Dephosphorylation of phytate compounds by means of acid phosphatase from Aspergillus niger

A non-purified preparation of intracellular acid phosphatase (EC 3.1.3.2) from a waste mycelium of Aspergillus niger was utilised for dephosphorylation of phytate compounds present in food and feed ingredients. The enzymic hydrolysis of p-nitrophenylphosphate was used for assaying acid phosphatase activity, expressed in standard units (u). The hydrolysis of phytate phosphorus in wheat bran, soya bean meal and fully formulated feedstuffs for broilers (Galus galus; ‘Cornish’ × White Rock') was carried out at 40°C, a pH value of 4.5 and an enzyme dosage ranging from 12 to 30 u g^?1. Complete dephosphorylation of soya bean protein isolates was performed at 60°C, a pH value of 4.5 and an enzyme dosage of 12 u g^?1. In the gastrointestinal tract of broilers the in-vivo dephosphorylation of phytates present in feed was observed when the preparation of acid phosphatase was added to the diet.     

Purification and Properties of a Thermostable Inulinase (β-D-Fructan Fructohydrolase) from Bacillus stearothermophilus KP1289

A thermophilic soil isolate, Bacillus stearothermophilus KP1289, that grew from 41 °C to 69 °C, produced extracellular inulinases in the presence of inulin. One (inulinase II) of these enzymes was purified to homogeneity. The molecular weight (M_r) and the isoelectric point of the enzyme were estimated as 54,000 and 5.0, respectively. The enzyme was active between 30 and 75 °C and at pH 4.5—8.6 with an optimum at 60 °C and pH 6.1. At 69 °C and pH 7.0 the half-life of the enzyme was 10 min. The enzyme released fructose exo-wise from the non-reducing end of inulin (M_r = 4,5000). The Michaelis constant, catalytic center activity, and specificity constant for inulin at 60 °C and pH 5.0 were 80 mM (360 mg/mL), 460 s^—1, and 5.8 s^—1 mM^—1, respectively. The ratio of specificity constants for inulin, sucrose, and raffinose was 1:0.50:0.16. The enzyme was classified as a thermophilic thermostable β-D -fructan fructohydrolase (EC 3.2.1.80).     

Phytate dephosphorylation by Lactobacillus pentosus CFR3

Studies on phytate‐degrading enzymes from lactobacilli are scarce, despite its potential in improving the nutritional quality of plant‐based foods. Therefore, the current investigation deals with the phytate‐degrading enzyme produced by a native Lactobacillus pentosus strain. Phytase activity was highest towards the end of the exponential phase. Activity increased in the presence of maltose (381.1%) compared with glucose. The presence of phytate in the media stimulated the enzyme production. The enzyme of interest was a 70 kDa protein with a pH and temperature optima of 5.0 and 55–60 °C, respectively. It retained 46% of activity after exposure to 70 °C for 20 min and also showed broad substrate specificity. It was completely inhibited by Hg²⁺, Fe²⁺ and PMSF while being activated by Co²⁺. This report is the first to show dephytinisation of autoclaved finger millet flour either by fermentation with L. pentosus or by treatment with the corresponding cell‐free extract.     

Properties of Trypsin from the Pyloric Ceca of Atlantic Cod (Gadus morhua)

Trypsin (EC 3.4.21.4) was isolated from the pyloric ceca of Atlantic cod and purified to homogeneity by affinity chromatography. The enzyme catalyzed the hydrolysis of benzoyl arginine p-nitroanilide (BAPA, pH 8.2 and 25°C) such that V_max was 250 BAPA units per micromole trypsin and K_m was 1.48 mM. For the hydrolysis of tosyl arginine methyl ester (TAME, pH 8.1 and 25°C), V_max was 18.2 × 10³ TAME units/micromole trypsin, and K_m 0.22 mM. The pH and temperature optima with BAPA substrate were 7.5 and 40°C, respectively. Atlantic cod trypsin was most active and stable at alkaline pH. The enzyme was heat labile, losing more than 50% of its activity after incubation at 50°C for 30 min. Amino acid analysis of Atlantic cod trypsin revealed that the enzyme was rich in residues such as serine, glycine, glutamate and aspartate, but poor in basic amino acid residues compared to trypsins from warm blooded animals.     

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).     

PURIFICATION AND PROPERTIES OF A PHYTASE FROM RYE

Phytase (myo-inositol hexakisphosphate phosphohydrolase) has been purified about 2,000-fold from ungerminated rye with a recovery of 6%. The enzyme behaves as a monomeric protein of a molecular mass of about 67 kDa. OptimalpH for the degradation of phytate has been found at pH 6.0 and 45C. Kinetic parameters for the hydrolysis of Na-phytate are KM300 μM and kcat 358 s^?1 at 35C and pH 6.0. The rye enzyme exhibits a broad affinity for various phosphorylated compounds and hydrolyses phytate in a stepwise manner; the pentakis- and tetrakisphosphate were identified as 1(1,2,3,4,5)P₅ and I(2,3,4,5)P4 Consequently, this enzyme is a 6-phytase (EC 3.1.3.26).     

Phytate Reduction in Brown Beans (Phaseolus vulgaris L.)

Brown beans (Phaseolus vulgaris L.) were subjected to treatments to evaluate effects of pH, temperature, CaCl₂, tannase and fermentation on degradation of phytate. Soaking was performed at 21°C, 37°C and 55°C at pH 4.0, 6.0, 6.4, 7.0, and 8.0. Optimal conditions for phytate degradation were pH 7.0 and 55°C. After soaking 4, 8 or 17 hr at these conditions 79%, 87% and 98% of phytate was degraded, respectively. Addition of tannase enhanced reduction of phytate. Fermentation of presoaked whole beans resulted in reduction of 88% of phytate after 48 hr.     

Dephytinization of food stuffs by phytase of Geobacillus sp. TF16 immobilized in chitosan and calcium-alginate

In this work, Geobacillus sp. TF16 phytase was separately immobilized in chitosan and Ca-alginate with the efficiency of 38% and 42%, respectively. These enzymes exhibited broad substrate specificity. Maximal relative phytase activity was measured at pH 5.0 and 95°C and pH 3.0 and 75°C for chitosan and Ca-alginate, respectively. The enzymes were highly stable in a wide pH and temperature range. Values of K_m and V_max were determined as 2.38 mM and 3401.36 U/mg protein for chitosan, and 7.5 mM and 5011.12 U/mg protein for Ca-alginate. The immobilized enzymes showed higher resistance to proteolysis. After 4 h incubation, hydrolysis capacities of chitosan- and Ca-alginate immobilized enzymes for soymilk phytate were calculated as 24% and 33%, respectively. The chitosan- and Ca-alginate immobilized phytases conserved its original activity after 8 and 6 cycles of reuse, respectively. The features of the enzymes were very attractive and they might be useful for some industrial applications.     

Gene cloning and characterization of a trehalose synthase from Corynebacterium glutamicum ATCC13032

Trehalose synthase (TreS) is an enzyme which produces trehalose from maltose through intramolecular transglycosylation. In this study, a gene (cg2529) encoding for TreS from Corynebacterium glutamicum (CgTS) was cloned and expressed in Escherichia coli. The hexahistidinetagged CgTS showed an optimum temperature and pH of 35°C and pH 7.0, respectively. This enzyme was not thermostable, but stable in a broad pH range from pH 5.0 to 8.5. Its activity slightly increased by 5 mM Mg²⁺ and Fe²⁺, while it was strongly inhibited by 5 mM sodium dodecyl sulfate (SDS). CgTS catalyzed the conversion from maltose into trehalose, and vice versa. Lowering reaction temperature by 5°C from the optimum temperature significantly reduced hydrolysis activity to produce glucose as a by-product compared to transglycosylation activity to produce trehalose, leading to increase in the conversion yields from maltose into trehalose. Consequently, the maximum conversion yield by CgTS reached 69% at 25°C after 9 hr of reaction.     

Production and Application of a Maltogenic Amylase by a Strain of Thermomonospora viridis TF-35

An extracellular and thermostable maltogenic amylase-producing moderate thermophile (Thermomonospora viridis TF-35), which grew well at 28–60°C, with optima at 45°C and pH 7, was isolated from soil. Maximal enzyme production was attained after aerobical cultivation for 32 h at 42°C with a medium (pH 7.3) composed of 2% (w/v) soluble starch, 2% gelatin hydrolyzate, 0.1% K₂HPO₄ and 0.02% MgSO₄ · 7H₂O. The partially purified enzyme, which was most active at 60°C and pH 6.0 and stabilized with Ca²⁺, converted about 65, 80, 75, 75, 65 and 60% of maltotriose, maltotetraose, maltopentaose, amylose, amylopectin and glycogen into maltose as a major product under the conditions used, respectively. Glucose and small amounts of maltooligosaccharides were also formed concomitantly as by-products. The molar ratio of maltose to glucose from maltotriose were larger than 1 during all stages of the hydrolysis. About 70 and 76% of 25% (w/v) potato starch liquefites having a 3.5 DE value were converted into maltose by the enzyme in the absence and presence of pullulanase during the saccharification, respectively. About 90 and 94% of the starch liquefites were also converted into maltose with relatively low contents of maltooligosaccharides by the cooperative 2 step reaction with the enzyme after obtaining starch hydrolyzates containing about 85 and 90% maltose by the simultaneous actions of soybean ß-amylase and debranching enzymes.     

Partial Purification and Characterization of Extracellular Fructofuranosidase with Transfructosylating Activity from <Emphasis Type="Italic">Candida</Emphasis> sp.

The present work was carried out with the aim to investigate some properties of an extracellular fructofuranosidase enzyme, with high transfructosylating activity, from Candida sp. LEB-I3 (Laboratory of Bioprocess Engineering, Unicamp, Brazil). The enzyme was produced through fermentation, and after cell separation from the fermented medium, the enzyme was concentrated by ethanol precipitation and than purified by anion exchange chromatography. The enzyme exhibited both fructofuranosidase (FA) and fructosyltransferase (FTA) activities on a low and high sucrose concentration. With sucrose as the substrate, the data fitted the Michaellis–Menten model for FA, showing rather a substrate inhibitory shape for fructosyltransferase activity. The K _m and v _max values were shown to be 13.4 g L⁻¹ and 21.0 μmol mL⁻¹ min⁻¹ and 25.5 g L⁻¹ and 52.5 μmol mL⁻¹ min⁻¹ for FA and FTA activities, respectively. FTA presented an inhibitory factor K _i of 729.8 g L⁻¹. The optimum conditions for FA activity were found to be pH 3.25–3.5 and temperatures around 69 °C, while for FTA, the optimum condition were 65 °C (±2 °C) and pH 4.00 (±0.25). Both activities were very stable at temperatures below 60 °C, while for FA, the best stability occurred at pH 5.0 and for FTA at pH  4.5–5.0. Despite the strong fructofuranosidase activity, causing hydrolysis of the fructooligosaccharides (FOS), the high transfructosilating activity allows a high FOS production from sucrose (44%).     

Purification and Characterization of a Pullulan-Hydrolyzing Glucoamylase from Sclerotium rolfsii

The pullulan-hydrolyzing enzyme from the culture filtrates of Sclerotium rolfsii grown on soluble starch as a carbon source has been purified by ultrafiltration (Amicon, PM-10), ion-exchange chromatography (DEAE-Cellulose DE-52) and gel filtration chromatography (Bio-Gel P-150). The enzyme moved as a single band in non-denaturing polyacrylamide gel electrophoresis carried out at pH 2.9 and 7.5. The relative molecular mass of the enzyme was estimated to be 64.000 D by SDS-PAGE and 66.070 D by gel filtration on Bio-Gel P150. The enzyme hydrolyzed pullulan optimally at 50°C between pH 4.0–4.5, whereas, soluble starch was optimally hydrolyzed at a pH of between 4.0–4.5 and at 65°C. The Michaelis constant (Km) for pullulan was 5.13mg·ml⁻¹ (V_max 1.0U · mg⁻¹) and for soluble starch, it was 0.6mg · ml⁻¹ (V_max 8.33 U · mg⁻¹). The enzyme was observed to be a glycoprotein (12–13% carbohydrate by weight) and had a strong affinity for Concanavalin A. The enzyme hydrolyzed α-D-glucans in an exo-manner, which resulted in the release of glucose as the sole product of hydrolysis. Acarbose, a maltotetraose analog, was found to be a potent inhibitor of both pullulan and starch hydrolysis (100% inhibition at 0.06 μM). The enzyme has been characterized as a glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) showing a significant action on pullulan.     

Optimization of Enzymatic Hydrolysis of Pearlmillet for Glucose Production

Commercial α‐amylase preparation (Biotempase) and crude glucoamylase from Aspergillus sp. NA21 were used to hydrolyse pearlmillet, a non‐conventional starchy substrate. Various concentrations of starch (15—35% w/v) were used for liquefaction; 25% slurry was found to be judicious and optimal. Liquefaction in steam under pressure 2.06—2.75 N/cm², 104—105 °C) was found to be more economical than in a water bath at 95 °C. In the first case a 25% (w/v) slurry was liquefied in 60 min. A pH of 5.0 was found to be optimum for liquefaction. Biotempase dose was cut down by 33% to the prescribed one by addition of 150 ppm CaCl₂ to the slurry. Ninety percent saccharification of liquefied pearlmillet occurred under optimum conditions (24 h and pH 5.0). The optimum temperature for saccharification of pearlmillet was found to be 45 °C. Additions of Ca²⁺, Mg²⁺ and Zn²⁺ were found to have no effect on saccharification. Glucose was found to be the main hydrolysis product as indicated by paper chromatography.     

Purification and Characterization of β-Glucosidase from Aspergillus niger

Aspergillus niger, an isolate of soil contaminated with effluents from cotton ginning mill was grown in Czapek-Dox medium containing sawdust, Triton-X 100 and urea for production of an extracellular β-glucosidase. β-Glucosidase enzyme was purified (86-fold) from culture filtrate of A. niger by employing ammonium sulphate precipitation and gel filtration on sephadex G-75. The molecular mass of the purified enzyme was estimated to be 95 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The enzyme had an optimal activity on p-nitrophenyl β-D-glucopyranoside at 50°C and pH 5.0. The K_m and V_max of the enzyme on p-nitrophenyl β-D-glucopyranoside at 50°C and pH 5 were 8.0 mM and 166 µmol/min/mg of protein, respectively. The enzyme could hydrolyze cellobiose and lactose but not sucrose. Heavy metals like Hg²⁺, Al³⁺, and Ag⁺ inhibited the activity, whereas Zn²⁺ and detergents such as Triton-X 100 and Tween-80 increased the activity at 0.01%. The enzyme activity increased in the presence of methanol and ethanol.     

ALPHA-GALACTOSIDASE OF GERMINATING SEEDS OF CASSIA SERICEA SW.

Germinating seeds of Cassia sericea Sw. contain two molecular forms of α-galactosidase which were partially purified and characterized. Both enzyme forms had an optimum pH of 5.0 and an optimum temperature of 50 °C. K_m values for the substrate p-nitrophenyl-α-D-galactoside (PNPG) were 0.91 mM and 1.05 mM for the two forms, and substrate inhibition was observed at high concentrations of PNPG. The two forms of the enzyme had molecular weights of 46,000 and 33,000 by gel filtration. The enzyme forms were inhibited completely by Ag⁺, Cu²⁺ and Hg²⁺ ions and by p-chloromercuribenzoate showing that thiol groups are probably involved in catalysis. Both α-galactosidases hydrolyzed melibiose and raffinose, although hydrolysis of stachyose could not be detected. The enzyme may find potential use in the food industry to hydrolyze flatulence-causing sugars in processed foods and in production of sucrose from high-raffinose sugar beets. That the source (C. sericea seeds) of the enzyme is inexpensive provides an added advantage over use of cultivated legumes as a source of α-galactosidase.     

PURIFICATION AND CHARACTERIZATION OF HOMOGENEOUS ACID PHOSPHATASE FROM NONGERMINATED BUCKWHEAT (FAGOPYRUM ESCULENTUM) SEEDS

A buckwheat acid phosphatase (orthophosphoric‐monoester phosphohydrolase, EC 3.1.3.2) was purified about 250‐fold from nongerminated buckwheat seeds to apparent homogeneity with a recovery of 4% from the acid phosphatase activity in the crude extract. It is the major acid phosphatase among eight different acid phosphatases identified in the crude extract. The purified enzyme behaved as a monomeric protein of molecular mass about 45 kDa. The purified enzyme exhibited a single pH optimum at 5.25. Optimum temperature for the degradation of p‐nitrophenyl phosphate was 50C. The kinetic parameters for the hydrolysis of p‐nitrophenyl phosphate were determined to be K_M= 76 μmol L^?1 and k_cat= 924 s^?1 at pH 5.25 and 37C. While the enzyme failed to act on phytate as a substrate, the enzyme exhibited a broad substrate selectivity. The purified enzyme showed no measureable carboxylesterase activity and no divalent metal ion requirement.     

Some properties of polyphenol oxidase from lily

A study of crude polyphenol oxidase (PPO) from lily bulbs was carried out to provide information useful for guiding food processing operations. Optimum pH for the enzyme activity in the presence of catechol, were 4.0 and 7.0 at room temperature(approximately 20 °C) and the enzyme was stable in the pH range from 5.0 to 6.5 at 4 °C for 10 h. Its optimum temperature was 40 °C and the heat inactivation of the enzyme followed first‐order kinetics. Lily PPO possessed a diphenolase activity toward catechol, catechin and gallic acid; catechin was the best substrate for the enzyme considering the V_max/K_m ratio. The most effective enzyme inhibitor was sodium sulphite, although ascorbic acid, l ‐cysteine and thiourea were also effective inhibitors at high concentration. But NaCl and citric acid were poor inhibitors of the enzyme. Data generated by this study might help to better prevent lily bulbs browning.     

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.     

Heat inactivation and kinetics of polyphenoloxidase from palmito (Euterpe edulis)

Polyphenoloxidase (PPO, EC 1.14.18.1)was extracted from palmito (Euterpe edulis Mart) using 0.1 M phosphate buffer, pH 7.5. Partial purification of the enzyme was achieved by a combination of (NH₄)₂SO₄precipitation (35–90% saturation) and Sephadex G-25 and DEAE-cellulose chromatography. The purified preparation gave five protein bands on polyacrylamide gel electrophoresis, three of them with PPO activity. The K_mvalues for chlorogenic acid, caffeic acid, catechol, 4-methylcatechol and catechin were 0.57, 0.59, 1.1, 2.0 and 6.25 mM , respectively. PPO has a molecular weight of 51 000 Da, maximum activity at pH 5.6 with chlorogenic acid as substrate, and was stable between pH 5.0 and 8.0. The enzyme was heat stable at 50–60°C and inactivated at 75°C. The heat stability of palmito PPO was found to be pH dependent; at 50°C and pH 4.0 the enzyme was fully inactivated after 30 min. The pH/activity studies showed two groups with pK values c 4.6 and 6.7 involved in PPO catalysis.