Immobilization and characterization of naringinase for the hydrolysis of naringin

The degree of hydrolysis of naringin was investigated at various temperatures (40, 50, 60 °C), enzyme concentrations (0.01–0.30 mg ml⁻¹), and pH values (2.5–5.5) for naringinase enzyme. Naringinase was immobilized on celite by simple adsorption. Naringin content was determined by HPLC method. The degree of hydrolysis of naringin showed a linear increase up to an enzyme concentration of 0.2 mg ml⁻¹ that corresponds to 82% hydrolysis. The optimum values of pH for the hydrolysis of naringin were 4.0 for free and 3.5 for immobilized enzymes. Maximum enzyme activities were found to be 70 and 60 °C for free and immobilized enzymes, respectively. The values of K _m,app and V _max,app calculated were 1.22 mM and 0.45 μmol min⁻¹ mg enzyme⁻¹ for free and 2.16 mM and 0.3 μmol min⁻¹ mg enzyme⁻¹ for immobilized enzyme, respectively. The mathematical modelling was applied to the experimental data for hydrolysis of naringin as a function of time at 30, 40 and 50 °C. The increase in temperature from 30 to 50 °C increased the rate constant 3.09 times for free enzyme. However, the rate constants found for immobilized enzyme applications did not increase in a similar trend as a function of temperature. The retained activity of celite-adsorbed naringinase was found to be 83% at their optimum conditions. The retained activity of immobilized enzyme was followed up to the fifth run and was found to be almost unchanged after the third use at optimum reaction conditions (pH 3.5, 60 °C).     

Immobilization of inulinase for sucrose hydrolysis

A commercial inulinase preparation immobilized on various supports was used for sucrose hydrolysis. Entrapment and encapsulation in Ca-alginate and entrapment in an alginate–silicate sol–gel matrix were evaluated. Best results were obtained with Ca-alginate beads. The influence of sodium-alginate concentration on the immobilization yield was assessed. Inulinase entrapped in Ca-alginate beads displayed high activity in the range 50–60 °C, whereas the optimum for the free enzyme was 60 °C. The optimum pH of the immobilized enzyme was slightly more acidic (4.0) than the one observed for the free form (4.5). The apparent K_M for sucrose of the immobilized inulinase was 184 mM, as compared to 82 mM for free inulinase, as a result of diffusion resistances.     

Purification and characterization of a novel thermostable phytase from the thermophilic Geobacillus sp. TF16

A novel phytase from thermophilic Geobacillus sp. TF16 was puri?ed approximately 5-fold using ammonium sulfate precipitation and ion exchange chromatography, and determined as a single band 106.04 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Optimum temperature and optimum pH were found to be 85°C and 4.0, respectively. The enzyme is highly thermostable and V_max and K_m values were calculated as 526.28 U/mg and 1.31 mM, respectively. It was also found that the enzyme exhibited a broad substrate selectivity and resistance toward proteases and effectively hydrolyzed soymilk phytate. These results suggest that this study provides an alternative phytase enzyme with enhanced properties.     

α‐Amylase immobilization on functionalized nano CaCO3 by covalent attachment

In this study, α‐amylase was immobilized on glutaraldehyde activated silanized calcium carbonate nanoparticles by a using covalent binding method. The surface modified nano calcium carbonate (CaCO₃) were characterized using FTIR and SEM. Immobilization yield was found as 199.43 mg/g of calcium carbonate nanoparticles. The maximum activity was observed at pH 6.5. The immobilized enzyme had a higher activity at elevated temperature (50–90°C) than the free one. Reuse studies demonstrated that the immobilized enzyme could reuse 25 times while retaining 18.2% of its activity. Free enzyme lost its activity completely within 15 days. V_max values for the free and immobilized enzymes were calculated as 10 and 0.35 mg/mL/min, respectively.     

Characterization of L-Arabinose Isomerase in Bacillus subtilis,a GRAS Host,for the Production of Edible Tagatose

L-Arabinose isomerase (AI; E.C. 5.3.1.4), a commercial enzyme for the production of edible tagatose in vitro and ribulose production in vivo, has been studied using enzymes expressed in an Escherichia coli system, which might cause noxious by-products in food. To ensure food safety in the tagatose manufacturing process, we studied an AI expression system in Bacillus subtilis. The AI gene from Geobacillus stearothermophilus (GSAI) was expressed in Bacillus subtilis, a GRAS host used in the production of fermented soybean in Korea, after subcloning into a Bacillus subtilis - E. coli shuttle vector, and was characterized after purification. The activities of the crude enzyme extract and a purified sample were 0.15 U/mg protein and 2.7 U/mg protein, respectively. The optimal pH and the optimal temperature for arabinose and galactose as substrates were pH 8.0 and 60°C, respectively, the same as those for GSAI in an E. coli expression system. Substrate affinities (K_m) for arabinose and galactose were 77 mM and 279 mM, respectively, whereas in the E. coli expression system, they were 100 mM and 578 mM, respectively. Catalytic efficiencies (k_cat/K_m) for arabinose and galactose were 58.3 and 11.4 mM^?1 min^?1, respectively. The potential use of GSAI expressed in a GRAS host for the production of edible tagatose is discussed in light of these results.     

Biochemical characterization of esterase from soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.)

Esterases are enzymes that present good potential in industrial application, and soybean seed can represent an alternative source for this enzyme. The extraction and esterase activity of Brazilian soybean seeds (Glycine max L.) were evaluated. Esterase activity was found in both the germinated and non-germinated seeds at 2.90 and 1.70 U/mg, respectively, with a concentration in the powdered extract (freeze dried) of 100 mg/mL. The enzyme showed a preference for the hydrolysis of short chain fatty acids (120.02 U/mL) and optimum pH for activity was pH 8.0 with optimum temperatures of 40 and 80°C. The enzyme showed stability at 70°C showing 60% of residual activity and activity increased with the addition of the following salts: NaNO₃, K₂SO₄, and Na₂SO₄ in the reaction medium.     

CHARACTERIZATION OF PHYTASE ACTIVITY IN LUPIN SEED

Lupin phytase (myo-inositol hexaphosphate phosphohydrolase) and the degradation of its substrate phytic acid were studied during seed germination. Phytase and acid phosphatase activities were detected in nongerminated seeds and increased from days 1 to 8 of germination, while the concentration of phytic acid decreased during the same period. Phytase was extracted with 2% CaCl₂ solution and partially purified by ammonium sulfate fractionation and HPLC-gel permeation chromatography. Substrate specificites were determined using pnitrophenylphosphate or phytic acid. The pH and temperature optima for the fraction obtained by gel permeation were 5.0 and 50C respectively, while the Km and V _max values were 0.33 mM and 0.13 μmol·mL^?1·min^?1, respectively. It was not possible to separate phytase from phosphatase by use of gel permeation chromatography due to similarities in apparent molecular mass, however resolution of the two enzymes was achieved by DEAE-cellulose chromatography.     

PURIFICATION AND CHARACTERIZATION OF CANOLA SEED (BRASSICA sp.) PHYTASE

Germinated Altex and Westar (Brassica napus) and Candle and Tobin (B. campestris) cultivars of Canola were screened for phytase activity. On the basis of this preliminary screening, 7-day germinated Altex seedlings were selected as a source for isolation and characterization of phytase. Partial purification of a crude extract (FI) by acetone precipitation resulted in an 8-fold increase in phytase activity. Ion-exchange chromatography of the partially purified preparation (FII) yielded two fractions (FIIIA and FIIIB) both of which demonstrated phytase and phosphatase activities. Further purification by gel filtration chromatography resulted in two fractions (FIVA1 and FIVA2) from fraction FIIIA and two fractions (FIVB1 and FIVB2) from fraction FIIIB. Fraction FIVB1 demonstrated both phytase and phosphatase activities, FIVA2 and FIVB2 demonstrated phosphatase activity but no phytase activity and FIVA1 showed phytase but no phosphatase activity. Fraction FIVB1, which showed highest phytase activity (5.3 IU/mg protein), had the following characteristics: temperature optimum of 50°C, pH optimum of 5.2, K_m of 0.36 mM and relative activity for pyrophosphate 232 times higher than for phytate.     

Investigation of a wild pear species (Pyrus elaeagnifolia subsp. Elaeagnifolia Pallas) from Antalya,Turkey: polyphenol oxidase properties and anti-xanthine oxidase,anti-urease,and antioxidant activity

Polyphenol oxidase properties and anti-xanthine oxidase, anti-urease, and antioxidant activity were investigated in Pyrus elaeagnifolia subsp. elaeagnifolia Pallas harvested from Antalya, Turkey. Optimum pH and temperature were 7.0 and 30°C, respectively. Selected kinetic properties of polyphenol oxidase was evaluated.The K_m and V_max-values, using 4-methylcatechol as substrate, were calculated as 3.57 mM and 4781 U/mg protein, respectively. IC₅₀-values ranged from 0.0036 to 4.0231 mM against sodium metabisulphite, ascorbic acid, sodium azide, and benzoic acid, while ascorbic acid was the strongest inhibitor. Aquatic extracts of the sample exhibited strong antioxidant capacity and substantial xanthine oxidase and urease inhibition, with IC₅₀-values of 10.75 ± 0.11 and 0.97 ± 0.03 mg/mL, respectively.     

Characterization of an extracellular β‐d‐fructofuranosidase produced by Aspergillus niveus during solid‐state fermentation (SSF) of cassava husk

β‐d ‐Fructofuranosidases are biotechnologically important enzymes produced by various organisms. Here, Aspergillus niveus produced an extracellular β‐d ‐fructofuranosidase during SSF of cassava husk. This enzyme was purified 8.5‐fold (recovery of 5.2%). A 37‐kDa protein band was observed after 8% SDS‐PAGE. Native molecular mass is 91.2 kDa. Optimal temperature and pH of activity were 55°C and 4.5, respectively. The enzyme was stable at 50°C for 1 hr, and 80% of its activity was retained after 1 hr at pH 8.0. The enzymatic activity was improved by Mn²⁺, was resistant to most solvents, and was inhibited by Triton X‐100 and Tween 20. K_m and V_max with sucrose were 22.98 mM and 120.48 U/mg of protein, respectively. With Mn²⁺, these values were 16.31 mM and 0.30 U/mg of protein. The enzyme did not hydrolyze inulin and for this reason can be considered a true invertase. Thus, A. niveus β‐d ‐fructofuranosidase holds promise for invert sugar production.

Practical applications

β‐d ‐Fructofuranosidase is an enzyme that can be applied to different industrial sectors, especially food and beverage industries. It is responsible for the hydrolysis of sucrose and yields an equimolar mixture of D‐glucose and D‐fructose, named as inverted sugar syrup, with broad applications in the confectionery industry. The Aspergillus niveus enzyme hydrolyzed only sucrose here and can be considered a true invertase, showing its potential for application to invert sugar production. Besides, the use of cassava husk for enzyme production means an interesting utilization route of this agroindustrial residue. Thus, characterization of this enzyme is an important step for identification of its potential for practical applications.     

Optimization of Covalent Immobilization of Extracellular Ice Nucleators from Erwinia herbicola on Magnetic Fe3O4/Chitosan Nanoparticles for Potential Application in Freeze Concentration

A magnetically separable extracellular ice nucleators (ECINs) immobilized system was constructed, ECINs from Erwinia herbicola were covalently immobilized on magnetic Fe₃O₄/chitosan nanoparticles by coupling via glutaraldehyde (GA) or epichlorohydrin (ECH). The immobilization process parameters were optimized, and under optimized conditions, ice nucleation activity (INA) of GA and ECH immobilized ECINs reached at 1.56?×?10⁶?±?1.54?×?10⁵ Units/mg and 1.62?×?10⁶?±?1.91?×?10⁵ Units/mg, respectively. The stability, reusability and cytotoxicity of GA and ECH immobilized ECINs were characterized and compared; both GA and ECH immobilized ECINs demonstrated good reusability performance after 15 freeze–thaw cycles and low cytotoxicity, while GA immobilized ECINs were recovered more easily and more stable at different pH compared to ECH immobilized ECINs. Application of the immobilized ECINs in freeze concentration was investigated, and an increase of ice nucleation temperature for water, seawater and apple juice (6.83?±?0.18 °C, 8.08?±?0.50 °C and 9.38?±?1.12 °C) was achieved by using GA immobilized ECINs. The results collected so far show that there are extensively promising applications for GA immobilized ECINs in freeze concentration industry fields.     

THE CHARACTERISTICS OF SOYBEAN PHYTASE

Soybean phytase was extracted with 2% CaCl₂ and partially purified by ammonium sulphate fractionation followed by dialysis in 0.01 M tris-maleate buffer, pH 6.5. The enzyme showed an optimum pH of 4.8 and optimum temperature of 60°C. The phytase was partially inhibited at high substrate concentration, with an optimum substrate concentration at 20 mM and a K_m value of 2.4 × 10^-3 M. V_max was 0.22 μmole P_i liberated/min/mL enzyme. The inactivation and activation energies for the hydrolysis of phytic acid were approximately 47,000 cal/mole and 11,100 cal/mole, respectively. Enzyme activity was inhibited by about 25%, 23% and 22% in the presence of 10^-3 M Zn⁺⁺, Cu⁺⁺ and Hg⁺⁺, respectively, and was also decreased by about 85% in the presence of 10^-1 M N-ethylmaleimide and sodium fluoride. Reducing and chelating agents at concentrations up to 10^-1 M inhibited activity by about 50% and by more than 90%, respectively.     

Expression,purification, and characterization of human intestinal maltase secreted from Pichia pastoris

A gene encoding human intestinal maltase (HMA) was successfully expressed in Pichia pastoris under the control of the methanol-induced alcohol oxidase (AOX1) promoter. The secreted recombinant HMA fused with a His₆-tag was produced (150 U/L) and was easily purified from culture supernatants in a 3-step diafiltration, ultrafiltration, and affinity column chromatography protocol. The specific activity of the purified HMA was 16.8 U/mg. Endoglycosidase H digestion of the protein showed that the recombinant HMA was N-glycosylated. The purified HMA was maximally active at pH 6.5 and stable (≥90%) up to 65°C. The kinetic parameters K _m and V _max were 3.3±0.25 mM maltose and 61.9±2 U/mg, respectively.     

Immobilization of β-galactosidase from Cicer arietinum (gram chicken bean) and its catalytic actions

β-Galactosidase (β-d-galactosidase galactohydrolase EC 3.2.1.23) isolated and purified from Cicer arietinum (gram chicken bean) was immobilized on two kinds of modified resin D202 with glutaraldehyde. Both the immobilized enzymes had high protein-binding capacity and high yield of enzyme activity. Kinetics results showed that the enzyme activity attained its maximum at 57°C, pH 6 for the immobilized β-galactosidase I and 52°C, pH 6 for the immobilized β-galactosidase II, respectively. The operational pH range was increased. Kinetic constants (K_m, V_max and E_a) for the free and bound enzymes, with ONPG as substrate, were studied. Results showed that K_m and V_max of immobilized enzymes were decreased while E_a of them was increased. The effects of some compounds and organic solvents for the free and immobilized enzymes were discussed. Inhibitory constants for raffinose, lactose and d-galactose, which were all reversible inhibitors of the enzymes, were also obtained.     

Effects of chitosan coating on oxidative stress in bruised Yali pears (Pyrus bretschneideri Rehd.)

The protective effects of chitosan on oxidative stress in bruised Yali pears (Pyrus bretschneideri Rehd.) were investigated. The fruit were treated with 1.5% chitosan before or after damage, respectively, and then stored at 16 °C and 90% relative humidity. Postharvest quality, reactive oxygen species (ROS) and antioxidant enzymes were analysed. Results showed that bruise induced the accumulation of H₂O₂ and O^2?. However, chitosan treatments reduced the levels of ROS and delayed the decease of glutathione (GSH) content in bruised fruit. Meanwhile, activities of antioxidant enzymes, including catalase, peroxidases, superoxide dismutase, ascorbate peroxidase or GSH reductase was 40.7%, 98.1%, 62.3%, 127.8% or 23.8% higher in chitosan‐bruised fruit and 41.1%, 80.8%, 18.5%, 102.9% or 45.2%, respectively, higher in bruised‐chitosan fruit than untreated bruised fruit on the 15th day.     

Characterization of a β-Glucosidase from an Edible Mushroom,Lycoperdon Pyriforme

A β-glucosidase from Lycoperdon pyriforme, a wild edible mushroom, was characterized biochemically. The enzyme showed a maximum activity at pH 4.0 and 50°C when p-nitrophenyl-β-D-glucoside was used as a substrate. K_m and V_max values were calculated as 0.81 mM and 1.62 U/mg protein, respectively. The enzyme activity was conserved about 85% over a broad range of pH (3.0–9.0) at 4°C after 24 h incubation. The activity was fully retained after 60 min incubation at 20–40°C. Na⁺, Li⁺, Mg²⁺, Mn²⁺, Zn²⁺, Co²⁺, Ca²⁺, and Cu²⁺ did not affect the enzyme activity and 0.25% sodium dodecylsulfate inhibited the enzyme activity approximately 76%. Ethylenediamine tetra-acetic acid, phenylmethanesulfonylfluoride, and dithiothreitol showed no or a little negative effect on the enzyme activity. The resistance of the enzyme to some metal ions, chemicals, and ethanol along with the pH stability, can make it attractive for future applications in industry.     

Production and Gelatin Entrapment of Laccase from Trametes versicolor and its Application to Quantitative Determination of Phenolic Contents of Commercial Fruit Juices

Laccase (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) is a particularly promising enzyme for several industrial fields, including food industries, since this enzyme catalyzes the oxidation of ortho and para-diphenols, amino-phenols, polyphenols, polyamines, lignins, and aryl diamines as well as some inorganic ions coupled to the reduction of molecular dioxygen to water. In this study, laccase was produced from one of the best laccase-producing organisms, Trametes versicolor. For this purpose, several phenolic acids were tested as laccase inducers. Caffeic acid and ferulic acid were determined to be the best inducers among the tested phenolic acids. Also, it was shown that laccase activity could be determined by using caffeic acid and ferulic acid as phenolic substrates by measuring the rates of oxygen consumption. Laccase was immobilized in gelatin under optimized conditions. Kinetic constants K _m and V _max for immobilized enzyme were estimated to be 74.758 μM and 0.744 μmol.ΔO₂/ml.min for caffeic acid and 0.999 μM and 57.80μ mol.ΔO₂/ml.min for ferulic acid, respectively. The immobilized enzyme exhibited the maximal activity at pH 4.5, and at 35°C. Immobilized enzymes were used for the determination phenolic contents of commercially prepared fruit juices. Caffeic acid contents of black cherry, apricot, and peach juice were determined to be 1640±33, 679±24 and 408±29 mg/L, and their ferulic acid contents were determined to be 1786±28, 800±30, and 444±28 mg/L, respectively.     

Characterization of glucoamylase immobilized on magnetic nanoparticles

Magnetic support was prepared by precipitation from an alkaline solution of divalent and trivalent iron ions and subsequently was modified with 3‐aminopropyltriethoxysilane. FTIR analysis showed existence of a new Si–O–Fe bond in obtained particles. Scanning electronic microscopy images shows that the nanoparticles of all samples have particle size below 30 nm. Glucoamylase AMG 300L was immobilized onto the modified magnetic support using glutaraldehyde as a coupling agent. Obtained preparations had specific activity of 148 U/g of the support when measured at 55°C using maltose as substrate. The immobilized enzyme exhibited mass transfer limitation as reflected by a higher apparent K_m value and a lower energy of activation. The immobilization was almost completely terminated after 30 min of the reaction at 30°C. The highest immobilization yield of the enzyme was achieved at glutaraldehyde concentration of 10 mM. The immobilization did not influence considerably on optimum pH and temperature of substrate hydrolysis catalyzed by investigated enzyme (55°C, pH 4.5). Moreover, immobilized glucoamylase was easily separated from the reaction medium by an external magnetic field and retained about 60% of initial activity after nine repeated cycles of enzyme reaction followed by magnetic separation.     

Polyphenol oxidase properties,anti-urease,and anti-acetylcholinesterase activity of Diospyros lotus L. (Plum Persimmon)

Diospyros lotus fruit polyphenol oxidase was purified using affinity chromatography, resulting in a 15-fold enrichment in specific activity. The purified enzyme, having 16.5 kDa molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibited the highest activity toward 4-methylcatechol. Maximum diphenolase activity was reached at pH 7.0 and 60°C in the presence of 4-methylcatechol. K_m and V_max values were calculated as 3.8 mM and 1250 U/mg protein, respectively. Ascorbic acid was a promising inhibitor with an IC₅₀ value of 0.121 µM. The activity of the purified enzyme was stimulated by Fe²⁺, Sr²⁺, Zn²⁺, and K⁺ and deeply inhibited by Hg²⁺, at 1 mM final concentration. Aqueous extract of Diospyros lotus L. fruit showed strong substantial urease and acetylcholinesterase inhibition, with IC₅₀ values of 1.55 ± 0.05 and 16.75 ± 0.11 mg/mL, respectively.     

Zinc and Phytate Distribution in Peas. Influence of Heat Treatment, Germination, pH, Substrate, and Phosphorus on Pea Phytate and Phytase

The largest proportions of zinc and phytate, 88.7 and 97.1%, respectively, were in the Garfield pea cotyledon; the greatest concentrations were in the germ. Cooking peas by two different methods resulted in 13% phytate reduction. Peas incubated 6.5 hr from 25 to 80°C yielded maximum phytate loss (25%) at 60°C due to phytase activated hydrolysis. Germination (10 d) decreased pea phytate 75% and increased phytase activity 12-fold. Semi-purified germinated pea phytase showed temperature optimum at 45°C, pH optimum of 5.2, 30% inhibition by 1 mM inorganic P, and substrate preference for pyrophosphate. Incubation of early germinated peas at optima pH and temperature is suggested for maximum phytate reduction.