KINETICS OF THE INTERACTION OF PANCREATIC α-AMYLASE WITH A KIDNEY BEAN (PHASEOLUS VULGARIS) - AMYLASE INHIBITOR

An amylase inhibitor isolated from black beans (Phaseolus vulgaris) can completely inhibit porcine pancreatic α-amylase forming a 1:1 stoichiometric complex. The kinetic pattern of complex formation is pH dependent. At pH 5.5 it follows a first order reaction with rate constant of 0.029 min^?1 and 0.017 min^?1 at 37°C and equimolar inhibitor and enzyme concentration, respectively, of 10^?8 M and 10^?9 M. At pH 6.9 it is a second order reaction, with a rate constant of 0.25 × 10⁶ M^?1 min^?1 at 37°C, with 4 × 10^?8 M concentrations of enzyme and inhibitor. The dissociation constants of the enzymeinhibitor complex are 1.7 × 10^?10 M at pH 5.5 and 4.4 × 10^?9 M at pH 6.9, at 37°C. The kinetic data obtained at pH 5.5 suggested the formation of an initial reversible complex followed by a conformational change step. The complex can be dissociated either in acid pH (4.3) or at pH values higher than 6, 5 with partial recovery of the amylase activity.     

Neutral and acidic ascorbate oxidases from satsuma mandarin (Citrus unshiu Marc): Isolation and properties

The neutral and acidic forms of ascorbate oxidase (AAO) were isolated and purified from young fruits of satsuma mandarin by a combination of (NH₄)₂SO₄ fractionation, ion-exchange and hydrophobic chromatography, and gel filtration. The neutral and acidic AAO were both diamers of two subunits with native molecular weights of 133 and 136 kDa, respectively. Neutral AAO was stable in the range of pH 5 and 8 and exhibited optimal activity at pH 5.5 and 45°C. In contrast, acidic AAO was stable between pH 5 and 10 and exhibited optimal activity at pH 5.5 and 50°C. When L-ascorbate and D-isoascorbate were used as substrates, K_m values of neutral AAO were 2.98 × 10^?4 M and 3.36 × 10^?4 M and those of acidic AAO were 1.99 × 10^?4 M and 1.86 × 10^?3 M, respectively. The activities of the two forms of AAO were totally inhibited by metabisulphite and cyanide, substantially suppressed by higher concentrations of diethyldithiocarbamate and fluoride, and not inhibited at all by monovalent and divalent metal ions tested.     

The regulatory role of the tetrapeptide AcSDKP in skin and hair physiology and the prevention of ageing effects in these tissues – a potential cosmetic role

The naturally occurring tetrapeptide acetyl‐N‐Ser‐Asp‐Lys‐Pro (AcSDKP) recognized as a potent angiogenic factor was shown recently to contribute to the repair of cutaneous injuries. In the current article, we report the ability of AcSDKP to exert a beneficial effect on normal healthy skin and scalp and to compensate for the ageing process. In vitro AcSDKP at 10^?11–10^?7 M significantly stimulates the growth of human keratinocytes, fibroblasts and follicle dermal papilla cells. Moreover, it enhances the growth of human epidermal keratinocyte progenitor and stem cells as shown in a clonogenic survival assay. Topical treatment of ex vivo cultured skin explants with 10^?5 M AcSDKP increases the thickness of the epidermis and upregulates the synthesis of keratins 14 and 19, fibronectin, collagen III and IV as well as the glycoaminoglycans (GAGs). In the ex vivo‐cultured hair follicles, AcSDKP promotes hair shaft elongation and induces morphological and molecular modifications matching the criteria of hair growth. Furthermore, AcSDKP at 10^?11–10^?7 M was shown to improve epidermal barrier, stimulating expression of three protein components of tight junctions (claudin‐1, occludin, ZO‐1) playing an important role in connecting neighbouring cells. This tetrapeptide exercises also activation of SIRT1 implicated in the control of cell longevity. Indeed, a two‐fold increase in the synthesis of SIRT1 by cultured keratinocytes was observed in the presence of 10^?11–10^?7 M AcSDKP. In conclusion, these findings provide convincing evidence of the regulatory role of AcSDKP in skin and hair physiology and suggest a cosmetic use of this natural tetrapeptide to prevent skin ageing and hair loss and to promote the cutaneous regeneration and hair growth.     

PARTIAL CHARACTERIZATION OF TRYPSIN-CHYMOTRYPSIN INHIBITORS FROM BEAN (PHASEOLUS VULGARIS L., VAR. ROSINHA G2): CHEMICAL AND PHYSICAL PROPERTIES

The three trypsin inhibitors A, B and C previously isolated from Brazilian pink bean (Phaseolus vulgaris L. var. Rosinha G2) had molecular weights of 18,200 to 18,500 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, 20,000 by gel filtration on Sephadex G-100 and 20,400 by sucrose density gradient ultracentrifugation with a Stokes molecular radius of 20 Å, a frictional coefficient of 1.14, a diffusion coefficient of 10.7 × 10^?7 cm²s^?1, a partial specific volume of 0.69 cm³g^?1 and a molar absorptivity of 5.5 × 10³ M^?1 cm^?1 at 280 nm. All three inhibitors bound two moles of trypsin and one mole of chymotrypsin. The K_i values for trypsin were: A, 8.5 × 10^?10 M; B, 1.8 × 10^?10 M and C, 6.8 × 10^?10 M while for chymotrypsin they were: A, 4.4 × 10^?7 M; B, 2.8 × 10^?8 M and C, 3.0 × 10^?8 M. Reductive methylation caused loss of inhibitor activity of all three inhibitors against trypsin without significantly affecting inhibitor activity against chymotrypsin (with exception of inhibitor B), indicating that the inhibitors have lysine in binding site for trypsin. Partial reduction of the disulfide bonds caused loss of inhibitor activity against both trypsin and chymotrypsin with some regain of inhibitor activity following dialysis. Cyanogen bromide cleaved all three inhibitors into two fragments with significant retention of inhibitor activity. Cyanogen bromide-treated inhibitor B had nearly twice the original inhibitor activity against trypsin with no loss of inhibitor activity against chymotrypsin.     

PURIFICATION AND CHARACTERIZATION OF LIPOXYGENASE ISOZYMES FROM CANOLA (Brassica napus cv, WESTAR) SEED

Four isozymes, I', II'a, II'b and III’ of lipoxygenase (EC 1.13.11.12) from Canola (Brassica napus, cv Westar) seed were purified by successive chromatography on ion-exchange and size-exclusion columns using a Fast Protein Liquid Chromatograph (FPLC). The homogeneity of each isozyme was demonstrated by a single protein band on SDS-polyacrylamide gel electrophoresis. The molecular weights of isozymes I', II'a, II'b and III’ were 72, 000, 106, 000, 78, 000 and 62, 000, respectively. The optimum pH for lipoxygenase activity was 6.5 for isozyme I’ and 6.0 for isozymes II'b, II'b and III'. Apparent K_m value for isozymes I', II'a, II'b and III’ were 5.5 × 10^?4 M, 3.4 × 10^?4 M, 4.0 × 10^?4 M and 3.8 × 10^?4 M, respectively. Isozyme I’ displayed preferential activity towards monolinoleate and dilinoleate, while isozyme II'a demonstrated preferential activity towards dilinoleate followed by mono- and trilinoleate. No enzymatic activity was observed with both isozymes I’ and II'a toward free linoleic acid. Isozyme II'b showed activity towards free linoleate as well as mono-, di- and trilinoleate. Isozyme III’ showed preferential activity towards free linoleate. The activity of isozymes I’ and II'a was inhibited completely by the addition of 10 mM and 4 mM KCN, respectively, while the addition of 3 mM and 10 mM KCN to isozymes II'b and III', respectively, increased activity by approximately 20%.     

Microarray studies on the genes responsive to the addition of spermidine or spermine to a Saccharomyces cerevisiae spermidine synthase mutant

The naturally occurring polyamines putrescine, spermidine or spermine are ubiquitous in all cells. Although polyamines have prominent regulatory roles in cell division and growth, precise molecular and cellular functions are not well‐established in vivo. In this work we have performed microarray experiments with a spermidine synthase, spermine oxidase mutant (Δspe3 Δfms1) strain to investigate the responsiveness of yeast genes to supplementation with spermidine or spermine. Expression analysis identified genes responsive to the addition of either excess spermidine (10^?5 M ) or spermine (10^?5 M ) compared to a control culture containing 10^?8 M spermidine. 247 genes were upregulated > two‐fold and 11 genes were upregulated >10‐fold after spermidine addition. Functional categorization of the genes showed induction of transport‐related genes and genes involved in methionine, arginine, lysine, NAD and biotin biosynthesis. 268 genes were downregulated more than two‐fold, and six genes were downregulated > eight‐fold after spermidine addition. A majority of the downregulated genes are involved in nucleic acid metabolism and various stress responses. In contrast, only a few genes (18) were significantly responsive to spermine. Thus, results from global gene expression profiling demonstrate a more major role for spermidine in modulating gene expression in yeast than spermine. Copyright © 2009 John Wiley & Sons, Ltd.     

STUDIES ON BLACK GRAM (Phaseolus mungo L.) TRYPSIN INHIBITOR

ABSTRACT A trypsin inhibitor isolated from black gram (Phaseolus mungo L.) had 75 amino acid residues with an estimated molecular weight of 7892. The kinetic constants K_m and V_max as evaluated by the Dixon and Cornish-Bowden plots were 2.7 × 10^?5and 6 × 10^?3M/min, respectively. The dissociation constants of the enzyme-inhibitor complex (Ki) and the enzyme-inhibitor-substrate complex (Ki') were respectively 4 × 10^?7M and 1.9 × 10^?6M. Trypsin inhibition by black gram trypsin inhibitor was of a linear-mixed type. Chemical modification studies suggested the possible involvement of lysine and arginine at the active site of the inhibitor.     

α-, γ- and δ-Tocopherol Effects on Chlorophyll Photosensitized Oxidation of Soybean Oil

The effects of 0, 1.0, 2.0 and 4.0 (x 10^?3 M) α-, γ- or δ-tocopherol on chlorophyll b photosensitized oxidation of soybean oil in methylene chloride were studied by peroxide values and headspace oxygen. As concentrations of tocopherols increased, peroxide values decreased and headspace oxygen increased (P < 0.05). At 1.0 × 10^?3 M, α-tocopherol showed highest antioxidant effect, γ-tocopherol second and then δ-tocopherol. α- and -γ-Tocopherols had similar effects and δ-tocopherol had lower effect at 2.0 × 10^?3 M (P < 0.05). However, the three tocopherols were not different (P > 0.05) at 4.0 × 10^?3 M. α-Tocopherol quenched singlet oxygen to reduce the photosensitized oxidation of oil. The quenching rate constants of α-tocopherol were 2.7 × 10⁷M^?1sec^?1 by peroxide value and 2.6 × 10⁷ M^?1sec^?1 by headspace oxygen.     

The influence of 2,4-dichlorophenoxyacetic acid on lactic acid bacteria

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) (4 × 10^?5M-4 × 10^?3M) on the growth and physiological activity of Streptococcus lactis subsp. diacetylactis and Lactobacillus casei has been investigated when cultivated in milk and lactose-peptone media. The concentrations of 2,4-D applied in milk did not cause complete growth inhibition of any of the strains. The degree of inhibition was dependent upon the bacterium species and the herbicide dose. Growth of the tolerant strain—L. casei W-10—was reduced by about 40% (in the presence of 4 × 10^?3M 2,4-D), whereas the the level of lactic acid production remained almost unchanged. Under the same conditions, growth of the sensitive strain S. lactis subsp. diacetylactis 239 was reduced by almost 25 times and lactic acid production inhibited by about 40% in comparison with the controls. Cultivation of S. lactis subsp. diacetylactis 239 in lactose-peptone medium with 2 × 10^?3M 2,4-D was characterised by a lengthened lag-phase, lengthened generation time (3 times) and an increased rate of herbicide metabolism. In addition, selective or mutative action of 2,4-D has been shown. Herbicide treatment inhibited the cells of c.f.u. producing the least acid and enhanced the contribution of the active ones. Cells of c.f.u. with activities not found in the control population have also been revealed. Both species of bacteria were capable of metabolism of 2,4-D to 2,4-dichlorophenol.     

Efficiency of modified skimmed milk base media to achieve high exopolysaccharide/cell ratios by Lactobacillus delbrueckii subsp. bulgaricus SZ2 in optimised conditions defined by the response surface methodology

Exopolysaccharide (EPS) production by Lactobacillus delbrueckii subsp. bulgaricus SZ2 was optimised in modified MRS (M‐MRS) using the response surface methodology (RSM). Maximum EPS production was 74.3 ± 2 mg/L, and the optimised values of the three variables predicted for maximum EPS production included a temperature of 38.7 °C, Bacto‐casitone and glucose concentrations of 24.5 and 29.6 g/L, respectively. To compare EPS production in MRS and skimmed milk (SM), the kinetics of EPS formation and growth were monitored in M‐MRS, SM, skimmed milk plus 2% additional sucrose (Suc‐SM) and skimmed milk containing Bacto‐casitone (20 g/L) and yeast nitrogen base (5 g/L) (BY‐SM). EPS production in all the media tested seemed to be growth‐related. The EPS/cell ratios were determined to be 3.12 × 10^?10, 1.43 × 10^?10, 4.42 × 10^?11 and 3.16 × 10^?11 mg/cell, in Suc‐SM, SM, M‐MRS and BY‐SM, respectively, clearly indicating the greater effect of C/N ratio when cell behaviour in EPS production is considered.     

Methyl jasmonate reduces chilling injury symptoms and enhances colour development of ‘Kent’ mangoes

Treatment of mango (Mangifera indica cv ‘Kent’) fruits with methyl jasmonate (MJ) vapour for 20 h at 20 °C reduced chilling injury (CI) symptoms and enhanced skin colour development. MJ at 10^?4 M was the most effective concentration for reducing CI and decay in fruits stored at 5 °C followed by 7 days at 20 °C (shelf‐life period). The use of 10^?5 M MJ enhanced yellow and red colour development of mangoes stored at 20 °C. These fruits possessed higher L*, a* and b* values than untreated fruits and those treated with 10^?4 M MJ. Ripening processes were inhibited by cold storage (5 °C) in control fruits. After cold storage and shelf‐life period, fruits treated with 10^?5 M MJ ripened normally and contained the highest total soluble solids (TSS). These fruits also maintained higher sugar and organic acid levels than fruits subjected to other treatments. We concluded that MJ treatment could be used to reduce decay and CI symptoms and also to improve colour development of mango fruits without adversely affecting quality. © 2001 Society of Chemical Industry     

Inactivation of yeast and filamentous fungi by the lactoperoxidase-hydrogen peroxide-thiocyanate-system

The antifungal activity of the lactoperoxidase (LPO) system with glucose oxidase (GOD) as source of hydrogen peroxide was determined in salt solution and in apple juice. The test organisms Rhodutorula rubra and Saccharomyces cerevisiae were cultivated aerobically in apple juice, Mucor rouxii was grown on wort agar adjusted to pH 4.5. Aspergillus niger and Byssochlamys fulva were kept on malt extract agar. Spores of the filamentous fungi were harvested by suspension in salt solution supplemented with Tween 80® and checked microscopically. The antifungal activity of the combined enzyme system was tested with initial counts of approx. 10⁵ cfu · ml^?1 (yeast cells or spores) suspended in salt solution supplemented with 25 mg · l^?1 thiocyanate and 20 g · l^?1 glucose or in apple juice supplemented with the same amount of thiocyanate. The tests were performed with 25 ml of the medium in 100 ml Erlenmeyer flasks shaken at 28 °C under aerobic conditions. Inactivation was achieved for all test organisms in both media. The yeast strains were found to be least stable while B. fulva was most resistant. A combination of 5 U · ml^?1 LPO with 0.5 to 1 U · ml^?1 GOD was sufficient for complete inactivation of this mold in salt solution within 2 h. The enzyme system also showed antifungal activity in apple juice at acid pH (3.2), although its effectiveness was reduced. In this medium, B. fulva was inactivated by 20 U · ml^?1 LPD and 1 U · ml^?1 GOD within 4 h. R. rubra and S. cerevisiae were unable to survive in apple juice at 5 U · ml^?1 LPO combined with 1 U · ml^?1 GOD. For inhibition by GOD alone, higher amounts of this enzyme were needed and even then only M. rouxii and R. rubra have been affected within the concentration range tested (maximum 3 U · ml^?1).     

Factors influencing permeability of the cell membrane of the osmotolerant yeast Saccharomyces rouxii grown in the presence and absence of 18% NaCl. I. Na+/K+-activated Mg2+-dependent ATPase

It was found that cells of Saccharomyces rouxii contain an ouabain-inhibited ATPase, assumed to be an Na⁺/K⁺-activated Mg²⁺-dependent ATPase, which could serve as a sodium pump protecting the cells in a high salt environment. Twenty-two cell homogenates or supernatants (centrifuged at 3000 × g) grown without added salt in the medium contained sufficient total ATPase activity to liberate (on average) 0.225 μM P_i min^?1 mg^?1 protein. The percentage of total ATPase inhibited by the addition of ouabain (1 × 10^?4 M) varied from 7 to 100%. Cell homogenates or supernatants from cells grown in the presence of 18% NaCl in the media contained sufficient ATPase activity to liberate (on average) 0.114 μM P_i min^?1 mg^?1 protein, about 50% of the total ATPase activity found in the non-salt grown cells. The percentage of total ATPase activity inhibited by ouabain ranged from 16 to 100%. Although the non-salt-grown cells contained approximately double the total ATPase activity of the salt-grown cells. there was evidence that the percentage of total ATPase that is ouabain sensitive (Na⁺/K⁺-activated ATPase) is higher in the salt-grown cells. Also, cells of S. rouxii grown in media without added NaCl, recovered by centrifugation and transferred to media containing 18% NaCl for 16 h and again recovered by centrifugation, homogenized and centrifuged at 10 000 × g contained 61.2% ouabain-sensitive ATPase compared with 21.3% ouabain-sensitive ATPase in the cells before adaptation to the high salt environment.     

Productions and monomer compositions of exopolysaccharides by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains isolated from traditional home-made yoghurts and raw milk

In this study, a total of forty‐five strains of lactobacilli and streptococci were determined exopolysaccharide (EPS) production in skim milk and Man Rogosa and Sharpe (MRS)/M17 medium, viscosity and proteolytic activity. The exopolysaccharide production by lactobacilli strains during growth in MRS medium was twenty‐one to 211 mg L^?1, while in skim milk was to thirty‐six to 315 mg L^?1. The EPS production by streptococci strains during growth in M17 medium was sixteen to 114 mg L^?1, while in skim milk was to twenty‐four to 140 mg L^?1. The EPS production of strains was lower in MRS/M17 medium than skim milk. Results showed that it was not clear correlation between the viscosity and EPS production of some strains. All strains were shown proteolytic activity. Positive correlations between exopolysaccharide production and proteolytic activity in skim milk were found some strains of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. These results indicated that the high exocellular protease‐producing strains can produce high EPS in skim milk. The monomer compositions of the EPSs formed by selected five strains were analysed. Mannose dominated (99–100%) on the EPS produced by L. delbrueckii subsp. bulgaricus and S. thermophilusstrains (except L. delbrueckii subsp. bulgaricus 22) in skim milk and MRS/M17 medium. Besides, the EPSs of strains in skim milk contained small amount of lactose.     

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     

Proteolysis of αs1-Casein by Papain in a Complex Environment. Influence of Ionic Strength on the Kinetic Constants

The influence of α_s1-casein concentration on the hydrolytic activity of papain was studied. High substrate concentration was inhibitory. In presence of NaCl, the papain affinity for α_s1-casein increased (the apparent Michaelis constant (K_m. app.) decreased from 9.4 10^?5 to 5.6 10^?5 M) and was lower for 0.08M NaCl. The maximum velocity (V_max app-) was independant from NaCl concentration below 0.34M but decreased above. The enzyme-substrate affinity was increased by the addition of urea, but the V_max app. decreased. The inhibitory effect of excess substrate was more important with the presence of both urea and NaCl. Acetylation of α_s1-casein showed that K_m. app. (21.2 10^?5M) was independant of salt concentration, while the V_max app. varied, and the substrate inhibitory activity was suppressed.     

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⁺).     

Effect of Electrical Stimulation on Postmortem Property Change of Myofibrillar Proteins

Changes in biochemical properties of myofibrillar proteins of rabbit muscle, which had been subjected to electrical stimulation soon after slaughter, during postmortem storage at 0°C were investigated. Myofibrillar ATPase activity and the ATPase activity of acto-heavy meromyosin (HMM) complex, reconstituted from actin and HMM which had been prepared from at-death and postmortem muscles, decreased at first and then increased slightly during 7 days storage. In addition, the change of the dissociation constant of acto-HMM complex of electrically stimulated muscle during postmortem storage was quite small, i.e., 1.59 ± 10^?4M for at-death muscle, 1.70 ± 10^?4M for muscle stored for 1 day and 1.49 ± 10^?4M for muscle stored for 7 days. This indicates that electrical stimulation treatment minimized the postmortem change of actin-myosin interaction.     

The effect of germination on the phytase activity,phytate and total phosphorus contents of some Nigerian‐grown grain legumes

BACKGROUND: Grain legumes are under‐exploited as possible sources of phytase for the poultry industry. The current study was conducted to assess the effect of germination on phytase activities, phytate and total phosphorus content in samples of Nigerian‐grown grain legumes. The legumes screened were African yambean (AYB, Sphenostylis stenocarpa), lima bean (Phaseolus lunatus), pigeon pea (Cajanus cajan), cowpea (Vigna unguiculata) and groundnut (Arachis hypogea). RESULTS: Phytase activity was low in AYB, lima bean and pigeon pea but high in cowpea and groundnut. Phytate content ranged between 3.01 g kg^?1 and 8.95 g kg^?1 while total phosphorus content ranged between 2.63 g kg^?1 and 5.93 g kg^?1. The grain legumes with higher phytase activity recorded the lowest phytate and phosphorus content. During germination there was an initial 4‐fold to 35‐fold increase in phytase activity after 6–7 days of germination followed by a decrease until 10 days (P < 0.05). The increase in phytase activity during germination was accompanied by a significant reduction in phytate (P < 0.05) and a small but significant increase in total phosphorus. CONCLUSION: The increase in phytase activity and the accompanying decrease in phytate content could have a positive implication for the nutrition of poultry and ruminants and for the environment. Copyright © 2010 Society of Chemical Industry     

Partial characterisation of a new barley amylase

A new amylolytic enzyme found in barley (Hordeum vulgare) grain was partially characterised with respect to physicochemical properties and enzymatic activity. The enzyme preparation showed one antigenically homogeneous amylolytic band. Isoelectric focusing resolved the new amylase into two components, one isoelectric at pH 4.5, the other at pH 3.0. During focusing the original activity of the new amylase decreased by 80%. The purified preparation was inactivated by pH-values below 4.5 and above 9.0 and also by temperatures above + 40°C for 1 h. The new amylase splits the 1,4-α-glycosidic linkages, clearly by endo-attack, of starch, amylopectin, amylose and β-limit dextrin optimally at pH 6.5 at +40°C giving K_m-values 8.9 × 10^?3, 4.4 × 10^?3, 6.6 × 10^?3 and 1.7 × 10^?3 g/ml, respectively. The hydrolysis products from β-limit dextrin were 24% glucose and 46% maltose in the total digest. Mercuric chloride, pCMB,^a EDTA^a and TRIS^a have no noticeable effect on the new amylase, indicating that it is stable under conditions where the other amylolytic enzymes are deactivated. The new amylase seems to be a hydrolase acting on o-glycosyl compounds, EC 3.2.1., but could not be identified with any of the amylolytic enzymes of vegetable origin studied previously.