Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8–11

A protease from sorghum malt variety KSV8–11 was purified by a combination of dialysis against 4 M sucrose, ion‐exchange chromatography on Q‐Sepharose (Fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 5‐fold to give a 14.1% yield relative to the total activity in the crude extract and a final specific activity of 1348.9 U mg^?1 protein. SDS‐PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 16 KDa. Using casein as substrate, the purified protease had optimal activity at 50°C and maximal temperature stability between 30°C and 40°C but retained over 64% of its original activity after incubation at 60°C for 30 min. The pH optimum was 5.0 with maximum stability at pH 6.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. The protease was inhibited by Ag⁺, Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺, iodoacetic acid (IAA) and p‐chloromercuribenzoate (p‐CMB), stimulated by Cu²⁺, Sr²⁺, phenylmethylsulfonyl‐fluoride (PMSF) and 2‐mercaptoethanol (2‐ME) while Mn²⁺ and ethylenediaminetetraacetic acid (EDTA) had no effect. The purified enzyme had a K_m of 18 mg·mL^?1 and a V_max of 11.1 μmol · mL^?1 · min^?1 with casein as substrate.     

Effects of physicochemical factors and in vitro gastrointestinal digestion on antioxidant activity of R‐phycoerythrin from red algae Bangia fusco‐purpurea

R‐phycoerythrin (R‐PE) was purified from the red algae Bangia fusco‐purpurea after 35–50% ammonium sulphate fractionation followed by ion‐exchange column chromatography on DEAE‐Sepharose, resulting in a purity (A₅₆₅/A₂₈₀) ratio of 5.1. The circular dichroism spectroscopy results suggested that the structure of R‐PE is predominately helical. The antioxidant activity of R‐PE was studied and revealed changes in conformation and antioxidant activity at different temperatures and pH values. After in vitro‐simulated gastrointestinal (GI) digestion of R‐PE, the scavenging activity of ABTS radical (EC₅₀, 769.9 μg mL^?1), DPPH radical (EC₅₀, 421.9 μg mL^?1), hydroxyl radical (EC₅₀, 32.4 μg mL^?1) and reducing power (A₇₀₀ = 0.5, 625.8 μg mL^?1) were measured. Gel filtration chromatography analysis showed that the molecular weight distribution of the final GI digest that still contained high antioxidant activity was <3 kDa. Our present results indicate that digestion‐resistant antioxidant peptides of R‐PE may be obtained by in vitro GI proteinases degradation.     

Isolation,purification and properties of the peroxidase from the hull of Glycine max var HH2

Soybean hull peroxidase (EC 1.11.1.7), an acidic peroxidase isolated from soybean (Glycine max var HH2) hulls was purified to electrophoretic homogeneity by a combination of ammonium sulphate fractionation, DEAE‐Sephadex A‐50 chromatography, concanavalin A‐Sepharose 4B affinity chromatography and Bio‐Gel P‐60 gel filtration. The specific activity of purified peroxidase was about 57‐fold higher than that of crude extract. The yield was about 16.4%. The molecular weight of the enzyme was estimated to be 38 000 by SDS‐polyacrylamide gel electrophoresis. The peroxidase was a glycoprotein containing about 18.7% carbohydrate, approximately one‐quarter of which was shown to be glucosamine residues. It was found to have an isoelectric point of 3.9. The enzyme was most active at pH 4.6 and 45°C, and was stable in the pH range 2.5–11.5. The enzyme could tolerate heating for 10 min at 75°C without being inactivated, and at 85°C, it took 40 min to inactivate the enzyme 50%, confirming that the peroxidase was a novel thermostable enzyme. Fe ²⁺, Fe³⁺, Sn²⁺, CN ⁻ and N₃⁻ inhibited enzyme activity, while Hg²⁺, Ag⁺, Pb ²⁺, Cr³⁺, EDTA and SDS were not significantly inhibitory. © 1999 Society of Chemical Industry     

In vitro effects of steroidal saponins from Yucca schidigera extract on rumen microbial protein synthesis and ruminal fermentation

In a first experiment, ground alfalfa hay and rolled barley grain were incubated in buffered ruminal fluid with and without Yucca schidigera extract (YE, 0 or 10 mg ml⁻¹). Gas and total VFA production from barley grain were increased (P < 0.05) by YE during the first 10 h of incubation; from alfalfa hay, these were reduced (P < 0.001) throughout the 24 h. Yucca extract reduced (P < 0.001) acetate/propionate ratios and ammonia concentrations, irrespective of substrate. In a second experiment, ground barley grain was incubated in a buffered suspension of mixed ruminal microbes obtained by low‐speed centrifugation of ruminal fluid. Steroidal saponins (SAP) isolated from YE were included at 0, 15, 75 or 225 µg ml⁻¹. Microbial incorporation of ¹⁵N was increased (P < 0.05) by 15 µg SAP ml⁻¹ but decreased (P < 0.05) by 225 µg SAP ml⁻¹. Gas and VFA production peaked with 75 µg SAP ml⁻¹ and were elevated (P < 0.05) relative to control in the presence of 75 µg SAP ml⁻¹. Microbial protein synthesis was increased (P < 0.05) by SAP at 15 µg ml⁻¹ and reduced (P < 0.05) by the higher concentrations. Acetate/propionate ratios were linearly reduced by SAP from 8 to 24 h incubation (P < 0.01). The effects of SAP on digestive microbes were less pronounced (P < 0.05) with barley grain digestion than with alfalfa. A YE × diet interaction was recorded. The biological activity of YE was shown to be attributable to its SAP and to be diet‐dependent. © 2000 Society of Chemical Industry. Contributions of Y Wang, T A McAllister, L J Yanke and Z Xu © Minister of Public Works and Government Services Canada 2000     

Antioxidant activity and characterization of volatile constituents of beechwood creosote

Volatile constituents of beechwood creosote were determined using gas chromatography (GC) and gas chromatography–mass spectrometry (GC‐MS). The major volatile constituents of creosote were 2‐methoxyphenol (guaiacol; 25.2%) 2‐methoxy‐4‐methylphenol (4‐methylguaiacol; 21.4%), 3‐methylphenol (m‐cresol; 8.3%) 4‐methylphenol (p‐cresol; 7.9%) 2‐methylphenol (o‐cresol; 4.6%) and phenol (2.8%). The antioxidant activity of creosote was evaluated by three different chemical assays. Beechwood creosote exhibited potent inhibitory effects on the formation of conjugated diene hydroperoxides (from methyl linoleate) at concentrations of 500, 750 and 1000 µg ml^?1. Creosote had a potent inhibitory effect on the oxidation of hexanal for 40 days at a level of 5 µg ml^?1 and also inhibited malonaldehyde (MA) formation from ethyl arachidonate by 92% at a level of 50 µg ml^?1. The antioxidative activity of creosote was comparable with that of the well‐known antioxidants α‐tocopherol and BHT in the hexanal assay. However, creosote displayed comparatively lower antioxidant activity in the other two assays. Copyright © 2005 Society of Chemical Industry     

Purification and characterization of pectin methylesterase from acerola (Malpighia glabra L.)

The enzyme pectinmethylesterase (PME) from acerola was extracted and purified by gel anion‐exchange chromatography (Q Sepharose) and filtration on Sephadex G‐100. The results showed two different PME isoforms (PME1 and PME2), with molecular masses of 25.10 and 5.20 kDa, respectively. PME1 specific activity increased by 9.63% after 60 min incubation at 98 °C, while PME2 retained 66% of its specific activity under the same conditions. The K_m values of PME1, PME2 and concentrated PME were 0.94, 0.08 and 0.08 mg mL^?1, respectively. The V_max value of PME1, PME2 and concentrated were 204.08, 2, 158.73 and 2.92 µmol min^?1 mg^?1 protein, respectively. Copyright © 2007 Society of Chemical Industry     

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⁺).     

Antigenotoxicity of extracts from Pleurotus citrinopileatus

While Pleurotus citrinopileatus is a widely used edible mushroom, little is known about its physiological effects. Extracts, including aqueous extract, water‐soluble polysaccharide (WSP), crude protein solution (CPS) and residue from chloroform–ethyl acetate–methanol elution (CEM), were obtained first from fruiting bodies, through a solid‐state culture, and then from the mycelium, through a submerged culture. This study explored the antigenotoxicity effects of these extracts from Pleurotus citrinopileatus via the Ames test and a spore rec‐Assay. The results showed that, regardless of where the extract came from, the fruiting body or the mycelium, the antigenotoxicity effect was highest for CEM, followed by CPS, aqueous extract and WSP. The results of the Ames test indicated that, among several mutagens, CEM had the highest inhibition rate against AFB_l in TA98 and TA100 and the lowest inhibition rate against NQNO. The concentrations of the various extracts were as follows: water extracts were 1 mg ml^?1 and 5 mg ml^?1 WSP, while CPS and CEM were 0.4 mg ml^?1 and 2 mg ml^?1, respectively; the higher the concentration of the extract, the higher the antimutagenicity effect. The results of the rec‐Assay indicated that CEM had the highest anti‐DNA‐damaging activity with or without the S9 mixture; the higher the concentration, the more significant the effect (p < 0.05). The anti‐DNA‐damaging activities were lower in the water extract concentrations, at 30 µg disc^?1 dry weight^?1, while the WSP, CPS and CEM at 12, 150 and 60 µg disc^?1, respectively, were high. Copyright © 2004 Society of Chemical Industry     

PURIFICATION AND CHARACTERIZATION OF A SERINE PROTEINASE FROM THE TUNA PYLORIC CAECA

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G‐100, ion‐exchange chromatography on a DEAE‐Sephadex α‐50 and gel filtration chromatography on a Sephadex G‐75 column. The purification and yield were 30.5‐fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH 7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg²⁺ and Zn²⁺) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K_m and V_max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.     

PURIFICATION AND PROPERTIES OF CYCLODEXTRIN GLUCANOTRANSFERASE FROM AN ALKALOPHILIC BACILLUS sp. 7-12

Cyclodextrin glucanotransferases (EC 2.4.1.19) (CGTase) are industrially important enzymes for production of cyclodextrin (CD) from starch. γ‐CD yield of CGTase from alkalophilic Bacillus species is usually much lower than β‐CD, while from alkalophilic Bacillus sp. 7‐12. γ‐CD yield is close to β‐CD. A CGTase from alkalophilic Bacillus sp. 7‐12 was purified and characterized. When purified by ammonium sulfate fractionation, DEAE‐cellulose column chromatography and Sepharose CL‐6B column chromatography, the enzyme obtained consisted of a single band that did not dissociate into subunits by SDS polyacrylamide gel electrophoresis. Molecular weight of the purified enzyme was determined to be 69,000 Da by SDS‐PAGE. The enzyme showed a K_mof 1.24 mg/mL and V_max0.101 µM/min when potato starch was used as substrate. The enzyme was stable below 70C with an optimum activity at 60C, and stable at pH range 6–10 with an optimum pH at 8.5. The enzyme activity was strongly inhibited by Ag⁺, Cu²⁺, Mg²⁺, Al³⁺, Co²⁺, Zn²⁺, Fe²⁺and slightly inhibited by Sn²⁺, Mn²⁺. The ions Ca²⁺and K⁺, EDTA and DTT had no influence on the enzyme activity.     

Isolation of a free radical‐scavenging antioxidant from water spinach (Ipomoea aquatica Forsk)

Ipomoea aquatica Forsk, a green leafy vegetable that is a rich source of vitamins and amino acids with many health benefits, has been explored for the isolation and identification of its bioactive compounds. Activity‐guided repeated fractionation of a methanol extract on a silica gel column followed by an XAD column yielded a compound that exhibited antioxidant activity with an EC₅₀ value of 83 ± 1.02 µg ml^?1 reaction mixture. It also showed very strong lipid peroxidation‐inhibitory activity in a liposome model system with an EC₅₀ value of 72.2 ± 0.9 µg ml^?1. However, it showed negligible metal‐chelating activity. Based on UV, 2D nuclear magnetic resonance and gas chromatography/mass spectrometry studies, the compound was tentatively identified to be 7‐O‐β‐D ‐glucopyranosyl‐dihydroquercetin‐3‐O‐α‐D ‐glucopyranoside. This is the first report on the antioxidant properties of I aquatica leaf extracts. Copyright © 2005 Society of Chemical Industry     

Formation of fumonisins and other secondary metabolites by Fusarium oxysporum and F. proliferatum: a comparative study

The principal aim of this study was to estimate the formation of fumonisins (FB₁ and FB₂), moniliformin (MON), and ergosterol (ERG) by Fusarium oxysporum and Fusarium proliferatum, while the formation of beauvericin (BEA) was estimated by the latter Fusarium species only. Moreover, the effect of temperature on the biosynthesis of mycotoxins was also evaluated. Fumonisins were formed by F. proliferatum, with the highest yield at 18°C (720.0–1976.6 µg g^?1 for FB₁, 74.2–670.8 µg g^?1 for FB₂) and only by three of four F. oxysporum strains at a very low level (0.02–4.77 µg g^?1 for FB₁, 0.02–2.15 µg g^?1 for FB₂). The amount of MON formed by F. proliferatum was the highest (p < 0.001) at 32°C (3056.87 µg g^?1), while MON biosynthesis by F. oxysporum was lower 227.54 µg g^?1 (p < 0.001). BEA was produced by F. proliferatum with the highest level at 25°C (p < 0.001). ERG–recognized as an indicator of fungal biomass development and as a consequence of mycotoxin formation–was found at the highest concentration at a biosynthesis temperature of 25°C for F. proliferatum and F. oxysporum (p < 0.001).     

Purification and enzymatic characteristics of a novel polyphenol oxidase from lotus seed (Nelumbo nucifera Gaertn.)

A polyphenol oxidase (PPO) from lotus seed was purified by the procedures including ammonium sulphate precipitation and affinity chromatography. The apparent molecular mass was 38.6 kDa by SDS‐PAGE. Kinetic studies showed that the K_m and V_max values for catechol were 6.04 mm and 416.67 U, respectively. The PPO performed optimal activity in 20 °C and pH 7.0. The enzymatic activity could be mainly maintained up to 50 °C and pH 4.0–7.0. The activity could be inhibited by various inhibitors including thiourea, urea, sodium hydrogen sulphite, EDTA·2Na, SDS, citric acid, guanidine hydrochloride, ascorbic acid, sodium sulphite and sodium thiosulphate. The metal ions Ba²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺ and Zn²⁺ could inhibit the activity of PPO, while Cu²⁺ performed obvious enhancement. The enzymatic properties of PPO could probably provide practical application in inhibiting the PPO activity and preventing enzymatic browning in the process of picking, transportation, processing and storage of fresh lotus seeds.     

Antioxidant and antimicrobial activities of Polygonum cognatum Meissn extracts

The antioxidant activities, reducing powers, 2,2‐diphenyl‐l‐picrylhydrazyl (DPPH) radical‐scavenging activities, total phenolic compound contents and antimicrobial activities of ether, ethanol and hot water extracts of Polygonum cognatum Meissn were studied in vitro. The highest antioxidant activity was found in the water extract. However, there were no statistically significant differences among 15 µg ml^?1 extract‐containing samples in linoleic acid emulsion (0.02 M , pH 7.0) during 120 h of incubation (P > 0.05). The reducing power of the water extract was the highest, but its reducing power was markedly lower than that of ascorbic acid. The highest DPPH radical‐scavenging activity was found in the water extract, with 50% DPPH radical scavenging at a concentration of 100 µg ml^?1 dried water extract, while at the same concentration of dried ethanol extract the value was 12%. Surprisingly, no DPPH radical‐scavenging activity was observed in the ether extract. The concentrations of phenolic compounds found were 0.48, 0.50 and 0.01 µg ml^?1 gallic acid equivalent in 10 µg ml^?1 water, ethanol and ether extracts respectively. The ether and ethanol extracts showed antimicrobial activity against Staphylococcus aureus and Bacillus subtilis. The water extract did not show antimicrobial activity against the studied micro‐organisms. © 2002 Society of Chemical Industry     

Purification and some properties of a novel raw starch‐digesting amylase from Aspergillus carbonarius

A medium was developed to obtain the maximum yield of raw starch‐digesting amylase from Aspergillus carbonarius (Bainier) Thom IMI 366159 in submerged culture with raw starch as the sole carbon source. The amylase was purified to apparent homogeneity by sucrose concentration and ion exchange chromatography on S‐ and Q‐Sepharose (fast flow) columns. SDS‐PAGE revealed two migrating protein bands corresponding to relative molecular masses of 31.6 and 32 KDa. The enzyme was optimally active at pH 6.0–7.0 and 40 °C, was uninfluenced across a relatively broad pH range of 3.0–9.0 and retained over 85% activity between 30 and 80 °C after 20 min incubation. The enzyme was strongly activated by Co²⁺ and only slightly by Fe²⁺, while Ca²⁺, Hg²⁺, EDTA and N‐bromosuccinamide elicited significant repression of the enzyme activity. The enzyme hydrolysed amylopectin (K_m 0.194 mg ml ⁻¹), glycogen (K_m 0.215 mg ml ⁻¹), pullulan (K_m 0.238 mg ml ⁻¹), amylose (K_m 0.256 mg ml ⁻¹) and raw potato starch (K_m 0.260 mg ml ⁻¹), forming predominantly maltose and relatively smaller amounts of glucose. © 2000 Society of Chemical Industry     

Purification and characterisation of β‐mannanase from Lactobacillus plantarum (M24) and its applications in some fruit juices

β‐Mannanase was purified 2619.05‐fold from the Lactobacillus plantarum (M24) bacterium by ammonium sulphate precipitation and ion exchange chromatography (DEAE‐Sephadex). The purified enzyme gave two protein bands at a level of approximately 36.4 and 55.3 kDa in the SDS‐PAGE. The purified mannanase enzyme has shown its maximum activity at 50 °C and pH 8, and it has been also determined that the enzyme was stable at 5–11 pH range and over 50 °C. The V_max and K_m values have been identified as 82 mg mannan mL^?1 and 0.178 mm , respectively. The effects of some metal ions such as Fe²⁺, Ca²⁺, Co²⁺, Ni²⁺, Mn²⁺, Cu²⁺ and Zn²⁺ on the mannanase enzyme have been also investigated, and it has been determined that all metal ions had significant effects on the activation of the mannanase enzyme. In addition, the effectiveness of the purified mannanase enzyme on the clarification of some fruit juices such as orange, apricot, grape and apple has been investigated. During the clarification processes, the enzyme was more effective than crude extracts on the clarification of the peach juice with a ratio of 223.1% at most.     

Antioxidant activity of bigeye tuna (Thunnus obesus) head protein hydrolysate prepared with Alcalase

The antioxidant activities of tuna head protein hydrolysate (THPH) prepared with Alcalase were evaluated. THPH showed evident radical scavenging activity in a dose‐dependent manner with the IC₅₀ values for 1,1‐diphenyl‐2‐pycrylhydrazyl (DPPH), superoxide and hydroxyl radicals being 1.34, 1.20 and 2.84 mg mL^?1, respectively, and its reducing power was 0.948 at 12.5 mg mL^?1. THPH showed good inhibitory activity in soybean oil peroxidation after accelerated oxidation at 60 °C, and the oils with 0.01%, 0.05% and 0.1% THPH had significantly (P < 0.05) lower peroxide values than the control, after storage at 60 °C. Moreover, the inhibited oxidation effect of 0.1% THPH was similar to that of 0.01% butylated hydroxytoluene (BHT). The molecular weight distribution of THPH revealed that 70.5% of the total amount was peptides with molecular weight lower than 5000 Da, composing mostly of low molecular weight peptides located at 1020–2585 Da (30.78%) and 241–1020 Da (37.15%).     

Polyphenol oxidase activity in grass and its effect on plant‐mediated lipolysis and proteolysis of Dactylis glomerata (cocksfoot) in a simulated rumen environment

Little is known about the level or activity of polyphenol oxidase (PPO) in grasses and its potential impact on proteolysis and lipolysis. Six grass species were initially screened for PPO activity (740.6, 291.9, 213.6, 119.0, 16.3 and 6.5 U g^?1 fresh weight (FW) for cocksfoot, hybrid ryegrass, Italian ryegrass, perennial ryegrass, timothy and tall fescue respectively). Cocksfoot, which expressed the highest activity, was then used to determine the effect of PPO on plant‐mediated proteolysis and lipolysis in a simulated rumen environment. Sourced cocksfoot was macerated and incubated in an antibiotic‐containing anaerobic medium with or without ascorbate to deactivate PPO in the dark at 39 °C over five time points. At each time point (0, 1, 2, 6 and 24 h), six replicate samples were destructively harvested; three of the replicates were used for lipid analysis and the other three for protein, free amino acid and bound phenol determination. Characterisation of the herbage showed PPO activities of 649.6 and 0 U g^?1 FW, which were reflected in the extent of phenol (derived from quinones) binding to protein after 24 h of incubation, namely 65.1 and 29.6 mg bound phenol g^?1 protein (P < 0.001) for cocksfoot and cocksfoot + ascorbate respectively. Proteolysis, measured as free amino acids released into the incubation buffer, was significantly reduced (P < 0.001) with increasing PPO activity, with values after the 24 h incubation of 0.03 and 0.07 mmol L^?1 g^?1 FW for cocksfoot and cocksfoot + ascorbate respectively. Lipolysis, measured as the proportional decline in the membrane lipid polar fraction, was likewise reduced (P < 0.001) with increasing PPO activity, with values after the 24 h incubation of 0.43 and 0.65 for cocksfoot and cocksfoot + ascorbate respectively. Changes that occurred in protein and the lipid fractions (polar fraction, monoacylglycerol + diacylglycerol, triacylglycerol and free fatty acids) during the incubations are also reported and discussed. These results support the selection of forages high in PPO activity to reduce protein and lipid losses in silo and potentially in the rumen. Copyright © 2006 Society of Chemical Industry     

Purification and biochemical characterisation of a novel glutamate decarboxylase from rice bran

BACKGROUND: Glutamate decarboxylase (GAD) is a useful enzyme whose main function is to catalyse the irreversible α‐decarboxylation of L ‐glutamate to produce γ‐aminobutyric acid. The cheap and abundant rice‐processing by‐product rice bran contains a high amount of GAD, the purification and characterisation of which have not yet been reported. In this study, research on rice bran GAD was initiated. RESULTS: Rice bran GAD was purified to homogeneity via a combined purification protocol of ammonium sulfate fractionation, ion exchange chromatography and two gel filtrations, with a purification fold of 128.6 and an activity recovery of 21.3%. The enzyme was active at pH 5.5 and 40 °C and retained 80% of its original activity in the pH range 5–9 and the temperature range 30–50 °C. GAD activity was significantly enhanced in the presence of Ca²⁺ but strongly inhibited by Ag⁺, Hg²⁺, sodium dodecyl sulfate and CH₃COOH. Kinetic determination of the apparent K_m for L ‐glutamate and pyridoxal 5′‐phosphate gave values of 27.4 mmol L^?1 and 1.16 µmol L^?1 respectively. CONCLUSION: Considering that rice bran is cheap and commercially available and that rice bran GAD is relatively stable, the development of cost‐effective rice bran GAD‐related functional foods would seem to be feasible. Copyright © 2010 Society of Chemical Industry     

Purification and characterisation of carrot (Daucus carota L) pectinesterase

A pectinesterase isoform with an alkaline isoelectric point of over 8.66 was detected in crude extracts of carrot. The enzyme was purified by ion exchange and molecular exclusion chromatography. The molecular weight of the isoform was 25 kDa, determined in native conditions by filtration through Sephadex G‐75 SF. The enzyme showed a high affinity for its substrate, with K_m and V_max values of 0.031 mg ml^?1 and 6.77 units respectively for apple pectin. The pectinesterase activity exhibited an optimum around pH 7.4 and was activated by metallic ions, with optimum activities at NaCl concentrations between 130 and 330 mM and at CaCl₂ concentrations between 15 and 50 mM . The enzyme was activated most by Ca²⁺ and exhibited a greater tolerance of high concentrations of Na⁺. Comparison of its heat stability with other pectinesterases of vegetable origin indicated that the purified isoform was very thermolabile, being rendered inactive by heating for 5 min at 70 °C. The enzyme was inhibited by high concentrations of polygalacturonic acid and competitively inhibited by D ‐galacturonic acid, with a K_i value of 1 mM . Copyright © 2003 Society of Chemical Industry