beet pectin). Initially, the optimum pH and pectin concentration for forming protein–polysaccharide complexes were established by mixing 0.5 wt% β-lactoglobulin solutions with beet pectin (0–0.5 wt%) at different pH values (3–7). Biopolymer complexes in the sub-micron size range (d = 100–300 nm) were formed at pH 5.0 and 0.1 wt% pectin. These particles were then subjected to a thermal treatment (30–90 °C at 0.8 °C min⁻¹). The presence of pectin increased the thermal aggregation temperature of the protein, although aggregate formation was still observed when the protein–polysaccharide systems were heated above about 70 °C. The impact of pH (3–7) on the properties of heat-treated biopolymer particles (83 °C, 15 min, pH 5) was then established. The biopolymer particles were stable to aggregation over a range of pH values, which increased as the amount of pectin was increased. The biopolymer particles prepared in this study may be useful for encapsulation and delivery of bioactive food components, or as substitutes for lipid droplets.     

Sucrose hydrolysis by gelatin-immobilized inulinase from Kluyveromyces marxianus var. bulgaricus

The crude cell-free medium from a culture of Kluyveromyces marxianus var. bulgaricus was immobilized in a gelatin-water support, with an immobilization yield of 82.60% for inulinase activity. The optimum pH for both free and immobilized inulinase was the same (3.5) and the optimum temperatures were 55 °C for the free and 60 °C for the immobilized enzyme. The Arrhenius plots were linear and activation energies were 56.20 (free enzyme) and 20.27 kJ/mol K (immobilized enzyme). The kinetic parameters were calculated by Lineweaver–Burk plots and the V_max and K_m were 37.60 IU/mg protein and 61.83 mM for the free inulinase and 31.45 IU/mg protein and 149.28 mM for the immobilized enzyme, respectively. The operational stability of the immobilized inulinase was studied in a continuous fixed-bed column reactor for 33 days, at the end of which the sucrose conversion was 58.12%.     

Pineapple fruit bromelain affinity to different protein substrates

Optimal pH and temperature conditions for proteolytic activity of pineapple fruit bromelain were determined using five different substrates: azocasein and azoalbumin (pH 3–10 at 20–70 °C), casein and sodium caseinate (pH 2–10 at 20–70 °C), and haemoglobin (pH 2–6.5 at 30–60 °C). Fruit bromelain has shown optimum activity at pH 7.5 for azoalbumin and at 6.5 for azocasein, all at 55 °C. Fruit bromelain activity determined with casein and sodium caseinate has shown optimum activity at 59 °C, while the optimum pH was 7.7 for casein and 6.5 for sodium caseinate. Optimum hydrolysis conditions of fruit bromelain towards haemoglobin showed a sharp peak at an acidic pH 2.9 at 37 °C. The lowest results of K_m and the highest results of V_max/K_m were found for azocasein and azoalbumin. These substrates are highly recommended for fruit bromelain activity determination.     

Extraction and characterisation of pectin methylesterase from black carrot (Daucus carota L.)

This study was carried out to determine some of the biochemical properties of pectin methylesterase (PME) from black carrot. The enzyme showed very high activity in a broad pH range of 6.5–8.5, with the optimum pH occurring at 7.5. The optimum temperature for maximal PME activity was found to be 55 °C. NaCl enhanced PME activity, particularly at 0.2 M. K_m and V_max values for black carrot PME using apple pectin as substrate were found to be 2.14 mg/ml (r² = 0.988) and 3.75 units/ml, respectively. The enzyme was stable between the temperatures of 30–50 °C/5 min whereas it lost nearly all of its activity at 70 °C/5 min. E_a and Z values were found to be 196.8 kJmol⁻¹ (r² = 0.996) and 2.16 °C (r² = 0.995), respectively.     

Polyacrylamide–gelatine carrier system used for invertase immobilization

Invertase was immobilized into polyacrylamide–gelatin carrier system by chemical cross-linking with chromium (III) acetate, chromium (III) sulphate, and potassium chromium (III) sulphate. The optimum conditions, namely substrate concentration, temperature, and pH were determined. The effect of polyacrylamide–gelatin ratio and cross-linker concentration on immobilized enzyme activity were analysed. Maximum immobilized enzyme activities were obtained with chromium (III) acetate (0.01 mol dm⁻³), chromium (III) sulphate (0.004 mol dm⁻³) and potassium chromium (III) sulphate (0.001 mol dm⁻³) for 0.177 (w/w) polyacrylamide–gelatin carrier ratio as 79%, 72% and 79%, respectively. The K_m values were 86 and 166 mM for free and immobilized enzyme, respectively. All immobilized samples were used 20 times over a period of 2 months without a considerable loss of activity.     

Immobilization of naringinase from Aspergillus niger CECT 2088 in poly(vinyl alcohol) cryogels for the debittering of juices

Naringinase, induced from Aspergillus niger CECT 2088 cultures, was immobilized into a polymeric matrix consisting of poly(vinyl alcohol) (PVA) hydrogel, cryostructured in liquid nitrogen, to obtain biocatalytically active beads. The effects of matrix concentration, enzyme load and pH on immobilization efficiency were studied. Between 95% and 108% of the added naringinase was actively entrapped in PVA cryogel, depending on the conditions of immobilization used. The optimal conditions were: 8% (w/v) PVA at pH 7 and 1.6–3.7 U ml⁻¹ of enzyme load. The pH/activity profiles revealed no change in terms of shape or optimum pH (4.5) upon immobilization of naringinase. However, the optimum temperature was shifted from 60 °C to 70 °C and the activation energy of reaction, E_a, was decreased from 8.09 kJ mol⁻¹ to 6.36 kJ mol⁻¹ by immobilization. The entrapped naringinase could be reused through six cycles (runs of 24 h at 20 °C), retaining 36% efficacy for the hydrolysis of naringin in simulated juice.     

Partial purification and preparation of bovine lactoperoxidase and characterization of kinetic properties of its immobilized form incorporated into cross-linked alginate films

Lactoperoxidase (LPS), purified directly from bovine rennet whey by Toyopearl-SP cation-exchange chromatography and lyophilized by using dextran as supporting material, maintained almost 70 and 60% of its activity after almost 2 and 5 months storage at −18 °C, respectively. Incorporation of the prepared LPS into alginate films between 0.08 and 0.69 mg/cm² (516–4325 U/cm²) caused the immobilization of most of the enzyme and gave films with LPS activity between 0.05 and 2.8 U/cm², determined in the presence of 8 μM H₂O₂. Between 2 and 24 μM H₂O₂ concentrations, a two-fold increase in H₂O₂ concentration caused 1.5–2.5-fold increase in LPS activity of films incorporated with 0.24–0.28 mg/cm² (1200 U/cm²) LPS. The Q₁₀ and E_a of immobilized enzyme activity between 4 and 16 °C were 1.69 and 34.6 kJ/mol, respectively. However, in the 16–30 °C range, the temperature change had almost no effect on LPS activity of films. The optimal activity of immobilized LPS was observed at pH 6.0, but the enzyme maintained 30–85% of its activity between pH 3.0 and 7.0. The immobilized LPS also had a high stability between pH 4.0 and 6.0. The results of this study showed the good potential of LPS-incorporated alginate films in forming a natural antimicrobial mechanism in different foods.     

Effects of temperature,solid content and pH on the stability of black carrot anthocyanins

Anthocyanin stability of black carrots was studied at various solid contents (11, 30, 45 and 64° Brix) and pHs (4.3 and 6.0) during both heating, at 70–90 °C, and storage at 4–37 °C. Monomeric anthocyanin degradation fitted a first-order reaction model. Degradation of monomeric anthocyanins increased with increasing solid content during heating, while it decreased during storage. For example, at pH 4.3, half-life periods for anthocyanins at 30, 45 and 64° Brix were, respectively, 8.4, 6.9 and 5.2 h during heating at 80 °C and 18.7, 30.8 and 35.9 weeks during storage at 20 °C. At 30–64° Brix, increasing pH from 4.3 to 6.0 enhanced the degradation of anthocyanins during heating. The effect of pH on thermal stability of anthocyanins was also studied at six different pHs (2.5–7.0) in citrate-phosphate buffer solutions and significant decrease in anthocyanin stability was observed at pHs above 5.0. Higher activation energies (E_a) were obtained during heating than during storage with increasing solid contents. At 30–64° Brix, E_a values ranged from 68.8 to 95.1 kJ mol⁻¹ during heating and from 62.1 to 86.2 kJ mol⁻¹ during storage. Q₁₀ values at 20–37 °C were as high as 3.1 at 45° Brix and 3.6 at 64° Brix.     

Pico molar assay of riboflavin in human urine using voltammetry

Using a new type of DNA and carbon nano tube (CNT) mixed paste electrode using cyclic and square wave anodic stripping voltammetric (SWASV) methods, this study presents an assay of riboflavin (RF) under optimum conditions. Results of the experiment yielded a low working concentration range of nanograms with 1–10 and 10–170 ng L⁻¹ and 5–105 μg L⁻¹, at an accumulation time of 80 s in a 0.1 M H₃PO₄ electrolyte solution. A relative standard deviation of 30 μg L⁻¹ was observed at an accuracy level of 0.1164% (n = 15) under optimum conditions. The detection limit (S/N) was pegged at 0.2 ng L⁻¹ (5.31 × 10⁻¹³ mol L⁻¹ RF). The proposed method was successfully applied to an actual human urine and drug sample, and can be applied to assays of other biological samples.     

A novel aspartic protease from the viscera of Sardinelle (Sardinella aurita): Purification and characterisation

A novel aspartic protease was extracted from the defatted viscera of sardinelle (Sardinella aurita) and purified, with a 9.5-fold increase in specific activity and 23.3% recovery. The molecular weight of the purified enzyme was estimated to be 17 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The purified enzyme appeared as a single band on native-PAGE. The optimum pH and temperature for protease activity were around 3.0 and 40 °C, respectively. The enzyme showed pH stability between 2.0 and 5.0 and retained more than 50% of its activity after heating for 30 min at 50 °C. The enzyme lost 90% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride. Its K_m value was determined to be 0.73 × 10⁻⁴ M using haemoglobin as a substrate. The N-terminal 12 amino acid sequence of the purified acidic protease was R V I I E D X D Q F C T. This sequence showed low homology with aspartic peptidases of several other species of fish, suggesting that the enzyme is a new aspartic protease.     

Development of polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for sodium saccharin residue in food samples

A sensitive and specific polyclonal antibody (PcAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) for sodium saccharin is described. 6-Amino saccharin was coupled to carrier protein for artificial antigen by diazotisation. New Zealand white rabbits were immunised to obtain anti-sodium saccharin PcAb and then icELISA was developed. The assay showed high sensitivity and specificity to sodium saccharin, with the 50% inhibition value (IC₅₀) of 0.243 μg mL⁻¹, workable range (IC₃₀–IC₇₀) of 0.050–12.8 μg mL⁻¹ and limit of detection (LOD, IC₂₀) of 0.021 μg mL⁻¹. The average recoveries of sodium saccharin in spiked food samples were estimated ranging from 70.7% to 98.8%. A statistically significant correlation of results was obtained between this new ELISA and previously established HPLC approaches with the food-relevant sodium saccharin concentration range 0–320 μg mL⁻¹ (R² = 0.9887–0.9975). These results indicated that the established ELISA was a potential and useful analytical tool for rapid determination of sodium saccharin residue in food samples.     

Determination of Ge(IV) in rice in a mercury-coated glassy carbon electrode in the presence of catechol

Electrochemical adsorptive cathodic stripping voltammetry determination of Ge(IV) using the catechol complex on a mercury-coated (MC) electrode was prepared using a glassy carbon electrode (GCE), the peak potential of which was −0.5 V vs. Ag/AgCl on MCGCE. The various parameters of the catechol concentration, its pH, and others were optimized. The linear working ranges were obtained in the concentration of 2–700 μg L⁻¹ Ge(IV). The relative standard deviation at the Ge(IV) concentration of 50 μg L⁻¹ was 1.37% (n = 15) using the optimum condition, and the detection limit was found to be 0.6 μg L⁻¹ (8.26 × 10⁻⁹ M) (S/N = 3), with an adsorption time of 180 s. The Ge(IV) response was highly linear. This developed method was applied to Ge(IV) to determine the presence of rice grains.     

Effect of modifiers for As,Cu and Pb determinations in sugar-cane spirits by GF AAS

Palladium plus magnesium nitrates with and without Ir, Ru and W were evaluated for the simultaneous determination of As, Cu and Pb in cachaça by graphite furnace atomic absorption spectrometry. For 20 μL of sample, 5 μL Pd(NO₃)₂ and 3 μL Mg(NO₃)₂ dispensed together onto the Ir-coated platform of the THGA, analytical curves in the 0–30.0 μg L⁻¹ As, 0–1.50 mg L⁻¹ Cu and 0–60.0 μg L⁻¹ Pb were built up and typical linear correlation coefficients were always better than 0.999. The limit of detection was 1.30 μg L⁻¹ As, 140 μg L⁻¹ Cu and 0.90 μg L⁻¹ Pb. As, Cu and Pb contents in 10 cachaça samples agreed with those obtained by ICP-MS. Recoveries of spiked samples varied from 96% to 106% (As), 97% to 112% (Cu) and 92% to 108% (Pb). The relative standard deviation (n = 12) was typically 2.7%, 3.3% and 1.9%.     

Immobilization of α-amylase onto cyclic carbonate bearing hybrid material

Under ambient conditions, 4-((3-trimethoxysilyl)propoxy)methyl)1,3-dioxolan-2-one was synthesized from 3-glycidyloxypropyl trimethoxysilane. Methacrylate and cyclic carbonate functional hybrid matrix was prepared by sol-gel method. α-Amylase was covalently bounded onto the matrix via cyclic carbonate functionality. Immobilization yield was found as 34.4 ± 2.6 mg per gram of hybrid matrix. The maximum activity was observed at pH 6.5. Immobilization did not change the pH-dependency of the enzyme. The immobilized enzyme had a higher activity at elevated temperature (50–80° C) than the free one. Immobilized enzyme exhibited 80% activity after 20 runs and 69% after 30 runs. Free enzyme lost its activity completely within 15 days. Immobilized enzyme lost only 30% of its activity in 25 days. V_max values for the free and immobilized enzymes were calculated as 58 × 10⁻³ and 5.2 × 10⁻³ mg/ml min⁻¹, respectively.     

Tungsten permanent chemical modifier with co-injection of Pd(NO3)2 + Mg(NO3)2 for direct determination of Pb in vinegar by graphite furnace atomic absorption spectrometry

A tungsten carbide coating on the integrated platform of a transversely heated graphite atomizer (THGA^®) used together with Pd(NO₃)₂ + Mg(NO₃)₂ as modifier is proposed for the direct determination of lead in vinegar by graphite furnace atomic absorption spectrometry. The optimized heating program (temperature, ramp time, hold time) of atomizer involved drying stage (110 °C, 5 s, 30 s; 130 °C, 5 s, 30 s), pyrolysis stage (1000 °C, 15 s, 30 s), atomization stage (1800 °C, 0 s, 5 s) and clean-out stage (2450 °C, 1 s, 3 s). For 10 μL of vinegar delivered into the atomizer and calibration using working standard solutions (2.5–20.0 μg L⁻¹ Pb) in 0.2% (v/v) HNO₃, analytical curve with good linear correlation (r = 0.9992) was established. The characteristic mass was 40 pg Pb and the lifetime of the tube was around 730 firings. The limit of detection (LOD) was 0.4 μg L⁻¹ and the relative standard deviations (n = 12) were typically <8% for a sample containing 25 μg L⁻¹ Pb. Accuracy of the proposed method was checked after direct analysis of 23 vinegar samples. A paired t-test showed that results were in agreement at 95% confidence level with those obtained for acid-digested vinegar samples. The Pb levels varied from 2.8 to 32.4 μg L⁻¹. Accuracy was also checked by means of addition/recovery tests and recovered values varied from 90% to 110%. Additionally, two certified reference materials were analyzed and results were in agreement with certified values at a 95% confidence level.     

Emulsifying properties of chickpea protein isolates: Influence of pH and NaCl

Structure and emulsifying properties of chickpea protein isolates (CPI) as a function of protein concentration, oil volume, pH and ionic strength were studied. The optimum protein concentration 2 g l⁻¹ used to determine the emulsifying properties was obtained. Emulsifying activity index (EAI) increased from 244 to 376 m² g⁻¹ with pH from 3.0 to 11.0 except the protein isoelectric point (pI 5.0), where the EAI was 20 m² g⁻¹ and emulsion droplet size was the largest. At lower ionic strengths (0.0–0.1 M NaCl, pH 7.0), EAI decreased from 253 to 72.4 m² g⁻¹; however, it increased from 72.4 to 231.4 m² g⁻¹ at higher ionic strengths (0.1–1.0 M NaCl). A positive relation between EAI and surface hydrophobicity (S₀) of CPI at various ionic strengths was obtained, while EAI was independent of S₀ under different pH values. α-Helix was the major configuration of CPI at the pI or lower ionic strength.