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.     

Naringinase Immobilization in Packaging Films for Reducing Naringin Concentration in Grapefruit Juice

Naringin, a bitter compound in citrus, may be converted to a nonbitter form by enzymic hydrolysis. Our objective was to determine the feasibility of immobilizing naringinase in a food contact approved packaging film. Naringinase from Penicillium sp. was immobilized in cellulose acetate films with up to 23% efficiency at 7°C. Kinetic studies showed that the free enzyme had an optimum pH=3.5 and the immobilized enzyme pH=4.0. Activation energy decreased upon immobilization (from 14.2 to 11.0 Kcal/mol), thus providing an increased catalytic efficiency for immobilized naringinase. The Michaelis constant for immobilized naringinase (K_m=2.1 mM) was lower than for free enzyme (K_mm=3.6 mM). Keeping films under dry storage for 1 mo at room temperature did not cause decreased enzyme activity. A film area/volume ratio (cm²/mL of 10° Brix grapefruit juice) of 7.2 hydrolyzed 60% of the naringin in 15 days at 7°C.     

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.     

TRICHODERMA REESEIα-GALACTOSIDASE ACTIVITY ON LOCUST BEAN AND GUAR GALACTOMANNANS

The effect of side-chain density on the kinetic parameters of a side-chain-cleaving hemicellulase was determined. Kinetic parameters were based on the rate of Trichoderma reesei α- galactosidase-catalyzed liberation of galactose from galactomannan (guar and locust bean) substrates. The focus enzyme was the predominant α-galactosidase obtained from the fungus'galactomannan-supplemented cell-free culture medium. Substrate concentrations were based on the number of galactosyl moieties per volume reaction mixture. The K_m values for the galactomannan substrates differed approximately 4.3-fold (28.36 and 121.16 μM), the more branched substrate having the higher K_m. In contrast, the corresponding V_max values were found to be essentially the same. The results indicate the enzyme preferentially acts at sites of low side-chain density .     

Performance of enzymatic wheat gluten hydrolysis in batch and continuous processes using Flavourzyme

The aim of this study was to investigate the process performance of wheat gluten hydrolysis with Flavourzyme in the conventional batch and in an enzyme membrane reactor system. The release of amino acids and peptides from wheat gluten using Flavourzyme in a batch process (T 50 °C, pH 5) is limited due to product inhibition. Investigation of Flavourzyme inhibition kinetics by l-amino acids revealed that the strongest inhibition was observed by l-cysteine. Thus, application of an enzyme membrane reactor with continuous product removal and substrate and enzyme retention was assessed. The highest productivity in the enzyme membrane reactor system was found using a 10 kDa molecular weight cut-off polyethersulfone membrane. This resulted in an increased product enzyme ratio from 2.1 mg product/nkat in the conventional batch process to 4.9 mg product/nkat with the enzyme membrane reactor after 20 h. The enzyme membrane reactor retained 80% of the initial space-time yield (STY_init 5 g/L/h) after nine days.     

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.     

Production of high degree polymerized chitooligosaccharides in a membrane reactor using purified chitosanase from Bacillus cereus

Crude chitosanase from Bacillus cereus NTU-FC-4 was separated by a cation exchanger to three fractions named CBCI, CBCII, and CBCIII. The CBCI hydrolyzed chitosan to yield dimers. The primary hydrolytic products of CBCII were low degree polymerized (DP) chitooligosaccharides. The CBCIII had the fastest reaction rate and yielded high DP chitooligosaccharides (heptamer and higher DP oligomers). When CBCIII was used in the ultrafiltration membrane reactor with enzyme/substrate ratio 0.06 unit/mg and 100 min of residence time (RT), the concentration of high DP oligomers was 9.78 mg/mL which occupied ca. 48% of total oligomers in the final product as compared to ca. 29% resulted from the crude enzyme. Decrease of RT to 50 min and 33 min, the high DP oligomers in the products were ca. 61% and 69%, respectively. This system could be operated for at least 24 h and kept a constant permeate flux and product output rate.     

The Flavonoid Eriodictyol as Substrate of Peach Polyphenol Oxidase

ABSTRACT: Spectral changes produced in the oxidation of eriodictyol by peach polyphenol oxidase were followed over time. A product with λ_max= 390 nm was seen to appear before another with λ_max= 475. The product absorbing at 390 nm must correspond to the o -quinone derived from eriodictyol. The compound absorbing at 475 nm must be derived from this eriodictyol-o-quinone. Progress curves at this wavelength revealed a lag, the length of which varied with enzyme and substrate concentrations. This lag must have been caused by chemical reactions taking place after the enzymatic reaction. When eriodictyol oxidation was studied in the presence of 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH), a potent nucleophilic reagent that reacts with the eriodictyol-o-quinone to form a dark pink product absorbing at 508 nm, the lag disappeared. When the kinetic parameter was evaluated in the presence of MBTH (K_m= 0.6 m M ), the results was similar to those obtained without MBTH. Eriodictyol oxidation was inhibited by tropolone, which behaved as a classic competitive inhibitor (K_I= 15 μ M ). The inhibition results reported show that eriodictyol oxidation was strictly dependent on the presence of polyphenol oxidase. In addition, other oxidase activities, such as laccase and H₂O₂ independent phenol oxidase, were not detected in the enzyme extract.     

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.     

PURIFICATION AND CHARACTERIZATION OF POLYPHENOL OXIDASE FROM POTATO: II. INHIBITION AND CATALYTIC MECHANISM

KCN and ascorbic acid showed competitive inhibition patterns with K_is values of 0.032 and 0.27 mM, respectively. Uncompetitive inhibition patterns were obtained with sodium azide, L-cysteine and NaCl with K_ii values of 3.3 mM, 0.12 mM and 0.3 M, respectively. A noncompetitive inhibition pattern was obtained for thiourea with 0.067 mM for K_is and 0.59 mM for K_ii. Cu₂⁺ increased the activity about 2.5 fold at or above 40 μM and K⁺ decreased the enzyme activity about 33% at 0.4 M. Other metal ions did not have any effects on the activity. Two pK values of 5.8 and 8.0 were obtained from V_max profile and two pK values of 5.9 and 8.1 from V_max/K_m profile. The data suggest that cysteine is likely to be involved in catalysis and histidine in binding. Data from chemical modification show that cysteine was completely inactivated at 1.74 mM o-methylisourea, and histidine and tryptophan were modified at much higher concentrations of diethylpyrocarbonate and N-bromosuccinimide, respectively. It is suggested that the protonated cysteine works as a general base, tryptophan as a substrate binding residue and histidine as a oxygen binding residue.     

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.     

Characterization of honey amylase

ABSTRACT:  The major α-amylase in honey was characterized. The optimum pH range and temperature were determined for the enzyme as 4.6 to 5.3 and 55 °C, respectively. The enzyme was stable at pH values from 7 to 8. The half-lives of the purified enzyme at different temperatures were determined. The activation energy for heat inactivation of honey amylase was 114.6 kJ/mol. The enzyme exhibited Michaelis–Menten kinetics with soluble starch and gave K _M and V _max values of 0.72 mg/mL and 0.018 units/mL, respectively. The enzyme was inhibited by CuCl (34.3%), MgCl₂ (22.4%), and HgCl₂ (13.4%), while CaCl₂, MnCl₂, and ZnSO₄ did not have any effect. Starch had a protective effect on thermal stability of honey amylase. Therefore, it might be critical to process or control the amylase in honey before incorporation into starch-containing foods to aid in the preservation of starch functionality. One step could involve heat treating honey with other ingredients, especially those that dilute and acidify the honey environment.     

Use of response surface methodology to optimise the hydrolysis of whey protein isolate in a tangential flow filter membrane reactor

Response surface methodology (RSM) was used to investigate the effects of substrate concentration (S), enzyme concentration (E) and initial permeate flow rate, (J_i), on permeate flux behaviour in a 10 kDa nominal molecular weight cut-off (NMWCO) tangential flow filter (TFF) enzyme membrane reactor (EMR) during 3 h hydrolysis of whey protein isolate (WPI) using Protease N Amano (IUB 3.4.24.28, Bacillus subtilis) at pH 7.0 and 45 °C. The average residual permeate flow rate (J_residual), residual enzyme activity (A_residual) and product recovered in permeate designated as apparent sieving (S_apparent) were monitored. The quadratic model regression equations obtained revealed that all the three factors had significant but dissimilar influences on permeate flux behaviour. J_residual, S_apparent and A_residual increased with increasing E, A_residual decreased with increasing J_i and there was substrate inhibition at low E. The optimised factors were S = 4.72% (w/v), E = 0.055% (w/v; hence E/S ≈ 1% w/w) and J_i = 6.91 mL/min (approximately 0.7% reactor volume per minute). The optimised values were 87.24%, 52.37% and 35.08% for J_residual, A_residual and S_apparent, respectively. The actual values for the responses agreed well with the predicted values implying that RSM is suitable for EMR optimisation. Covariance values showed that J_residual and S_apparent increased concomitantly while A_residual decreased with increasing S_apparent and J_residual.     

SELECTED p-NITROANILIDES AS SUBSTRATES FOR SENSITIVE PAPAIN ASSAY

N-acetyl-Leu-Leu-Gly-p-nitroanilide and N-succinyl-Ala-Ala-(S-benzyl) Cys-p-nitroanilide were found to be very sensitive papain substrates, making it possible to detect 0.5 or 0.7 μg of technical papain in 1 mL of beer in a short time. This detection limit is about 10 times lower than the usual papain level used for chillproofing. The estimated kinetic constants (k_cat, K_m) for papain, bromelain and trypsin permit certain conclusions about papain specificity for oligopeptides, which will help to prepare even more specific p-nitroanilide substrates.     

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.     

Purification and Characterization of an α-L-Rhamnosidase from Aspergillus terreus of Interest in Winemaking

ABSTRACT: An enzyme with α-L-rhamnosidase activity was purified to homogeneity from a culture filtrate of Aspergillus terreus after growth in a medium containing L-rhamnose as the sole carbon source. The biosynthesis of this enzyme was repressed by glucose. The enzyme had a molecular mass of 96 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 4.6 as determined by analytical isoelectric focusing. The pH and temperature optima for the enzyme were found to be 4.0 and 44 °C, respectively. Using p-nitrophenyl-α-L-rhamnopyranoside as a substrate, the enzyme exhibited Michaelis-Menten kinetics with K_M and V_max values of 0.17 mM and 84 U/mg, respectively. The enzyme was inhibited competitively by L-rhamnose (K₁ 2.5 mM). Divalent cations such as Ca²⁺ Mg²⁺ Zn²⁺ and Co²⁺ stimulated the a-L-rhamnosidase activity, whereas this was inhibited by Hg2+ and Cd2+. Ethanol (12% v/v) and glucose (21% w/v) decreased enzyme activity by approximately 20%, while this was not affected by SO₂.     

PURIFICATION AND CHARACTERIZATION OF JERUSALEM ARTICHOKE (HELIANTHUS TUBEROSUS L) POLYPHENOL OXIDASE

Polyphenol oxidase (EC 1.14.18.1) has been purified from Jerusalem artichoke tubers by immobilized copper affinity chromatography. The enzyme is primarily an o-dihydroxyphenol oxidase with apparent K_m values of 1.9, 3.5 and 3.9 mM for chlorogenic acid, 4-methylcatechol, and catechol, respectively. Several compounds exhibited inhibitory action for the enzyme in the order of: sodium metabisulfite > sodium diethyldithiocarbamate > 2,3-naphthalenediol > thioglycollate. Multiple forms were identified by gel filtration and SDS-gradient polyacrylamide gel electrophoresis: two aggregates with apparent MW of 120 and 86 K and two monomeric subnits of 40–42 and 32–34 K, respectively. Concentration dependent association-dissociation phenomena most likely determine the multimeric state of this enzyme. While the aggregated forms exhibited specificity towards mono-, di- and polyhydroxyphenols, the low MW subunits were found active only with o-dihydroxyphenols. The isoelectric points of the various enzyme species were within the range of 4.0 to 10.0. The enzyme was found to contain appreciable amounts of associated carbohydrate material.     

Enzyme membrane reactor in isolation of antioxidative peptides from oil industry waste: A comparison with non-peptidic antioxidants

Edible oil industries suffer from the problem of seed meal utilization, which is recognized as a byproduct of edible oil. Present work has investigated production of peptide antioxidants, from oil seed meals to meet the increasing crave for natural antioxidants in food and pharmaceutical industries. Metallo-endopeptidase ‘Protease A Amano 2G’ (Aspergillus oryzae) was used to hydrolyze seed protein isolate in enzyme membrane reactor (EMR) and protein hydrolysate was sequentially fractionated by ultrafiltration to obtain potential peptide fraction. Degree of hydrolysis was varied within enzyme to substrate ratio 0.1–2 g/100 g and hydrolysis time 10–60 min to maximize peptide yield and antioxidant activity of peptides in vitro. Controlled hydrolysis with enzyme dose of 2 g/100 g, exhibited peptide yield of 4.60 ± 0.08 mg/100 mg meal protein in membrane reactor (DH 30.7%) and 4.23 ± 0.22 mg/100 mg meal protein in batch mode of hydrolysis (DH 29.3%). Antioxidant potential of peptide fractions were compared with commercial non-peptidic antioxidants and major findings confirm superior activity for protein fragments (IC₅₀-0.062 mg/mL) as compared to protein isolate (IC₅₀-0.038 mg/mL), with enhanced product stability by preventing oxidative deterioration. Results substantiate the prospect of EMR in obtaining peptides from edible oil industry waste, having comparable antioxidative potential with commercial non-peptidic antioxidants.     

Optimization of beta-carotene production by Rhodotorula glutinis using high hydrostatic pressure and response surface methodology

ABSTRACT:  Rhodotorula glutinis RG6 was treated by high hydrostatic pressure (HHP) of 300 MPa for 15 min for improving its ability of β-carotene production. After the treatments of 5 repeated cycles, the mutant strain RG6p was obtained, β-carotene production of which reached 10.01 mg/L, increased by 57.89% compared with 6.34 mg/L from parent strain RG6. To optimize the medium for β-carotene fermentation by mutant RG6p, a response surface methodology (RSM) approach was used in conjunction with a factorial design and a central composite design, and the maximum yield of β-carotene (13.43 mg/L), an increase of 34.17% compared to the control, was obtained at a pH 6.7 with an optimum medium (40 mL/250 mL) of yeast extract (4.23 g/L), glucose (12.11 g/L), inoculum (30 mL/L), tomato extract (2.5 mL/L), peanut oil (0.5 mL/L), and (NH₄)₂SO₄ (5 g/L).     

Respiration Rate of Sweet Cherries: 'Burlat', 'Sunburst' and 'Sweetheart' Cultivars

ABSTRACT: Oxygen depletion and CO₂ formation were measured using a closed system method at various temperatures. No CO₂ inhibition effect was observed. Respiration rates were related to O₂ concentration by a simple Michaelis-Menten equation. Lineweaver-Burk plots of respiratory rate versus O₂ concentration were nonlinear over the whole O₂ concentration range explored. However, this type of analysis allowed us to differentiate two O₂ concentration intervals (above and below 10%) in which the double reciprocal plots are linear. Respiration rates decreased with decreasing temperature following the Arrhenius law. The Michaelis-Menten equation constants V_m and K_m for the 3 cultivars at 2, 5, and 20°C are tabulated.