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.     

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%.     

Cell wall invertase immobilization within calcium alginate beads

Yeast cell wall invertase (CWI) was immobilized within calcium alginate beads. The result of entrapment was the complete immobilization of all the added CWI. Three types of biocatalysts were prepared: CWI-S, CWI-155 and CWI-157. The optimum pH values were 4.5 and 5.0 for free and immobilized invertase, respectively. The optimum temperature was 60 °C, for both free CWI and CWI-S immobilizate. 80 °C was the optimum temperature for CWI-155 whereas the optimum temperature for CWI-157 ranged from 50 °C to 80 °C. Immobilized CWI was more stable than was free CWI above optimum activity temperatures. The activation energies were 32 kJ/mol for free CWI and 45, 21 and 25 kJ/mol for CWI-S, CWI-155 and CWI-157, respectively. The K_m values of free and immobilized CWI-157 were 28.4 mM and 72 mM, respectively. The V_max values were estimated as 4.5 mM/min and 0.42 mM/min, respectively. Immobilized CWI-157 was tested in a batch reactor using 70% sucrose (w/v). Complete sucrose conversion was achieved after 55 h. After 40 consecutive cycles, CWI-157 retained 90% of its activity.     

Immobilization of pepsin on chitosan beads

In this study, chitosan beads were prepared by using a cross-linking agent and the resulting beads were employed in immobilization process. Studies on free and immobilized pepsin systems for determination of optimum temperature, optimum pH, thermal stability, pH stability, operational stability, storage stability and kinetic parameters were carried out. The optimum temperature interval for free pepsin and immobilized pepsin were 30–40 and 40–50 °C, respectively. Free and immobilized pepsin showed higher activity at pH 2.0–4.0. Apparent K_m = 12.0 g L⁻¹ haemoglobin (1.56 mM tyrosine) and V_max = 5220 μmol (mg protein min)⁻¹ values were obtained for free pepsin, while apparent K_m = 20.0 g L⁻¹ haemoglobin (2.16 mM tyrosine) and V_max = 2780 μmol (mg protein min)⁻¹ values were obtained for immobilized pepsin. Thermal stability and storage stability of immobilized pepsin were higher than that of free pepsin. Milk clotting activity was used for evaluation of the applicability of pepsin immobilization to industrial process. Optimum milk clotting temperature was found as 40 °C for free pepsin and 50 °C for immobilized pepsin.     

Immobilization and stabilization of pectinase by multipoint attachment onto an activated agar-gel support

Pectinase was immobilized on an activated agar-gel support by multipoint attachment. The maximal activity of immobilized pectinase was obtained at 5 °C, pH 3.6, with a 24 h reaction time at an enzyme dose of 0.52 mg protein/g gel, and the gel was activated with 1.0 M glycidol. These conditions increased the thermal stability of the immobilized pectinase 19-fold compared with the free enzyme at 65 °C. The optimal temperature for pectinase activity changed from 40 to 50 °C after immobilization; however, the optimal pH remained unchanged. The immobilized enzyme also exhibited great operational stability, and an 81% residual activity was observed in the immobilized enzyme after 10 batch reactions.     

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.     

Inhibition of polyphenoloxidase from apple by Maillard reaction products prepared from glucose or fructose with l-cysteine under various conditions of pH and temperature

The effects of Maillard reaction products (MRPs), synthesized from equimolar glucose or fructose with l-cysteine (1 mol l⁻¹) aqueous model mixtures, by modulating pH and temperature of heating, according to a two-factor and five-level experimental design, were investigated on polyphenoloxidase (PPO) activity from apple. Final pH and absorbance measurements at 350 nm were also selected as indicators of the Maillard reaction development and checked. In general, inhibitory potency (IP) of the mixtures increased with the increase in temperature (80-120°C) and the decrease in pH (pH 2.0-12.0) of the reaction medium. A linear relationship between the IP and heating time (0-48 h) or Abs.350 nm (0-70 AU) was demonstrated for glucose/cysteine system heated from 80°C to 120°C. Polarographic and spectrophotometric data were used to calculate kinetic constants and activation energy (Ea) values of inhibitory MRPs formation versus PPO activity and of those compounds absorbing at 350 nm. Ea values for these reactions were close, being 191 and 124 kJ mol⁻¹, respectively. The experimental design allowed to conclude that linear effects of both factors as well as a quadratic effect of pH were significant, leading to optimum conditions for the production of glucose-derived MRPs inhibitors. In most cases, glucose produced MRPs with higher IP compared to counterpart fructose-cysteine MRPs.     

Comparison of the effects of sucrose and hexose on furfural formation and browning in cookies baked at different temperatures

The influence of the type of sugar and baking temperature on sugar degradation, hydroxymethylfurfural (HMF) formation and browning was studied in model cookies. The baking process was characterised by the temperature in the cookie and the water content and activity. A reference browning was selected to compare the differently processed cookies. The accumulation of HMF was modelled at three temperatures for three formulas (sucrose (S-CK), glucose (G-CK) or fructose (F-CK)). HMF started to accumulate at a_w between 0.5 and 0.7 depending on the temperature and followed a first order kinetic, highly dependent on the baking temperature and type of sugar. Cookies baked at 200 °C accumulated 10–100 times less HMF than those baked at higher temperatures. Below 250 °C, S-CK produced less HMF than G- or F-CK, but the inverse was observed at 300 °C.     

Hydrolysis of oligosaccharides in soybean products by Debaryomyces hansenii UFV-1 α-galactosidases

α-Galactosides are abundant sugars found in legumes such as soybean. Since humans and monogastric animals lack α-galactosidase in the digestive tract, they are unable to digest these sugars, which induce flatulence. The use of α-galactosidases is promising as a means to overcome this problem, and to increase the consumption of soy products. Immobilized α-galactosidase, derived from Debaryomyces hansenii UFV-1, exhibited an activity of 40 U per g of silica and an activity yield of 50%. The optimum pH of free and immobilized α-galactosidase was 5.0 and the optima temperatures were 60 and 80 °C, respectively. The soymilk stachyose was completely hydrolyzed by different enzyme forms after incubation for 4 h at 60 °C, while raffinose was reduced by 100%, 25% and 68% by free, immobilized enzymes and permeabilized cells, respectively. The soy molasses treatment with free enzyme for 6 h promoted reduction in stachyose and raffinose contents by 100% and 50%, respectively.     

Electrochemical biosensor based on gold coated iron nanoparticles/chitosan composite bound xanthine oxidase for detection of xanthine in fish meat

A xanthine oxidase was immobilized covalently onto chitosan bound gold coated iron nanoparticles (CHIT/Fe-NPs@Au) electrodeposited on the surface of pencil graphite electrode (PGE). A xanthine biosensor was fabricated using XOD/CHIT/Fe-NPs@Au/PGE as working, Ag/AgCl as reference and Pt as auxiliary electrode connected through potentiostat. The enzyme electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS). The biosensor exhibited optimum current response within 3 s at pH 7.4, 35 °C and working range 0.1–300 μM, when polarized at 0.5 V vs Ag/AgCl. The sensitivity of the biosensor was 0.001169 mAμ M^–1 cm^–2 with detection limit of 0.1 μM (S/N = 3). The biosensor showed only 25% loss in its initial activity after its 100 uses over 100 days, when stored at 4 °C.     

Galacto-oligosaccharide synthesis by immobilized Aspergillus oryzae β-galactosidase

Aspergillus oryzae β-galactosidase was immobilized by three different techniques, namely adsorption on celite, covalent coupling to chitosan and aggregation by cross-linking. These techniques were compared in terms of the yield of immobilized preparation, enzymatic characteristics, stability and efficiency in oligosaccharide synthesis. Immobilization led to increase in K_m in each case. Immobilization on chitosan gave maximum enzyme yield and oligosaccharide synthesis. At 60 °C, the chitosan-immobilized enzyme was stabilized (by 1.6-fold) due to protection effect of the matrix. However, at 65 °C, the t_1/2 of cross-linked enzyme aggregates (CLEA) of β-galactosidase was 1.07 h as compared to 0.79 h in the case of free enzyme. Both chitosan-immobilized enzyme and CLEA were used for oligosaccharide synthesis. Using 20% (w/v) lactose, the chitosan-immobilized enzyme gave maximum oligosaccharide yield (17.3% of the total sugar) as compared to free enzyme (10.0%) in 2 h at 40 °C. CLEA were instead found effective in lactose hydrolysis yielding 78% monosaccharide in 12 h.     

Galacto-oligosaccharides production during lactose hydrolysis by free Aspergillus oryzae β-galactosidase and immobilized on magnetic polysiloxane-polyvinyl alcohol

The synthesis of galacto-oligosaccharides (GOS) by the action of Aspergillus oryzae β-galactosidase free and immobilized on magnetic polysiloxane-polyvinyl alcohol (mPOS-PVA) was studied. A maximum GOS concentration of 26% (w/v) of total sugars was achieved at near 55% lactose conversion from 50%, w/v lactose solution at pH 4.5 and 40 °C. Trisaccharides accounted for more than 81% of the total GOS produced. GOS formation was not considerably affected by pH and temperature. The concentrations of glucose and galactose encountered near maximum GOS concentration greatly inhibited the reactions and reduced GOS yield. GOS formation was not affected by enzyme immobilization in the mPOS-PVA matrix, indicating the absence of diffusional limitations in the enzyme carrier. Furthermore, this water insoluble magnetic derivative was reutilized 10-times and retained about 84% of the initial activity. In addition, the kinetic parameters for various initial lactose concentrations were determined and compared for the free and immobilized enzyme.     

Amperometric assessment of glucose electrode behaviour in mixed solvents and determination of glucose in dairy products

An amperometric biosensor based on a ruthenium(III), nickel(II) and iron(II) hexacyanometallate (HCM)-modified graphite electrode and immobilized glucose oxidase has been used for the determination of glucose in water-miscible organic solvent/aqueous buffer mixtures. Although the specific activity of biochemically active molecule such as enzyme is reduced in organic environment, it was established that the presence of water soluble organic solvents such as methanol, ethanol and acetonitrile (φ = 10%) enhance the biosensor response. Hydrogen peroxide, produced by enzyme-catalysed reduction of glucose, was measured in phosphate buffer solution (pH = 6.86) at −50 mV against a reference Hg|Hg₂Cl₂|3 M KCl electrode to determine the concentration of glucose. The influence of the addition of different volume fractions (φ = 10–60%) of methanol, ethanol, acetone, acetonitrile and isopropanol on biosensor response was investigated. The obtained amperometric signals were fast, reproducible and linearly proportional to glucose concentrations in the range of 0.1–0.8 mM, with a squared correlation coefficient of 0.9994 for buffer solution. With the addition of ethanol (φ = 10% and 40%) the plateau on I/c curve was obtained for concentrations of glucose higher than 0.8 and 1.1 mM, respectively. The biosensor proved to be stable for several months. The recoveries of added glucose (0.200 and 0.300 mM) from aqueous solution and from solution with ethanol φ = 10% ranged from 96.0% to 108.0%. The biosensor was used for the determination of glucose in some food samples of dairy industry, and the results were consistent with those obtained with the commercially available glucose enzyme photometric kit.     

Production of highly enantioselective (−)-menthyl butyrate using Candida rugosa lipase immobilized on epoxy-activated supports

Optically active (−)-menthyl butyrate was synthesized by enantioselective esterification of racemic (±)-menthol and butyric anhydride using lipase from Candida rugosa immobilized onto epoxy-activated supports of Eupergit C and Eupergit C 250 L through physical adsorption method. The effects of various temperature, storage condition, stability in organic solvent and lipase recyclability were investigated for their influence on the enzymatic enantioselective formation of (−)-menthyl butyrate. The immobilized lipases retained high catalytic activity of up to 31% yield and 100% enantiomeric excess of the desired product, and showed better stability compared to the native lipase. They also exhibited about 50% retained activity even after incubation at higher temperatures, storage at room temperature and after long incubation in hexane. Immobilized lipases also showed considerably efficient reusability.     

Characteristics and antioxidative activity of Maillard reaction products from a porcine plasma protein–glucose model system as influenced by pH

Maillard reaction products (MRPs) were prepared by heating the solution containing 2% porcine plasma protein (PPP) and 2% glucose adjusted to various pHs (8, 9, 10, 11 and 12) at 100 °C for different times (0, 2, 4, 6 and 8 h). The pH of all MRPs markedly decreased within 2 h of heating time. Browning and intermediate products, as monitored by absorbance at 420 nm and absorbance at 294 nm, sharply increased within 2 h (P < 0.05). Thereafter, slight increases were observed up to 8 h. Fluorescence intensity (Ex 347 and Em 415 nm) sharply increased within the first 2 h with a subsequent decrease when heating time increased (P < 0.05). Increases in browning and formation of intermediate products were concomitant with the decreases in free amino group and reducing sugar contents. Among all MRPs tested, those derived from the PPP–glucose system at pH 12 rendered the highest browning and intermediate products. However, no differences in reducing power or DPPH radical-scavenging activity of MRPs with initial pH ranges of 10–12 were noticeable. Electrophoretic study revealed that cross-linked proteins with high molecular weight were formed in the PPP–glucose model system to a greater extent at pHs 8 and 9, than at pHs 10–12. Nevertheless, heating times had no pronounced effect on protein pattern of glycated proteins.     

Interaction between calcium chloride and some carbohydrates as seen by mutual diffusion at 25 °C and 37 °C

Ternary mutual diffusion coefficients measured by Taylor dispersion method (D₁₁, D₂₂, D₁₂ and D₂₁) are reported for aqueous solutions of calcium chloride (CaCl₂) and some carbohydrates (glucose, fructose and sucrose) at 25 °C and 37 °C at carrier concentrations from 0.000 to 0.100 M, for each solute, respectively.     

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.     

Changes in the antioxidant activity of loach (Misgurnus anguillicaudatus) protein hydrolysates during a simulated gastrointestinal digestion

A two-stage in vitro digestion model system (a pepsin treatment for 2 h followed by a pancreatin treatment for 2 h, both at 37 °C) was used to simulate the process of human gastrointestinal (GI) digestion to determine the changes in antioxidant activities of loach peptide previously prepared by papain digestion. Results showed that the final GI digests contained 38.1% free amino acids, with short chain peptides (<500 Da) making up the rest of the biomass. Enzymatic breakdown of the GI digests increased their hydroxyl (12% increase), 2,2′-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) (5% increase) radical scavenging activity, the reducing power (77% increase) and the chelating ability of Cu²⁺ (12% increase), compared to the blank. The results showed that the digestion by gastrointestinal proteases can be used to produce antioxidant peptides, with the advantage that the peptides formed will resist physiological digestion in GI tract.     

Aglycone production by Lactobacillus rhamnosus CRL981 during soymilk fermentation

Lactobacillus rhamnosus CRL981 showed the highest levels of β-glucosidase and was selected to characterize this enzyme system, among 63 strains of different Lactobacillus species. The maximum activity was obtained at pH 6.4 and 42 °C. The enzyme showed weak resistance to thermal inactivation maintaining only 20% of the initial activity when it was exposed at 50 °C for 5 min. It also, showed stability when stored at 4 °C for 60 days. Afterwards, L. rhamnosus was evaluated for hydrolysis of isoflavones to aglycones, cell population, residual sugars and organic acid produced during fermentation on soymilk (37 °C for 24 h). Higher viable counts were obtained after 12 h of fermentation (8.85 log CFU ml⁻¹) followed by a drop of pH and an increase of acidity during fermentation due the production of organic acids. L. rhamnosus CRL981 was able to proliferate in soymilk and produce a high β-glucosidase activity achieving a complete hydrolysis of glucoside isoflavones after 12 h of fermentation. The present study indicates that L. rhamnosus CRL981 could be used in the development of different aglycone-rich functional soy beverages.     

Immobilization of recombinant thermostable β-galactosidase from Bacillus stearothermophilus for lactose hydrolysis in milk

A recombinant thermostable β-galactosidase from Bacillus stearothermophilus was immobilized onto chitosan using Tris(hydroxymethyl)phosphine (THP) and glutaraldehyde, and a packed bed reactor was utilized to hydrolyze lactose in milk. The thermostability and enzyme activity of THP-immobilized β-galactosidase during storage was superior to that of free and glutaraldehyde-immobilized enzymes. The THP-immobilized β-galactosidase showed greater relative activity in the presence of Ca²⁺ than the free enzyme and was stable during the storage at 4°C for 6 wk, whereas the free enzyme lost 31% of the initial activity under the same storage conditions. More than 80% of lactose hydrolysis in milk was achieved after 2 h of operation in the reactor. Therefore, THP-immobilized recombinant thermostable β-galactosidase from Bacillus stearothermophilus has the potential for application in the production of lactose-hydrolyzed milk.