MITOCHONDRIAL—ASSOCIATED CDP—DIACYLGLYCEROL SYNTHASE ACTIVITY IN GERMINATING SOYBEANS

The mitochondrial-associated CTP: phosphatidic acid cytidylyltransferase (CDP-diacylglycerol synthase, EC 2.7.7.41) was studied from germinating soybeans. The enzyme exhibited a broad pH optimum between pH 6.5 and pH 8.0. Activity was dependent on the addition of magnesium ions (20 mM) and the nonionic detergent Triton X-100 (5 mM). The apparent K_m values for CTP and phosphatidic acid were 0.58 mM and 0.12 mM, respectively. A V_max of 0.12 U/mg was found for the mitochondrial-associated enzyme. Phospholipids generally stimulated activity while ADP and CDP strongly inhibited activity. Thiore-active agents inhibited activity indicating that a sulphydryl group is essential for activity. The enzyme was thermally inactivated at temperatures above 35°C.     

Comparison of kinetic profile of free and immobilized glucose oxidase, immobilized on low-density polyethylene using UV polymerization

ABSTRACT:  Bioactive packaging is an important area of active packaging in which an active component is incorporated into the food contact surface of the package to interact with the food components without itself migrating into the food. Embedding bioactivity in a UV polymerizable resin is a novel and versatile technique for incorporating bioactive components into food packaging. In a previous article, glucose oxidase was immobilized in a packaging material using a UV curable resin. The relevance of this model system for deoxygenation of fruit juices was discussed. Though the technique efficiently immobilized enzymes in packaging material, during polymerization and immobilization the catalytic ability of the enzyme was not specifically explored. This article compares and contrasts the catalytic ability in terms of the kinetic profile of free and immobilized enzyme for the same model system: deoxygenation of juices. Kinetic behavior of immobilized and free glucose oxidase enzyme was evaluated at both shelf stable (room temperature) and refrigerated storage conditions to simulate the actual package life. It was observed that both the free enzyme and the immobilized enzymes follow the Michaelis–Menten kinetics model. There was no significant difference between the catalytic ability ( k_cat / K_m ) of free and immobilized enzymes at treatment temperatures (30, 25, and 10 °C); however, at refrigeration temperature (5 °C), the values for free enzyme were significantly higher than the immobilized enzyme.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF α-GALACTOSIDASE FROM ASPERGILLUS ORYZAE

A crude extract of α-galactosidase obtained by fermenting Aspergillus oryzae on wheat bran was purified 35 fold by ethanol precipitation, gel filtration and ion-exchange chromatography. The final preparation was free of protease activity but contained invertase activity. The molecular weight of the enzyme was estimated as 64,000 daltons. The pH and temperature optima were 4.0 and 60°C, respectively. The enzyme was stable over the pH range 3–7.5 and at temperatures up to 55°C (pH 4.0). The K_m values for p-nitrophenyl α-Dgalactopyranoside (PNPG) and raffinose were 4.0 × 10⁻⁴M and 1 × 10⁻²M, respectively. Divalent cations were not required for activity. More than 80% of the oligosaccharides in soy milk were hydrolyzed after 3 h at 50°C using 0.113 PNPG units/mL milk.     

PURIFICATION AND CHARACTERIZATION OF POLYPHENOL OXIDASE FROM POTATO: I. PURIFICATION AND PROPERTIES

Polyphenol oxidase (Isozyme I) from potato was extracted and purified with ammonium sulfate, cation-exchange (Bio-Rad Bio-Scale S2) and Sephadex G-100 column chromatography. The enzyme was purified 11.8 fold resulting in a specific activity of 250.3 units/mg. Optimum pH of the enzyme was 6.6. Optimum temperature of the enzyme was 40C and its half-life was 0.8 min at 70C. The K_mfor catechol, pyrogallol, 4-methyl catechol, caffeic acid and L-DOPA were 4.11 mM, 0.61 mM, 0.78 mM, 0.50 mM and 32 mM, respectively. However, monophenols such as tyrosine, p-cresol and 1-naphtol did not show any activity. Data for V_max/K_m which represents catalytic efficiency show that 4-methyl catechol has the highest value. The molecular weight of the active enzyme was 86,000 Da, composed of two identical subunits. The number of Cu²⁺ ions bound was found to be 2 per enzyme molecule.     

PURIFICATION AND CHARACTERIZATION OF TWO TOMATO POLYGALACTURONASEISOENZYMES

The properties of tomato polygalacturonases at two ripening stages were investigated. Two isoenzymes, PG I and II, were isolated from underripe fruits with an orange skin color. Fully ripe fruits contained only polygalacturonase II. PG I and II were purified by chromatography on DEAE-Sephadex A-50, Sephacryl S-200 and CM-agarose chromatography. PG I had a M_r of 199,500 as determined by Sephacryl S-300 gel filtration and was 50% inactivated at 66.5°C and pH 4.5 after incubation for 5 min. It had an activation energy (E_a) of 16.8 Kcallmol (70.3 times 10³ Jlmol), V_max of 27.7 units/mg protein and K_m value of 7.5 times 10⁻² mM polygalacturonic acid. PG II had a M_r of 45,700 and was 50% inactivated at 58°C under the same conditions. Both isozymes had a pH optimum of 4.6. PG II had an E_a value of 14.8 Kcallmol (61.9 times 10³ Jlmol), V_max value of 58.8 units/mg protein and K_m value of 3.8 times 10⁻² mM polygalacturonic acid. PGI gave rise to only one band during electrophoresis in polyacrylamide gels, whereas PG II showed one major and one minor band both with PG activity. Gel electrophoresis in the presence of sodium dodecyl sulfate resulted in two major bands (M_r= 47,500 and 41,000) for PG I and only one major band (M_r= 47,500) for PG II. PG I is composed of several subunits, all of which are glycoproteins.     

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).     

THE β-N-ACETYLGLUCOSAMINIDASE ACTIVITY OF EGG WHITE. 1. Kinetics of the Reaction and Determination of the Factors Affecting the Stability of the Enzyme in Egg White

SUMMARY– Enzymatic activity of β-N-acetylglucosaminidase, which occurs naturally in chicken egg white, was characterized to establish conditions suitable for routine assay for this enzyme in egg products. A variation in enzyme content of approximately 3-fold was observed in individual fresh eggs. The enzyme has a pH optimum between 3.0 and 3.4, and a K_m of approximately 0.6 mM for the substrate p-nitrophenyl-N-acetyl-β-D-glucosaminide. Activation energy for hydrolysis of this substrate is 10.7±0.8 kcal/mole. The enzyme is stable for at least several hours at ambient temperature from pH 6.8 to approximately 8.8. Above pH 8.8, inactivation is first order with respect to time. Enzyme activity in shell eggs decreases fairly rapidly at ambient temperature; loss of activity probably results from increase in pH of egg white, which occurs normally upon loss of carbon dioxide. Eggs held at 4°C retain activity much longer.     

CHARACTERIZATION OF PHYTASE OF BEANS (PHASEOLUS VULGARIS)

Phytase from California Small White beans was extracted, concentrated and enriched by heat treatment and ammonium sulfate fractionation. Crude enzyme fractions tenaciously retained endogenous phytate which could be removed by autolysis at 45°C and pH 5.2. Characterization of the phytase preparation showed: (1) increase in activity with increasing temperature from 37 to 60°C, (2) an activation energy of approximately 9,200 cal/mole for the hydrolysis of inositol hexaphosphate; (3) pH optimum of 5.2; (4) a K_m value of 2.22 × 10^-4M; (5) apparently partial inhibition at high substrate concentrations; (6) fully competitive inhibition by one of the reaction products, inorganic phosphate, with K_i=3.1 × 10⁴M. These properties and kinetic constants are comparable to those reported for phytase preparations from similar sources and they adequately account for in situ autolytic loss of phytic acid from these beans.     

DEVELOPMENT AND DISTRIBUTION OF ACTINIDIN IN KIWIFRUIT (ACTINIDIA CHINENSIS) AND ITS PARTIAL CHARACTERIZATION

The actinidin activity in kiwifruit increased 3-fold after 3 weeks postharvest ripening at 5°C with a 15% decrease in specific activity. Actinidin activity is highest in the pulp of ripe fruit at 27,600 U/kg fruit. The thiol protease actinidin from kiwifruit was extracted and purified 26-fold to a specific activity of 57 U/mg with K_m= 91 μM and k_cat= 101 s⁻¹ towards N-α-CBZ-lysine p-nitrophenyl ester at pH 6.0 and 25°C.     

Encapsulation and stimulated release of enzymes using lecithin vesicles

Lecithin vesicles prepared by dehydration-rehydration (DR) were used to encapsulate enzymes (lysozyme and pepsin). The encapsulating efficiency was highest when the pH was close to the isoelectric point of each enzyme. Vesicles stored in suspension at 10°C for up to 21 days showed no release of enzymes. Acidic pH at 10°C and 25 mM Ca²⁺ at 10°C or 37°C produced pulse-like release of 17–35%, while acidic pH at 37°C produced pulse-like release followed by slow release up to 100%, and Tween 80 induced steady release from the beginning. The hydrolysis pattern of a protein by pepsin released from DR vesicles for 142 hr was similar to that obtained by the same total amount of fresh pepsin solution added stepwise, in proportion, indicating that the pepsin retains its activity throughout the period of encapsulation.
Vesicles prepared by processing of lecithin-enzyme solution by a homogenizer (Microfluidizer^TM) were also characterized and found effective.     

Pacific Cod Muscle 5'-Nucleotidase

SUMMARY: A 5'-nucleotidase, widely distributed in teleost fish muscles, was purified about 20-fold from Pacific cod (Gadus macrocephalus) by chromatography of a dialyzed aqueous extract of the muscle on DEAE-cellulose. The enzyme was unstable and lost 85% of its activity in 1 hr at 37°C 53% in 10 min at 42°C and 40% in 1 hr at 30°C. It was stable for 6 days at 0°C, could be dialyzed for up to 3 days at 0°C against 1 mM tris buffer pH 7.5 and quickly frozen and thawed without loss of activity. However, it was inactivated rapidly when held at −30°C. Brief exposure to pH 4.0 or 5.0 effected marked destruction. Attempts at further purification by means of chromatography on hydroxylapatite, adsorption using alumina Cγ and starch gel electrophoresis failed due to instability.
The enzyme was strongly inhibited by EDTA, pyrophosphate, KF and ZnCl₂ (1-10 mM); less markedly inhibited by GSH, 2-mercaptoethanol, carbonate and CaCl₂ (10 to 100 mM). It was strongly activated by Mn⁺⁺ and weakly activated by Mg⁺⁺. The optimum pH was 7.6, and the Km was 5 × 10⁻⁴M with UMP and 8 ⁻⁴M with IMP. It hydrolyzed, in order of effectiveness, LJMP, IMP, CMP, d-AMP, GMP, d-IMP, d-GMP, d-UMP and AMP, but not p-nitro phenylphosphate, sugar phosphates or a number of other compounds including 2',3'-nucleotides.     

交联海藻酸钠固定化柚(皮)苷酶

以海藻酸钠为载体,戊二醛为交联剂,采用交联-包埋-交联法对柚（皮）苷酶进行了固定化。在单因素实验基础上,通过正交实验得到海藻酸钠固定化柚（皮）苷酶的最优工艺条件:海藻酸钠质量浓度3.0%,给酶量为0.01mg/g载体,前交联戊二醛体积分数2.0%,前交联时间1.5h,后交联戊二醛体积分数0.025%,后交联时间2h,制备的固定化酶最高活力5.07U/g。同时,对固定化柚（皮）苷酶的稳定性进行了研究,结果表明:固定化酶的温度耐受性与存储稳定性较游离酶有较大幅度的提高;固定化酶重复使用7次（60℃,pH4.0）后,活力仍然保持在60%。      

Operational Stability and Kinetic Study of a Membrane Reactor with Pectinases from Aspergillus niger

ABSTRACT: The kinetic and operational behavior of a free enzyme membrane reactor (FEMR) were analyzed. High conversion (83%) of the pectin could be achieved with a substrate-to-enzyme ratio of 23.3 at 46°C and pH 4.8, with a transmembrane pressure of 34.5 kPa and recycling flow rate of 36 L/h. The FEMR showed a 1st-order deactiva-tion profile with a calculated half life (t_1/2) of 21.7 d in the operation conditions. Akinetic study of the hydrolysis of pectin by free endopectinases from Aspergillus niger was done by the simultaneous use of a stirred-tank reactor and a FEMR. Kinetic data were fit to a model based on the Michaelis-Menten rate expression and the design equation for a continuous membrane reactor. The kinetic characteristics observed in the hydrolysis of pectin included a product competitive inhibition. The K_m and K_i values for the membrane reactor were 2 to 3 times greater those obtained in a stirred-tank reactor. On the basis of the V_max values, the configuration of the FEMR was found to have excellent catalytic efficiency when compared with the stirred-tank reactor.     

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.     

MCM-41固定化柚苷酶脱苦葡萄柚汁

以介孔分子筛MCM-41为载体,戊二醛为交联剂,采用吸附-交联法进行了柚苷酶的固定化。研究了酶液浓度、戊二醛浓度、吸附交联时间和固定化pH对固定化效果的影响,对影响固定化效果的因素进行了分析。确定最佳的固定化条件为:酶液浓度为0.4mg/mL,戊二醛浓度2.0%,吸附交联时间为6h,固定化pH为4.0(醋酸缓冲液)。采用海藻酸钠和聚乙烯醇对制备的固定化酶进行二次包埋处理,并应用于葡萄柚汁的脱苦。结果显示,游离酶和MCM-41固定化酶对果汁的脱苦率分别为96.65%和92.90%,海藻酸钠和聚乙烯醇二次固定化的柚苷酶脱苦率分别为72.64%和70.90%。脱苦后的果汁营养成分与理化指标均有一定程度降低。为柚苷酶的固定化提供了一种新型的载体材料,为固定化柚苷酶在果汁脱苦中的工业化应用提供了理论基础。     

Treatment of Grapefruit Juice for Bitterness Removal by Amberlite IR 120 and Amberlite IR 400 and Alginate Entrapped Naringinase Enzyme

ABSTRACT: Treatment of grapefruit ( Citrus aurantium ) juice with Amberlite IR 400 resulted in 69.23% naringin removal after 5 min exposure with significant clarification and 89.41% tartness (acidity) reduction. Amberlite IR 120 removed only 9% naringin in 1 min with increased shelf life of the juice without any change in clarity, quality, and naringin content. Alginate entrapped naringinase treatment of the juice resulted in 83.84% naringin hydrolysis at 55°C and 220 rpm in 180 min with 1.98 enzyme units/mL of juice. The soluble enzyme hydrolyzed only 65.53% naringin under similar conditions. Kinetic constant values of the immobilized and soluble enzymes were found to be 9.75 mM and 20 mM, respectively.     

Process Characteristics of Hydrolysis of Chitosan in a Continuous Enzymatic Membrane Reactor

ABSTRACT: Crude enzyme from Bacillus cereus NTU-FC-4 was used to hydrolyze chitosan of 66% deacetylation in a membrane reactor, operated at 45 °C and pH 5, to continuously produce chitooligosaccharides. Major oligomers in the product from the reactor were chitobiose, chitotriose, chitotetraose, chitopentaose, and chitohexaose. When the membrane reactor was operated at an enzyme/substrate ratio of 0.2 (unit/mg) and residence time of 100 min, it reached steady state in 2.5 h. The system could be operated for 15 h and still maintained a stable product composition. When the volume replacement exceeded 2.5, the productivity of the membrane reactor became higher than that of the batch reactor, and the difference between them became even greater when the volume replacement was further increased. The apparent Michaelis constant (K_m) for the enzyme in the membrane reactor was 18.8 mg/mL, but the apparent K_m was 5.4 mg/mL for the batch reactor, suggesting that the affinity of the enzyme for chitosan was lower in the membrane reactor compared with the enzyme in the batch reactor. The estimated values of apparent V_max were 0.18 and 0.20 mg reducing sugar/mL/min for the enzyme in the membrane reactor and in the batch reactor, respectively, indicating that the enzyme activity was not greatly altered when used in the membrane reactor.     

Optimization of fermentation parameters for enhanced production of naringinase by soil isolate Aspergillus niger VB07

Aspergillus niger VB07 was isolated from the soil of citrus fruit market used to produce extracellular naringinase in a liquid medium. Various parameters of fermentation have been examined in order to improve overall enzyme yield. Naringinase yield of 17.28 IU/mL in an optimized medium containing naringin (0.1%), rhamnose (0.5%), peptone (0.25%), glycine (10 mM), and pH 4.5 at 28°C for 7 days has been obtained at shake flask level. Compared to the initial medium, the highest naringinase yield observed is approximately increased by 1.8 times under the optimized conditions. Amino acids mediated naringinase synthesis was reported.     

交联壳聚糖载体固定化柚苷酶工艺

以戊二醛为交联剂,壳聚糖为载体,采用交联-吸附偶联法固定柚苷酶,通过单因素和正交试验优化确定最佳固定化工艺。结果表明,柚苷酶的最佳固定化条件为：以质量浓度为3.5g/100mL的壳聚糖制备的凝胶微球为载体,凝结剂NaOH质量浓度1.0g/100mL、戊二醛体积分数7.0%、交联时间2.0h、pH 4.0、酶液质量浓度2.0mg/mL、25℃时吸附交联3.0h,得到固定化酶最高酶比活力为7.37U/g;与游离酶相比而言,固定化酶最适pH值与最适反应温度均无明显变化;固定化酶在不同温度(40、50、60℃)条件下重复使用7次,相对酶活力仍能保持在70%、60%和50%以上。     

Acrylamide–Sepiolite Based Composite Hydrogels for Immobilization of Invertase

ABSTRACT:  Novel composite hydrogels, poly(acrylamide)–sepiolite (PAS), poly(acrylamide/acrylic acid)–sepiolite (PAAS), and poly(acrylamide/itaconic acid)–sepiolite (PAIS) were prepared and used for the immobilization of invertase. The parameters of equilibrium swelling, diffusional exponent, and diffusion coefficient of these hydrogels were calculated from swelling experiments. Invertase was immobilized onto PAS, PAAS, and PAIS and immobilized invertases (PASI, PAASI, and PAISI) were prepared. Optimum pH values for free invertase, PASI, PAASI, and PAISI are found to be 5, 5.5, 4.5, and 6, respectively, and the optimum temperatures were 30, 50, 50, and 35 °C for free invertase PASI, PAASI, and PAISI. It was found that  K_m  values of free invertase, PASI, PAASI, and PAISI were 11.3, 41.0, 94.5, and 56.0 mM, respectively.  V _max values were 2 μmol/min for free invertase, 8.10 μmol/min for PASI, 1.30 μmol/min for PAASI, and 0.42 μmol/min for PAISI, respectively. The invertase immobilized hydrogels showed excellent, temperature, storage, and operational stability.