Characteristics of an Acidic Phytase from Aspergillus aculeatus APF1 for Dephytinization of Biofortified Wheat Genotypes.

Phytases are the special class of enzymes which have excellent application potential for enhancing the quality of food by decreasing its inherent anti-nutrient components. In current study, a protease-resistant, acidic phytase from Aspergillus aculeatus APF1 was partially purified by ammonium sulfate fractionation followed by chromatography techniques. The molecular weight of partially purified phytase was in range of 25-35 kDa. The purified APF1 phytase was biochemically characterized and found catalytically active at pH 3.0 and 50 °C. The Km and Vmax values of APF1 phytase for calcium phytate were 3.21 mM and 3.78 U/mg protein, respectively. Variable activity was observed with metal ions and among inhibitors, chaotropic agents and organic solvents; phenyl glyoxal, potassium iodide, and butanol inhibited enzyme activity, respectively, while the enzyme activity was not majorly influenced by EDTA, urea, ethanol, and hexane. APF1 phytase treatment was found effective in dephytinization of flour biofortified wheat genotypes. Maximum decrease in phytic acid content was noticed in genotype MB-16-1-4 (89.98%) followed by PRH3-30-3 (82.32%) and PRH3-43-1 (81.47%). Overall, the study revealed that phytase from Aspergillus aculeatus APF1 could be effectively used in food and feed processing industry for enhancing nutritional value of food.

Effect of Flavonoid-Rich Extract of Glycyrrhiza glabra on Gut-Friendly Microorganisms, Commercial Probiotic Preparations, and Digestive Enzymes.

Flavonoid-rich extract prepared from Glycyrrhiza glabra has been found to be beneficial in patients with functional dyspepsia and was reported to possess some gut health-promoting properties such as antioxidant, anti-inflammatory and anti-Helicobacter pylori activities. In the present study, the flavonoid-rich extract of Glycyrrhiza glabra was evaluated for its compatibility with probiotic strains (Lactobacillus casei, Lactobacillus fermentum, Lactobacillus plantarum, and Streptococcus thermophilus), commercial probiotic drinks, and digestive enzymes (pancreatic α-amylase, α-glucosidase, phytase, xylanase, and pancreatic lipase). Results of this study indicated that the flavonoid-rich extract of Glycyrrhiza glabra is compatible with the tested probiotic strains, probiotic drinks and digestive enzymes.

Purification and characterization of extracellular phytase from a marine yeast Kodamaea ohmeri BG3.

The extracellular phytase in the supernatant of cell culture of the marine yeast Kodamaea ohmeri BG3 was purified to homogeneity with a 7.2-fold increase in specific phytase activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadextrade mark G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow Anion-Exchange). According to the data from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 98.2 kDa while the molecular mass of the purified enzyme was estimated to be 92.9 kDa and the enzyme was shown to be a monomer according to the results of gel filtration chromatography. The optimal pH and temperature of the purified enzyme were 5.0 and 65 degrees C, respectively. The enzyme was stimulated by Mn(2+), Ca(2+), K(+), Li(+), Na(+), Ba(2+), Mg(2+) and Co(2+) (at a concentrations of 5.0 mM), but it was inhibited by Cu(2+), Hg(2+), Fe(2+), Fe(3+), Ag(+), and Zn(2+) (at a concentration of 5.0 mM). The enzyme was also inhibited by phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid (at a concentration of 1.0 mM), and phenylgloxal hydrate (at a concentration of 5.0 mM), and not inhibited by EDTA and 1,10-phenanthroline (at concentrations of 1.0 mM and 5.0 mM). The K (m), V (max), and K (cat) values of the purified enzyme for phytate were 1.45 mM, 0.083 micromol/ml . min, and 0.93 s(-1), respectively.

Identification and characterization of a phytase of potential commercial interest.

Phytases catalyse the hydrolytic degradation of phytic acid and its salts and are added to monogastric animal feed to ameliorate the negative environmental and nutritional consequences of dietary phytate. Screening of 58 microbial strains identified a phytase produced by Rhizopus oligosporus ATCC 22959 that displayed physicochemical characteristics likely to render it of potential industrial interest. The 124 kDa enzyme was purified to homogeneity by anion exchange chromatography, gel filtration and chromatofocusing. The monomeric glycosylated enzyme (30.5% total carbohydrate) displayed maximum activity at 65 degrees C and pH 5.0. It displayed a Km of 10.4 microM, a Vmax of 1.32 nmols(-1) and a Kcat of 51 s(-1). It is acid tolerant, retaining full activity after incubation at pH 2.0 for 6h. HPLC analysis indicated the enzyme's ability to almost completely degrade phytate. Substrate specificity studies showed its ability to dephosphorylate several additional phosphorylated molecules. Activity was unaffected or moderately stimulated by a range of metal ions with only Ca2+ exerting a modest (13%) inhibitory effect. The enzyme is significantly more thermostable at 80 degrees C and retains a significantly greater proportion of maximal activity at physiological temperatures than do two commercial phytases tested for comparative purposes. This may render it of industrial interest.

Dietary P regulates phosphate transporter expression, phosphatase activity, and effluent P partitioning in trout culture.

Phosphate utilization by fish is an important issue because of its critical roles in fish growth and aquatic environmental pollution. High dietary phosphorus (P) levels typically decrease the efficiency of P utilization, thereby increasing the amount of P excreted as metabolic waste in effluents emanating from rainbow trout aquaculture. In mammals, vitamin D3 is a known regulator of P utilization but in fish, its regulatory role is unclear. Moreover, the effects of dietary P and vitamin D3 on expression of enzymatic and transport systems potentially involved in phosphate utilization are little known. We therefore monitored production of effluent P, levels of plasma vitamin D3 metabolites, as well as expression of phosphatases and the sodium phosphate cotransporter (NaPi2) in trout fed semipu diets that varied in dietary P and vitamin D3 levels. Mean soluble P concentrations varied markedly with dietary P but not with vitamin D3, and constituted 40-70% of total effluent P production by trout. Particulate P concentrations accounted for 25-50% of effluent P production, but did not vary with dietary P or vitamin D3. P in settleable wastes accounted for <10% of effluent P. The stronger effect of dietary P on effluent P levels is paralleled by its striking effects on phosphatases and NaPi2. The mRNA abundance of the intestinal and renal sodium phosphate transporters increased in fish fed low dietary P; vitamin D3 had no effect. Low-P diets reduced plasma phosphate concentrations. Intracellular phytase activity increased but brushborder alkaline phosphatase activity decreased in the intestine, pyloric caeca, and gills of trout fed diets containing low dietary P. Vitamin D3 had no effect on enzyme activities. Moreover, plasma concentrations of 25-hydroxyvitamin D3 and of 1,25-dihydroxyvitamin D3 were unaffected by dietary P and vitamin D3 levels. The major regulator of P metabolism, and ultimately of levels of P in the effluent from trout culture, is dietary P.

[Insect juvenile hormone enhancing gene expression in silkworm baculovirus vector system].

5th instar silkworms were infected with recombinant baculovirus containing phytase gene or wild type BmNPV at 48 hr after ecdysis, then treated with 100 ppm Juvenile hormone. It showed that the expression level of phytase gene and polyhedrin gene per silkworm was increased by 30% and 40%, respectively. The LT50 was lengthened for more than 4 h, and the average weight of sick silkworm was increased by 10%. The results indicated that the improvement of expression efficiency of phytase gene and polyhedrin gene was mainly caused by longer time of virus replication in silkworm after the treatment of Juvenile hormone.

Phytase activity in the small intestinal brush border membrane of the chicken.

The kinetics, mineral dependency, and pH dependency of phytate hydrolysis by preparations of chicken small intestinal brush border membrane vesicles were determined. Substantial phytate hydrolysis occurred over the pH range from 5 to 6.5 with a maximum hydrolysis at pH of 6. Inclusion of 25 mM MgCl2 in the media doubled the rate of phytate hydrolysis. The brush border was shown to have no nonspecific acid phosphatase activity and excess phytate had no effect on alkaline phosphatase activity at pH 11. Under optimal conditions of pH 6 plus 25 mM MgCl2, a kinetic model of a single Michaelis-Menten type of enzymatic activity with a Km of 0.160 +/- 0.008 mM and a Vmax of 42.5 +/- 1.0 nmol/mg vesicle protein per min plus a small unsaturable component converged to the data (P < 0.05). The specific and total activities of intestinal brush border phytase were highest in the duodenum (P < 0.05) and decreased progressively down the length of the gut. Intestinal brush border vesicles prepared from broiler chicks and mature laying hens had comparable specific phytase activity. However, the total activity of brush border phytase was 35% higher in the small intestine of laying hens (P < 0.05). Intestinal brush border phytase could contribute to phytate-phosphorus digestibility and may be subject to regulation in response to the dietary phosphorus and vitamin D status of the chicken.

Phytase activity, phosphorus and calcium retention, and performance of single comb White Leghorn layers fed diets containing two levels of available phosphorus and supplemented with direct-fed microbials.

The presence of phytase activities in condensed cane molasses solubles (CCMS) and CCMS-Lactobacillus (Lacto) were determined. Single Comb White Leghorn layers were fed .25 and .45% available P (AP) diets supplemented with CCMS and CCMS-Lacto for nine 28-d periods to determine phytase activities of the gastrointestinal (GI) tract contents and intestine, liver, and pancreatic tissues, the GI tract pH, the P and Ca retention, and layer performance. Six dietary treatments were corn-soybean (C-S) control, C-S+CCMS, and C-S+CCMS-1,100 mg Lacto/kg diet (ppm) [4.4 x 10(7) cfu/mg Lacto] each with .25 and .45% AP. The CCMS were used as a carrier for the Lacto, and the CCMS and CCMS-Lacto premix were incorporated at 2% of the diets. Phytase activity was much higher in CCMS-Lacto premix than in CCMS. Phytase activities of the crop contents were higher with the CCMS-Lacto diets regardless of the AP level. Intestinal phytase activity was higher with the .45% AP CCMS-Lacto diet than the unsupplemented .45% AP diets. Lactobacillus supplementation did not stimulate phytase activities in the intestinal contents or liver and pancreatic tissues. The pH of the crop and intestinal contents were much lower for the Lacto-fed layers than the layers fed unsupplemented diets regardless of dietary AP levels. No differences in Ca retentions were observed with Lacto supplementation regardless of the dietary AP levels. However, higher P retentions were observed with the Lacto supplementation in the .25% AP diet. Layers fed .25 and .45% AP Lacto-supplemented diets had lower hen-day egg production, poorer feed conversion value, consumed slightly more feed, produced less egg mass, and laid larger eggs than the layers fed .25 and .45% AP unsupplemented diets. Lacto supplementation to .25% AP diet produced eggs with higher specific gravity than the unsupplemented .45% AP diet, but not different from unsupplemented .25% AP diet. Layers fed the .25% AP diets had lower BW gains then layers fed the .45% AP diets regardless of lacto supplementation. Phytase activity was present in the lacto source, and the presence of phytase and Lacto supplementation to a .25% AP diet improved P retention in layers.