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 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 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.     

Some characteristics of polyphenol oxidase purified from edible yam (Dioscorea cayenensis-rotundata cv. Longbô) cultivated in Côte d'Ivoire

Polyphenol oxidase (PPO_Y) of edible yam ( Dioscorea cayenensis-rotundata cv. Longbô ) cultivated in Côte d'Ivoire was purified to homogeneity by a procedure that included ion exchange chromatography, ammonium sulphate fractionation, gel filtration and hydrophobic chromatography using dopamine as a substrate. The relative molecular weight of the PPO_Y was estimated to be 94.75 ± 0.63 and 151.56 ± 2.75 kDa by SDS–PAGE and gel filtration on Sephacryl S100 HR, respectively, indicating this PPO_Y as a multimer. Maximal PPO_Y activity occurred at 30 °C and pH 6.6. The enzyme was stable at 25 °C and its pH stability was in the range of 6.0–7.0. The values V _max/ K _m showed that the enzyme had the greatest reactivity towards dopamine among the substrates used. PPO_Y showed no activity toward the monophenols and was completely inhibited by beta-mercaptoethanol, sodium thiosulphate, l -ascorbic acid, sodium bisulphite and l -cysteine. Data generated by this study might help to better prevent edible yam browning.     

CHARACTERIZATION OF LIPOXYGENASE FROM MACKEREL (SCOMBER SCOMBRUS) MUSCLE

The polyunsaturated fatty acids (PUFA) in fish render them potentially susceptible to enzymatic oxidation. Lipoxygenase (LOX) activity from mackerel (Scomber scombrus) was characterized with respect to pH stability and activity, temperature stability, buffer and substrate preferences and inhibition patterns. The partially purified enzyme was stable over the pH range 4.0–11.0 and had a pH-activity optimum pH of 5.6. Mackerel muscle LOX (mmLOX) was stable at 0–50C but lost activity at higher temperatures. Enzymatic assays using different buffers revealed that MES-NaOH and Bistris-HCl were the suitable buffers for reaction. Substrate preference was for unoleic acid compared to linolenic, arachidonic, docosahexaenoic oroleicacid. K_m values were 0.69 and 0.57 mM and V_max was 0.058 and 0.019 μmol mm^-1, for linoleic and linolenic acid, respectively. mmLOX was inhibited by classical LOX inhibitors such as BHA, BHT, NDGA, esculetin and also by the heme protein inhibitor KCN. Values for the IC₅₀ (concentration for half maximal inhibition) was between 0.02 μM and 41 μM.
Normal phase high performance liquid chromatography (HPLC) analyses revealed that both 13- and 9-hydroperoxides were formed during mmLOX catalyzed oxidation with Unoleic acid substrate although a higher proportion of the 13-isomer was formed. With enzymes from different mackerel tissues (skin, gills and muscle), mmLOX had the highest hydroperoxide forming ability.     

PURIFICATION AND PROPERTIES OF PHOSPHOLIPASE A2 ISOZYMES FROM PYLORIC CECA OF THE STARFISH (ASTERINA PECTINIFERA)

Phospholipase A₂ isozyme II (PLA₂ II), which showed different mobility on native PAGE from that of the PLA₂ isozyme I (PLA₂ 1) isolated previously, was purified from pyloric ceca of the starfish (Asterina pectinifera). The PLA₂ II mainly released oleic acid from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. N-terminal amino acid sequence of the PLA₂ II was SVYQF. Temperature and pH optima of the PLA₂ II were at around 50C and pH 9.0, respectively, and the enzyme activity was enhanced by sodium deoxycholate and 1 mM or higher concentration of Ca²⁺. The PLA₂ II did not show fatty acid specificity for hydrolysis of phosphatidylcholine (PC). Specific activity of the PLA² II was about 10 times higher than that of commercially available porcine pancreatic PLA₂. The PLA₂ II hydrolyzed PC more effectively than phosphatidylethanolamine. These characteristics of the PLA₂ II were the same as those of the PLA₂ I.     

PURIFICATION AND PROPERTIES OF GLUCOSE 6-PHOSPHATE DEHYDROGENASE FROM CORIANDER (CORIANDRUM SATIVUM) LEAVES

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP⁺ oxidoreductase, EC 1.1.1.49; G6PD) was purified from coriander (Coriandrum sativum) leaves; the kinetic behavior and some properties of the enzyme were also investigated. The purification was done at 4C and involved two steps: ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 26.4% and had a specific activity of 1.826 U/mg protein. Optimum pH, stable pH, optimum temperature, molecular weight, K_M and V_max values for NADP⁺ and glucose 6-phosphate (G6-P) were also determined.
The overall purification was about 74-fold. SDS-PAGE of the purified enzyme showed a single band. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm.
The molecular mass was estimated to be 74.4 kDa by SDS-PAGE and 73.2 kDa by Sephadex G-200 gel filtration column chromatography. The enzyme had an optimum pH at 8.5 and was stable at pH 8.0 in 0.1 M Tris-HCl buffer. The optimum temperature was at 30C. The K_M values for NADP⁺ and G6-P were 0.026 mM and 0.116 mM, respectively. The V_max values for these substrates were 0.035 EU/mL and 0.038 EU/mL, respectively.     

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.     

Antimicrobial Activity of Lactoperoxidase System Incorporated into Cross-Linked Alginate Films

ABSTRACT:  In this study, the antimicrobial effect of lactoperoxidase (LPS) incorporated alginate films was investigated on Escherichia coli (NRRL B-3008), Listeria innocua (NRRL B-33314), and Pseudomonas fluorescens (NRRL B-253) in presence of different concentrations of H₂O₂ (0.2, 0.4, and 0.8 mM) and KSCN (1, 2, and 4 mM). The incorporation of 70 nmol ABTS/min/cm² LPS into alginate films gave 0.66 to 0.85 nmol ABTS/min/cm² enzyme activity at 0.2 to 0.8 mM H₂O₂ concentration range. The antimicrobial activity of LPS system on target bacteria changed according to the concentrations of KSCN and H₂O₂. The growth of all tested bacteria was prevented for a 6-h period by applying LPS system in presence of 0.4 or 0.8 mM H₂O₂ and 4 mM KSCN. At 0.8 mM H₂O₂ and 4 mM KSCN, the LPS system also inhibited growth of L. innocua and P. fluorescens for a 24-h incubation period, whereas E. coli growth could not be inhibited for 24 h under these conditions. At 0.2 mM H₂O₂ and 1 to 4 mM KSCN, a considerable inhibitory effect was obtained only on P. fluorescens . The decreasing order of the resistance of studied bacteria to LPS system is as follows: E. coli , L. innocua , and P. fluorescens . The developed antimicrobial system has a good potential for use in meat, poultry, and seafood since alginate coatings are already used in these products. Further studies are needed to test the LPS incorporated edible films in real food systems.     

OXIDATION OF INDOLEACETIC ACID BY AN APPARENTLY HOMOGENEOUS PEROXIDASE FROM THE FLAVEDO OF WASHINGTON NAVEL ORANGES (CITRUS SINENSIS)

Orange flavedo peroxidase was purified by ammonium sulfate fractionation, gel filtration and ion-exchange chromatography. The anionic fraction, representing 51% of the total peroxidase activity, was an apparently homogeneous isoenzyme with an pI of 4.0. The cationic fraction was composed of two isoenzymes with pI of 10.0 and 11.5.
The anionic peroxidase oxidized indoleacetic acid in the presence of H₂O₂ and 2,4-dichlorophenol. This reaction was inhibited by Mn²⁺ and enzyme activity decreased at increasing pH in the range of 4.0 to 5.5. This isoenzyme also oxidized indoleacetic acid in the reaction mediated by 2,4-dichlorophenol, Mn²⁺ and phosphate. In this case the optimum pH was 4.5 and the reaction was inhibited by H₂O₂.
Study of the characteristics of both reactions indicates that, even though they could share some common steps and have similar end products, they probably differ in their reaction mechanism. The possible physiological significance of the H₂O₂ mediated oxidation of indoleacetic acid is briefly discussed.     

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.     

Natural maize phenolic acids for control of aflatoxigenic fungi on maize

ABSTRACT:  Natural phytochemicals may be an alternative to synthetic chemicals for controlling fungal growth and mycotoxin production in stored maize. A key to progress in this field is to select the best natural maize phytochemicals to be applied in a storage maize ecosystem. This research was undertaken to evaluate the effects of the natural phytochemicals trans-cinnamic acid (CA) and ferulic acid (FA) alone at concentrations of 20 to 30 mM and in 5 combinations on Aspergillus flavus Link and A. parasiticus Speare populations and aflatoxin B₁ production. Studies on Aspergillus population and aflatoxin B₁ production were carried out in maize grain in relation to a water activity a_w of 0.99, 0.97, 0.95, and 0.93. CA and FA at concentrations of 25 to 30 mM, respectively, and CA-FA mixture T9 (25 + 30 mM) were the treatments most effective at inhibiting A. flavus and A. parasiticus population at all a_w assayed after 11 d of incubation. At all a_w values, the mixture CA-FA T9 (25 + 30 mM) completely inhibited (100%) aflatoxin B₁ production by both strains at a_w= 0.99, 0.97, 0.95, and 0.93. Decreased aflatoxin B₁ levels in comparison with the control were observed with mixtures CA-FA T6 (10 + 25 mM), T7 (20 + 20 mM), and T8 (20 + 30 mM) of both strains in the majority of a_w assayed. The data show that CA and FA could be considered as effective fungitoxicants for A. flavus and A. parasiticus in maize in the a_w range 0.99 to 0.93. The information obtained shows promise for controlling aflatoxigenic fungi in stored maize.     

Partial purification and characterisation of banana [Musa (AAA Group) 'Gros Michel'] polyphenol oxidase

Polyphenol oxidase (PPO) from pulp of banana [Musa (AAA Group) 'Gros Michel'] was extracted and precipitated with 80% saturated ammonium sulphate followed by conventional column chromatography on Sephacryl S-200 HR and fast protein liquid chromatography on Mono Q column. The lyophilised PPO obtained from Sephacryl S-200 HR column was used for characterisation and inhibition studies. The partially purified PPO obtained from the Mono Q column exhibited at least three isoenzymes. The banana PPO had optimum pH for activity at 7 and it was stable around the same pH. Only 48% of initial enzyme activity was lost after heating at 70 °C for 30 min. The enzyme was completely inhibited by 2 m m sodium metabisulphite, 2 m m l -cysteine, 4 m m ascorbic acid, and 100 m m 4-hexylresorcinol. The K _m and V _max of banana PPO for dopamine were 2.08 m m and 0.124 m m  min⁻¹ respectively.     

INHIBITION OF LISTERIA MONOCYTOGENES BY CINNAMIC ACID: POSSIBLE INTERACTION OF THE ACID WITH CYSTEINYL RESIDUES

The effects of cinnamic acid (C₆H₅CH=CHCOOH) on L. monocytogenes strain Scott A and listeriolysin O (LLO) activity were studied. In addition, a possible mode of action of the acid was investigated. L. monocytogenes was more susceptible to cinnamic acid than to benzoic acid, and lowering the broth pH increased the inhibition. LLO activity was inhibited in the presence of 0.1% cinnamic or benzoic acid, and western blot analysis showed reduced intensity of listeriolysin bands in the presence of 0.2% of the acids. Addition of cysteine (5 mM) to cinnamic acid in PBS, pH 7.0, caused gradual reduction in UV absorption of the acid during incubation for 12 h. Cinnamic acid (>0.5%, pH 7.0) inhibited glucose uptake and ATP production in resting cells of L. monocytogenes. Results suggest a role of the cinnamate anion in the antilisterial and anti-LLO activity and potential applications in the food industry.     

POLYPHENOL OXIDASE FROM BARHEE AND ZAHDI DATES. II. CHARACTERIZATION

Purified polyphenol oxidase (PPO) from Barhee and Zahdi date cultivars had a molecular weight of 17,500 and 17,000, respectively, as determined by exclusion gel chromatography. The enzyme possessed activity on o-diphenols but not on monophenols or m-diphenols. The highest activity was on catechol with K_m of 3.5 and 8.75 mM for PPO from Barhee and Zahdi dates, respectively. PPO from the two cultivars showed optimal pH for activity and stability around 6.0 and 7.0, respectively. The enzyme was completely inactivated when incubated at 70°C for 10 min. Activation energy for conversion of substrate to product was 3400 and 3600 cal/mole for PPO from Barhee and Zahdi dates, respectively. However, the activation energy for denaturation was 28000 and 27000 cal/mole for the enzyme from Barhee and Zahdi cultivars, respectively. Ascorbic acid, oxalic acid and sodium metabisulfite caused non-competitive inhibition for PPO activity, while sodium chloride was an uncompetitive inhibitor. Sodium metabisulfite was the most potent inhibitor with Ki values of 0.025 and 0.030 mM for PPO from Barhee and Zahdi dates, respectively.     

GAMMA GLUTAMYL TRANSPEPTIDASE OF SPROUTED ONION

SUMMARY –An enzyme capable of liberating p-nitroaniline from γ- l -glutamyl p-nitroanilide has been purified 800-fold from sprouted onions. The enzyme acts optimally at pH 9.0, is activated by amino acids and inhibited by borate and competitively by γ-glutamyl derivatives. With the p-nitroanilide as substrate it exhibits a K_m of 14.3 mM whereas the K_i values with glutathione and γ-glutamyl-S-methyl- l -cysteine as inhibitors are 0.20 and 2.14 mM respectively. From chromatography of the reaction products and from kinetic considerations it is concluded that the purified enzyme is a transpeptidase (E.C.2.3.2.1) whereas crude onion extract may, in addition, possess a true γ-glutamyl hydrolase. It is suggested that such enzymes may play a role in the disappearance of peptides in post-dormant onions and may be useful evoking the full flavor potential of onion.     

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.     

Effect of carbon dioxide on improving the keeping quality of raw milk

Fresh cows' milk samples were treated with CO₂ to give a calculated CO₂ content of 77mM. The treated samples as well as the control were stored at 7° and 20 ± 5°C for three days and analysed periodically. The increase in the total count as well as the counts of psychrotrophic, lactic acid and coliform bacteria during storage at 7°C was found to be inhibited by the addition of CO₂. The treated samples had higher titratable acidity and lower pH values than the controls but satisfied the clot-on-boiling test. The keeping quality of cooled milk can therefore be improved by the addition of CO₂.     

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.     

MEASURING THE WATER HOLDING CAPACITY OF NATURAL ACTOMYOSIN FROM CHICKEN BREAST MUSCLE IN THE PRESENCE OF PYROPHOSPHATE AND DIVALENT CATIONS

The water holding capacity (WHC) of natural actomyosin (NAM) extracted at pH 9.2 in 0.6M KCl was measured in the presence and absence of various combination of sodium pyrophosphate (PP_i), MgCl₂ and CaCl₂ using a modification of the classical centrifugation technique. Samples, in the presence of 0.15M NaCl and 20 mM sodium phosphate buffer pH 6, were spun at 30,900 X G (as measured at the bottom of the centrifuge tube) for 15 min at 2–4C. The results show that between 17 and 20 g water/g protein were bound over a wide range of NAM concentrations. In each case the amount of water held by the experimental sample was equal to or less than the amount held by a control run at the same time: 5 mM PP_i= 100%; 5 mM PP_i+ 5 mM MgCl₂= 58%; 5 mM MgCl₂= 85%; 5 mM PP_i+ 5 mM CaCl₂= 68% and 5 mM CaCl₂= 92% of the control. Thus polyphosphate addition in the presence of divalent cations lowers the WHC of NAM. The absence of the organized structure of muscle in NAM is postulated to be the reason that polyphosphate plus divalent cation reduced WHC in these samples. A series of preliminary experiments were run in order to determine the effect of experimental parameters on WHC.