Characterization of a glycerol/H+ symport in the halotolerant yeast Pichia sorbitophila

Pichia sorbitophila is a halotolerant yeast capable of surviving to extracellular NaCl concentrations up to 4 M in mineral medium when glucose or glycerol are the only carbon and energy sources. Evidence is presented here that glycerol, the main compatible solute this yeast accumulates so as to maintain osmotic balance, is actively co-transported with protons. This transport system was shown to be constitutive, not needing induction by either glycerol or salt, and was not repressible by glucose. In glucose- or glycerol-grown cells, a simple diffusion was detectable, and iterative calculations were performed to calculate kinetic parameters, in the presence and in the absence of NaCl. At 25°C, pH 5·0, in glucose-grown cells these were: K_m = 0·81 ± 0·11 mM and V_max = 634·2 ± 164·8 μmol h^?1 per g (glycerol); K_m = 1·28 ± 0·60 mM and V_max = 558·6 · 100·6 μmol h^?1 per g (protons). Correspondent stoichiometry was approximately 1, either for these conditions or in the presence of 1 M -NaCl. An increase in acumulation capacity was evident when different concentrations of NaCl were present. This capacity was shown to be dependent on ΔpH and membrane potential, consistently with an electrogenic character. We suggest that the main role of this system is in osmoregulation, by keeping glycerol accumulated inside the cells, compensating for leakage, due to its liposoluble character.     

Molecular cloning,expression, and enzymatic characterization of <Emphasis Type="Italic">Solanum tuberosum</Emphasis> hydroperoxide lyase

A cDNA encoding hydroperoxide lyase (HPL) was isolated from Solanum tuberosum, cloned into pQE-30 vector, and expressed in E. coli. The recombinant protein was purified by nickel affinity chromatography and showed an approximate molecular weight of 54 kDa by SDS–PAGE analysis, which was similar to the predicted value based on the putative amino acid sequences (53.9 kDa). 13-Hydroperoxy-linolenic acid (13-HPOT) was the preferred substrate for the enzyme compared with 13-hydroperoxy-linoleic acid (13-HPOD). The corresponding volatile products were 2(E)-hexenal and n-hexanal tested by headspace-gas chromatography, respectively. The enzyme was optimally active at 25 °C and pH 6.5. The K _m, V _max, and the catalytic efficiency (V _max/K _m) for 13-HPOT were 56.6 μM, 71.3 units/mg, and 1.26 units/mg · μM, respectively. Activity of the recombinant potato HPL increased when Triton X-100, sodium chloride, or potassium chloride was added in the reaction mixture, while calcium chloride decreased activity of the recombinant enzyme.     

Survival of Escherichia coli O157:H7 in dried beef powder as affected by water activity,sodium chloride content and temperature

A study was carried out to determine the rate of inactivation of Escherichia coli O157:H7 in beef powder as affected by a_w(0·34±0·06±0·01), sodium chloride content (0·5, 3·0 and 20%) and temperature (5 and 25° C) over an 8-week storage time. Retention of viability of acid-adapted, acid-shocked, and control cells was determined. Overall, there were no significant differences (P≤0·05) in survival among the three types of cells subjected to the same test parameters, suggesting that mechanisms associated with induction of acid adaptation or acid shock do not result in cross protection against dehydration or osmotic stresses. At each a_wand within cell type, an increase in sodium chloride concentration resulted in significant reductions in the number of viable cells after a given storage time. Regardless of cell type, survival was significantly higher in beef powder containing 0·5 or 3% sodium chloride compared to powder containing 20% salt. The rate of inactivation was enhanced at a_w0·34±0·06 compared to a_w0·68±0·01 and at 25° C compared to 5° C.     

Effect of pH and Ionic Strength on the Heat Stability of Rapeseed 12S Globulin (Cruciferin) by the ANS Fluorescence Method

The heat stability of rapeseed 12S globulin (cruciferin) was examined using 8-anilinonaphthalene-1-sulphonic acid (ANS) as a fluorescence probe. Heating cruciferin (0·06–0·3 mg ml⁻¹ in 10 mM glycyl–glycyl piperizine buffer, pH 7·0, with 0·1–1·0 M NaCl) for 20 min increased its hydrophobicity as monitored by ANS fluorescence measurements. The mid-point temperature for the heat effect (T_m) increased linearly with increasing solvent pH (T_m (°C)=4·16 pH+41 (μ=0.1)) or sodium chloride concentration (T_m (°C)=14·7 [NaCl]+71 (pH=7·0)). The range of T_m values for cruciferin was 45–96°C. At 20°C cruciferin was unstable at pH<3·0 but relatively stable under alkaline conditions (pH 8–10). Though possessing an oligomeric structure, cruciferin appears to heat denature in accordance with the two-stage deactivation model for simple globular proteins.     

Kinetic studies on strawberry anthocyanin hydrolysis by a thermostable anthocyanin-β-glycosidase from Aspergillus niger

Several kinetic characteristics of a thermostable anthocyanin-β-glycosidase from Aspergillus niger have been evaluated. With strawberry anthocyanins as substrate, at pH optimum (4·0) and t = 30°C, K_m was found to be $\text{123 ± 4 μ}\text{m}$ and V_max, 1·16 ± 0·06 μmol min^?1mg^?1 protein. Temperature optimum was observed at about 68°C. The apparent energy of activation was calculated to be 11 ± 1 kcal/mol. The inhibitory effect of different sugars and sugar derivatives was examined. Glucono-deltalactone ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 2·3 ± 0·1 μ}\text{m}$), gluconic acid ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 82 ± 2 μ}\text{m}$) and glucose ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 1·3 ± 0·1}\text{m}\text{m}$) appeared to be competitive inhibitors of this enzyme.     

Purification,properties and heat inactivation of pectin methylesterase from apple (cv Golden Delicious)

Pectin methylesterase from apple (cv Golden Delicious) was extracted and purified by affinity chromatography on a CNBr‐Sepharose®‐PMEI column. A single pectin methylesterase peak was observed. Isoelectric points were higher than 9. Kinetic parameters of the enzyme were determined as K_m = 0.098 mg ml⁻¹ and V_max = 3.86 µmol min⁻¹ ml⁻¹ of enzyme. The optimum pH of the enzyme was above 7.5 and its optimum temperature was 63 °C. The purified PME required the presence of NaCl for optimum activity, and the sodium chloride optimum concentration increased with decreasing pH (from 0.13 M at pH 7 to 0.75 M at pH 4). The heat stability of purified PME was investigated without and with glycerol (50%), and thermal resistance parameters (D and Z values) were calculated showing that glycerol improved the heat resistance of apple PME. © 2000 Society of Chemical Industry     

The enzymic release of fatty acids from phosphatidylcholine in green peas (Pisum sativum)

‘Phospholipid acyl-hydrolase’ (PLAH), an enzymic activity releasing fatty acid from phosphatidylcholine (PC), has been identified and characterised in green peas. The K_m value for PC dipalmitoyl ester was 0·167 mm. The enzymic activity possessed a pH optimum of 5·6 and was stable for 20 min only at that pH value. The optimum temperature was 45°C and thermal sensitivity was indicated by a 94% decrease in activity upon exposure of the enzyme to 55°C for 3 min, and by an exponential decrease in activity upon storage at 4°C for 1 week. The enzyme was optimally activated by 2·0 mm calcium chloride at pH 5·6, and the optimal concentration of sodium dodecyl sulphate was 0·75 mg ml^?1. Pea PLAH was non-competitively inhibited by sodium cyanide, EDTA and p-chloromercuribenzoate, with no activity in the presence of mercuric chloride. The results from this study are related to those of other workers on lipid-degrading enzymes in peas, and a pathway is proposed for the enzymic degradation of endogenous lipids in fresh or unblanched frozen peas during post-harvest storage.     

Isoelectric fractionation and some properties of a protease from soyabean seeds

A protease, capable of hydrolysing benzoyl DL -arginine p-nitroanilide(BAPA), and L-amino acid β-naphthylamide derivatives, was purified, by isoelectric focusing in the region pH 3–6, from dormant and 6-day germinated soyabean seeds. The enzyme was focused at pH 4·80. The K_m value using BAPA as substrate was found to be 5·03 × 10⁻⁴M . Maximum activity of the enzyme towards BAPA was obtained in the pH 8·2–8–5 region. Slight activation was observed in the presence of 0·05 M concentration of Ca²⁺ and Mg²⁺ ions. The protease lacked caseinolytic activity, and was not inhibited by Kunitz soyabean trypsin inhibitor.     

Purification and some properties of polyphenoloxidase in eggplant (Solanum melongena)

Polyphenoloxidase (EC 1.10.3.1) in eggplant (Solatium melongena L) was purified by ammonium sulphate fractionation, DEAE-Cellulofine and DEAE-Toyopearl chromatography and Sephadex G-100 gel filtration. The enzyme was purified about 110-fold with a recovery of 5%. The purified enzyme more quickly oxidised chlorogenic acid (5-caffeoylquinic acid, IUPAC) than 10 other substrates used. The K_m value for the enzyme was found to be 0·50 mM with respect to chlorogenic acid; the optimum pH of the enzyme was about 4 with enzyme stability between pH 5 and 8. The enzyme was completely inactivated after heat treatment at 75°C for 30 min or 80°C for 5 min. Sodium metabisulphite, potassium cyanide and sodium fluoride markedly inhibited the enzyme activity.     

Reactivity of sorbate and glycerol in some model intermediate moisture systems

Model systems were developed to study the rôle of selected humectants and antimycotics in non-enzymic browning, haemoprotein breakdown and collagen degradation reactions at a_w 0·85 and initial pH 5·5. a_w adjustment was made using sodium chloride and the solutions contained 0·5% glucose, 0·5% sorbate, 0·5% propionate, 30% glycerol or 30% glycerol plus 0·5% sorbate. During aerobic storage at 38°C or 65°C, 10% lysine or glutamate solutions all exhibited increased browning and 0·01% or 0·02% haemoglobin solutions increased loss of haemoprotein from solution in the presence of glycerol and/or sorbate. During anaerobic storage only the glucose-containing solutions exhibited any reactivity.The pH and concentration of reactive carbonyls in the systems were also monitored and the rôle of sorbate and glycerol oxidation products in non-enzymic browning is discussed in the light of these results.Breakdown of the collagen in heat-treated tendon during storage at 38°C under similar conditions was also studied. Although the results were not unequivocal, it was apparent that, at least initially, storage in 30% glycerol caused increased degradation.     

KINETIC PROPERTIES OF PHOSPHATIDYLINOSITOL SYNTHASE FROM GERMINATING SOYBEANS

ABSTRACT CDP-1,2-diacyl-sn-glycerol (CDP-diacylglycerol): myo-inositol phosphatidyltransferase (EC. 2.7.8.11) catalyzes the final step in the de novo synthesis of phosphatidylinositol in the microsome fraction of germinating soybeans. The Michaelis constants for CDP-diacylglycerol and myo-inositol were determined for the reaction using a Triton X-100-CDP-diacylglycerol mixed micelle substrate. The K_mvalues for CDP-diacylglycerol and myo-inositol were 30μM and 0.11 mM, respectively.     

Studies on potassium nutrition of plants III.—Effects of potassium concentration on growth and mineral composition of vegetable seedlings in solution culture

Because of diffusion, the effective concentration of nutrients in contact with plant roots in sand culture is much less than the concentration of the nutrient solution supplied. For comparison with earlier observations in sand culture, solution culture experiments have been made with cabbage and red beet seedlings using readily available equipment for the control, within reasonable limits, of the potassium concentration of the root environment. Weight responses and K content of tops and roots, Na content of tops, top: root weight ratios, percentage dry residue of top and root and potassium flux through the roots were influenced in similar ways by increase of K⁺ concentration in the medium. There were steep slopes to about K_0·1 followed by an abrupt change of slope and levelling off or a slight fall at the highest potassium concentrations. The weight responses, K content of top and root and potassium flux increased with increasing nutrient medium K⁺ concentration whereas the other relationships were in the reverse direction. Root potassium contents increased in a similar manner to those of the tops but were lower than the latter at corresponding medium K⁺ concentrations. Root sodium contents rose to a maximum at a medium concentration of 0·1 mequiv./1 of K⁺ and thereafter fell with increasing potassium in the medium. The sums of the cation equivalents (K, Na, Mg, Ca) in the tops and roots were approximately constant. The limiting potassium concentration was determined from the response curves as the lowest concentration at which maximum yield was obtained. For beet in solution culture, the value was 0·15 and the corresponding concentration in sand culture was 0·7 mequiv./1 (ratio, sand/solution = 4·7); the values for cabbage were, respectively, 0·13 and 0·5 mequiv./1 (ratio, 3·8). The rate of removal of potassium by beet seedlings from solutions containing initial concentrations in the range 0·01-5 mequiv./1 was determined. The flux of potassium through the root surfaces varied from 0·35–1·89 μg K/g root/sec with increasing initial concentration in the medium.     

Spectroscopic and molecular docking studies of the interactions of sunset yellow and allura red with human serum albumin

Binding interactions of human serum albumin (HSA) with sunset yellow (SY) and allura red (AR), two food colorants, were investigated at the molecular level through fluorescence and UV absorption as well as molecular docking. The collective results of the study under the simulated physiological conditions proposed a static type of binding occurring between the two dyes and HSA. When compared with AR (293 K: Ksv = (4.21 ± 0.36) × 10⁴ L·mol⁻¹; K_b = (0.30 ± 0.23) × 10⁶ L·mol⁻¹), SY (293 K: Ksv = (6.80 ± 0.10) × 10⁴ L·mol⁻¹; K_b = (3.11 ± 2.01) × 10⁶ L·mol⁻¹) had stronger quenching ability and higher affinity for HSA due to less steric hindrance. It can be deduced that the energy transfer from HSA to the two dyes occurred with high probability based on the Förster resonance energy transfer theory (r < 7 nm, 0.5 R₀ < r < 2.0 R₀). The spectral analysis suggested that the formation of the dye-HSA complex resulted in the change in microenvironment around Tyr and Trp residues and in the secondary structure of the protein. According to molecular docking simulation, the two structural analogs almost bound to the same site of HSA, near Sudlow's Site I, but significant difference existed in the number and location of hydrogen bond (H-bond) formed between the dyes and HSA. From the molecular docking along with the thermodynamic parameters (AR: ΔH^o = −(58.79 ± 15.24) kJ·mol⁻¹, ΔS^o = −(115.1 ± 31.10) J·mol⁻¹·K⁻¹; SY: ΔH^o = −(52.24 ± 3.15) kJ·mol⁻¹, ΔS^o = −(50.07 ± 11.14) J·mol⁻¹·K⁻¹), it could be inferred that H-bond and van der Waals forces were the major binding forces involved in formation of the dye-HSA complexes.     

Acid phosphatase in the tubers of Dioscorea species and its purification from the white yam (D. rotundata poir)

Acid phosphatase activity was determined in 15 cultivars from four species of yam. A 12-fold purification of the enzyme from Dioscorea rotundata (cv. chikakwondo) gave a homogeneous preparation as demonstrated by polyacrylamide gel electrophoresis. This enzyme preparation has an apparent molecular weight of 115 000 ±2000 and an optimum activity at a pH of 5·20 and a temperature of 50°C. The K_m of the enzyme is 3·81 mM with disodium p-nitrophenylphosphate (p-NNP) as a substrate. The energy of activation, heat of activation, energy of inactivation and heat of inactivation are 7·0, 6·4, 4·41 and 4·34 kcal M^?1, respectively. Although it has very little activity with most organic phosphoric acid esters, it is significantly inhibited by Ca²⁺, Hg²⁺ and EDTA and activated by Mg²⁺. The enzyme has a half-life of 50,17 or 13 days, respectively, when stored at 6-8°C, 0°C or room temperature (29±2°C).     

Urea and Heat Unfolding of Cold-Adapted Atlantic Cod(Gadusmorhua)Trypsin and Bovine Trypsin

The reversible unfolding reactions for phenylmethylsulphonyl fluoride (PMSF)-modified trypins from Atlantic cod (cod PMS-trypsin) and cattle (bovine PMS-trypsin) were monitored by fluorescence spectrophotometry as a function of urea concentration and temperature. For urea unfolding at 25°C, the free energy change at zero concentration of urea (ΔG(H₂O)) for cod PMS-trypsin was 11(±4·4) kJ mol⁻¹ compared with 18(±1·14) kJ mol⁻¹ for bovine PMS-trypsin, while the mid-point concentration for urea unfolding curve ([urea]_1/2) was 3·0(±0·57) M and 4·1(±0·16) M, respectively. From studies of enzyme heat unfolding, the mid point temperature of the thermal unfolding curve ( T _m ) was 46(±1·4)°C for cod PMS-trypsin compared with 57(±2)°C for bovine PMS-trypsin. The standard free energy change (Δ G° ) for reversible thermal unfolding of cod PMS-trypsin was 9(±1) kJ mol⁻¹ compared with 19(±1) kJ mol⁻¹ for bovine PMS-trypsin. Values for the enthalpy (Δ H _m ), entropy (Δ S _m ) and heat capacity (Δ C _p ) for heat unfolding are compared. Results from urea and thermal unfolding studies show that cod PMS-trypsin has a significantly lower conformational stability than bovine PMS-trypsin.     

Characterisation of a trypsin/chymotrypsin inhibitor from jack fruit (Artocarpus integrifolia) seeds

Jack fruit seed (Artocarpus integrifolia Hook f) trypsin inhibitor (JSTI) was found to be rich in acidic amino acids and devoid of free thiol groups. The N-terminal and C-terminal amino acids of JSTI were aspartic acid and scrine, respectively. The inhibitor was stable under conditions of extremes of pH (3·0–12·0), at high temperatures and in the presence of denaturing agents. JSTI showed a non-competitive type of inhibition with K_i values of 0·48 ± 0·17 nM and 0·16 ± 0·04 nM for trypsin and chymotrypsin, respectively. The JSTI–trypsin complex exhibited chymotrypsin inhibitory activity suggesting the ‘double-headed’ nature of the inhibitor. Chemical modification of lysine residues resulted in loss of trypsin and chymotrypsin inhibitory activities of JSTI indicating that amino groups are essential for activity.     

Purification and characterization of highly active and stable polyphosphatase from Saccharomyces cerevisiae cell envelope

Saccharomyces cerevisiae cell envelope polyphosphatase was isolated in highly active and stable form by extraction from cells with zwittergent TM-314 followed by chromatography of the extract on phosphocellulose and QAE-Sephadex in the presence of 5 mM -MgCl₂, 0·5 mM -EDTA and 0·1% Triton X-100. The enzyme possessed a specific activity of 220 U/mg and after 30 days retained 87% of its activity at ?20°C. Polyphosphatase molecular mass was determined to be about 40 kDa by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme hydrolysed polyphosphates with various chain lengths (n = 3–208), had low activity for GTP and did not split pyrophosphate, ATP and p-nitrophenylphosphate. On polyphosphates with chain lengths n = 3, 9 and 208, K_m values were 1·7 × 10^?4, 1·5 × 10^?5 and 8·8 × 10^?7 M respectively. Polyphosphatase was most active and stable at pH 6·0–8·0. The enzyme showed maximal activity at 50°C. The time of half inactivation of polyphosphatase at 40, 45 and 50°C was 45, 10 and 3 min, respectively. In the absence of divalent cations and also with Ca²⁺ or Cu²⁺, the enzyme showed practically no activity. The ability of divalent cations to activate polyphosphatase was reduced in the following order: Co²⁺ > Mg²⁺ > Mn²⁺ > Fe²⁺ > Zn²⁺. Polyphosphatase was completely inhibited by 1 mM -ammonium molybdate and 50 μM -Zn²⁺ or Cu²⁺ (in the presence of Mg²⁺).     

ACID INVERTASES FROM WHITE YAM, (DIOSCOREA ROTUNDATA) TUBER: PURIFICATION AND CHARACTERIZATION OF TWO ISOENZYMES

ABSTRACT White yam invertase (E.C. 3.2.1.26) was extracted from Dioscorea rotundate tuber and purified 30 fold. The enzyme consists of two molecular forms, termed invertases I and II, with average molecular weights of 263,000 ± 1000 and 230,000 ± 2000, respectively. Both enzymes had a temperature optima of 40C, and pH optima of 4.7 and 6.0 for invertases I and II, respectively. The K_m values for invertases I and II were 4.65 mM and 9.25 mM, respectively, with sucrose as substrate. The enzymes were less specific for maltose, lactose, raffinose, melezitose, planteose, and lychnose than for sucrose.     

Myricetin,an antioxidant flavonol,is a substrate of polyphenol oxidase

The present study demonstrates the antiradical efficiency of myricetin, a flavonol widely distributed in fruits and vegetables, by testing its ability to react with two different free radicals, ABTS^+· and DPPH^·. The polyphenolic nature of myricetin led us to consider the possibility of its oxidation by polyphenol oxidase (PPO). The results reported show that myricetin can be oxidised by PPO extracted and partially purified from broad bean seeds. The reaction was followed by recording spectral changes with time, maximal spectral changes being observed at 372 nm. The presence of two isosbestic points (at 274 and 314 nm) suggested that only one absorbing product was formed. The spectral changes were not observed in the absence of PPO. The oxidation rate varied with the pH, reaching its highest value at pH 5.5. The myricetin oxidation rate increased in the presence of SDS, an activing agent of polyphenol oxidase. Maximal activity was obtained at 1.3 mM SDS. The kinetic parameters were also determined: V _m = 1.35 µM min⁻¹, K _m = 0.3,mM , V _m/ K _m = 4.5 × 10⁻³ min⁻¹. Flavonol oxidation was inhibited by a selective PPO inhibitor such as cinnamic acid (K_I = 1 mM ). The results reported show that myricetin oxidation was strictly dependent on the presence of polyphenol oxidase. © 1999 Society of Chemical Industry     

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.